Search

Introduction/Background This first-in-human, phase 1 study aimed to characterize the safety, tolerability, maximum tolerated dose (MTD), pharmacokinetic (PK) profile, and antitumor activity of RAD140, an oral selective androgen receptor (AR) modulator (SARM). Patients and Methods This dose-escalation study with a 3 + 3 design and PK expansion cohort enrolled postmenopausal women with ER+/HER2- metastatic breast cancer (mBC). Serum sex hormone-binding globulin (SHBG) and prostate-specific antigen (PSA) were used as surrogate markers of AR engagement. Results Twenty-two (21 AR+) heavily pretreated mBC patients were enrolled. Dose levels included 50 mg (n = 6), 100 mg (n = 13), and 150 mg (n = 3) once daily (QD). Most frequent (> 10%) treatment-emergent adverse events (TEAEs) were elevated AST (59.1%), ALT (45.5%), and total blood bilirubin (27.3%), and vomiting, dehydration, and decreased appetite and weight (27.3% each). Grade 3/4 TEAEs occurred in 16 (72.7%) patients and included elevations in AST/ALT and hypophosphatemia (22.7% each). Treatment-related TEAEs occurred in 17 per 22 patients (77.3%); 7 (31.8%) were Grade 3; none were Grade 4. The half-life (t1/2) of 44.7 hours supported QD dosing. At the MTD of 100 mg/day, 1 patient with an ESR1 mutation at baseline had a partial response. Overall, clinical benefit rate at 24 weeks was 18.2%, and median progression-free survival was 2.3 months. SHBG decreased in 18 per 18 patients, and PSA increased in 16 per 20 patients. Paired baseline and on-treatment tumor biopsies demonstrated AR engagement. Conclusion RAD140 is a novel oral AR-targeted agent for the treatment of AR+/ER+/HER2- mBC with an acceptable safety profile and preliminary evidence of target engagement and antitumor activity.

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.

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

To investigate the activity and myeloprotective properties of erufosine, a novel alkylphosphocholine (APC), on human malignant cells and normal bone marrow cells.Human or mouse bone marrow cells were exposed to erufosine, miltefosine, perifosine, or edelfosine in CFU-GM assays. Human MDA-MB-231 breast carcinoma, Panc-1 pancreatic carcinoma, and RPMI8226 multiple myeloma cells were exposed to erufosine in colony formation assays. Colony formation of Panc-1 tumor cells and mouse bone marrow cells ex vivo were quantified following intravenous administration of erufosine to tumor-bearing mice. Western blotting methods were applied to human U87 glioblastoma cells exposed to erufosine to investigate Akt inhibition.Erufosine was less toxic to human and mouse bone marrow cells than perifosine, miltefosine, and edelfosine and was equally toxic to human and mouse CFU-GM. The human cancer cells MDA-MB-231 breast, Panc-1 pancreatic, and RPMI8226 MM cells were more sensitive to erufosine in a colony formation assay than were human bone marrow cells generating an approximately tenfold differential in IC(90) values. Erufosine injected intravenously significantly reduced Panc-1 tumor cell colony formation ex vivo but not mouse bone marrow CFU-GM. Erufosine inhibited Akt phosphorylation in human U87 glioblastoma cells.Erufosine offers potential as a novel therapeutic for cancer with a reduced toxicity profile to bone marrow cells compared with other agents in this class. Human cancer cells were more sensitive to erufosine than human or mouse bone marrow cells indicating a favorable therapeutic window for erufosine.

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.

