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Although high-dose therapy and autologous stem cell transplant (ASCT) is superior to conventional chemotherapy for treatment of myeloma, most patients relapse and the time to relapse depends upon the initial prognostic factors. The administration of non-cross-resistant chemotherapies during the post-transplant period may delay or prevent relapse. We prospectively studied the role of consolidation chemotherapy (CC) after single autologous peripheral blood stem cell transplant (auto-PBSCT) in 103 mostly newly diagnosed myeloma patients (67 patients were < or =6 months from the initial treatment). Patients received conditioning with BCNU, melphalan+/-gemcitabine and auto-PBSCT followed by two cycles of the DCEP+/-G regimen (dexamethasone, cyclophosphamide, etoposide, cisplatin+/-gemcitabine) at 3 and 9 months post-transplant and alternating with two cycles of DPP regimen (dexamethasone, cisplatin, paclitaxel) at 6 and 12 months post-transplant. With a median follow-up of 61.2 months, the median event-free survival (EFS) and overall survival (OS) are 26 and 54.1 months, respectively. The 5-year EFS and OS are 23.1 and 42.5%, respectively. Overall, 51 (49.5%) patients finished all CC, suggesting that a major limitation of this approach is an inability to deliver all planned treatments. In order to improve results following autotransplantation, novel agents or immunologic approaches should be studied in the post-transplant setting.

Myelosuppression is one of the major side effects of most anticancer drugs. To confer myeloprotection, our laboratory generated drug-resistant mutants of select target human enzymes for gene transfer to the bone marrow. Mutants of two of these enzymes, dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S), were previously shown to confer resistance to methotrexate and 5-FU, respectively, and recently a fusion cDNA of both mutant enzymes (DHFR F/S-TS G52S) was shown to confer dual resistance to both antimetabolites. In this study, we examined the sensitivity of the DHFR F/S-TS G52S fusion protein to the multitargeted antifolate, pemetrexed (LY231514, Alimta), which targets both DHFR and TS and is currently in phase III trials for the treatment of solid tumors and in combination with cisplatin has been shown to be an advance in the treatment of mesothelioma. The K(i) for the DHFR F/S portion of the purified fusion protein to pemetrexed was increased by greater than 9000-fold when compared to wtDHFR (8000 versus 0.86 nM), while the K(i) for the TS G52S portion of the fusion protein to pemetrexed was similar to that of wtTS (2.8 versus 3.1 nM). When the fusion gene was retrovirally transduced into NIH 3T3 fibroblasts, the IC(50) to pemetrexed was three- to four-fold higher than cells transduced with DHFR F/S or TS G52S alone (163 versus 53 and 45 nM, respectively). Similarly, expression of the DHFR F/S-TS G52S fusion gene in retrovirally transduced mouse marrow cells resulted in an increased survival of CFU-GM colonies when compared to cells transduced with either of the mutants alone. Co-expression of mutant DHFR and TS enzymes has additive effects in conferring resistance to pemetrexed-induced toxicity. This construct may be useful for conferring myeloprotection to patients receiving this drug.

This study was conducted to define a new maximum tolerated dose and the dose-limiting toxicity (DLT) of melphalan and autologous hematopoietic stem cell transplantation (AHSCT) when used with the cytoprotective agent amifostine. Fifty-eight patients with various types of malignancy who were ineligible for higher-priority AHSCT protocols were entered on a phase I study of escalating doses of melphalan beginning at 220 mg/m(2) and advancing by 20 mg/m(2) increments in planned cohorts of 4 to 8 patients until severe regimen-related toxicity (RRT) was encountered. In all patients, amifostine 740 mg/m(2) was given on 2 occasions before the first melphalan dose (ie, 24 hours before and again 15 minutes before). AHSCT was given 24 hours after the first melphalan dose. Melphalan was given in doses up to and including 300 mg/m(2). Hematologic depression was profound, although it was rapidly and equally reversible at all melphalan doses. Although mucosal RRT was substantial, it was not the DLT, and some patients given the highest melphalan doses (ie, 300 mg/m(2)) did not develop mucosal RRT. The DLT was not clearly defined. Cardiac toxicity in the form of atrial fibrillation occurred in 3 of 36 patients treated with melphalan doses/=280 mg/m(2) and was deemed fatal in 1 patient given melphalan 300 mg/m(2). (Another patient with a known cardiomyopathy was given melphalan 220 mg/m(2) and died as a result of heart failure but did not have atrial fibrillation.) Another patient given melphalan 300 mg/m(2) died of hepatic necrosis. The maximum tolerated dose of melphalan in this setting was thus considered to be 280 mg/m(2), and 27 patients were given this dose without severe RRT. Moreover, 38 patients were evaluable for delayed toxicity related to RRT; none was noted. Tumor responses have been noted at all melphalan doses and in all diagnostic groups, and 21 patients are alive at median day +1121 (range, day +136 to day +1923), including 16 without evidence of disease progression at median day +1075 (range, day +509 to day +1638). Amifostine and AHSCT permit the safe use of melphalan 280 mg/m(2), an apparent increase over the dose of melphalan that can be safely administered with AHSCT but without amifostine. Further studies are needed not only to confirm these findings, but also to define the antitumor efficacy of this regimen. Finally, it may be possible to evaluate additional methods of further dose escalation of melphalan in this setting.

The purpose of the study was to examine the yield of CD34(+) cells, response rates, and toxicity of high-dose cyclophosphamide with or without etoposide in patients with multiple myeloma. In total, 77 myeloma patients received either cyclophosphamide 4.5 g/m(2) (n=28) alone or with etoposide 2 g/m(2) (n=49) in a nonrandomized manner, followed by G-CSF 10 microg/kg/day for the purpose of stem cell mobilization. The effects of various factors on CD34(+) cell yield, response rate and engraftment were explored. A median of 22.39 x 10(6) CD34(+) cells/kg were collected on the first day of leukapheresis (range 0.59-114.71 x 10(6)/kg) in 71 (92%) of patients. Greater marrow plasma cell infiltration (P=0.02) or prior radiation therapy (P=0.02) adversely affected CD34(+) cell yield. In total, 45% of patients receiving cyclophosphamide and 56% of those receiving cyclophosphamide/etoposide had at least a minimum response by EBMT criteria. In all, 25% of patients who received cyclophosphamide alone vs 75.5% of patients who received combined chemotherapy required hospitalization mainly for treatment of neutropenic fever. Cyclophosphamide alone is associated with impressive CD34(+) cell yields and clear antimyeloma activity. The addition of etoposide resulted in increased toxicity without significant improvement in CD34(+) cell yield or response rates.

High-dose chemotherapy using melphalan (HDMEL) is an important component of many conditioning regimens that are given before autologous hematopoietic stem cell transplantation (AHSCT). In contrast to the situation in myeloma, and to a lesser degree acute leukemia, only a very limited published experience exists with the use of HDMEL conditioning as a single agent in doses requiring AHSCT for lymphoma, both Hodgkin lymphoma (HL) and especially non-Hodgkin lymphoma (NHL). Thus, we report results of treating 26 lymphoma patients (22 with NHL and four with HL) with HDMEL 220-300 mg/m(2) plus amifostine (AF) cytoprotection and AHSCT as part of a phase I-II trial. Median age was 51 years (range 24-62 years); NHL histology was varied, but was aggressive (including transformed from indolent) in 19 patients, indolent in two patients and mantle cell in one. All 26 patients had been extensively treated; 11 were refractory to the immediate prior therapy on protocol entry and two had undergone prior AHSCT. All were deemed ineligible for other, 'first-line' AHSCT regimens. Of these 26 patients, 22 survived to initial tumor evaluation on D +100. At this time, 13 were in complete remission, including four patients who were in second CR before HDMEL+AF+AHSCT. Responses occurred at all HDMEL doses. Currently, seven patients are alive, including five without progression, with a median follow-up in these latter patients of D +1163 (range D +824 to D +1630); one of these patients had a nonmyeloablative allograft as consolidation on D +106. Conversely, 14 patients relapsed or progressed, including five who had previously achieved CR with the AHSCT procedure. Two patients, both with HL, remain alive after progression; one is in CR following salvage radiotherapy. Six patients died due to nonrelapse causes, including two NHL patients who died while in CR. We conclude that HDMEL+AF+AHSCT has significant single-agent activity in relapsed or refractory NHL and HL. This experience may be used as a starting point for subsequent dose escalation of HDMEL (probably with AF) in established combination regimens.

