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Targeting critical epigenetic regulators reverses aberrant transcription in cancer, thereby restoring normal tissue function1–3. The interaction of menin with lysine methyltransferase 2A (KMT2A), an epigenetic regulator, is a dependence in acute leukaemia caused by either rearrangement of KMT2A or mutation of the nucleophosmin 1 gene (NPM1)4–6. KMT2A rearrangements occur in up to 10% of acute leukaemias and have an adverse prognosis, whereas NPM1 mutations occur in up to 30%, forming the most common genetic alteration in acute myeloid leukaemia7,8. Here, we describe the results of the first-in-human phase 1 clinical trial investigating revumenib (SNDX-5613), a potent and selective oral inhibitor of the menin–KMT2A interaction, in patients with relapsed or refractory acute leukaemia (ClinicalTrials.gov, NCT04065399). We show that therapy with revumenib was associated with a low frequency of grade 3 or higher treatment-related adverse events and a 30% rate of complete remission or complete remission with partial haematologic recovery (CR/CRh) in the efficacy analysis population. Asymptomatic prolongation of the QT interval on electrocardiography was identified as the only dose-limiting toxicity. Remissions occurred in leukaemias refractory to multiple previous lines of therapy. We demonstrate clearance of residual disease using sensitive clinical assays and identify hallmarks of differentiation into normal haematopoietic cells, including differentiation syndrome. These data establish menin inhibition as a therapeutic strategy for susceptible acute leukaemia subtypes.

sFLT01 is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domain of human VEGFR1/Flt-1 (hVEGFR1) fused to the Fc portion of human IgG1 through a polyglycine linker. It binds to both human VEGF (hVEGF) and human PlGF (hPlGF) and to mouse VEGF (mVEGF) and mouse PlGF (mPlGF). In vitro, sFLT01 inhibited the proliferation of human umbilical vein endothelial cells and pericytes stimulated by either hVEGF or hPlGF. In vivo, sFLT01 had robust and significant antitumor activity in numerous preclinical subcutaneous tumor models including H460 non–small cell lung carcinoma, HT29 colon carcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia), 786-O, and RENCA renal cell carcinoma (RCC). sFLT01 also increased median survival in the orthotopic RENCA RCC model. sFLT01 had strong antiangiogenic activity and altered intratumoral microvessel density, blood vessel lumen size and perimeter, and vascular and vessel areas in RCC models. sFLT01 treatment resulted in fewer endothelial cells and pericytes within the tumor microenvironment. sFLT01 in combination with cyclophosphamide resulted in greater inhibition of tumor growth than either agent used alone as a monotherapy in the A673 Ewing's sarcoma model. Gene expression profiling indicated that the molecular changes in the A673 sarcoma tumors are similar to changes observed under hypoxic conditions. sFLT01 is an innovative fusion protein that possessed robust antitumor and antiangiogenic activities in preclinical cancer models. It is a dual targeting agent that neutralizes both VEGF and PlGF and, therefore, has potential as a next generation antiangiogenic therapeutic for oncology. Mol Cancer Ther; 10(3); 404–15. ©2011 AACR.

Supplementary Table 1 from sFLT01: A Novel Fusion Protein with Antiangiogenic Activity

The recombinant fusion protein aflibercept (ziv-aflibercept in the United States) binds VEGF-A, VEGF-B, and placental growth factor (PlGF). The monoclonal antibody bevacizumab binds VEGF-A. Recent studies hypothesized that dual targeting of VEGF/PlGF is more beneficial than targeting either ligand. We compared activity of aflibercept versus bevacizumab in 48 patient-derived xenograft (PDX) colorectal cancer models. Nude mice engrafted subcutaneously with PDX colorectal cancer tumors received biweekly aflibercept, bevacizumab, or vehicle injections. Differential activity between aflibercept and bevacizumab, determined by mouse (m), human (h), VEGF-A, and PlGF levels in untreated tumors, was measured. Aflibercept induced complete tumor stasis in 31 of 48 models and bevacizumab in 2 of 48. Based on statistical analysis, aflibercept was more active than bevacizumab in 39 of 48 models; in 9 of 39 of these models, bevacizumab was considered inactive. In 9 of 48 remaining models, aflibercept and bevacizumab had similar activity. Tumor levels of hVEGF-A (range 776–56,039 pg/mg total protein) were ∼16- to 1,777-fold greater than mVEGF-A (range 8–159 pg/mg total protein). Tumor levels of mPlGF (range 104–1,837 pg/mg total protein) were higher than hPlGF (range 0–543 pg/mg total protein) in 47 of 48 models. Tumor cells were the major source of VEGF; PlGF was primarily produced by tumor stroma. Because tumor levels of hVEGF-A were far greater than mVEGF-A, bevacizumab's inability to bind mVEGF-A is unlikely to explain higher and more consistent aflibercept activity. Neutralizing PlGF and VEGFR-1 activation may be a factor and should be investigated in future studies. In these colorectal cancer PDX models, aflibercept demonstrated greater antitumor activity than bevacizumab. Mol Cancer Ther; 13(6); 1636–44. ©2014 AACR.

PDF - 6591K, Tumor growth curves utilizing median ATVs of the 48 CRC PDXs: Tumor growth curves for all 48 patient-derived CRC tumors in mice engrafted SC, and which were treated with aflibercept SC injection (2x/week, 25 mg/kg), vehicle (SC injection, 2x/week), or bevacizumab (IV injection, 2x/week, 25 mg/kg) for a total of 3 weeks. Tumor measurements were recorded twice per week. Error bars represent MAD.

PDF - 44K, Distribution of the tumor origin across PDX models: Of the 48 PDX tumors, 8 were derived from primary patient tumors, 36 from metastatic sites, 1 from a site of recurring disease, and 1 of unknown anatomical origin. All of the primary tumors (8/8) were associated with phenotype A where aflibercept is more efficacious than bevacizumab. PDX tumors that originated from metastatic tumors were represented in both phenotypes A (30/39) and B (9/9).

PDF - 107K, Genomic characterization of PDX models: 48 PDX tumors comprising similar mutational patterns of KRAS, BRAF, PIK3CA, and PTEN have been reported in several other publications.

PDF - 6683K, Tumor growth curves utilizing mean ATVs of the 48 CRC PDXs: Tumor growth curves for all 48 patient-derived CRC tumors in mice engrafted SC, and which were treated with aflibercept SC injection (2x/week, 25 mg/kg), vehicle (SC injection, 2x/week), or bevacizumab (IV injection, 2x/week, 25 mg/kg) for a total of 3 weeks. Tumor measurements were recorded twice per week. Error bars represent standard error of the mean.

Supplementary Figure 1 from sFLT01: A Novel Fusion Protein with Antiangiogenic Activity

PDF - 115K, Legends to Supplementary Figures. Supplementary Table S1. Characteristics of 48 patient-derived colon cancer tumor xenografts. Supplementary Table S2. Evaluation of statistical differences between treatment(s) groups at the terminal point.

Breast cancer; Cancer Càncer de mama; Càncer Cáncer de mama; Cáncer The METRIC study (NCT#0199733) explored a novel antibody–drug conjugate, glembatumumab vedotin (GV), targeting gpNMB that is overexpressed in ~40% of patients with triple-negative breast cancer (TNBC) and associated with poor prognosis. The study was a randomized, open-label, phase 2b study that evaluated progression-free survival (PFS) of GV compared with capecitabine in gpNMB-overexpressing TNBC. Patients who had previously received anthracycline and taxane-based therapy were randomized 2:1 to receive, GV (1.88 mg/kg IV q21 days) or capecitabine (2500 mg/m2 PO daily d1–14 q21 days). The primary endpoint was RECIST 1.1 PFS per independent, blinded central review. In all, 327 patients were randomized to GV (213 treated) or capecitabine (92 treated). Median PFS was 2.9 months for GV vs. 2.8 months for capecitabine. The most common grade ≥3 toxicities for GV were neutropenia, rash, and leukopenia, and for capecitabine were fatigue, diarrhea, and palmar-plantar erythrodysesthesia. The study did not meet the primary endpoint of improved PFS over capecitabine or demonstrate a relative risk/benefit improvement over capecitabine. Funding provided by Celldex Therapeutics, Inc.