109 Background: gpNMB is an internalizable transmembrane glycoprotein expressed in melanoma and multiple other tumor types. The ADC GV (CDX-011) delivers the potent cytotoxin MMAE to gpNMB+ cells. GV has shown promising activity in advanced melanoma and breast cancer. Methods: This Phase II study (CDX011-05) assessed the efficacy and safety of GV monotherapy (1.9 mg/kg q3w) for patients (pts) with advanced melanoma progressive after ≤1 chemotherapy, ≥1 checkpoint inhibitor (CPI) and if BRAFV600mutated, ≥1 BRAF/MEK inhibitor. Central IHC determined gpNMB expression in archival and/or pre-treatment tumor. Primary endpoint was objective response rate (ORR) (RECIST 1.1) with ≥6 responders out of 52 evaluable pts as threshold for antitumor activity. Additional endpoints: progression free survival (PFS), overall survival (OS), duration of response (DOR), safety, PK/PD and correlation of tumor gpNMB expression with efficacy. Results: 62 pts enrolled (all evaluable) had median age of 67 years; 55% male; 21% BRAFV600mutated; 63% with ≥3 lines prior therapy; 100% had prior CPI; 100% Stage IV; 89% M1c. One confirmed complete response (CR) and 6 confirmed partial responses (PR, including 1 unconfirmed CR) were seen (confirmed ORR = 11%, p = 0.035 comparing to reference ORR 5%); 33 pts had stable disease including 3 unconfirmed PR. Median DOR = 6.0 (range: 4.1, 14+) months (mos), median PFS = 4.3 mos and median OS = 9.8 mos; 26 pts continue to be followed for survival. All pts with available tissue (60/60) had gpNMB+ tumors; 47/60 had 100% gpNMB+ epithelial cells; no clear correlation with outcome was seen in this population with consistent high expression. Toxicities included alopecia, neuropathy, rash, fatigue and neutropenia. Treatment-related rash in cycle 1 was associated with improved ORR (rash = 22%; no rash = 7%), PFS (p = 0.007) and OS (p = 0.035). Conclusions: GV has promising activity (primary endpoint of ORR was met) with a manageable safety profile in heavily pre-treated melanoma pts. Additional cohorts evaluating GV with either varlilumab, an activating anti-CD27 monoclonal antibody, or PD-1 inhibitors are open to accrual to provide further insights into the synergy of ADC and immunotherapy. Clinical trial information: NCT02302339.

34 Background: TIM-1 (KIM-1, HAVcr-1) is a type 1 transmembrane glycoprotein. Expression is limited in healthy tissues but upregulated after kidney injury, in ovarian cancer and renal cell carcinoma (RCC). TIM-1 is associated with a more malignant RCC phenotype and immune response regulation. CDX-014, an antibody drug conjugate, contains a fully human IgG1 monoclonal antibody (mAb) conjugated to the potent cytotoxin MMAE. CDX-014 is in a Ph 1/2 trial for RCC (NCT02837991). The prevalence of TIM-1 in other cancers was investigated to explore the broader potential for CDX-014. Methods: Tumors and normal adjacent tissues (NAT) were provided (Mosaic Labs) via IRB-reviewed protocol MOS001 for in vitro analysis of remnant, anonymized human samples. The study included 188 tumors: 30 renal, 45 ovarian, 34 endometrial, 2 cervical, 2 vaginal, 15 bile duct, 15 CRC, 30 NSCLC and 15 thymoma plus 75 NAT. Mosaic performed a validated immunohistochemistry assay on FFPE tissues. A FITC-conjugated, anti-TIM-1 mAb was applied after slide deparaffinization and antigen retrieval. Anti-FITC (Novus Bio), Envision+ HRP and DAB (Dako) were used for visualization. A pathologist scored hematoxylin-counterstained slides for % positive cells and staining intensity. A cut-off of > 2% of cells staining at any intensity was deemed positive. Results: TIM-1 was universally prevalent in renal papillary (14/14) and clear cell carcinoma (CCC [13/13]) plus vaginal CCC (2/2). TIM-1 was also seen in ovarian CCC (12/15; 80%), endometrial CCC (9/21; 43%), adeno NSCLC (2/15; 13%), serous ovarian (1/10; 10%), adeno endometrial (1/13; 8%) and CRC (1/15; 7%). There was no staining in chromophobe RCC (3/3), cervical CCC, endometrioid or mucinous ovarian (10 each), bile duct carcinoma, squamous NSCLC or thymoma. There was minimal to no TIM-1 in NAT except for 7/10 kidney samples (possibly from unappreciated kidney injury). Conclusions: The data suggest that in addition to renal and ovarian cancer, TIM-1 targeted therapy such as CDX-014 warrants exploration in patients with endometrial CCC, vaginal CCC and adeno NCSLC. These results support the ongoing first in human Ph 1/2 study in RCC and potential broader clinical development of CDX-014 in other indications.