Hypermethylation of the O(6)-methylguanine DNA methyltransferase (MGMT) promoter region leads to transcriptional repression of the MGMT gene and is a common event in primary human neoplasia. The purpose of this study was to determine the frequency and clinical relevance of MGMT gene promoter hypermethylation in curatively resected non-small cell lung cancer (NSCLC).MGMT hypermethylation, expressed as the ratio between methylated MGMT to unmethylated MYOD1 in genomic DNA, was analyzed in normal and matching tumor tissue from 90 patients with NSCLC, and a control group of 10 patients without cancer using a methylation-specific fluorogenic Real-Time PCR (Taqman) system.Hypermethylation of the MGMT promoter was detected in 34 of 90 (38%) tumor specimens and 16 of 90 (18%) matching normal lung tissues of patients with NSCLC, and in 0 (0%) cases of the control group without lung cancer. The mean MGMT methylation level was significantly higher in tumor than in matching normal tissue (P0.001). MGMT methylation in normal tissue was always accompanied with MGMT methylation in matching tumor tissue. Patients without MGMT promoter hypermethylation showed a significantly better survival than patients with MGMT promoter hypermethylation (P = 0.017). Multivariate analysis revealed MGMT promoter methylation as an independent unfavorable prognostic factor (P = 0.030).MGMT promoter hypermethylation is a common event in patients with primary NSCLC. This epigenetic alteration is associated with inferior survival, suggesting that MGMT promoter hypermethylation might be an important biomarker for a biological aggressive disease in NSCLC.

Increased intracellular concentrations of the initiator protein Rep (or RepA) interfere with pSC101 DNA replication, and mutated Rep proteins that result in an increase in plasmid copy numbers do not inhibit the replication. A rep mutant (rep(inh)) defective in the inhibitory activity was isolated and found to be a new high copy number mutant. The inhibitory function of Rep was enhanced by the coexistence of directly repeated sequences (DR; iterons) in the replication origin region (ori), but not by the inverted repeat sequences (IR) in ori and the rep promoter. This synergistic effect of Rep and DR sequences for the replication inhibition was dependent on their intracellular concentrations. Considering that DR sequences are the specific binding sites of the Rep monomer form, the Rep monomer-DR complex might be responsible for the inhibition of the plasmid replication. Furthermore, the Rep monomer in the crude cell extracts facilitated dimerization of DR DNA fragments by DNA ligase. Neither synergistic inhibitory function with DR nor Rep mediated dimerization of DR DNA was observed in high copy number mutant Rep proteins. The role of the Rep-iteron complex in the copy number control of pSC101 is discussed.

Targeting the Notch signaling pathway for cancer treatment is currently the subject of early clinical trials. However, treatment-related toxicities, particularly gastrointestinal adverse events, have limited clinical development of this class of agents. The development of efficacy biomarkers to gamma-secretase inhibitors and antibodies to Notch ligands and receptors could speed clinical testing. Mutations in Notch1, a dominant Notch receptor, are rare in human solid tumors, and clinical activity has not been observed in patients with tumors bearing Notch1 activating mutations, including T-cell acute lymphoblastic leukemia from available clinical data. Identification of tumors with abundant target expression and/or active Notch signaling status characteristic of Notch intracellular domain (NICD) expression by immunohistochemistry may have clinical utility. All levels of tumor Notch1 expression comprised of low, moderate and high detected by immunohistochemistry and quantitated by digital imaging technology were observed in 52% (34/66) of lung cancers, 51% (35/68) of ovarian cancers, and 11% (7/63) of breast cancers (lung or ovary vs. breast, P Citation Format: Dat Nguyen, Larry Rubinstein, Mark E. Sherman, Joseph E. Tomaszewski, Naoko Takebe, Percy Ivy, James H. Doroshow, Sherry X. Yang. Differential expression of Notch1 in lung, ovarian and breast cancers. [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 3838. doi:10.1158/1538-7445.AM2014-3838

BACKGROUND: New therapeutic approaches are urgently needed for recurrent GBM (rGBM). Recent studies suggest that GBM oncogenesis and recurrence are regulated by glioma initiating cells (GIC), a minority population of quiescent cells driven by embryonic signaling pathways and resistant to radiation and chemotherapy. Our hypothesis was that interrupting the sonic hedgehog signaling pathway (SHh), an integral mediator of GIC proliferation, would slow tumor progression and improve six month progression free survival (PFS6) in correlation with decreased GSC proliferation and self-renewal. METHODS: A two armed, randomized phase 0/II study of Vismodegib, an inhibitor of SMO was performed in 40 patients undergoing resection for rGBM. Arm I was randomized to receive Vismodegib for 7 days pre-operatively; Arm II was not treated with drug pre-operatively. All patients were treated with Vismodegib alone post-operatively until progression or death. Our primary objective was to estimate six month progression-free survival (PFS6), with secondary endpoints of median overall survival (mOS), response and toxicity. Secondary objectives included determination of: intra-tumoral PK and PD; inhibition of SHh signaling by RT-PCR and immunohistochemistry; and inhibition of GIC proliferation and self-renewal by neurospheres proliferation and limited dilution assays. RESULTS: PK/PD data and tumor specimens obtained from 39/40 patients demonstrated plasma and tissue levels for patients in Arm I (treated pre-operatively) were within therapeutic range. SHh signaling intermediates (Gli-1, Gli-2, PTCH-1b) as determined by RT-PCR and IHC were significantly lower in Arm I vs. Arm II (p < 0.0002). Finally, proliferating CD133 + neurospheres in serum free-media were derived from 17 of the 39 cultures assessed. Array CGH was consistent with GIC (rather than NSC) as the cells of origin, and the proportion of tumor-derived CD133+ neurospheres undergoing proliferation and self-renewal was decreased in Arms I vs. II (p

2552 Background: The primary objective of phase I clinical trials is to evaluate the tolerability and pharmacokinetics of a new agent. Phase I trials in oncology remain ethically controversial. Our previous analysis from year 1991 through 2002 reported a response rate of 10.6 percent and a toxicity-related death rate of 0.49 percent. An increasing number of molecular targeted agents are under investigation and these results may not reflect current phase I oncology trials. Methods: We reviewed all non-pediatric phase 1 oncology trials sponsored by the Cancer Therapy Evaluation Program at the National Cancer Institute between January 1, 2001 and December 31, 2012. We report the rates of response to treatment, of stable disease, of grade 4 toxic events, and of treatment-related deaths. Results: We analyzed 286 trials, comprising 8314 participants. All 8314 participants were evaluated for toxic events, whereas 6604 participants were evaluated for response. The overall response rate (complete and partial respo...

2026 Background: The prognosis for GBM, which appears to be driven by glioma initiating cells (GIC), remains poor. Our hypothesis was that interrupting sonic hedgehog signaling (SHh) would inhibit ...

10521 Background: Few effective treatments (tx) exist for patients (pts) with tyrosine kinase inhibitor (TKI)-resistant gastrointestinal stromal tumors (GIST) and other advanced soft tissue sarcoma...

e22123 Background: The Notch signaling pathway is crucial in context-dependent cell fate determination that has been implicated in tumorigenesis in an increasing number of human malignancies includ...

In Japan, it is quite rare for an investigator to submit an investigational new drug application to initiate a clinical trial and obtain approval of a drug on the basis of clinical trial results. This means that development of new therapies is currently driven almost entirely by pharmaceutical companies as opposed to independent investigators. Here, we provide our perspective on the reasons for this situation and advocate investigator-initiated cancer drug development as a means of increasing access to better therapies for Japanese cancer patients.

We previously reported the protection of hematopoietic cells from methotrexate (MTX) toxicity using an N2-based double copy vector containing serine 31 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whether the use of SFG-based dicistronic vectors will lead to improvement in gene transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells, the 50% effective dose values of MTX conferred by the SFG vector were 8-fold higher than those obtained with the DC/SV6 vector. DHFR mRNA levels were 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respectively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was the same. These results indicate that the in vitro transduction efficiency and transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors.