PURPOSE This phase I study (RAD1901-005; NCT02338349) evaluated elacestrant, an investigational oral selective estrogen receptor degrader (SERD), in heavily pretreated women with estrogen receptor–positive, human epidermal growth factor receptor 2–negative metastatic breast cancer, including those with estrogen receptor gene alpha ( ESR1) mutation. The primary objective was to determine the maximum tolerated dose and/or recommended phase II dose (RP2D). METHODS The study consisted of a 3 + 3 design (elacestrant capsules) followed by expansion at RP2D (400-mg capsules, then 400-mg tablets) for the evaluation of safety and antitumor activity. Elacestrant was taken once daily until progression or intolerability. RESULTS Of 57 postmenopausal women enrolled, 50 received RP2D (400 mg once daily): median age, 63 years; median three prior anticancer therapies, including cyclin-dependent kinase 4,6 inhibitors (CDK4/6i; 52%), SERD (52%), and ESR1 mutation (circulating tumor DNA; 50%). No dose-limiting toxicities occurred; the most common adverse events at RP2D (400-mg tablet; n = 24) were nausea (33.3%) and increased blood triglycerides and decreased blood phosphorus (25.0% each). Most adverse events were grade 1-2 in severity. The objective response rate was 19.4% (n = 31 evaluable patients receiving RP2D), 15.0% in patients with prior SERD, 16.7% in patients with prior CDK4/6i, and 33.3% in patients with ESR1 mutation (n = 5/15). The clinical benefit rate (24-week) was 42.6% overall (n = 47 patients receiving RP2D), 56.5% (n = 23, ESR1 mutation), and 30.4% (n = 23, prior CDK4/6i). Elacestrant clinical benefit was associated with decline in ESR1 mutant allele fraction. CONCLUSION Elacestrant 400 mg orally once daily has an acceptable safety profile and demonstrated single-agent activity with confirmed partial responses in heavily pretreated patients with estrogen receptor–positive metastatic breast cancer. Notably, responses were observed in patients with ESR1 mutation as well as those with prior CDK4/6i and prior SERD. A phase III trial investigating elacestrant versus standard endocrine therapy is ongoing.

Introduction: The androgen receptor (AR) is expressed in ~90% of ER+ BC. Androgen-based therapies have demonstrated response rates from 20-25% in advanced/metastatic breast cancer (mBC). RAD140 is an oral nonsteroidal SARM with tissue-selective AR agonist activity in BC cells but attenuated activity in other androgen-responsive tissues. In in vivo and in vitro models recapitulating various stages of AR/ER+ BCs, RAD140 exhibited potent anti-tumor activity as a single agent and in combination with inhibitors (i) of CDK4/6 or mTOR. Methods: This is a first-in-human, phase 1 dose escalation study with a 3+3 design and pharmacokinetic (PK) expansion cohort. The primary endpoint is safety, tolerability, and to establish the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary endpoints are PK and antitumor activity by RECIST. In the dose escalation phase, patients (pts) were treated with RAD140 once daily (QD) in 28-day cycles. In the subsequent PK expansion (PKE) cohort, pts were treated with a single dose of 100 mg followed by 1 wk of PK sampling before entering continuous 28-day treatment cycles with 100 mg QD dosing. Dose-limiting toxicity (DLT) was assessed during the first 28-day cycle. Key eligibility criteria included: ER+/HER2- and inoperable/mBC, post-menopausal, and ineligible for standard therapy. Archival tumor samples within 2 years or fresh tumor biopsies collected at the time of enrollment were analyzed retrospectively by immunohistochemistry (IHC) for AR, ER, progesterone receptor (PR), HER2 and Ki67. Serum sex hormone-binding globulin (SHBG) and prostate specific antigen (PSA) were used to assess AR engagement. Results: Dose escalation and PKE enrollment (n=22) is complete. Median age = 60 years, 100% stage IV, 86% visceral disease, 95% AR+, 91% AR+/ER+, and 82% AR+/ER+/HER2-. Median number of prior lines of therapy for mBC = 4; prior fulvestrant 86%, aromatase inhibitor 96%, CDK4/6i 91%, mTORi/PI3Ki 46%, chemotherapy 91%. Starting dose levels were 50 mg (n=6), 100 mg (n=13), and 150 mg (n=3) QD. Median time on treatment = 9 wk (range 30%) treatment-emergent adverse events were elevated AST (27% grade [G] 1; 9% G2; 18% G3; 5% G4) and elevated ALT (18% G1; 23% G3). DLTs at 150 mg were hypophosphatemia (n=2). DLTs at 100 mg were hypophosphatemia (n=1), elevated ALT (n=3), and elevated AST (n=1). All DLTs were grade 3 and reversible. No drug-related deaths occurred. PK samples collected for 144 hrs after the 100 mg single dose showed variable absorption with a half-life of approximately 60 hrs. The observed human steady-state PK exposure at 100 mg exceeds the PK exposure of the efficacious dose in mouse of 10 mg/kg. Of 9 evaluable pts at the MTD of 100 mg, there was 1 partial response (PR; ORR=11%) and 3 pts (33%) with stable disease ≥12 wk. Time to PR was 15.9 wk with duration 18.6+ wk. Three pts remain on treatment. SHBG decreased in 18/18 pts and PSA increased in 15/20 pts with paired samples available, indicative of AR target engagement, most notably in the pt with PR. In 1 pt with paired pre- and on-treatment tumor biopsies, a strong induction of AR by IHC, predominantly in the nucleus was seen, further suggesting AR activation by RAD140 at the tissue level. Conclusions: The MTD and recommended dose for RAD140 is 100 mg QD. RAD140 is a novel oral AR-targeted agent for the treatment of AR+/ER+ mBC with an acceptable safety profile and preliminary evidence of target engagement and antitumor activity. This study is ongoing. NCT03088527 Citation Format: Erika Hamilton, Patricia LoRusso, Cynthia Ma, Neelima Vidula, Rebecca G Bagley, Steven Troy, Miriam Annett, Ziyang Yu, Aaron Weitzman, Maureen G Conlan, Amy Weise. Phase 1 dose escalation study of a novel selective androgen receptor modulator (SARM), RAD140, in estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-), metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-01.

Background gpNMB is an internalizable transmembrane protein overexpressed in ˜40% of TNBC, and associated with a worse prognosis. Preclinical data implicates gpNMB in tumor invasion and metastasis. GV is a novel ADC designed to deliver monomethyl auristatin E (MMAE) to gpNMB-overexpressing cells. Prior phase 1/2 studies suggested promising activity of GV in gpNMB-overexpressing TNBC. Methods In the METRIC trial (NCT#01997333), patients (pts) with mTNBC were randomized 2:1 to GV (1.88 mg/kg IV q21 days) or capecitabine (2,500 mg/m2 PO daily d1-14 q21 days) until disease progression or intolerance. Key eligibility criteria included: gpNMB over-expression (>25% tumor cells positive by central immunohistochemistry of archival tissue); estrogen and progesterone receptor expression Results From Feb 2014 to Aug 2017, 327 women were randomized at 120 institutions to GV (n=218) or capecitabine (n=109). Pretreatment characteristics were well balanced between arms: median age of 55 years; 77% visceral disease; 2.4 years median duration of disease at study entry; 1 median prior anticancer regimen for metastatic disease. At the cut-off for final data analysis (Nov 30, 2017), 21 pts remained on treatment and 98 pts were alive. For GV vs. capecitabine based on independent central review, median PFS was 2.9 (95% CI: 2.8, 3.5) vs. 2.8 (95% CI: 1.6, 3.2) months (HR=0.95; p=0.76); median OS was 8.9 (95% CI: 7.9, 10.5) vs. 8.7 (95% CI: 6.9, 10.8) months (HR=1.06; p=0.73); ORR was 16% (95% CI: 11.1, 22.4) vs. 15% (95% CI: 8.6, 23.5); and median DOR was 5.6 (95% CI: 3.0, 7.8) vs. 4.2 (95% CI: 3.0, 12.2) months. A post-hoc subgroup analysis suggested the greatest benefit from GV was in potentially taxane-sensitive disease (i.e., not previously rechallenged or >6 months progression-free interval following last taxane). The incidence of grade ≥ 3 treatment-related adverse events (TRAEs) was 58% in the GV arm and 37% in the capecitabine arm. The most common grade ≥ 3 TRAEs were neutropenia (27%), rash (15%), and leukopenia (9%) in the GV arm, and diarrhea (14%) and palmar-plantar erythryodysesthesia (8%) in the capecitabine arm. There was 1 fatal TRAE of neutropenic sepsis in the GV arm and none in the capecitabine arm. Conclusion The METRIC study evaluating GV in mTNBC did not meet the primary efficacy endpoint of improved PFS over capecitabine. While anticancer activity was seen with GV, it was comparable to capecitabine with no therapeutic advantage of GV in terms of ORR, PFS, or OS. The safety profile of GV was consistent with prior experience with no new safety signals identified. Citation Format: Vahdat LT, Forero-Torres A, Schmid P, Blackwell K, Telli ML, Melisko M, Holgado E, Moebus V, Cortes J, Fehrenbacher L, Montero AJ, Ma C, Nanda R, Wright GS, He Y, Bagley RG, Halim A, Turner CD, Yardley DA. METRIC: A randomized international phase 2b study of the antibody-drug conjugate (ADC) glembatumumab vedotin (GV) in gpNMB-overexpressing, metastatic, triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-01.