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.

Antiangiogenesis has been validated as a therapeutic strategy to treat cancer, however, a need remains to identify new targets and therapies for specific diseases and to improve clinical benefit from antiangiogenic agents. Tumor endothelial marker 7 (TEM-7) was investigated as a possible target for therapeutic antiangiogenic intervention in cancer. TEM-7 expression was assessed by in situ hybridization or by immunohistochemistry (IHC) in 130 formalin-fixed paraffin-embedded (FFPE) and 410 frozen human clinical specimens of cancer plus 301 normal tissue samples. In vitro TEM-7 expression was evaluated in 4 human endothelial cell models and in 32 human cancer cell lines by RT-PCR and flow cytometry. An anti-TEM-7 antibody was tested in vitro on human SKOV3 ovarian and MDA-MB-231 breast carcinoma cells that expressed TEM-7 in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. In frozen tumor tissues, TEM-7 mRNA and protein was detected in all but one of the cancer types tested and was infrequently expressed in normal frozen tissues. In FFPE tumor tissues, TEM-7 protein was detected by IHC in colon, breast, lung, bladder, ovarian and endometrial cancers and in sarcomas. TEM-7 protein was not detected in head and neck, prostate or liver cancers. TEM-7 expression was restricted to the vasculature and was absent from tumor cells. In vitro, TEM-7 was not detected in human microvascular endothelial cells (HMVEC) or human umbilical vein endothelial cells (HUVEC) but was induced in endothelial precursor/progenitor cells (EPC) in the presence of the mitogen phorbol ester PMA. An anti-TEM-7 antibody mediated ADCC and phagocytosis in SKOV3 and MDA-MB-231 cell lines infected with an adenovirus expressing TEM-7. These data demonstrate that TEM-7 is a vascular protein associated with angiogenic states. TEM-7 is a novel and attractive target for antiangiogenic therapy.

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.