Interleukin-1 (IL-1) produced in peripheral blood mononuclear cell (PBMC) cultures or added exogenously has been shown to upregulate HIV expression in vitro. Inhibition of IL-1 in HIV-infected individuals may inhibit HIV activation and slow disease progression. Recombinant human IL-1 receptor (rHu-IL-1R), the soluble extracellular portion of the human type I IL-1 receptor, inhibits HIV expression in acutely infected primary PBMCs and in the chronically infected promonocytic cell line, U1. We, therefore, conducted a phase I/II trial of the soluble rHu-IL-1R in HIV-1-infected individuals with CD4 T cell counts300/microl to evaluate its safety and activity. Twelve evaluable patients were enrolled at three rHu-IL-1R dose levels:125 (n=3), 500 (n=3), and 1250 (n=6) microg/m2 per dose by subcutaneous (s.c.) injection three times a week for 8 weeks, followed by a 4 week observation period. rHu-IL-1R was safe and well tolerated. There were no deaths, no treatment-related grade 3/4 events, and no premature study discontinuations because of adverse events. The maximum tolerated dose was not reached. Seven patients reported improvements in one or more symptoms, including weight gain (3), improved energy level (4), decreased diarrhea (1), decreased night sweats (1), improvement in psoriatic arthritis (1), and improvement in a nonspecific chronic diffuse skin rash (1). Of 3 evaluable patients with Kaposi's sarcoma, 1 remained stable and 2 showed minimal progression. No consistent trends in absolute CD4 counts or percentages, quantitative HIV cultures, or serum p24 antigen, beta2-microglobulin, or triglyceride levels were observed. rHu-IL-1R is safe and well tolerated at the doses tested but induced no consistent changes in objective markers of HIV disease. Symptomatic improvements will require confirmation in randomized, placebo-controlled trials.

A major obstacle in eradicating unresectable or disseminated tumor cells with chemotherapy is thought to be due to inherent drug resistance, which in some instances may be overcome by the use of higher doses (Hryniuk and Bush 1984). Currently available chemotherapy, however, is often associated with various toxicities, in particular, bone marrow toxicity which is limiting for many of these agents. Use of recombinant hematopoietic growth factors or cytokines has enabled a modest increase in the dose chemotherapy that is tolerated. Most commonly used cytokines are granulocyte colony—stimulating factor (G—CSF) and granulocyte macrophage colony—stimulating factor (GM—CSF). Clinical trials using interleukin—3 (IL—3) and PIXY 321 (fusion protein of GM—CSF and IL—3) did not show a significant advantage over control groups (Ellis et al. 1996; Hofstra et al. 1997). Thrombopoietin, alone or in combination with other cytokines, is currently being evaluated in clinical trials. Another means of overcoming myelosuppression is the use of autologous bone marrow or peripheral stem cell transplantation after high doses of chemotherapy. This approach is currently being used in Hodgkin’s disease, breast and ovarian carcinomas (Bierman et al. 1993; Rosti et al. 1992; Reece et al. 1992; Chopra et al. 1993; Barnett et al. 1993).

A potential clinical use of gene transfer technology would be to introduce via a retroviral vector, cDNAs encoding mutant forms of dihydrofolate reductase (DHFR) into hematopoietic progenitor cells, thus allowing high doses of methotrexate MTX to be safely administered. Mice bearing a chemotherapy sensitive breast cancer tumor, treated with high dose cyclophosphamide treatment and transplantated with bone marrow cells transfected with a mutant DHFR cDNA, tolerate high doses of MTX post transplant, leading to cure.

We have previously shown that transfer of a mutated dihydrofolate reductase (DHFR) confers resistance to methotrexate (MTX) to infected cells. We report herein the construction of a retrovirus vector, DC/SV6S31tk, which carries the herpes simplex virus thymidine kinase gene (HSVtk) as well as the mutated Serine 31 DHFR (S31) cDNA. 3T3 cells infected with DC/SV6S31tk are more resistant to MTX than cells infected with DC/SV6S31, which carries the S31 and Neo gene. In DC/SV6S31tk-infected cells, a fraction of cells (20-40%) were more resistant to MTX compared with DC/SV6S31-infected cells, and these cells survived a 5-day exposure to 200 microM of MTX. The mechanism of this augmented resistance is attributed to the salvage of thymidine by HSVtk, as the augmentation is reversed when dialyzed serum is used for cytotoxicity assays. The cells that survive high-dose MTX selection have high levels of expression of S31 DHFR and HSVtk, although copy numbers of the proviral sequences do not increase significantly. Transduction of cells with the DC/SV6S31tk vector also sensitizes cells to ganciclovir (GCV). Co-expression of a metabolically related gene in a retroviral vector to potentiate the resistance imparted by a drug resistance gene may be useful for gene therapy for cancer patients.

10558 Background: Our group previously reported that PDGFRA is the most highly overexpressed kinase gene in synovial sarcoma. Our preclinical studies also demonstrated synergistic anti-tumor activity by inhibiting both PDGFRA and mTOR signaling with imatinib and rapamycin respectively in PDGFRA + synovial sarcoma cell lines. Based on these data, a phase Ib/II study to evaluate the toxicity and efficacy of imatinib and everolimus was undertaken. Methods: The primary endpoint of the phase 1b portion of the study was to determine the maximum tolerated dose (MTD) of everolimus administered with imatinib. Starting dose of everolimus and imatinib was 5 mg/day and 400 mg/day respectively. Response rate (RR) by RECIST was the primary end-point of the Phase II study. The phase II study used a Simon two stage design. 9 patients (pts) were to be accrued initially. If there were no responses, further accrual would be stopped and treatment declared ineffective. If there was at least 1 response, an additional 15 pts would be accrued for a total of 24. Key eligibility: metastatic disease, any number of priors. Pre and post treatment tumor biopsies were mandated. Results: 12 pts were treated.5 M and 7 F, median age 44 (range: 22-71), median priors 4 (range: 0 - 6). Two DLTs were observed at dose level 2 (10 mg everolimus /400 mg imatinib) with grade 3 transaminases and hypophosphatemia. Everolimus 5 mg/ imatinib 400 mg was the MTD in the phase II study. 10 pts evaluable for response, included 4 pts treated at the MTD in the Phase 1b study. There were no RECIST responses. Stable disease was observed in 3 pts (7, 7, 19 mos.). Western blot and IHC analysis of matched pair tumor biopsies indicate inhibition of p-AKT, p-S6 and decreases in Ki 67. Conclusions: Imatinib and everolimus failed to achieve its primary response endpoint. However, prolonged stable disease in 3 pts in association with inhibition of PDGFRA and mTOR suggest clinical benefit and biological effect for this drug combination. Clinical trial information: CTEP 8603.

7508 Background: Targeted inhibition of the Hedgehog (HH) pathway by V & Insulin-like Growth Factor type-1 Receptor (IGF-1R) by Cx enhances efficacy of chemotherapy, and also demonstrates activity against the tumor cell fraction responsible for disease recurrence in SCLC. Methods: Patients (Pts) with newly diagnosed ES-SCLC with measurable disease, ECOG PS 0-1 were randomized to receive (four 21-day cycles) CE alone [(C 75 mg/m2 D1 & E 100 mg/m2 D1-3) Arm A] or in combination with either V [(150 mg/day PO) Arm B] or Cx [(6mg/kg weekly IV) Arm C]. Pts with responsive or stable disease on Arms B & C were continued on V or Cx respectively until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS), stratified on gender. Circulating tumor cells (CTCs) were isolated/enumerated by Veridex Cell Search Platform at baseline, after 1or 2 cycles of chemotherapy, at completion of 4 cycles & 3 months (m). thereafter for correlation with efficacy parameters. The study was designed to detect a PFS HR of 0.58 with 90% power & an overall one-sided type I error rate of 0.10 for each of the comparisons of the V and Cx arms to CE alone. Results: 155 eligible pts were treated; 136 have died & 149 have experienced a PFS event. Pt. demographics & disease characteristics are well-balanced between the three arms except higher rate of PS 0 on Arm B (p=0.03). The median PFS on Arms A, B & C are 4.7, 4.4 & 4.6 m, the median OS 9.1, 9.8 & 10.1 m, & the RR are 43%, 52% & 49% respectively. None of the comparisons of these outcomes are statistically significant. PFS HR for V+CE vs. CE: 1.32, p=0.21, and for Cx+CE vs. CE: 1.12, p=0.58. The median OS among those with low CTC count (≤100 per 7.5ml) at baseline is 10.7 m vs. 7.2 m for those with high CTC count [HR1.70, p=0.01]. Toxicities in all three arms are as expected with the combination of CE alone. Conclusions: There is no significant improvement in PFS or OS with the addition of either V or Cx to CE vs.CE alone in pts with ED-SCLC. Low baseline CTC count is associated with improved OS, suggesting its role as a prognostic biomarker in SCLC. Clinical trial information: NCT00887159.