Introduction: Translocations involving the MLL gene on chromosome 11q23 result in fusions with ≥10 partner genes that act as drivers in relatively chemotherapy resistant MLL-rearranged (MLLr) leukemias. These chimeric proteins form part of protein complexes that aberrantly upregulate transcription of the leukemogenic HOX and MEIS1 genes. Menin is a key member of the complex and localizes the complex to chromatin. Similarly, in NPM1 mutant (mNPM1) AML, the interaction between wild-type MLL and menin leads to a HOX and MEIS1-mediated leukemogenic transcriptional program. In preclinical models of MLLr and mNPM1 leukemias, inhibition of the interaction between MLL and menin downregulates HOX and MEIS1 transcription and reverses leukemogenesis (Uckelmann 2020; Krivstov 2019). SNDX-5613 (5613) is a potent, selective protein-protein interaction inhibitor of menin being evaluated in the AUGMENT-101 study, a first-in-human (FIH), Ph 1/2 study in pts with R/R acute leukemia. Here, we report the results of the completed, Ph 1 component. Methods: The study includes 2 parallel dose-escalation cohorts: pts not taking (Arm A) or taking (Arm B) strong CYP3A4 inhibitors. An early amendment restricted enrollment to MLLr or mNPM1 leukemias. Dose escalation used a "rolling 6" design with expansion at efficacious doses. 5613 is dosed orally q12h in continuous 28-day cycles. Dose levels evaluated in Arm A were 113 (n=1), 226 (n=6), 276 (n=10), and 339 mg (n=8), and in Arm B 113 (n=16), 163 (n=6), and 226 mg (n=7). Primary objectives of Ph 1 were to determine the safety, MTD, recommended Ph 2 dose (RP2D), and PK profile of 5613. Exploratory endpoints included antileukemic activity and pharmacodynamics of 5613. Results: As of 29Jun2021, 54 pts had received at least 1 dose of 5613; 13 pts remained on treatment. Baseline characteristics are detailed in Table 1. The median age was 49 years; 82% (n=44) of pts had AML; 65% (n=35) had MLLr leukemia, and 19% (n=10) had mNPM1 leukemia. Pts had a median of 3 prior therapies (range, 1-12); 57% and 44% had prior venetoclax or transplant, respectively; 59% of pts had not responded to their most recent line of therapy. 5613 exhibited dose-proportional PK, and exposure increased ~2-3-fold when given with a strong CYP3A4 inhibitor. Measurement of target occupancy and gene expression showed that 5613 disrupted menin binding to chromatin and decreased leukemogenic gene expression consistent with the established mechanism of action. The only DLTs were Grade 3 prolonged QTc; all events were clinically asymptomatic. The MTD was 276 mg q12h in Arm A and 163 mg q12h in Arm B; 2 doses in each arm met the protocol-defined criteria (safety, percentage of planned dose intensity, and pharmacokinetic exposure) for a RP2D. The frequency of Grade 3 prolonged QTc at these doses was 8% (3/38). No ventricular arrhythmias were reported, and no pts discontinued 5613 due to a treatment-related event. Other common (>10%) treatment-related AE (TRAE) were nausea (n=12; 22%), vomiting (n=9; 17%), differentiation syndrome (n=8; 15%), and diarrhea (n=6;11%). Grade ≥3 TRAE are shown in Table 2. In the 45 pts with MLLr or mNPM1 leukemias, responses were seen in both Arms A and B and at multiple dose levels. The composite CR (CRc: CR+CRh+CRp+CRi/MLFS) rate was 44% (20/45 pts) (Table 3). Among pts with MLLr leukemia, the CRc rate was 49% (17/35 pts) and in pts with mNPM1 leukemia, the CRc rate was 30% (3/10 pts); 14/20 (70%) pts with CRc achieved MRD negativity assessed locally by flow cytometry or PCR. Of 22 pts who received prior transplant, 10 (45%) achieved CRc. The CR/CRh rate was 22% (10/45) in pts with MLLr or mNPM1 leukemia. Median time to CR/CRh was 2 months (mo). With a median follow-up of 3.2 mo, the median duration of response (DoR) for patients achieving a CR/CRh was 5.2 mo. Responding patients were censored at the time they discontinued the study to proceed to an allogeneic stem cell transplant (n=6). Consistent with the proposed preclinical mechanism of menin inhibition, the 9 patients with wild-type MLL and NPM1 did not respond to 5613. Conclusion: In this FIH Ph 1 study, SNDX-5613 demonstrates an acceptable safety profile and promising antileukemic activity in pts with heavily pretreated R/R MLLr and mNPM1 acute leukemia. Two dose levels in each arm met the prespecified criteria for RP2D. Ph 2 expansion includes cohorts of pts with MLLr AML, MLLr ALL, and mNPM1 AML. Figure 1 Figure 1. Disclosures Stein: Foghorn Therapeutics: Consultancy; Blueprint Medicines: Consultancy; Gilead Sciences, Inc.: Consultancy; Abbvie: Consultancy; Janssen Pharmaceuticals: Consultancy; Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; PinotBio: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Novartis: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Syros Pharmaceuticals, Inc.: Consultancy; Syndax Pharmaceuticals: Consultancy. Stone: Syndax: Membership on an entity's Board of Directors or advisory committees; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy; Innate: Consultancy; Janssen: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Boston Pharmaceuticals: Consultancy; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy, Research Funding; Jazz: Consultancy; Onconova: Consultancy; AbbVie: Consultancy; Novartis: Consultancy, Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Aprea: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Actinium: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Agios: Consultancy, Research Funding; Celgene: Consultancy; Macrogenics: Consultancy. Arellano: KITE Pharma, Inc: Consultancy; Syndax Pharmaceuticals, Inc: Consultancy. Rosen: Syndax: Current Employment. Meyers: Syndax Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Patents & Royalties; Nuvalent: Consultancy, Membership on an entity's Board of Directors or advisory committees. Huang: Syndax: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Serapta: Ended employment in the past 24 months. Smith: Syndax: Consultancy; Tempest: Current holder of stock options in a privately-held company; Athira: Current holder of stock options in a privately-held company. Bagley: Syndax: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Radius Health: Ended employment in the past 24 months. Thirman: AbbVie: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Adaptive Biotechnologies: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Pharmacyclics: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Merck: Research Funding; Syndax: Research Funding; TG Therapeutics: Research Funding. Patel: Ignyta: Research Funding; Incyte: Research Funding; Jacobio: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jounce Therapeutics: Research Funding; Klus Pharma: Research Funding; Kymab: Research Funding; Loxo Oncology: Research Funding; LSK Biopartners: Research Funding; Lycera: Research Funding; Mabspace: Research Funding; Macrogenics: Research Funding; Merck: Research Funding; Millennium Pharmaceuticals: Research Funding; Mirati Therapeutics: Research Funding; ModernaTX: Research Funding; ORIC Pharmaceuticals: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Phoenix Molecular Designs: Research Funding; Placon Therapeutics: Research Funding; Portola Pharmaceuticals: Research Funding; Prelude Therapeutics: Research Funding; Qilu Puget Sound Biotherapeutics: Research Funding; Revolution Medicines: Research Funding; Ribon Therapeutics: Research Funding; Seven and Eight Biopharmaceuticals: Research Funding; Syndax: Research Funding; Synthorx: Research Funding; Stemline Therapeutics: Research Funding; Taiho: Research Funding; Takeda: Research Funding; Tesaro: Research Funding; TopAlliance: Research Funding; Vedanta: Research Funding; Verastem: Research Funding; Vigeo: Research Funding; Xencor: Research Funding; Exelixis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Alexion, AstraZeneca Rare Disease: Other: Study investigator; Hutchinson MediPharma: Research Funding; Hengrui: Research Funding; H3 Biomedicine: Research Funding; Gilead: Research Funding; GlaxoSmithKline: Research Funding; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forma Therapeutics: Research Funding; Evelo Biosciences: Research Funding; Eli Lilly: Research Funding; EMD Serono: Membership on an entity's Board of Directors or advisory committees, Research Funding; Effector Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Cyteir Therapeutics: Research Funding; Curis: Research Funding; Clovis: Research Funding; Ciclomed: Research Funding; Checkpoint Therapeutics: Research Funding; Calithera: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; BioNTech: Research Funding; Bicycle Therapeutics: Research Funding; AstraZeneca: Research Funding; Artios Pharma: Research Funding; Aileron Therapeutics: Research Funding; Agenus: Research Funding; ADC Therapeutics: Research Funding; Acerta Pharma: Research Funding; Florida Cancer Specialists: Research Funding. Issa: Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding; Kura Oncology: Consultancy, Research Funding.