Table of Contents Introduction - Cancer Stem Cells vs. Normal Stem Cells Rebecca Bagley, MS, Genzyme Corporation Chapter 1: Stem Cell Microenvironment - Hematopoietic Stem Cells Linheng Li, PhD, Stowers Institute for Medical Research Tannishtha Reya, PhD, Duke University Medical Center David Scadden, MD, Center for Regenerative Medicine, Mass General Hospital Sean Morrison, PhD, University of Michigan Medical School Chapter 2: Tumor Niche/Microenvironment - Mesenchymal Stem Cells Paul Simmons, PhD, University of Texas Health Science Center Ajeeta Dash, PhD, Genzyme Corporation Chapter 3: Cancer Stem Cells - Brain Tumors Peter Dirks, PhD, MD, University of Toronto Hospital for Sick Children Jeremey Rich, MD, Duke University Medical Center Nobuko Uchida, PhD, Stem Cells, Inc. Sheila Singh, MD, University of Toronto Hospital for Sick Children Chapter 4: Cancer Stem Cells - Breast Carcinoma Jane Visvader, PhD, Victorian Breast Cancer Research Consortium Max Wicha, MD, University of Michigan Comprehensive Cancer Center Michael Clarke, MD, Stanford University Stem and Regenerative Medicine Institute Connie Eaves, PhD, British Columbia Cancer Research Centre Chapter 5: Cancer Stem Cells - Lung Cancers Carla Bender Kim, PhD, Children's Hospital/Harvard University Joan Schiller, MD, Southwestern Medical School/Simmons Comprehensive Cancer Center Jaclyn Hung, PhD, British Columbia Cancer Research Centre Chapter 6: Cancer Stem Cells - Pancreatic Cancer Diane Simeone, MD, University of Michigan Medical Center Chapter 7: Cancer Stem Cells - Ovarian Paul Szotek, MD, Mass General Hospital, Harvard Medical School Chapter 8: Cancer Stem Cells - Colon Michael Clarke, MD, Stanford University Stem and Regenerative Medicine Institute Hans Clevers,MD/PhD, Netherlands Institute for Developmental Biology Chapter 9: Cancer Stem Cells - Prostate Cancer Owen Witte/Susan Kasper Chapter 10: Epidermal Stem Cells Elaine Fuchs, PhD, Rockefeller University Fiona Watt, PhD, Cancer Resarch UK London, Cambridge Institute Chapter 11: Hematopoietic Stem Cells in Blood Cancers: Leukemia / Myeloma / Lymphoma Catriona Jamieson, MD/PhD, University of California Medical Center Daniel Tenen, MD, Beth Israel Deaconess Medical Center, Harvard Medical School Dominique Bonnet, PhD, London Research Institute Michael Becker, MD, University of Rochester Medical Center John Dick, PhD, Toronto General Research Institute Frederick Alt, PhD, Howard Hughes Medical Institute Michael Cleary, MD, Stanford University School of Medicine Irving Weissman, MD/PhD, Stanford University School of Medicine Craig Jordan, PhD, University of Rochester Medical Center Chapter 12: Epigenetic Control and Chromosomal Instability Laurie Jackson-Grusby, PhD, Children's Hospital, Harvard Medical School Kornelia Polyak, MD/PhD, Dana-Farber Cancer Institute Bradley Bernstein, MD/PhD, Mass General Hospital, Harvard Medical School Stephen Baylin, MD, Johns Hopkins School of Medicine Chapter 13: Signaling Pathways Roeland Nusse, PhD, Stanford University Andreas Androutsellis-Theotokis, PhD, National Institute of Health Ivan Maillard, PhD, University of Pennsylvania School of Medicine Tannishtha Reya, PhD, Duke University Medical Center Linheng Li, Stowers, PhD, Institute for Medical Research Chapter 14: Targeting Cancer Stem Cells: A New Therapeutic Approach Tessa Holyoake, PhD, University of Glasgow D. Gary Gilliland, MD/PhD, Howard Hughes Medical Institute Jeffrey Molldrem, MD, M. D. Anderson Cancer Center Vera Donnenberg, PhD, McGowan Institute for

129 Background: Glycoprotein NMB (gpNMB) is an internalizable transmembrane protein overexpressed in ~20% of BC. gpNMB promoted BC metastases in a murine model and is a poor prognostic marker in BC patients (pts) (Rose 2010). Glembatumumab vedotin (GV, CDX-011) is a novel antibody-drug conjugate targeting gpNMB+ cancer cells with the potent cytotoxin monomethyl auristatin E. In a Phase I/II and in the Phase II EMERGE study, GV was well-tolerated (treatment-related toxicity included rash, neutropenia, and neuropathy) with promising activity in gpNMB+ BC tumors including TNBC. In EMERGE, GV vs. investigator’s choice chemotherapy, demonstrated an objective response rate of 30% (7/23) vs. 9% (1/11) in pts with tumor gpNMB overexpression (gpNMB in > 25% of tumor cells); 18% (5/28) vs. 0% (0/11) in TNBC; and 40% (4/10) vs. 0% (0/6) in gpNMB-overexpressing TNBC. Improvements in progression-free survival (hazard ratio (HR) = 0.11) and overall survival (HR = 0.14) in gpNMB-overexpressing TNBC were noted. Methods: This retrospective analysis of the EMERGE study examined frequency of gpNMB overexpression by various baseline and disease characteristics. Tumors were centrally assessed by immunohistochemistry (IHC) and included data for 328 pts. Results: Tumor gpNMB overexpression was present in 21% (69/328) of screened and 40% (38/96) of TNBC pts. gpNMB overexpression was consistent in progesterone, estrogen, or HER2+ expressing tumors at rates of 12-15%. gpNMB overexpression was not observed among 17 lobular tumors, but was present in 21% (57/270) of ductal tumors. gpNMB overexpression was seen across organ sites, including lung (43%, 3/7), lymph node (31%, 12/39), chest wall (23%, 3/13), breast (21%, 44/209), bone (13%, 1/8), and liver (13%, 3/24). There were no apparent differences in gpNMB overexpression by age, race or disease setting (early vs. advanced) at tissue collection. Analysis is ongoing to determine if prior treatments may upregulate gpNMB expression. Conclusions: gpNMB overexpression in BC, especially in TNBC, appears consistent in archival primary and/or metastatic BC, regardless of age. A randomized multicenter study of GV in gpNMB overexpressing metastatic TNBC (METRIC) is ongoing. Clinical trial information: NCT01156753.