2553 Background: Angiogenesis inhibitors promote autophagy, a response to nutrient deprivation in which autophagosomes (AP) and lysosomes fuse to recycle intracellular constituents, leading to sustained tumor viability. We hypothesized that the VEGF-R2, c-kit, PDGFR inhibitor Su induces autophagy. The autophagy inhibitor HCQ may then interfere with autophagy dependent tumor survival, possibly improving patient (pt) outcomes. Methods: This trial determined the MTD of Su+HCQ in pts with advanced malignancies using a 3+3 design. Su 50 mg qd was given in 4 week on/2 week off cycles (C) with daily HCQ in escalating dose cohorts. A MTD expansion cohort of 12 pts was also enrolled. Modulation of autophagy was measured by changes in the AP marker light chain-3 (LC3)II/I ratio in paired PBMC samples from C1 and C2. Results: 21 pts, median age 59, PS 0 (7), 1 (13) or 2 (1) enrolled, including 5 colon, 2 renal and 5 sarcomatous tumors. 4 DLTs were observed: 3 DLTs (gr 3 confusion, gr 3 colon fistula, gr 3 thrombocytopenia) in DL2 (50 mg Su, 200 mg bid HCQ), and 1 DLT (gr 3 dehydration) in DL1 (50 mg Su, 200 mg HCQ). DL1 was thus declared the MTD. Gr 3/ 4 toxicities included: related to Su, thrombocytopenia (14%), fatigue (19%), neutropenia (14%), hypertension (14%), intestinal perforation (10%); related to HCQ, fatigue (14%). PK data of Su alone, its active metabolite SU012662 and the total were evaluated using noncompartmental analysis. Cmax and AUCs of the active metabolite SU012662 significantly increased (p 3 cycles (median 73 days). Inconsistent autophagy modulation was seen in PBMCs. LC3 immunohistochemistry on baseline tumor blocks to assess individual tumor autophagy levels is in progress. Conclusions: pK interaction between Su and HCQ resulting in accumulation of Su metabolites may be responsible for the predominantly Su-associated toxicities observed which prohibited further HCQ dose escalation. Lack of evidence for autophagy modulation is likely due to the inability to escalate HCQ to doses necessary to induce autophagy inhibition. Further study of this combination seems unwarranted. Clinical trial information: NCT00813423.

6022 Background: ACC is a rare disease, accounting for 1/3rd of MSGT, in which 90% of cases express the protein product of the ckit proto-oncogene. Dasatinib is a potent and selective inhibitor of five oncogenic PTKs/kinase families including ckit. We conducted a phase II study to determine the antitumor activity of dasatinib in ACC and non-ACC MSGT. Methods: In a two-stage design, adult patients (pts) with recent radiographic progressive, recurrent or metastatic ACC, + cKIT, were treated with dasatinib 70 mg PO BID. Pts with non-ACC MSGT of other histologic types, were treated as a separate cohort. Response was assessed every 8 wks by imaging using RECIST criteria. The study design stipulated enrollment of n=40 ACC patients (20 in stage I and 20 in stage II) and 25 non-ACC patients (14 in stage I and 11 in stage II). Results: Fifty-four pts were enrolled, 40 ACC and 14 non-ACC. One additional pt was a screen failure. Baseline data on 54 pts are: M:F = 28:26, median age 56.6 yrs (range 20-82), PS 0:1:2 = 24:28:2, prior radiation:chemotherapy = 44:21. The most frequent adverse events experienced (as % of pts, worst grade 2 or higher and at least possibly related to study drug) were: fatigue (28%), nausea (19%), headache (15%), lymphopenia (11%), dyspnea (11%), alanine aminotransferase increased (7%), anorexia (7%), vomiting (7%), alkaline phosphatase increased (6%), diarrhea (6%), and non-cardiac chest pain (6%). No grade 4 adverse events occurred and only 15 pts experienced a grade 3 adverse event (at least possibly attributed to study drug), primarily dyspnea (4 patients) and fatigue (3 patients). Significant cardiac toxicity was observed in one pt (grade 3 prolonged QT corrected interval). Among ACC pts, best response to dasatinib: 0 pts (0%) had PR, 21 pts (52%) had SD (range 2.8-13.8 months), 12 pts (30%) had PD, and 2 died prior to cycle 2. Median PFS was 4.8 mos. For 14 evaluable non-ACC pts, none had an objective response, triggering early stopping. 7 had SD (range 1.4-6.6 months), and 4 PD. Conclusions: Although there were no objective responses, dasatinib is well tolerated, with tumor stabilization achieved by 52% of ACC pts. Dasatinib demonstrated no activity in non-ACC MSGT. Clinical trial information: NCT00859937.

4011 Background: The HH pathway is overexpressed in gastroesophageal (GE) tumors. Pre-clinically, HH inhibitors have demonstrated a reduction in GE tumor growth, cell motility and invasiveness. V, an oral small-molecule antagonist of the Hh pathway, has previously been safely combined with FOLFOX chemotherapy. Methods: Pts with untreated metastatic or locally advanced gastric or GEJ adenocarcinoma were randomized 1:1, stratified by institution and disease status (with or w/o distant mets) to FOLFOX (ox 85 mg/m2, LV 200 mg/m2, 5-FU bolus 400 mg/m2, 5-FU infusion 2400 mg/ m2 over 48 hrs) q14d plus V or placebo (P) (150mg PO daily). Cycle defined as 2 weeks and no crossover allowed at progression. FFPET and blood were collected for biomarker analyses. Response assessed every 8 weeks (RECIST 1.1). Primary endpoint was progression-free survival (PFS), secondary objectives were overall survival (OS), response rate (RR), and toxicity. Results: 124 pts enrolled at 20 sites between 10/09-2/12. 123 pts eligible for analysis (V/P 60/63). Pt characteristics (V/P): median age 58/62; ECOG PS 0: 24 (40%) / 30 (48%); male 39 (65%) / 52 (83%); GEJ 37 (62%) / 39 (61%); diffuse or mixed histology 19 (32%) / 10 (16%), recurrent disease 10(17%) / 16 (25%). Median number of FOLFOX cycles 10/11. Most common Grade ≥3 toxicities: (% pts V/P) neutropenia 50/32 (p=0.07), neuropathy 19/13 (p=0.49), fatigue 15/10 (p=0.50), thrombosis 14/11 (p=0.92), anemia 10/10 (p=0.99), hemorrhage-GI 8/11 (p=0.77), hypokalemia 10/5 (p=0.76), nausea 8/8 (p=0.99). Death on or within 30 days of treatment 6.7%/15.6% (p=0.20). Median PFS in intent to treat population 7.3/8.0 mo (95% CI 4.6-10.1/5.1-11.0; p=0.64) and median OS 11.5/14.9 mo (95% CI 9.6-13.4/11.3-18.4; p=0.23). Overall RR (%) 35/35 (p=0.99). Conclusions: Addition of V to FOLFOX did not improve PFS in an unselected advanced GE carcinoma population. Blood and tissue biomarker analyses are ongoing to determine if there is a subset of patients who may derive benefit from V. Supported by: N01-CM-62204, -62201, -62207, -62206, -62209, -62208 and 2UL1 TR000457-06. Clinical trial information: NCT00982592.