Background: The majority of patients (pts) with advanced ER+ breast cancer (BC) will experience progression of disease after endocrine therapy (ET) due to acquired resistance, including development of ESR1 mutations (mut). Response to conventional ET agents, including aromatase inhibitors and fulvestrant, in late lines of therapy is poor with overall response rates (ORR) less than 10% and median progression free survival (mPFS) of 2-3 months. Elacestrant is a novel, nonsteroidal, oral SERD with marked antitumor activity documented in multiple in vivo pt derived xenograft models of BC, including those derived from heavily pretreated pts and those with ESR1 mut. Methods: RAD1901-005 is a multi-part phase 1 study of elacestrant with the primary objective to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D). Secondary objectives are safety, tolerability, pharmacokinetics, and preliminary antitumor activity. Part A was dose escalation with 3+3 design (capsule; doses 200, 400 and 600 mg QD); Part B was safety expansion (capsule); Part C was expansion (tablet); Part D expansion (tablet) was added by amendment to study later line pts including prior CDK4/6 inhibitor (i) use (NCT02338349). Key eligibility criteria: advanced or metastatic (m) ER+, HER2- BC; postmenopausal status. For Parts A-C: ≤2 prior chemotherapy regimens (CT) for mBC and >6 mo ET with progression (PD). For Part D: ≤1 prior CT for mBC, ≥2 prior lines of ET including PD on fulvestrant, and CDK4/6i. We present updated and final results from the trial (all parts). Results: From April 2015 to March 2018, 57 pts were enrolled: Part A: 13, B: 20, C: 14, D: 10 (of 36 planned; enrollment was terminated due to change in regulatory strategy). MTD/RP2D was 400 mg QD. For all pts treated at 400 mg (n=50): stage IV 100%; visceral disease 70%; median prior lines of tx 3 (Part D: 4), including median of 1 prior line of CT and 3 prior lines of ET; prior CDK4/6i 52% (Part D: 100%); prior fulvestrant 50% (Part D: 100%); prior mTORi 26%. At baseline, 51% of pts had detectable ESR1 mut, including D538G, Y537S/N/C, L536H/P/R, S436P and E380Q. Among pts treated with 400 mg tablet (n=24), treatment-emergent adverse events in ≥20% were nausea, hypertriglyceridemia, hypophosphatemia, vomiting, dyspepsia, constipation, hypokalemia, fatigue, back pain, myalgia, headache, and urinary tract infection; the majority were grade 1 and 2. There were no treatment-related deaths. 13% of pts discontinued due to AEs. Antitumor activity is summarized in Table 1. There were 6 confirmed partial responses; median duration of response was 24.9 wk; median time to response was 8.2 wk. One pt with ESR1 mut with best response of SD remains on treatment at 3 yr. Conclusion: Elacestrant at the RP2D of 400 mg QD has an acceptable safety profile and demonstrated significant single-agent anti-tumor activity in heavily pretreated (mostly 4th line or greater) ER+, HER2- mBC pts. Activity was seen following prior fulvestrant, prior CDK4/6i, and in pts with ESR1 mut. The ORR of 19.4% and mPFS of 4.5 mo compare favorably with those reported for fulvestrant and AIs given in earlier treatment settings. Elacestrant monotherapy vs. standard of care ET monotherapy is currently being studied in ER+, HER2- mBC with 1 or 2 lines of prior tx including prior CDK4/6i, in the phase 3 “EMERALD” study (NCT03778931). Table 1. Efficacy OutcomesParts A-CPart DOverallORR, % (RECIST-evaluable pts) (n/N)27.3% (6/22)0% (0/9)19.4% (6/31)Clinical Benefit Rate, % (PR+SD ≥24 wk) (n/N)47.4% (18/38)22.2% (2/9)42.6% (20/47)mPFS (95% CI), mo, N=50 (ITT population)5.4 (3.7, 11.2)1.9 (1.8, 8.6)4.5 (1.9, 7.4) Citation Format: Virginia Kaklamani, Aditya Bardia, Sharon Wilks, Amy Weise, Donald Richards, Wael Harb, Cynthia Osborne, Robert Wesolowski, Meghan Karuturi, Paul Conkling, Rebecca Bagley, JungAh Jung, Teeru Bihani, Maureen Conlan, Peter Kabos. Final analysis of phase 1 study of elacestrant (RAD1901), a novel selective estrogen receptor degrader (SERD), in estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-) advanced breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-07.

Background Glembatumumab vedotin is an antibody-drug conjugate that produced preliminary clinical activity against advanced melanoma in a phase 1 dose-escalation trial. The objective of the current study was to investigate further the antitumor activity of glembatumumab vedotin at the recommended phase 2 dose in heavily pretreated patients with melanoma. Methods This single-arm, phase 2 study enrolled patients with stage IV melanoma who were refractory to checkpoint inhibition and to B-raf proto-oncogene, serine/threonine kinase (BRAF)/mitogen-activated protein kinase kinase (MEK) inhibition (in the presence of a BRAF valine mutation at codon 600). Patients received 1.9 mg/kg glembatumumab vedotin intravenously every 3 weeks until they developed disease progression or intolerance. The primary endpoint was objective response rate (ORR), which was determined according to Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary endpoints included progression-free survival (PFS), duration of response, overall survival (OS), safety, and clinical efficacy versus tumor glycoprotein NMB (gpNMB) expression. Tumor expression of gpNMB was assessed using immunohistochemistry. Results In total, 62 patients received treatment. The ORR was 11% and the median response duration was 6.0 months (95% confidence interval [CI], 4.1 months to not reached). The median PFS was 4.4 months (95% CI, 2.6-5.5 months), and the median OS was 9.0 months (95% CI, 6.1-11.7 months). For patients who developed rash during the first cycle versus those who did not, the ORR was 21% versus 7%, respectively, and there was an overall improvement in PFS (hazard ratio, 0.43; P = .013) and OS (hazard ratio, 0.43; P = .017). The most frequent adverse events were alopecia, neuropathy, rash, fatigue, and neutropenia. With one exception, all evaluable tumors were positive for gpNMB, and 46 of 59 tumors (76%) had 100% gpNMB-positive epithelial cells. Conclusions Glembatumumab vedotin had modest activity and an acceptable safety profile in patients with advanced melanoma who were refractory to checkpoint inhibitors and MEK/BRAF inhibition. Treatment-related rash may be associated with response.

JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post-PV myelofibrosis (PPMF) and rapid post-essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2(V617F) mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2-independent. Interestingly, we originally found a decreased in the JAK2(V617F) allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.

Background Glycoprotein NMB (gpNMB) is an internalizable transmembrane protein overexpressed in approximately 20% of breast cancer (BC), including approximately 40% of TNBC. gpNMB is a poor prognostic marker in BC (Rose CCR 2010) and preclinically has been implicated in tumor invasion, metastasis, and angiogenesis. GV is a novel antibody-drug conjugate targeting the potent cytotoxin monomethylauristatin E (MMAE) to gpNMB overexpressing cancer cells. In a Phase I/II study and the Phase II "EMERGE" study, GV demonstrated promising activity with TNBC patients (pts) deriving the greatest benefit and exhibiting the highest degree of gpNMB overexpression. GV was well-tolerated with the most frequent treatment-related toxicities consisting of rash, neutropenia, and neuropathy. In subset analyses of the EMERGE trial, objective response rate (ORR) was 30% (7/23) for GV vs. 9% (1/11) for investigator's choice in tumors with gpNMB overexpression (>25% of tumor epithelium); 18% (5/28) vs. 0% (0/11) in TNBC; and 40% (4/10) vs. 0% (0/6) in gpNMB-overexpressing TNBC for GV and IC respectively, with apparent improvements in progression-free survival (PFS; hazard ratio (HR) = 0.11) and overall survival (OS; HR = 0.14). Trial design The METRIC Trial (NCT#01997333) is an international (USA, CA, Aus), two-arm phase II study. Pts are randomized 2:1 to GV (1.88 mg/kg IV q 21 days) or capecitabine, a current standard of care for this population (2,500 mg/m2 daily for d1-14, q21 days) until progression or intolerance. Crossover is not permitted. Eligibility criteria Key eligibility criteria include: >25% of tumor epithelium gpNMB+ by central immunohistochemistry (IHC) screening of archival tissue; estrogen receptor and progesterone receptor 2 toxicity. Specific aims The primary endpoint is PFS per independent, blinded central review committee according to RECIST 1.1. Secondary endpoints are ORR, duration of response, OS, safety, pharmacokinetics and pharmacodynamics. Exploratory endpoints are quality of life and/or cancer-related pain. Statistical methods and target accrual The trial has 85% power to detect a PFS HR of 0.64 with two sided α = 0.05. The hypothesized median PFS is 4.0 months for capecitabine and 6.25 months for GV. Target accrual is open for 300 pts. Citation Format: Melisko M, Yardley DA, Blackwell K, Forero A, Ma C, Montero A, Daniel BR, Wright G, Fehrenbacher L, Chew H, Ferrario C, Nanda R, Seiler Jr M, Guthrie T, Vance K, Ouellette G, He Y, Bagley RG, Zhang J, Vahdat LT. The METRIC trial: A randomized international study of the antibody-drug conjugate glembatumumab vedotin (GV or CDX-011) in patients with metastatic gpNMB-overexpressing triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-03-15.