Anti-angiogenic strategies have largely focused on endothelial cells and progenitors. However, pericytes are also an important component of vasculature. Perivascular cells from normal tissues have been widely reported, yet have not been extensively studied from human tumors. We have investigated pericytes from tumors of patients with lung cancer, the leader of cancer-related deaths in both men and women. Antibodies and magnetic beads were used to isolate cells from non-small cell lung carcinomas (NSCLC). The morphology of the pericytes was distinct with multiple elongated cytoplasmic extensions. Molecular expression of angiogenic genes was quantified by RT-PCR. Flow cytometric analysis shows that NSCLC pericytes express antigens such NG2 and VEGFR1 and present the ganglioside 3G5. The value of pericytes as models of tumor vasculature was demonstrated in cell-culture-based angiogenesis assays such as tube formation and proliferation. Results show that pericytes from some NSCLC but not all were able to maintain tubes networks on Matrigel. Pericyte function can be influenced by angiogenic growth factors or anti-angiogenic agents. Pericytes displayed invasive action against NSCLC clusters in the absence of other cell types. Perivascular cells contribute to the progression of disease and are an attractive target for anti-angiogenic therapy.

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.

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

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.

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.

Choriocarcinomas are an aggressive form of cancer that develops in the uterus from tissue that would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and the tumors are highly angiogenic. Human placental growth factor (hPlGF) is an angiogenic growth factor that promotes the growth of blood vessels in the placenta. hPlGF levels can be higher under pathological conditions such as cancer. Some human tumor specimens have higher PlGF levels than normal tissue, thus hPlGF is a target for anti-angiogenic therapy. The human choriocarcinoma cell lines BeWo, JAr, and JEG-3 were evaluated as preclinical models of hPlGF overexpression. The levels of hVEGF-A and hPlGF secreted by the choriocarcinomas and 11 human and mouse cancer cell lines in culture were measured by ELISA. The choriocarcinomas secreted higher levels of hPlGF and lower levels of hVEGF-A than 10/11 other cancer cell lines. The hPlGF serum levels in mice bearing subcutaneous choriocarcinoma tumors correlated with tumor volume and reached >2,000 pg/ml. By comparison, no hPlGF was detected in the serum of mice bearing H460 non-small lung carcinoma, HT29 colon carcinoma or MOLM-13 acute myeloid leukemia xenograft tumors. Although choriocarcinoma cells secreted both hPlGF and hVEGF-A in culture, serum levels of hVEGF-A in mice bearing choriocarcinoma xenografts were undetectable or Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1596. doi:10.1158/1538-7445.AM2011-1596