2035 Background: Almost 80% of adult medulloblastoma are of the SHH subtype. Second line therapy for adult MB is limited; therefore we tested the efficacy of vismodegib, a small molecule inhibitor of Smoothened (SMO) among patients with this disease. Methods: Adult patients with refractory or recurrent MB and who had measureable disease were enrolled on the study. Immunohistochemistry (IHC) was used to stratify patients to Stratum A (non-SHH group); Stratum B (SHH tumors) and Stratum C (indeterminate). All patients were treated with vismodegib at 150 mg/day PO daily. Tumor response, which had to be maintained for 8 weeks to meet protocol definition of sustained response, was assessed using RECIST criteria and central imaging review. Separate but identical Simon 2-stage MinMax designs (α = 0.10) were used in strata A and B to test for evidence that sustained response rates exceeded 5% with 90% power to detect 25% sustained response rates. Thus, 3/20 sustained responses were needed to declare potential activity of vismodegib. Results: 32 patients with a median age of 30 years (range 22.4-51.9) were enrolled on the study [Stratum A (n = 8); Stratum B (n = 20); Stratum C (n = 4)], including 18 males and 14 females. No responses were observed in Strata A or C and the median duration of treatment was 1.5 months (range 0.66-2.33). Three of 20 patients enrolled on Stratum B had sustained responses. The median duration of therapy for Stratum B patients was 2.76 months (range 0.33- 13.61). Six patients were on treatment for ≥6.44 months and 3 remain on treatment after 5.42, 9.34 and 13.61 months. During course 1, 2 patients experienced grade 3 decrease in lymphocytes; 1 experienced a grade 4 thromboembolic event; and 2 experienced grade 3 toxicities (back pain & syncope). During course 2, 3 patients experienced grade 3 toxicities (decrease in lymphocytes; myalgia & seizure). One other patient experienced grade 3 hypophosphatemia in courses 1 and 2. Conclusions: Vismodegib has activity against recurrent or refractory adult ‘SHH-subtype’ medulloblastoma and should be considered as a therapeutic option for newly diagnosed patients with this disease. Clinical trial information: NCT00939484.

TL32711 is a novel small molecule peptidomimetic of the SMAC (Second Mitochondria-derived Activator of Caspases) that promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Normally SMAC is located in the mitochondria; however it is released into the cytosol when cells undergo apoptosis. Release into the cytosol and cleavage of SMAC promotes caspase-9 activation by binding to inhibitors of apoptosis proteins, IAPs. Removing the inhibitory activity of IAPs increases cells’ sensitivity to apoptotic stimuli and abrogates NF-κB survival signaling. Thus, by targeting and eliminating IAPs, SMAC mimetics re-activate death signals in cancer cells, inhibit resistance mechanisms and induce tumor death by apoptosis. This study was undertaken to determine radiation sensitization potential of TL32711 in GBM using in vitro and in vivo approaches. TL32711 alone did not cause any killing effects in GBM cells. However, in vitro apoptosis was increased when TL32711 was combined with radiation in GBM cell lines. In vivo data demonstrated that TL32711 alone delayed the growth of implanted xenograft tumors that were further delayed when combined with radiation. The survival of mice was greatly increased in TL32711 plus radiation treated GBM intracranial orthotopic model when compared to TL32711 or radiation treatment alone. Mechanistically, TL32711 confers radiation sensitization by abrogating NFκB activity that is elevated by radiation-induced TNF-α. Taken together, this is the first report that provides strong evidence that TL32711 in combination with ionizing radiation has potential to increase the effectiveness of radiotherapy. Funded by NCI Contract No. HHSN261200800001E. Citation Format: David Cerna, Donna Carter, Naoko Takebe, C. Norman Coleman, Mansoor M. Ahmed, Stephen Yoo. Radiosensitization of GBM by a novel peptidomimetic of the Second Mitochondria-derived Activator of Caspases (SMAC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1599. doi:10.1158/1538-7445.AM2013-1599

67 Background: The HH pathway is overexpressed in gastroesophageal (GE) tumors. Pre-clinically, HH inhibitors have demonstrated a reduction in GE tumor growth, cell motility and invasiveness. V, an oral small-molecule antagonist of the Hh pathway, has previously been safely combined with FOLFOX chemotherapy. Methods: Pts with untreated, metastatic or locally advanced gastric or GEJ adenocarcinoma were randomized 1:1, stratified by institution and disease status (with or w/o distant mets) to FOLFOX (ox 85 mg/m2, LV 200 mg/m2, 5-FU bolus 400 mg/m2, 5-FU infusion 2400 mg/ m2 over 48 hrs) q14d plus V or placebo (P) (150mg PO daily). Cycle defined as 2 weeks and no crossover allowed at progression. FFPET and blood were collected for biomarker analyses. Response assessed every 8 weeks (RECIST 1.1). Primary endpoint was progression-free survival (PFS), secondary objectives were overall survival (OS), response rate (RR), and toxicity. Results: 124 pts (V/P 60/64) enrolled at 20 sites between 10/09-2/12. Pt characteristics (V/P): median age 58/62; ECOG PS 0: 23 (40%) / 30 (47%); male 39 (65%) / 53 (83%); GEJ 37 (62%) / 39 (61%); diffuse histology 20 (43%) / 14 (30%). Median number of FOLFOX cycles 9.5/11. Most common Gr ≥3 toxicities: (% pts V/P) neutropenia 50.0/31.7 (p=0.07), neuropathy 23.1/14.3 (p=0.33), fatigue 15.4/9.5 (p=0.50), thrombosis 13.5/11.1 (p=0.92), anemia 9.6/9.5 (p=0.99), hypokalemia 9.6/4.8 (p=0.52), nausea 7.7/9.5 (p=0.99). Death on or within 30 days of treatment 6.7%/15.6% (p=0.20). Median PFS in ITT population 11.5/9.3 mo (95% CI 8.5-14.4/6.7-11.9; p=0.34) and median OS 12.2/13.9 mo (95% CI 10.2-14.3/11.5-16.3; p=0.48). Non-statistically significant trends toward improved PFS with V were noted in female pts, diffuse histology, and PS 1 (p≤ 0.08). RR (%) 33/27 (p=0.64). Conclusions: Addition of V to FOLFOX did not improve PFS in an unselected advanced GE carcinoma population. Blood and tissue biomarker analyses are ongoing to determine if there is a subset of patients who may derive benefit from V. Supported by: N01-CM-62204, -62201, -62207, -62206, -62209, -62208 and 2UL1 TR000457-06 Clinical trial information: NCT00982592.

3078 Background: Dasatinib (D) is a first in class Src kinase inhibitor, and inhibits BCR-Abl, c-Kit, PDGFR-beta, EPHA2 and Src family kinases including Src, Lck, Yes, Fyn at nanomolar concentrations. Initially FDA approved for use in imatinib resistant CML. It is a small molecule targeted therapy hepatically metabolized primarily by CYP3A4. We conducted a phase I study to determine maximum tolerated dose (MTD) and pharmacokinetics (PK) of D in patients (pts) with liver dysfunction (LD). Methods: Pts with advanced solid tumors or lymphoma, Zubrod ≤2, no baseline ascites or pleural effusions, adequate renal and bone marrow function, received PO D daily. Cycles q28 days. Pts stratified into 4 LD groups: normal, mild, moderate, severe, using Child-Pugh classification (CPC). Data also collected for NCI ODWG Organ Dysfunction Working GroupCriteria. D dose was escalated in sequential cohorts of pts within each LD category. Blood analysis for D concentrations were determined during cycle 1 using a validated LC-MC/MS assay. Study objectives included characterizing safety, tolerability, PK, identifying the MTD and obtaining preliminary evidence of efficacy. Results: 54 registered pts, 51 pts received 51 cycles of D at doses starting at 100 mg in mild LD (50-140 mg). Median age 60, male 55%, Zubrod 1 70%. CRC 27%. Groups: normal-17, mild-20, moderate-13, severe-1 pt(s). Related AEs include fatigue 35%, diarrhea 27%, anemia 27%, nausea 25%, vomiting 21%, lymphopenia 13%, rash 13%, pleural effusion 8%. 1 DLT of increased CK in a pt in moderate LD with past history of similar episode with previous sorafenib. Previous linear PK disposition, and no accumulation. No apparent PK differences between normal and mild groups. Cycle 1 Day 1 D 140 mg Normal Group: Cmax 129 ng/mL. Mild Group 140mg: Cmax 157 ng/mL. Prolonged disease stabilization (≥4 cycles) in 6 pts, 3 CRC (4,5,8); 1 Pancreas, HCC, Bladder (4,5,6). Conclusions: Recommended dose for Dasatinib given PO QD for pts with mild, moderate, or severe LD using clinical criteria with CPC and no baseline ascites, are 140 mg, 70 mg, insufficient pts, respectively. Dose adjustment not necessary in pts with mild LD.