See related article by Folkman, [Cancer Res 1974;34:2109–13][1] . Visit the Cancer Research 75th Anniversary [timeline][2]. In a seminal publication by Dr. Judah Folkman ([1][3]), the concept of tumor angiogenesis via the identification of a then undefined tumor angiogenesis factor transformed

Ewing/PNET (peripheral neuroepithelioma) tumors are rare aggressive bone sarcomas occurring in young people. Rare-disease clinical trials can require global collaborations and many years. In vivo models that as accurately as possible reflect the clinical disease are helpful in selecting therapeutics with the most promise of positive clinical impact. Human Ewing/PNET sarcoma cell lines developed over the past 45 years are described. Several of these have undergone genetic analysis and have been confirmed to be those of Ewing/PNET sarcoma. The A673 Ewing sarcoma line has proven to be particularly useful in understanding the biology of this disease in the mouse. The chromosomal translocation producing the EWS/FLI1 fusion transcript characterizes clinical Ewing sarcoma. Cell lines that express this genetic profile are confirmed to be those of Ewing sarcoma. The A673 Ewing sarcoma line grows in culture and as a xenograft in immunodeficient mice. The A673 model has been used to study Ewing sarcoma angiogenesis and response to antiangiogenic agents. Many Ewing sarcoma clinical specimens express the cell surface protein endosialin. Several Ewing sarcoma cell lines, including the A673 line, also express cell surface endosialin when grown as subcutaneous tumor nodules and as disseminated disease; thus the A673 is a useful model for the study of endosialin biology and endosialin-directed therapies. With the advent of tools that allow characterization of clinical disease to facilitate optimal treatment, it becomes imperative, especially for rare tumors, to develop preclinical models reflecting disease subsets. Ewing PNET sarcomas are a rare disease where models are available.

Purpose: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. Experimental Design: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. Results: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. Conclusions: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.

Topoisomerase I (TopoI), an established anticancer target, is an enzyme producing a single-strand DNA break during transcription. Several noncamptothecin TopoI inhibitors have been identified. One of these, ARC-111, was compared with two clinically used camptothecins, topotecan and irinotecan/SN-38. In mouse and human bone marrow colony formation [colony-forming units granulocyte-macrophage (CFU-GM)] assays, the IC90 values were 519 and 331 nmol/L for topotecan and SN-38 mouse CFU-GM and were 19 and 26 nmol/L for human CFU-GM, giving mouse to human differentials of 28- and 13-fold. ARC-111 produced IC90 values of 28 nmol/L in mouse and 6.2 nmol/L in human CFU-GM, thus only a 4.5-fold differential between species. Human bone marrow CFU-GM was more sensitive to topotecan than were several human cancer cell lines, but ARC-111 cytotoxicity was similar for human bone marrow CFU-GM and the seven human tumor cell lines tested. In HCT-116 xenografts, tumor growth delays (TGD) were 17 days for irinotecan and 20 days for ARC-111. In HT-29 xenografts, the TGD was 9 days for both irinotecan and ARC-111. Both ARC-111 and docetaxel had a TGD of 21 days in NCI-H460 xenografts, and both ARC-111 and gemcitabine had a TGD of 7 days in MiaPaCa2 xenograft. Current TopoI inhibitors have broad antitumor activity in human tumor xenografts that is not achieved in the clinic. This may be due to greater sensitivity of human bone marrow than mouse to the cytotoxicity of these agents. It may be possible to achieve similar levels of ARC-111 in patients as in mice allowing improved antitumor activity. [Mol Cancer Ther 2008;7(10):3212–22]

The recombinant fusion protein aflibercept (ziv-aflibercept in the United States) binds VEGF-A, VEGF-B, and placental growth factor (PlGF). The monoclonal antibody bevacizumab binds VEGF-A. Recent studies hypothesized that dual targeting of VEGF/PlGF is more beneficial than targeting either ligand. We compared activity of aflibercept versus bevacizumab in 48 patient-derived xenograft (PDX) colorectal cancer models. Nude mice engrafted subcutaneously with PDX colorectal cancer tumors received biweekly aflibercept, bevacizumab, or vehicle injections. Differential activity between aflibercept and bevacizumab, determined by mouse (m), human (h), VEGF-A, and PlGF levels in untreated tumors, was measured. Aflibercept induced complete tumor stasis in 31 of 48 models and bevacizumab in 2 of 48. Based on statistical analysis, aflibercept was more active than bevacizumab in 39 of 48 models; in 9 of 39 of these models, bevacizumab was considered inactive. In 9 of 48 remaining models, aflibercept and bevacizumab had similar activity. Tumor levels of hVEGF-A (range 776–56,039 pg/mg total protein) were ∼16- to 1,777-fold greater than mVEGF-A (range 8–159 pg/mg total protein). Tumor levels of mPlGF (range 104–1,837 pg/mg total protein) were higher than hPlGF (range 0–543 pg/mg total protein) in 47 of 48 models. Tumor cells were the major source of VEGF; PlGF was primarily produced by tumor stroma. Because tumor levels of hVEGF-A were far greater than mVEGF-A, bevacizumab's inability to bind mVEGF-A is unlikely to explain higher and more consistent aflibercept activity. Neutralizing PlGF and VEGFR-1 activation may be a factor and should be investigated in future studies. In these colorectal cancer PDX models, aflibercept demonstrated greater antitumor activity than bevacizumab. Mol Cancer Ther; 13(6); 1636–44. ©2014 AACR.

Introduction: NY-ESO-1 is a cancer testis antigen frequently expressed in a broad range of tumors: lung, breast, ovarian, bladder, liver cancer, melanoma, sarcoma and myeloma. CDX-1401, a fully human monoclonal antibody linked to full length NY-ESO-1, binds to dendritic cell (DC) receptor DEC-205 to stimulate NY-ESO-1 specific CD4 and CD8 responses. Immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) assays were developed to determine NY-ESO-1 expression in human tumors and normal adjacent tissues (NAT). A qRT-PCR assay for the detection of LAGE-1, a cancer testis antigen with significant DNA homology to NY-ESO-1, was also developed but no commercially specific antibody was available for IHC. Methods: Formalin fixed, paraffin embedded tumors and NAT were procured from a commercial source or obtained from Mosaic Laboratories; split anonymized samples were analyzed for NY-ESO-1 by IHC at Mosaic and for NY-ESO-1 and LAGE-1 by qRT-PCR at MolecularMD (OR) under IRB-approved protocols allowing in vitro analysis of remnant human samples. The study included: 18 each melanoma and ovarian cancers; 13 each colorectal (CRC), head & neck (H&N), and non-small cell lung cancers (NSCLC); and 38 NAT. HT1080 fibrosarcoma and SKOV3 ovarian cancer cell lines (ATCC, VA) and normal testis were used as controls. The qRT-PCR assays were validated using in vitro transcribed RNA on NY-ESO-1 and LAGE-1 kits provided by Life Technologies and the NY-ESO-1 IHC was developed using a mouse monoclonal antibody (Sigma, MO). Results: The NY-ESO-1 and LAGE-1 qRT-PCR assays had a linear range of 100-1,000,000 copies/reaction with limit of detection between 1-10 copies. An excess of LAGE-1 copies did not impact the specificity of the NY-ESO-1 assay and vice-versa. NY-ESO-1 protein was detected by IHC and NY-ESO-1 transcripts and LAGE-1 transcripts were detected in 9 (12%), 12 (16%) and 16 (21.3%), respectively, of the 75 tumor tissues while only 2 of the 38 normal samples were positive for LAGE-1. When stromal staining was considered in addition to epithelial staining, 16 (21.3%) tumor samples were positive by IHC. NY-ESO-1 protein was more prevalent in NSCLC (38%), ovarian cancer (11%) and melanoma (11%) vs. other tumor types. Positive staining by IHC was achieved in tumor tissues shown positive for NY-ESO-1 and negative for LAGE-1 transcripts. In general, tumor tissues obtained from Mosaic were of better quality, as assessed by the pathologist, and more positive than commercially procured samples. Conclusions: Two qRT-PCR assays were developed to specifically quantify NY-ESO-1 or LAGE-1 mRNA without cross reaction. The antibody used in IHC assay seemed specific to NY-ESO-1. Inadequate sample quality impacted target prevalence. Implementing these assays in clinical trials may aid in prospectively identifying patients with NY-ESO-1 positive malignancies who could derive benefit from investigational NY-ESO-1 vaccines designed to induce anti-tumor immunity, such as CDX-1401. Citation Format: Abdel Halim, Rebecca G. Bagley, Lisa Dauffenbach, Eric P. Olsen, Tibor Keler. IHC and RT-PCR assays for detection of cancer antigen NY-ESO-1 in human tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1373.