Introduction: PlGF and VEGF stimulate angiogenesis and promote the growth of tumor vasculature. PlGF is a member of the VEGF family and binds to VEGFR1. sFLT01 is a novel fusion protein comprised of the Fc portion of human IgG1 and the PlGF- and VEGF-binding domain of VEGFR1/Flt-1. The properties of sFLT01 and the potential of sFLT01 as an anti-angiogenic agent to inhibit tumor growth were investigated in several in vitro assays and in multiple xenograft tumor models. Methods: The binding kinetics of sFLT01 for both the human and murine homologues of PlGF and VEGF were assessed by Biacore. The abilities of recombinant human PlGF and VEGF to induce endothelial cell and pericyte proliferation and of sFLT01 to inhibit this stimulation were investigated in cell-based assays. The secretion of human VEGF and PlGF in culture by the HT29 colon carcinoma, H460 lung carcinoma, and A673 sarcoma human cell lines was quantified by ELISA. In efficacy studies, sFLT01 was administered by intraperitoneal injection twice per week to immunodeficient mice bearing HT29, H460, or A673 subcutaneous tumors. Antibodies specific for human IgG and VEGR2 were applied to A673 sarcoma tumor sections from mice treated with sFLT01 to visualize sFLT01 in the tumors and determine VEGFR2 expression in the cellular components. Pericytes and endothelial cells were identified with antibodies against NG2 and CD31. Results: The Biacore results indicated that sFLT01 has high affinity for human and murine PlGF and VEGF. Human recombinant PlGF and VEGF each induced the proliferation of human pericytes and endothelial cells in culture. This stimulation was inhibited by sFLT01. A673 sarcoma, HT29 colon and H460 lung carcinoma cells secreted higher levels of VEGF than PlGF in culture. In vivo, 10 mg/kg sFLT01 was effective at significantly slowing the growth of HT29 colon carcinoma and A673 sarcoma tumors compared to controls. Further analysis of the A673 sarcoma tumors in sFLT01-treated mice by immunohistochemistry revealed that sFLT01 penetrated multiple areas of the tumor. sFLT01 was detected in the vasculature, stroma, necrotic areas, and adjacent to malignant cells. sFLT01 treatment in the A673 model disrupted vessel integrity with a lack of association between endothelial cells and pericytes. A673 sarcoma cells expressed VEGFR2 in vivo. Conclusion: sFLT01 neutralizes the angiogenic activity of multiple vasculogenic VEGF family members in vitro and inhibited the proliferation of cells that form blood vessels, endothelial cells and pericytes. In vivo, sFLT01 treatment resulted in disorganized tumor vasculature thereby slowing the growth of xenografts tumors. The expression of VEGFR2 in A673 sarcoma tumors suggests that VEGF may play a role in autocrine signaling in some malignant cells. sFLT01 has antitumor and antiangiogenic activity in several human tumor xenografts and may offer therapeutic benefit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1388.

Mapping the long arm of Chromosome (Chr) 4 has assumed medical relevance with the establishment of linkage of facioscapulohumeral muscular dystrophy (FSHD) to distal 4q markers. We have constructed a multipoint linkage map using DNA markers that map to the long arm of Chr 4. Segregation data were collected for 17 DNA markers on the multigenerational CEPH mapping families, and data for one marker were taken from the published CEPH database. Genotypic information for six of these markers was also collected from a set of 24 families that exhibited inheritance of FSHD. Multipoint analyses allowed us to construct a map of 12 loci, connecting two previously separate linkage groups. Significant sex-specific differences in recombination were found for some genetic intervals. Four loci from the distal region of this map showed linkage with FSHD. A map using these terminal markers gave the strongest support for FSHD in the most distal position over all other possible positions.

Introduction: Anti-angiogenic agents have shown clinical value in combination with chemotherapy by targeting the VEGF pathways. The development of tumor vasculature in human renal cell carcinomas (RCC) is stimulated not only by VEGF but by other angiogenic growth factors such as placental growth factor (PlGF) [1,2]. In the current study, we used a novel fusion protein, sFLT01, that includes human VEGFR1 domain 2 and an IgG1 Fc, which binds to both human VEGF and PlGF. The anti-angiogenic activity of sFLT01 was evaluated in several preclinical models of RCC. Methods: The binding affinity of sFLT01 for PlGF was evaluated in vitro in binding assays quantified by ELISA and Octet and in proliferation assays where endothelial cells are stimulated by PlGF. The ability of sFLT01 to slow tumor growth was evaluated in the 786-O human RCC xenograft model as well as in the mouse RENCA RCC orthotopic and subcutaneous tumor models. sFLT01 was delivered by intraperitoneal injection twice per week at doses ranging from 5–25 mg/kg. Microvessel density (MVD) and other vascular parameters were analyzed in the syngeneic and xenograft tumors by immunohistochemical methods with an antibody against mouse CD31 to identify endothelial cells. Results: sFLT01 bound to human PlGF with great affinity and inhibited HUVEC proliferation with an IC90 of approximately 2.3 nM. Subcutaneous 786-O RCC tumors were inhibited at a dose of 25 mg/kg and median survival time was increased by 33% in the orthotopic RENCA model. sFLT01 reduced MVD in subcutaneous RENCA tumors by approximately 50% and lumen area by approximately 66%. In the 786-O xenograft model, 5 or 25 mg/kg sFLT01 reduced the blood vessel area, lumen size, and vessel perimeter compared to control. Conclusions: sFLT01 is effective at inhibiting blood vessel growth in tumors by binding VEGF and PlGF thereby preventing the development of new vasculature and decreasing existing vasculature. sFLT01 reduces intratumoral blood vessel count, prolongs survival, and delays tumor progression in the RENCA and 786-O renal cell carcinoma models. As an anti-angiogenic agent, sFLT01 may be useful in treating human renal cell carcinomas. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A14.