10005 Background: Chondrosarcomas (CS) exhibit a strong activation of hedgehog (Hh) signaling which plays a crucial role in cartilage tumorigenesis. Preclinical data show that hedgehog blockade reduces strongly chondrosarcoma cell proliferation and tumour size. GDC-0449 is a small-molecule antagonist of the Hh pathway. Methods: This is a multicentric single-arm phase 2 clinical trial based on two-stage Simon’s design which assesses safety and efficacy of GDC-0449 in patients (pts) with advanced CS. All pts had to have documented progressive disease (PD) as per RECIST 1.1 before study entry. Pts receive GDC-0449 150mg (oral route), daily until PD or unacceptable toxicity. The primary endpoint is the 6-month non-PD rate according to RECIST. Based on the following hypotheses: 20% 6-month non-PD rate (H0), 40% acceptable 6-month non-PD rate (H1), 10% type I error rate, 90% power, a total of 37 assessable pts are necessary (17 for the first stage + 20 for the second stage). Following the inclusion of the first 17 pts, if ≥ four pts are progression-free at 6 months, the accrual will continue. In order to account for not assessable pts (+/- 10%), 41 pts will be recruited. Accrual started in February 2011 in six centers of the FSG. Results: As of January 24 2012, 40 pts (28 males, 12 females) have been included in the study. Median age is 59 years (30-86). The most frequent histological subtype is conventional CS (n=32). Twenty eight pts (70%) had grade 1 or 2 adverse events (AE) possibly related to the drug whereas 3 pts (7.5%) had grade 3 or 4 AE. The planned interim statistical analysis performed after central histological and radiological review showed that four patients out of the first 17 evaluable patients had stable disease indicating that GDC-0449 had reached the primary endpoint to justify continuing accrual after the 1st step of the study. 15 pts are still on treatment. Molecular analyses are available for 21 pts. No mutation of SMO was detected. qRT-PCR experiments and PTCH sequencing are ongoing. Conclusions: GDC-0449 is well tolerated and shows some activity in a subset of pts with advanced CS. Final clinical and molecular data will be presented at the meeting.

e13106 Background: GDC-0449 (Vismodegib, Genentech), an orally bioavailable small-molecule SMO (Smoothened) inhibitor, was recently approved for advanced basal cell carcinoma (BCC) and is being evaluated in other solid tumors. This study sought to determine whether there is a significant effect of food on the PK of G. Methods: In part I, patients (pts) were randomized 1:1 to fasting overnight (FO) or a high fat meal (HF) before a single dose of G 150 mg. The study was later amended to 1:1:2 randomization to FO, HF or a low fat meal (LF). Plasma concentrations of G were determined by a validated liquid chromatography-tandem mass spectrometry assay. PK endpoints were observed Cmax, observed Tmax, and AUC (0-24 and 0-168 hours). In part II, pts randomized to FO or HF in part I took G 150 mg/day in a fasting state, while pts randomized to LF took G 150 mg/day in a fed state. PK endpoints after two weeks were steady state (ss) Cmax, ss Tmax, and ss AUC (0-24 hours). 60 pts enrolled 10/09-11/11, of which 43 were evaluable for PK: 18 FO; 14 HF; 11 LF. Pt characteristics: Female 53%; median age 59; PS 0: 62%, 1: 38%. Tumor types: 43% colorectal; 17% pancreatic; 40% other. Results: PK data were log-transformed for analysis. AUC (0-168 hours) after a single dose was higher in HF than in FO pts (p = 0.049); ratio of geometric means = 1.46 (90% CI = 1.07-1.99). There was a trend toward higher Cmax after a single dose in HF than in FO pts (p = 0.071); ratio of geometric means = 1.39 (90% CI = 1.03-1.88). There were no significant differences between groups for single dose AUC (0-24 hours) and Tmax. There were no significant differences between fasting and fed groups for ss Cmax, ss Tmax, or ss AUC (0-24 hours). The frequencies of drug-related Grade ≥ 3 toxicities were similar in the fasting and fed groups. There were no objective responses; 11 pts (18%) had stable disease for a median duration of 16 weeks and a maximum of 104 weeks (BCC). Conclusions: A high fat meal increases plasma exposure to a single dose of G, but there are no significant PK or safety differences between fasting and fed groups at ss. GDC-0449 can be taken with or without food for daily dosing. This study design is an efficient way to evaluate the effect of food on oral anti-cancer drugs. Supported by NCI grant 5U01-CA69852-12.

Abstract 2865 Obatoclax is a small molecule mimetic of the BH3 domain of Bcl-2 family proteins. Obatoclax is broadly specific, with activity against Bcl-2, Bcl-X, Bcl-w and Mcl-1. CLL cells overexpress Bcl-2, Bcl-XL and Mcl-1 in particular, and obatoclax can induce apoptosis of CLL cells in vitro. A phase 1 study of single-agent obatoclax in heavily pretreated largely refractory CLL patients demonstrated that the dose-limiting toxicity was neurologic, including euphoria and ataxia, leading to a maximum tolerated dose of 28 mg/m2 given over 3 hours every 3 weeks. One PR was observed along with biologic activity demonstrated by reductions in lymphocyte counts and improvements in cytopenias. We therefore undertook this phase 1 study of the combination of obatoclax with FR in CLL patients relapsed after at least one prior therapy and in need of therapy again. Obatoclax was given as a three hour infusion on days 1 and 3 at three dose levels, 10, 14, and 20 mg/m2 per dose. Fludarabine was given at 25 mg/m2 days 1–5, and rituximab 375 mg/m2 day 1 following an option to split the dose in cycle 1. Thirteen patients were enrolled, seven men and six women, with median age 58. 5 (38%) had stage 3–4 disease. FISH showed one patient with del17p, one with complex karyotype, and five with del11q. Six of nine patients evaluable had high risk unmutated IGHV, and nine of ten patients evaluable were positive for ZAP-70. The median number of prior therapies was two, with 9 patients having had prior fludarabine-based combination chemotherapy, 10 patients having had prior rituximab, and 8 patients having had prior alkylator-based combination chemotherapy. The study therapy was well-tolerated, with a median of five cycles administered. One dose-limiting toxicity (DLT) was observed at the 20 mg/m2 obatoclax dose; this DLT was a greater than two week treatment delay for persistent grade 2–3 neutropenia in a patient who had had a similar event previously with FR alone. This DLT led to expansion of the third and highest cohort, which enrolled seven patients with no further DLTs observed. Other grade 3–4 toxicities have been limited and include neutropenia (n=5), thrombocytopenia (n=2), fever without neutropenia (n=2), increased ALT/AST (n=1), and dizziness (n=1). Neurologic side effects were easily managed and resembled alcohol intoxication, including grade 1–2 euphoria (n=6), ataxia (n=5), dizziness (n=6), anxiety (n=4), speech impairment (n=4) and confusion (n=3). The ORR by NCI-WG criteria was 85% (11/13; 90% CI 59–97%) with 15% CR (2/13; 90% CI 3–41%) and 38% nPRs (5/13; 90% CI 17–65%). With the addition of CT scan measurement of lymphadenopathy the ORR declined to 54% (7/13; 90% CI 29–78%) with no CRs. With a median follow-up of 26 months from the start of the study, three patients are in ongoing remission, six have relapsed with three receiving further therapy to date, two patients have gone on to stem cell transplantation, and two patients have died of disease. The median time to progression is 20 months (95% CI 9, 35 mos). We were able to demonstrate increased apoptosis compared to baseline in peripheral blood CLL lymphocytes during cycle 1 therapy in 9 of 13 patients, using Annexin V propidium iodide staining. We conclude that the FR-obatoclax regimen is well-tolerated and highly active in a relapsed CLL population. An extension of this study to increase the frequency of obatoclax dosing to days 1–3, and to change the chemotherapy backbone to FCR, is planned pending the availability of obatoclax. Disclosures: Brown: Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding; Pharmacyclics: Consultancy.