Introduction: gpNMB is an internalizable transmembrane protein overexpressed in 20% of breast cancers, 40% of triple-negative breast cancer (TNBC) and 80% of melanomas. Glembatumumab vedotin (GV; CDX-011) is a novel antibody-drug conjugate (ADC) that delivers the potent cytotoxin monomethyl auristatin E to cancer cells expressing gpNMB. GV is in Phase II clinical trials for TNBC (the pivotal “METRIC” study; NCT01997333) and melanoma (NCT02302339). We investigated the prevalence of gpNMB overexpression in other human cancers to explore the potential for therapeutic benefit of GV beyond TNBC and melanoma. Methods: Tumor and normal tissues were procured (Mosaic Laboratories, CA) under an IRB-reviewed protocol (MOS001) allowing in vitro analysis of remnant, anonymized human samples. The study included 20 pancreatic tumors, 21 normal pancreatic tissues, 20 head & neck (H&N) tumors, 21 normal H&N tissues, 20 lung squamous cell carcinoma (SCC), 20 lung adenocarcinoma, 20 normal lung and 21 osteosarcoma tissues but no normal bone tissues were available. A validated immunohistochemistry assay was performed centrally on formalin fixed, paraffin embedded tissues. A goat polyclonal anti-gpNMB antibody (R&D Systems, MN) was applied to slides after deparaffinization and antigen retrieval. Visualization was achieved with a rabbit anti-goat antibody (Vector Laboratories, CA) and EnVision+ anti-rabbit HRP detection system (Dako, CA). Hematoxylin-counterstained slides were scored by 2 pathologists for percent of positive cells and staining intensity (0, 1+, 2+, 3+). Considering only cells strongly stained (2+ and/or 3+), the highest percent of cells stained in normal corresponding tissues was used as a cut-off. Results: Tumor epithelial cells staining exceeded the cut-off in 55%, 40%, 85% and 45% of pancreatic, H&N, lung SCC and lung adenocarcinoma respectively, and 62% of osteosarcoma samples stained in 1-90% of tumor epithelial cells. In pancreatic, lung, and H&N tumors, staining was both cytoplasmic and membranous but only cytoplasmic and 1+ intensity for normal tissue, normal tumor-adjacent tissue, and non-tumor stromal cells. The pattern of gpNMB staining in osteosarcomas was obviously cytoplasmic. However, both cytoplasmic and membranous staining resulted in a broader panel of 67 osteosarcoma tissues and glembatumumab vedotin had strong activity in 3/6 osteosarcoma xenografts (Roth, PBC, 2015; Kolb, PBC, 2014). Conclusions: As compared to normal tissue, differential expression of gpNMB in human osteosarcoma, pancreatic, lung, and H&N tumor samples suggests that patients with these cancers may derive a benefit from treatment with the investigational agent glembatumumab vedotin that targets cell surface gpNMB. Clinical studies are being initiated in squamous cell lung cancer, osteosarcoma and uveal melanoma with ongoing studies in TNBC and melanoma. Citation Format: Abdel Halim, Rebecca G. Bagley, Tibor Keler. Glycoprotein NMB (gpNMB) overexpression is prevalent in human cancers: pancreatic cancer, non-small cell lung cancer, head and neck cancer, and osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5032.

Overexpression of TMPRSS4, a cell surface-associated transmembrane serine protease, has been reported in pancreatic, colorectal and thyroid cancers, and has been implicated in tumor cell migration and metastasis. Few reports have investigated both TMPRSS4 gene expression levels and the protein products. In this study, quantitative RT-PCR and protein staining were used to assess TMPRSS4 expression in primary non-small cell lung carcinoma (NSCLC) tissues and in lung tumor cell lines. At the transcriptional level, TMPRSS4 message was significantly elevated in the majority of human squamous cell and adenocarcinomas compared with normal lung tissues. Staining of over 100 NSCLC primary tumor and normal specimens with rabbit polyclonal anti-TMPRSS4 antibodies confirmed expression at the protein level in both squamous cell and adenocarcinomas with little or no staining in normal lung tissues. Human lung tumor cell lines expressed varying levels of TMPRSS4 mRNA in vitro. Interestingly, tumor cell lines with high levels of TMPRSS4 mRNA failed to show detectable TMPRSS4 protein by either immunoblotting or flow cytometry. However, protein levels were increased under hypoxic culture conditions suggesting that hypoxia within the tumor microenvironment may upregulate TMPRSS4 protein expression in vivo. This was supported by the observation of TMPRSS4 protein in xenograft tumors derived from the cell lines. In addition, staining of human squamous cell carcinoma samples for carbonic anhydrase IX (CAIX), a hypoxia marker, showed TMPRSS4 positive cells adjacent to CAIX positive cells. Overall, these results indicate that the cancer-associated TMPRSS4 protein is overexpressed in NSCLC and may represent a potential therapeutic target.

Choriocarcinomas are a rare form of cancer that develops in the uterus from tissue which would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and have been studied largely to investigate conditions related to pregnancy such as preeclampsia. Choriocarcinomas are highly angiogenic and produce high levels of placental growth factor (PlGF) to promote the development of blood vessels. Upregulation of PlGF expression also occurs during the development of other human malignancies such as breast cancer and melanoma. Both tumor specimens and plasma samples have higher levels of PlGF than normal tissues. Hence, PlGF has emerged as a valid target for anti-angiogenic therapy. The cell lines BeWo, JAR and JEG-3, derived from human choriocarcinomas, were investigated in vitro and in vivo for suitability as PlGF-dependent models. BeWo, JAR and JEG-3 cells were characterized in culture and were implanted into immunodeficient mice to generate subcutaneous tumors. The PlGF and VEGF angiogenic profiles of the choriocarcinoma cells and tumors were investigated by ELISA and by immunohistochemical methods. Double immunofluorescence methods were applied to choriocarcinoma xenograft sections to characterize the cellular components of the blood vessels. sFLT01, a fusion protein that neutralizes PlGF, was assessed in cell culture experiments and xenograft studies. The novel results presented here validate the importance of human choriocarcinoma cell lines and xenografts in further exploring the role of PlGF in tumor angiogenesis, for evaluating PlGF as an anti-angiogenic target, and for developing therapies that may provide clinical benefit.

We previously surveyed the expression of endosialin/ CD248/TEM-1 by immunohistochemistry in human clinical specimens of sarcomas and documented expression in tumor cells, stromal cells and vasculature. In the present study, we completed a retrospective analysis of the diagnostic reports available for these same samples in order to identify high-grade and metastatic disease. Our results show that endosialin can be detected in advanced disease. We screened human sarcoma cell lines in vitro for endosialin expression and developed preclinical human xenograft models of disseminated sarcoma. We found that 22 out of 42 human sarcoma cell lines were positive for endosialin with a positive correlation between mRNA and protein levels. When implanted in vivo, endosialin was expressed at all sites of dissemination. These data provide clinical and preclinical evidence that endosialin can be detected in advanced sarcoma. These results demonstrate for the first time that endosialin is a suitable therapeutic target for poor prognosis and advanced disease.

Purpose: Genz-644282 [8,9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one] has emerged as a promising candidate for antitumor agents. This report describes the bone marrow colony-forming unit, granulocyte macrophage (CFU-GM) and tumor cell CFU activity of topoisomerase I (Top1) inhibitors, such as Genz-644282, topotecan, irinotecan/SN-38, and ARC-111, and examines their activity in several human tumor xenograft models. Experimental Design: Colony-forming assays were conducted with mouse and human bone marrow and eight human tumor cell lines. In addition, 29 human tumor cell lines representing a range of histology and potential resistance mechanisms were assayed for sensitivity to Genz-644282 in a 72-hour exposure assay. The efficacy of Genz-644282 was compared with standard anticancer drugs (i.e., irinotecan, docetaxel, and dacarbazine) in human tumor xenografts of colon cancer, renal cell carcinoma, non–small cell lung cancer, and melanoma. Results: Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than was mouse bone marrow CFU-GM. The ratio of mouse to human IC90 values was more than 10 for the camptothecins and less than 10 for Genz-644282, which had more potency as a cytotoxic agent toward human tumor cells in culture than the camptothecins in the colony-forming and 72-hour proliferation assays. Genz-644282 has superior or equal antitumor activity in the human tumor xenografts than the standard drug comparators. Conclusions: On the basis of preclinical activity and safety, Genz-644282 was selected for development and is currently undergoing phase 1 clinical trial. Clin Cancer Res; 17(9); 2777–87. ©2011 AACR.