TMPRSS4, a cell surface transmembrane serine protease, is over expressed at the transcriptional level in pancreatic, colorectal and thyroid cancers compared with normal tissues. Recent studies have indicated a role for TMPRSS4 in tumor cell migration and tumor metastasis. We examined TMPRSS4 expression in dissected non-small cell lung cancer (NSCLC) tissues by quantitative RT-PCR and immunohistochemisty. At the transcriptional level, TMPRSS4 expression was elevated in adeno and squamous cell (SC) carcinomas when compared to the matching normal tissues (p=0.0035). At the protein level, over 100 tumor and normal specimens were examined with rabbit polyclonal anti-TMPRSS4 antibodies via immunohistochemistry. Adeno and SC carcinomas were positive for TMPRSS4 (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C166.

20520 Background: Tumors grow more slowly in endosialin/TEM1 KO mice compared to WT, suggesting that host endosialin/TEM1-positive stroma promotes malignancy (Nanda, PNAS, 2006). Methods: We surveyed 92 human clinical samples by IHC (52 epithelial tumors, 40 sarcomas) to measure the extent of endosialin/TEM1 expression and assess cellular localization. Expression was also studied in primary cell cultures and 49 cancer lines. Results: 46/52 adenocarcinomas and carcinomas (88.5%) expressed endosialin/TEM1 mainly in pericytes (7/7 bladder, 7/11 breast, 5/5 colon, 7/8 ovarian, 7/7 renal, 2/2 liver, 4/5 lung, and 7/7 gastric). In some tumors, endosialin/TEM1 was present in pericytes and in fibroblasts (2/7 bladder, 4/11 breast, 5/5 colon, 5/8 ovarian, 2/7 renal, 2/2 liver, 1/5 lung and 7/7 gastric cancers). 32/40 sarcomas (80%) expressed endosialin/TEM1 mainly in pericytes (10/11 synovial sarcomas, 9/10 malignant fibrous histiocytomas, 3/5 Ewing's sarcomas, 3/5 angiosarcomas, 3/4 rhabdomyosarcomas, 2/2 pleiomorphic sarcomas, 1/1 Kaposi's sarcoma, 0/1 leiomyosarcoma and 1/1 liposarcoma). Synovial sarcomas displayed intense fibroblast staining. Endosialin/TEM1 was expressed by sarcoma cells in a few samples. In culture, we previously showed expression in human fibroblasts, endothelial precursor cells, pericytes and mesenchymal stem cells (MSC). We now report that endosialin/TEM1 mRNA is present in MSC during osteogenic and adipogenic differentiation. In 49 human tumor lines, expression was restricted to sarcoma cells (7/15 sarcoma, 0/5 melanoma, 0/2 neuroblastoma, 0/4 breast, 0/1 ovarian, 0/3 prostate, 0/1 renal, 0/1 liver, 0/3 pancreatic, 0/6 colorectal and 0/8 hematopoietic cancer lines). Conclusions: Endosialin/TEM1 is frequently expressed in clinical cancer (85% of 92 samples), both in epithelial and mesenchymal tumors. Cellular expression is mesenchymal in clinical samples and in culture. In tissues, endosialin/TEM1 is present in pericytes and fibroblasts, with some expression in sarcoma cells. The importance of endosialin/TEM1-positive stroma in experimental animals and prevalence in patient samples warrant further investigation. Endosialin/TEM1 may be a useful therapeutic target. [Table: see text]