Context Some melanomas arising from acral, mucosal, and chronically sun-damaged sites harbor activating mutations and amplification of the type III transmembrane receptor tyrosine kinase KIT. We explored the effects of KIT inhibition using imatinib mesylate in this molecular subset of disease. Objective To assess clinical effects of imatinib mesylate in patients with melanoma harboring KIT alterations. Design, Setting, and Patients A single-group, open-label, phase 2 trial at 1 community and 5 academic oncology centers in the United States of 295 patients with melanoma screened for the presence of KIT mutations and amplification between April 23, 2007, and April 16, 2010. A total of 51 cases with such alterations were identified and 28 of these patients were treated who had advanced unresectable melanoma arising from acral, mucosal, and chronically sun-damaged sites. Intervention Imatinib mesylate, 400 mg orally twice daily. Main Outcome Measures Radiographic response, with secondary end points including time to progression, overall survival, and correlation of molecular alterations and clinical response. Results Two complete responses lasting 94 (ongoing) and 95 weeks, 2 durable partial responses lasting 53 and 89 (ongoing) weeks, and 2 transient partial responses lasting 12 and 18 weeks among the 25 evaluable patients were observed. The overall durable response rate was 16% (95% confidence interval [CI], 2%-30%), with a median time to progression of 12 weeks (interquartile range [IQR], 6-18 weeks; 95% CI, 11-18 weeks), and a median overall survival of 46.3 weeks (IQR, 28 weeks-not achieved; 95% CI, 28 weeks-not achieved). Response rate was better in cases with mutations affecting recurrent hotspots or with a mutant to wild-type allelic ratio of more than 1 (40% vs 0%, P = .05), indicating positive selection for the mutated allele. Conclusions Among patients with advanced melanoma harboring KIT alterations, treatment with imatinib mesylate results in significant clinical responses in a subset of patients. Responses may be limited to tumors harboring KIT alterations of proven functional relevance. Trial Registration clinicaltrials.gov Identifier: NCT00470470

Induction of epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSCs) can occur as the result of embryonic pathway signaling. Activation of Hedgehog (Hh), Wnt, Notch, or transforming growth factor-β leads to the upregulation of a group of transcriptional factors that drive EMT. This process leads to the transformation of adhesive, non-mobile, epithelial-like tumor cells into cells with a mobile, invasive phenotype. CSCs and the EMT process are currently being investigated for the role they play in driving metastatic tumor formation in breast cancer. Both are very closely associated with embryonic signaling pathways that stimulate self-renewal properties of CSCs and EMT-inducing transcription factors. Understanding these mechanisms and embryonic signaling pathways may lead to new opportunities for developing therapeutic agents to help prevent metastasis in breast cancer. In this review, we examine embryonic signaling pathways, CSCs, and factors affecting EMT.

TPS173 Background: The hedgehog (Hh) pathway is an evolutionally highly conserved signaling cascade important for control of many developmental processes. It is critical in gastroesophageal (GE) embryogenesis and normal cell growth and survival. The Hh pathway is also implicated in GE cancer formation and progression.(Berman, 2003; Rubin, 2006; Ma, 2005; Ohta, 2005) Pathway overexpression has been demonstrated preclinically in GE tumors and the degree of activation correlates with clinical and histologic features. Hh antagonists have demonstrated a dose dependent reduction in GE tumor growth as well as inhibition of cell motility and invasiveness. (Yanai, 2007; Yoo, 2008) GDC-0449 is an oral small-molecule antagonist of the Hh pathway which binds to and inhibits SMO thereby blocking Hh signal transduction. This study will provide data on efficacy and safety of GDC-0449 plus FOLFOX relative to a current standard of care (FOLFOX alone) as 1st line treatment in pts with advanced GE cancers. Methods: This is ...

Background: The Hh pathway is an evolutionally highly conserved signaling cascade important in gastroesophageal embryonic pattern formation and stem cell response to tissue damage. Its aberrant activation has also been implicated in gastric cancer formation and progression. Multiple therapeutics targeting the Hh pathway have been developed and in order to test their activity and pharmacodynamic responses, routine and consistent gene expression measurement from clinical samples must be available. Aims and Methods: The aim of this work was to measure levels of Hh pathway expression in both gastric tumors and normal gastric tissue, using a novel protocol of processing RNA from formalin fixed, paraffin-embedded tissue (FFPET). Total RNA was extracted from FFPET using a combination of Qiagen and RecoverAllTM kits according to the manufacturer's protocol(s). cDNA was synthesized using the iScriptTMcDNA Synthesis Kit and then a minimum of 20 ng RNA was pre-amplified using an Applied Biosystems pre-amplification cDNA kit. The primers for the Shh and Gli1genes were then added to the pre-amplification reaction and TaqManPreAmp master mix, amplified for 14 cycles on a thermal cycler and then real time PCR carried out for 40 cycles in duplicate. The relative quantification of the Shh and Gli1 genes were compared to the reference gene succinate dehydrogenase complex subunit A (SDHA) where the ratio for the change in expression for the selected gene = 2−ΔΔCT. Results: The levels of gene expression in gastric adenocarcinomas for Shh and Gli1 were respectively 3.89 ± 0.49 (mean ± SEM, N = 11) and 4.57 ± 1.46. For 8 non-tumor gastric specimens, the CT values for Shh ranged from 30.24 to 35.81 and for Gli1 from 31.21 to 34.81 while for SDHA the mean CT was 18.4 ± 1.87. Based on these CT values, no expression of Shh and Gli1 was present in these non-tumor gastric tissues. Conclusion: Hh pathway signaling can be quantitatively measured using as little as 20ng of RNA obtained from FFPET, making this methodology valuable in the clinic where tissue acquisition is often limited, as for gastric tumors. Using our methodology we found that there is increased Hh pathway expression in gastric carcinomas as compared to normal tissue, a finding consistent with previous reports in the literature. Supported by NO1-CM-07103-74 RECOVERY – ACTNOW Clinical Trial 8376 and NCI 2P30 CA16087-23, Tissue Acquisition and Banking Core. 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 2230. doi:10.1158/1538-7445.AM2011-2230

The authors report an unusual case of herpes simplex type 2 (HSV) hepatitis which presented as part of a systemic HSV infection accompanied by disseminated intravascular coagulation (DIC). The patient was a 49-year-old Japanese male who three months prior to admission underwent surgical resection of his thymus for an invasive thymoma. Postoperatively, he received a course of chemotherapy which included prednisone, cyclophosphamide, vincristine, and pinorubicin. After discharge from the hospital, he was put on a maintenance dosage of prednisone and cyclophosphamide. Two weeks prior to this admission, the patient developed rhinorrhea, chills and general fatigue. Routine follow-up laboratory tests revealed markedly elevated liver enzymes which led to his immediate hospitalization. The tentative diagnosis on admission was fulminant hepatitis with DIC. The patient’s condition steadily worsened during his hospitalization and acyclovir was initiated on the 4th hospital day due to the possibility of HSV hepatitis. He died on the same day. Histopathology performed on the liver at autopsy revealed hepatic inclusion bodies of HSV with positive immunohistochemical detection of the HSV type 2 antigen. Our case is the first report of HSV hepatitis associated with the removal of the thymus secondary to thymoma. It supports previous observations of disseminated HSV infection being prevalent in those patients with disorders of cell mediated immunity.

Cancer therapy strategies that selectively target tumors while sparing normal tissues would provide enormous clinical benefit to cancer patients. Central to developing these strategies is identification of tumor specific vulnerabilities and molecular targeted agents targeting these vulnerabilities. GMX1777/1778, a novel inhibitor of NAD+ biosynthesis, is a potent and specific inhibitor of the nicotinamide adnine dinucleotide (NAD+) biosynthesis enzyme, phosphoribosyl transferase (NAMPRT). Since cancer cells have a high rate of NAD+ turnover, modulation of NAD+ biosynthesis would be an attractive target for GMX 1777/1778. In addition to the depletion of NAD+ level, we present evidence of inducing other metabolic and oxidative stresses in a tumor specific manner with minimal damage to normal tissues in vitro and in vivo. Taken together, the cancer therapeutic strategy presented here would provide a novel way of specific killing of tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B98.