Purpose: Placental growth factor (PlGF) is an angiogenic protein. Upregulation of PlGF has been observed in the clinic following antiangiogenic regimens targeting the VEGF pathway. PlGF has been proposed as a therapeutic target for oncology. sFLT01 is a novel fusion protein that neutralizes mouse and human PlGF (mPlGF, hPlGF) and mouse and human VEGF-A (mVEGF-A, hVEGF-A). It was tested in syngeneic and xenograft tumor models to evaluate the effects of simultaneously neutralizing PlGF and VEGF-A and to investigate changes observed in the clinic in preclinical models. Experimental Design: Production of PlGF and VEGF-A by B16F10 and A673 cancer cells in vitro was assessed. Mice with subcutaneous B16F10 melanoma or A673 sarcoma tumors were treated with sFLT01. Tumor volumes and microvessel density (MVD) were measured to assess efficacy. Serum levels of hVEGF-A, hPlGF, and mPlGF at early and late time points were determined by ELISA. Results: Exposure of cancer cell lines to sFLT01 caused a decrease in VEGF secretion. sFLT01 inhibited tumor growth, prolonged survival, and decreased MVD. Analysis of serum collected from treated mice showed that sFLT01 administration caused a marked increase in circulating mPlGF but not hPlGF or hVEGF. sFLT01 treatment also increased circulating mPlGF levels in non–tumor-bearing mice. Conclusion: With the tumor cell lines and mouse models we used, antiangiogenic therapies that target both PlGF and VEGF may elicit a host response rather than, or in addition to, a malignant cell response that contribute to therapeutic resistance and tumor escape as suggested by others. Clin Cancer Res; 17(5); 976–88. ©2011 AACR.

sFLT01 is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domain of human VEGFR1/Flt-1 (hVEGFR1) fused to the Fc portion of human IgG1 through a polyglycine linker. It binds to both human VEGF (hVEGF) and human PlGF (hPlGF) and to mouse VEGF (mVEGF) and mouse PlGF (mPlGF). In vitro, sFLT01 inhibited the proliferation of human umbilical vein endothelial cells and pericytes stimulated by either hVEGF or hPlGF. In vivo, sFLT01 had robust and significant antitumor activity in numerous preclinical subcutaneous tumor models including H460 non–small cell lung carcinoma, HT29 colon carcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia), 786-O, and RENCA renal cell carcinoma (RCC). sFLT01 also increased median survival in the orthotopic RENCA RCC model. sFLT01 had strong antiangiogenic activity and altered intratumoral microvessel density, blood vessel lumen size and perimeter, and vascular and vessel areas in RCC models. sFLT01 treatment resulted in fewer endothelial cells and pericytes within the tumor microenvironment. sFLT01 in combination with cyclophosphamide resulted in greater inhibition of tumor growth than either agent used alone as a monotherapy in the A673 Ewing's sarcoma model. Gene expression profiling indicated that the molecular changes in the A673 sarcoma tumors are similar to changes observed under hypoxic conditions. sFLT01 is an innovative fusion protein that possessed robust antitumor and antiangiogenic activities in preclinical cancer models. It is a dual targeting agent that neutralizes both VEGF and PlGF and, therefore, has potential as a next generation antiangiogenic therapeutic for oncology. Mol Cancer Ther; 10(3); 404–15. ©2011 AACR.

Biomarkers have been the focus of investigations to diagnose disease, track response to therapy and predict prognosis. Meanwhile, the identification of new targets for therapeutic intervention is an ongoing quest in the field of oncology. The recognition of endosialin as an antigen that is selectively overexpressed in human tumor tissues offers new strategies for treating cancer. Not only do the tumor vasculature and stromal compartments upregulate endosialin but, importantly, the malignant cells of sarcomas strongly express endosialin as well. A diagnostic assay that measures the intensity of endosialin expression in malignant tissues would assist in selecting patients that could benefit from an antiendosialin therapy. Thus, endosialin holds potential value both as a therapeutic target and as a biomarker for certain human cancers.

Nucleoside analogs are rationally designed anticancer agents that disrupt DNA and RNA synthesis. Fludarabine and cladribine have important roles in the treatment of hematologic malignancies. Clofarabine is a next generation nucleoside analog which is under clinical investigation. The bone marrow toxicity, tumor cell cytotoxicity and human tumor xenograft activity of fludarabine, cladribine and clofarabine were compared. Mouse and human bone marrow were subjected to colony forming (CFU-GM) assays over a 5-log concentration range in culture. NCI-60 cell line screening data were compared. In vivo, a range of clofarabine doses was compared with fludarabine for efficacy in several human tumor xenografts. The IC90 concentrations for fludarabine and cladribine for mouse CFU-GM were >30 and 0.93 microM, and for human CFU-GM were 8 and 0.11 microM, giving mouse to human differentials of >3.8- and 8.5-fold. Clofarabine produced IC90s of 1.7 microM in mouse and 0.51 microM in human CFU-GM, thus a 3.3-fold differential between species. In the NCI-60 cell line screen, fludarabine and cladribine showed selective cytotoxicity toward leukemia cell lines while for clofarabine there was no apparent selectivity based upon origin of the tumor cells. In vivo, clofarabine produced a dose-dependent increase in tumor growth delay in the RL lymphoma, the RPMI-8226 multiple myeloma, and HT-29 colon carcinoma models. The PC3 prostate carcinoma was equally responsive to clofarabine and fludarabine. Bringing together bone marrow toxicity data, tumor cell line cytotoxicity data, and human tumor xenograft efficacy provides valuable information for the translation of preclinical findings to the clinic.

Tumor development is a complex and dynamic process that involves malignant, vascular, and stromal cells. Endosialin is a tumor endothelial marker (TEM) present in the microvasculature and stroma of human tumors. Cancer-associated fibroblasts (CAF) have been implicated in promoting tumor development and have been associated with mesenchymal stem cells (MSC). Since stem/progenitor cells recruited either from bone marrow or residing in nearby tissues can contribute to pathological processes we investigated endosialin in MSC using a novel monoclonal antibody. Endosialin is highly expressed by CAF and human bone marrow-derived MSC. MSC can form networks in a tube formation assay that is inhibited by an anti-endosialin antibody. Immunohistochemistry for human endosialin in xenograft tumors following co-injection of MSC and cancer cells identified MSC in tumor stroma. MSC are a potential target for anticancer therapeutic intervention and endosialin expression offers a new tool for the identification of MSC. Endosialin expression by both CAF and MSC further implies the potential contribution of MSC to tumor stroma via differentiation into tumor stromal fibroblasts.

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45−/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow–derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC. [Mol Cancer Ther 2008;7(8):2536–46]

Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor–stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy. [Mol Cancer Ther 2006;5(2):219–29]

VEGF inhibitors aflibercept (ziv-aflibercept in USA) and bevacizumab target vascular endothelial growth factor A (VEGF-A), and are approved for the 2nd line treatment of metastatic colorectal cancer (CRC) in combination with distinct chemotherapy regimens and after different 1st line therapies. Although both aflibercept and bevacizumab bind to VEGF-A (herein referred to as VEGF), aflibercept blocks additional VEGF family ligands PlGF and VEGF-B, which bind VEGF receptor 1 (VEGFR1), whereas bevacizumab does not. We recently utilized patient-derived xenograft (PDX) models of CRC to compare the pharmacological activity of aflibercept and bevacizumab. In a screening study in nude mice, aflibercept showed more potent anti-tumor activity in 39/48 CRC PDX compared to bevacizumab (Chiron M. et al, AACR-NCI-EORTC 2013). In these PDX, tumor levels of tumor-derived human VEGF (hVEGF) were much higher than host murine VEGF (mVEGF), however, a potential limitation was that bevacizumab does not bind mVEGF. To further rule out any contribution from mVEGF and to determine whether tumor cell expression of VEGFR1 played a role in the differential responses, we compared the effects of aflibercept and bevacizumab on selected PDX CRC tumors in SCID mice genetically engineered to express hVEGF from the mVEGF locus. In humanized VEGF mice, aflibercept was more potent in 3/7 PDX compared with bevacizumab but equivalent in 4/7 PDX. Tumors in PDX IMM-Colo-0018 and IMM-Colo-0010, with high and low VEGFR1 expression, respectively, regressed in response to aflibercept while bevacizumab resulted in growth stasis. PDX model CXF280 with high VEGFR1 expression, consistently showed significantly greater response to aflibercept in comparison to bevacizumab. In contrast, PDX model CFX260, also with high VEGFR1 expression, responded equally well to both drugs. There was no CRC PDX in humanized VEGF mice in which the activity of bevacizumab was greater than aflibercept. Immunohistochemistry analysis revealed that improved efficacy of aflibercept treatment over bevacizumab was associated with a greater reduction of tumor vessel density and sprouting in CFX280 tumors. Cumulatively, these results suggest that aflibercept is more potent than bevacizumab in certain PDX CRC tumor models. In this limited subset of PDX CRC models, differences in pharmacological activity observed between aflibercept and bevacizumab in humanized VEGF mice do not appear to correlate with tumor cell expression of VEGFR1, but instead are associated with a greater reduction of tumor blood vessels. We are further investigating the role of VEGFR1 and other factors that may explain enhanced anti-tumor efficacy of aflibercept in PDX CRC models in humanized mice. The results suggest that CRC patient subsets may exist where aflibercept is more active than therapies targeting only VEGF-A. Citation Format: Cristina L. Abrahams, Laura D. Winters, Baosheng Li, Asma A. Parveen, Tatjana Kloss, Rebecca G. Bagley, Donald A. Bergstrom, Gavin Thurston, Marielle Chiron. Enhanced activity of aflibercept compared to bevacizumab in a patient-derived xenograft colorectal cancer model is associated with reduced tumor vascularity in humanized VEGF mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4809. doi:10.1158/1538-7445.AM2014-4809