3504 Background: Bcl-2 is a rational target in SCLC since it is overexpressed in 60%-90% of tumors and may play a role in resistance of SCLC to chemotherapy. Obatoclax is a small molecule BH3 mimetic that blocks bcl-2 binding to proapoptotic family members. Obatoclax has growth inhibitory effects in several solid tumor cell lines and xenografts, with at least additive effects in combination with topotecan. The primary objective of this study was to evaluate the safety profile and maximum tolerated dose (MTD) of obatoclax plus topotecan in patients with relapsed SCLC and other solid tumors. Methods: We conducted a phase I dose escalation study using a standard “3+3” design. Obatoclax was administered at 14 or 20 mg/m2 over 3 hours on day 1 every 21 days. A subsequent cohort received obatoclax 14mg/m2 on days 1 and 3. Topotecan was given at 1.25 mg/m2 days 1–5. All patients received pegfilgrastim on day 8. Eligible patients were adults with solid tumors appropriate for treatment with topotecan. Patients with neurologically stable, treated brain mets were eligible. Results: 14 patients have been treated including 8 SCLC, 3 extrapulmonary small cell, 1 carcinoid, 1 Merkel cell and 1 melanoma previously treated with 1 or 2 lines of chemotherapy. Nearly all patients experienced neurologic toxicities during the obatoclax infusion which included ataxia, dysarthria, somnolence and/or mood alteration; these typically resolved 1–2 hours after completion of the infusion. The MTD of obatoclax was 20 mg/m2 on day 1 with Dose Limiting Toxicities (DLT) including grade 3 neurotoxicity (2 pts) and febrile neutropenia. Hematologic toxicity included grade 3/4 anemia (6 pts), thrombocytopenia (5 pts) and neutropenia (5 pts). Other toxicities included mild nausea/vomiting, fatigue, pruritus, and constipation. Clinical activity was seen in patients with SCLC including 1 PR and 4 SD out of 7 evaluable. The median TTP for these SCLC patients was 11 weeks. Conclusions: The recommended phase II dose is obatoclax 14 mg/m2 on days 1 and 3 with topotecan 1.25 mg/m2 on days 1–5 in 21 day cycles. A phase II study in second-line SCLC is open. Supported by NCI U01-CA69856. No significant financial relationships to disclose.

e13502 Background: AT-101 [R-(-)-gossypol acetic acid] (AT) is an orally-administered BH3 mimetic that lowers the threshold for apoptosis by direct binding to Bcl-2, Bcl-xL, Mc1–1, Bcl-W, and through upregulation of the proapoptotic proteins Noxa and Puma. Bcl- 2 is over-expressed in >80% of SCLC. In vitro study using SCLC cells showed that treatment with EP had synergistic cytotoxic effects in suppression of Bcl-2. This is a phase I dose-escalation study of AT in combination with EP with an expanded cohort of patients with ES-SCLC. This study is being conducted to determine the maximum tolerated dose (MTD), pharmacokinetics and activity of AT with EP ± pegfilgrastim (F) in patients with advanced, refractory solid tumors and/or ES-SCLC. Methods: This study used standard eligibility criteria except patients must not have received prior therapy that inhibits the Bcl-2 family. At dose level 1, patients received P 60 mg/m2 on day 1 and E 100 mg/m2 on days 1, 2, and 3 every 21 days. AT was administered 30 mg orally BID on days 1, 2 and 3 of each cycle. Results: 10 patients have been enrolled; 7 men, 3 women. Tumor types: 6 lung; 2 prostate; 1 head & neck; 1 unknown primary. 2 of 5 patients enrolled at dose level 1 experienced a DLT of neutropenic fever in cycle 1. Three subsequent patients were enrolled to dose level -1 (20 mg BID x 3 days, d1–3) which was well tolerated. Additional patients are being enrolled at dose level 1a (EP+AT with F). Grade 3/4 toxicities related to AT without F at dose level 1 and -1 were as follows: ANC (8), leucopenia (7), febrile neutropenia (2), low hemoglobin (1), thrombocytopenia (1), elevated AST (1), cardiac ischemia/MI (1), diarrhea (1). There were no reported grade 3/4 toxicities in two patients at level 1a. Four patients had stable disease; two progressive disease and four patients were unevaluable. Conclusions: The MTD without F was established at AT 20mg orally BID, P 60 mg/m2 on day 1, and E 100 mg/m2 on days 1, 2, and 3 every 21 days. The MTD with F has not yet been established. Accrual continues at dose level 1a. Subjective and objective evidence of clinical activity has been observed in patients with refractory solid tumors. This study was supported by NCI, UO1 CA062491, SAIC 25XS097 and 1ULRR025011. No significant financial relationships to disclose.

Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem2004,47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.

Human brain endothelial cells (HUBEC), a U.S. Navy proprietary cell line, was reported previously by Chute et al as a promising co-culture ex vivo expansion system for both adult bone marrow (ABM) and cord blood (CB) hematopoietic stem cells (HSC).a,b,c We report here our results of using HUBEC in ex vivo expansion and in vivo engraftment assay using NOD-SCID mice. CD34+ enriched fresh ABM was obtained using the method as described previously.a,b However, we used frozen CB and the same cytokines for both ABM and CB expansion whereas Chute et al used fresh CB and different cytokines. Ex vivo expansion studies for both ABM and CB were performed for 7 days in the HUBEC coated plates with previously reported cell density and cytokine cocktail containing GM-CSF, IL-3, IL-6, SCF, and flt-3 (GM36SF) in IMDM 10% FBS media.a HSC injections and BM harvesting of NOD-SCID mice as well as flow cytometric analysis were performed using the methods of Chute et al.a NOD-SCID mice were transplanted with limiting doses of either fresh ABM CD34+ cells or freshly thawed CB CD34+. The progeny of the identical doses of ABM CD34+ or the progeny of the identical doses of CB CD34+ cells was then transplanted. Culture with GM36SF alone resulted in a 15.5-fold and 70-fold increase in total cells, a 3.4-fold and 32-fold increase in CD34+ cells, and a 4.8-fold and 4.1-fold increase in CD34+/CD38- cells for ABM and CB, respectively. In contrast, HUBEC co-culture with GM36SF yielded a 25-fold and 48-fold increase in total cells, a 8.9-fold and 13-fold increase in CD34+ cells, and 114-fold and 106-fold increase in CD34+/CD38- cells for ABM and CB, respectively. HUBEC co-culture without GM36SF supported a 1.0-fold and 1.0-fold increase in total cells, a 0.06-fold and 0.1-fold increase in CD34+ cells, and 0.25-fold and 0.2-fold increase in CD34+/CD38- cells for ABM and CB. HUBEC co-culture with GM36SF and transwell (non-contact culture) resulted in a 20-fold and 48-fold increase in total cells, a 6-fold and 8-fold increase in CD34+ cells, and a 32-fold and 38-fold increase in CD34+/CD38- cells for ABM and CB. Overall, the transwell expansion of CD34+/CD38- population in both ABM and CB was reduced to 30% of that achieved in the contact culture. ABM CD34+ cells (5 x 105) engrafted 60% and the progeny of 5 x 105 cultured in the HUBEC monolayer with GM36SF engrafted in 90% of transplanted mice. CB CD34+ cells (1 x 104) engrafted 27% and the progeny 1 x 104 CB CD34+ cells cultured in the HUBEC monolayer with GM36SF engrafted 64% of NOD-SCID mice. SRC frequencies calculated as a 3.12-fold and 2.7-fold increase in CD34+ enriched ABM and CB, respectively, which was less than reported previously.a,b In summary, HUBEC supports and expands SRC mainly through cell-to-cell contact between HSC and endothelial cells, with HUBEC-secreted factors playing a minor role.

HeLa cells expressed 3.4- and 1.6-kilobase (kb) transcripts of the integrated human papillomavirus (HPV) type 18 genome. Two types of cDNA clones representing each size of HPV type 18 transcript were isolated. Sequence analysis of these two types of cDNA clones revealed that the 3.4-kb transcript contained E6, E7, the 5' portion of E1, and human sequence and that the 1.6-kb transcript contained spliced and frameshifted E6 (E6*), E7, and human sequence. There was a common human sequence containing a poly(A) addition signal in the 3' end portions of both transcripts, indicating that they were transcribed from the HPV genome at the same integration site with different splicing. Furthermore, the 1.6-kb transcript contained both of the two viral TATA boxes upstream of E6, strongly indicating that a cellular promoter was used for its transcription.

A genomic DNA sample from cervical cancer tissue, containing human papillomavirus (HPV) type 16, was found to induce malignant transformation of NIH 3T3 cells when it was tested by transfection assays using the calcium phosphate coprecipitation technique. The primary and secondary transformants contained the HPV type 16 DNA sequences and human specific Alu family sequences. To the best of our knowledge, it has not been reported previously that HPV type 16 DNA sequences in total genomic DNA from a cervical cancer have transforming activity.