Aflibercept (ziv-aflibercept in USA), a new fusion protein, targets vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF). Aflibercept + FOLFIRI is approved for treating metastatic colorectal cancer (mCRC) that is resistant to or has progressed following an oxaliplatin containing regimen. We investigated the pharmacological activity of aflibercept in CRC patient derived xenografts (PDX), most metastases-derived, and characterized PDX molecular and phenotypic properties. In 48 CRC PDX, Whole Exome Sequencing revealed: 85% APC, 79% TP53, 51% KRAS, 3% NRAS, 25% PIK3CA, 11% PTEN and 8% BRAF mutants. There was no significant association between angiogenic factors, tumor mutation profiles, and response to aflibercept. In tumors, protein levels of human (h) VEGF-A were significantly higher than mouse (m) VEGF-A, hPlGF, and mPlGF (Chiron et al, ESMO, 2013). Affymetrix probes detected VEGF-A and PIGF but not VEGF-B mRNA in most PDX. There was no significant association between expression of angiogenic factors and response to aflibercept. Histological sections of CRC tumors from PDX, xenografts created from cell lines, and patients’ tumor specimens indicated that stromal involvement in human tumors was recapitulated in PDX but not in tumors from cell lines. PlGF expression was analyzed by immunohistochemistry on colon cancer tissue arrays. A significant number of colon cancer specimens were positive for PlGF, expressed in tumor cells and stroma. Aflibercept resulted in anti-tumor activity in 47/48 CRC PDX models and complete tumor stasis in 31/48 models. Efficacy data from 5 PDX models responsive to aflibercept and CRC standard of care (SOC) drugs, all administered as monotherapies, are below. The robust activity of aflibercept in CRC PDX models that are sensitive to SOC drugs suggest that CRC patients would benefit from aflibercept treatment, independently of mutational status or expression of angiogenic factors. In Vivo Sensitivity Profile (optimal Tumor/Control (%))CRC PDX ModelPrior TreatmentsAflibercept 25 mg/kg, SC, 2x/wk5-FU100 mg/kg, IP, 1x/wkIrinotecan 50 mg/kg, IV, 1x/wkOxaliplatin 10 mg/kg, IP, Q2WCetuximab30 mg/kg, IV,1x/wkKRAS StatusCFX 1991Not available-2.531.51.856.342.1MutantCFX 2032Not available3.147.76.760.535.7MutantCFX 2048Not available-10.326.221.350.12.6Wild-typeCFX 20655-FU, leucovorin-11.920.614.471.82.2Wild-typeCFX 2067None6.253.045.158.914.2Wild-type Citation Format: Rebecca G. Bagley, Zakia Amalou, Marielle Chiron, Vincent Vuaroqueaux, Anne Caron, Nina Baltes, Heinz H. Fiebig. Aflibercept is highly active in clinically relevant, patient derived xenografts of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4808. doi:10.1158/1538-7445.AM2014-4808

Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function–associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit.

Identification of appropriate models for in vivo and in vitro preclinical testing of inhibitors of tumor angiogenesis and progression is vital to the successful development of anticancer therapeutics. Although the focus is on human molecular targets, most preclinical in vivo efficacy testing occurs in mice. The goal of the current studies was to identify a murine endothelial cell line to model tumor endothelium for studying the antiangiogenic activity of therapeutic compounds in vitro. In situ hybridization was performed on three s.c. grown syngeneic murine tumors (B16 melanoma, Lewis lung carcinoma, and CT26 colon carcinoma) to assess expression of murine homologs of human tumor endothelial cell markers in the vasculature of these tumor models. Seven murine endothelial cell lines were characterized for expression of the murine homologs of recognized endothelial cell surface markers as well as for tumor endothelial cell surface markers. The seven murine endothelial cell lines had similar generation times and five of the seven lines were able to form tubes on Matrigel. Real-time-PCR and flow cytometry analysis were used to evaluate relative mRNA and protein expression of murine homologs of several recognized endothelial cell surface markers in the seven cell lines. The expression of the mRNA for the murine homologs of five tumor endothelial cell surface markers was also evaluated. The 2H11 cell line expressed all five of the tumor endothelial cell surface markers as well as several well-recognized endothelial cells markers. The 2H11 cell line responds to known and novel antiangiogenic agents by inhibition of proliferation and tube formation. These cells can be used in in vitro angiogenesis assays for evaluating the potential antiangiogenic properties and interspecies cross-reactivity of novel compounds.

Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) have been the standards for cell-based assays in the field of angiogenesis research and in antiangiogenic drug discovery. These normal mature endothelial cells may not be most representative of human tumor endothelial cells. Human AC133+/CD34+ bone marrow progenitor cells were established in cell culture media containing vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and heparin to drive differentiation toward the endothelial phenotype. The resulting cells designated endothelial precursor cells (EPC) have many of the same functional properties as mature endothelial cells represented by HUVEC and HMVEC. By SAGE analysis, the genes expressed by EPC are more similar to the genes expressed by endothelial cells isolated from fresh surgical specimens of human tumors than are the genes expressed by HUVEC and HMVEC. Analysis of several cell surface markers by flow cytometry showed that EPC, HUVEC, and HMVEC have similar expression of P1H12, vascular endothelial growth factor 2, and endoglin but that EPC have much lower expression of ICAM1, ICAM2, VCAM1, and thrombomodulin than do HUVEC and HMVEC. The EPC generated can form tubes/networks on Matrigel, migrate through porous membranes, and invade through thin layers of Matrigel similarly to HUVEC and HMVEC. However, in a coculture assay using human SKOV3 ovarian cancer cell clusters in collagen as a stimulus for invasion through Matrigel, EPC were able to invade into the malignant cell cluster, whereas HMVEC were not able to invade the malignant cell cluster. In vivo, a Matrigel plug assay where human EPC were suspended in the Matrigel allowed tube/network formation by human EPC to be carried out in a murine host. EPC may be a better model of human tumor endothelial cells than HUVEC and HMVEC and, thus, may provide an improved cell-based model for second generation antineoplastic antiangiogenic drug discovery.

Background: The recombinant fusion protein aflibercept (afl) binds VEGF-A, -B, and placental growth factor (PlGF). Based on an OS benefit observed in the phase 3 VELOUR trial, aflibercept in combination with FOLFIRI was approved for patients with metastatic colorectal cancer (mCRC) following a prior oxaliplatin-containing regimen. In vitro, afl inhibited VEGFR signaling more potently and blocked cell migration more effectively vs. bevacizumab (bev) which only binds VEGF-A. Recent in vivo studies comparing the anti-tumor activity of afl vs. bev were conducted in patient-derived xenograft (PDX) CRC models. The PDX models are clinically relevant as they represent common genetic backgrounds in CRC and were derived from primary tumors or from metastases. A sequential treatment regimen of aflibercept post-bev vs. continuation of bev treatment was investigated. Methods: Forty eight different CRC adenocarcinoma tumors were engrafted subcutaneously (SC) into NMRI nude mice to generate 48 distinct PDX CRC models. ELISA and Luminex measured the expression of mouse and human VEGF-A and mouse and human PlGF in untreated tumors. Tumor-bearing mice (8 mice/group) were dosed with afl, bev, or placebo 2x/wk for 3 wks and SC tumor measurements were recorded 2x or 3x/wk. In 3 PDX models, mice that were treated with bev were randomized and either switched to afl treatment or continued to receive bev. Activity was expressed as relative change in tumor volume between treatment (ΔT) and control (ΔC) at 3 wks. A ΔT/ΔC ≤0% indicated complete tumor stasis. Student's t tests evaluated statistical differences in final tumor volumes. Results: Aflbercept induced complete tumor stasis in 31/48 PDX CRC models and bev in 2/48 PDX CRC models. The PDX models were characterized into 2 phenotypes based on statistical analysis: phenotype A, where afl activity was greater than bev activity (39 /48 PDX), and phenotype B, where there was no significant difference between the 2 drugs (9/48 PDX). Three models in which bev treatment resulted in eventual tumor escape vs. continuous inhibition by afl were selected for sequential treatment of bev followed by afl. Afl treatment began after bev treatment resulted in a greater than 2-fold increase in tumor volume. Greater anti-tumor activity occurred when afl was administered after bev vs. continuous bev treatment. At the end of the study, the tumor volumes were statistically significantly different between the afl and bev groups (T test p values < 0.0001). Analysis of angiogenic factors revealed that the tumor cells were the major source of VEGF whereas PlGF was primarily produced by the tumor stroma. Conclusions: Aflibercept induced complete tumor stasis more frequently in CRC PDX models than bevacizumab. As tumor levels of hVEGF-A were far greater than mVEGF-A, the inability of bevacizumab to bind mVEGF-A is unlikely to explain the higher and more consistent activity of aflibercept. In all 3 PDX models tested, switching to aflibercept treatment after bev treatment resulted in a greater tumor response than continuous bev treatment. The greater response to aflibercept than bev after an initial treatment with bev suggests that individual patients may derive more benefit from aflibercept than bev in second line treatment after an initial bev-based regimen. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B2. Citation Format: Marielle Chiron, Rebecca G. Bagley, Jack Pollard, Christophe Henry, Parminder Mankoo, Loic Vincent, Catherine Geslin, Tatjana Kloss, Donald A. Bergstrom. Switching to aflibercept treatment resulted in greater tumor responses than continuous bevacizumab treatment in patient-derived xenograft models of colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B2.

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.