Marked Tumor Research Articles

Abstract P5-08-06: PI3K blockade enhances the antitumor activity of eribulin in PIK3CA-mutant eribulin-resistant tumor xenografts

Abstract Eribulin is a recently approved microtubule-targeting agent (MTA) for the management of heavily pretreated metastatic breast cancer (BC) patients. Constitutive PI3K/Akt/mTOR survival pathway activation, either via mutational activation of the PI3K catalytic subunit (PIK3CA) or via inactivation of the tumor suppressor PTEN, may confer resistance to MTAs. Hence, we hypothesized that PI3K-pathway activation limits the antitumor activity of eribulin in HER2-negative BC and that PI3K inhibition enhances the efficacy of this chemotherapeutic agent. The predictive value of PIK3CA mutation or PTEN loss towards eribulin response was interrogated in vitro, using a panel of fourteen HER2-negative BC cell lines, and in vivo using six tumor models from cell-line or patient-derived tumors. While PIK3CA mutation did not seem to be predictive in vitro, the PIK3CA-mutated xenograft models underwent tumor progression upon single-agent eribulin therapy (Table 1). In the absence of concomitant PIK3CA mutation, PTEN loss was neither predictive in vitro nor in vivo. Moreover, eribulin induced PI3K-pathway activation in tumor xenografts, a potential escape mechanism to MTA-therapy. To validate the negative predictive value of PIK3CA mutation, BC tumors from patients treated with eribulin in the neoadjuvant and in the metastatic setting are being queried, as the pharmacodynamic activation of the PI3K-pathway upon eribulin treatment. Class I pan-PI3K (BKM120) or PI3K-a-specific (BYL719) inhibitors were used in vitro to block the PI3K-pathway concomitantly with eribulin treatment, resulting in enhanced antiproliferative and proapoptotic activity. Strikingly, in PIK3CA mutant xenograft models, while eribulin alone exhibited limited antitumor activity compared to PIK3CA-wild type models, co-administration of a PI3K inhibitor induced marked tumor regression (BKM120 data in Table 1, BYL719 data will be reported). Moreover, addition of the PI3K inhibitor at progression with eribulin single-agent also resulted in tumor regression. Of note, PIK3CA-wild type models also exhibited increased antitumor activity with the combined therapy compared to single-agent treatments. The precise mechanism by which the combination of eribulin and a PI3K-targeting agent results in tumor regression is currently under investigation, embracing both the induction of mitotic catastrophe in tumor cells and the regularization of the tumor vasculature. These results support the clinical development of therapeutic regimens combining PI3K-inhibitors to the approved MTA eribulin and might be predictive of clinical benefit both in the PIK3CA-mutant and -wild type breast cancer population. Table 1. Percentage change in tumor volume.StatusPIK3CA mutantPIK3CA mutant and PTEN-lowPTEN-lowWTModel/ TreatmentMCF7LPDX44CAL51MDA-MB-468PDX88PDX98Eribulin39 ± 9683 ± 51364 ± 127-47 ± 14-26 ± 34-36 ± 33BKM120501 ± 169116 ± 111119 ± 73-9 ± 23128 ± 3787 ± 112Eribulin + BKM120-70 ± 27-80 ± 1051 ± 46-75 ± 12-94 ± 5-65 ± 13Patient- (PDX) and cell line-derived tumor xenografts were treated with eribulin mesylate (0.1mg/kg, 3IW) and/or BKM120 (27.5 mg/kg, 6IW) for 26-31 days.. IW, in week. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-06.

The Enhanced In Vivo Activity of the Combination of a MEK and a PI3K Inhibitor Correlates with [18F]-FLT PET in Human Colorectal Cancer Xenograft Tumour-Bearing Mice

Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3′-deoxy-3′-[18F]-fluorothymidine ([18F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [18F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [18F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [18F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.

Walker 256/B malignant breast cancer cells improve femur angioarchitecture and disrupt hematological parameters in a rat model of tumor osteolysis

This study was designed to assess femur angioarchitecture and hematological effects of Walker 256/B cells in a rat model of tumor osteolysis. Tumor osteolysis was induced by in situ inoculation of Walker 256/B malignant cells. Six other rats were sham operated and served as control. Twenty days later, rats were euthanized, and femurs were collected than radiographed. Angioarchitecture [mean lumen diameter (MLD), wall thickness (WTh), Vessel number, volume, and separation (VNb, VV, and VSp respectively)] was studied by histomorphometry at 2 different positions (P1: diaphysis, and P2: metaphysis) of the operated femora. Some hematological parameters were also assessed. Walker 256/B induced marked tumor osteolysis, with cortical perforation and trabecular destruction, associated increase in bone vascularization (increases of VNb and VV and decrease of VSp). Angioarchitecture of W256/B rats was disorganized and showed large MLD and lower WTh. These effects were more prominent in P2. When compared to Sham group, significantly decreases at levels of red blood cell (RBC), hemoglobin (Hb), hematocrit (Ht), and white blood cell (WBC) were observed in W256/B rats. These results suggest that Walker 256/B cells induced tumor osteolysis, improve hypervasculature especially near the tumoral foci (P2) associated hematological disruption. Besides, tumor vessels showed abnormal (enlarged and thinner) and disorganized morphology.

The Novel VEGF Receptor/MET–Targeted Kinase Inhibitor TAS-115 Has MarkedIn VivoAntitumor Properties and a Favorable Tolerability Profile

VEGF receptor (VEGFR) signaling plays a key role in tumor angiogenesis. Although some VEGFR signal-targeted drugs have been approved for clinical use, their utility is limited by associated toxicities or resistance to such therapy. To overcome these limitations, we developed TAS-115, a novel VEGFR and hepatocyte growth factor receptor (MET)-targeted kinase inhibitor with an improved safety profile. TAS-115 inhibited the kinase activity of both VEGFR2 and MET and their signal-dependent cell growth as strongly as other known VEGFR or MET inhibitors. On the other hand, kinase selectivity of TAS-115 was more specific than that of sunitinib and TAS-115 produced relatively weak inhibition of growth (GI50 > 10 μmol/L) in VEGFR signal- or MET signal-independent cells. Furthermore, TAS-115 induced less damage in various normal cells than did other VEGFR inhibitors. These data suggest that TAS-115 is extremely selective and specific, at least in vitro. In in vivo studies, TAS-115 completely suppressed the progression of MET-inactivated tumor by blocking angiogenesis without toxicity when given every day for 6 weeks, even at a serum-saturating dose of TAS-115. The marked selectivity of TAS-115 for kinases and targeted cells was associated with improved tolerability and contributed to the ability to sustain treatment without dose reduction or a washout period. Furthermore, TAS-115 induced marked tumor shrinkage and prolonged survival in MET-amplified human cancer-bearing mice. These data suggest that TAS-115 is a unique VEGFR/MET-targeted inhibitor with improved antitumor efficacy and decreased toxicity.

P53-Based Strategy for Protection of Bone Marrow From Y-90 Ibritumomab Tiuxetan

IntroductionThough radiolabeled anti-CD20 antibody has expanding roles in the management of B-cell lymphoma, its main drawback has been radiation-induced damage to the bone marrow leading to acute grade 3 and 4 hematological toxicity and potential contribution to the development of myelodysplastic syndrome and secondary leukemia. Arsenic trioxide is currently used to treat acute promyelocytic leukemia and known as a cytotoxic agent. However, we have recently demonstrated that arsenic trioxide can act as a cytoprotective agent at much lower dose. This is due to its ability to temporarily and reversibly suppress p53 activation caused by DNA-damaging treatments such as chemotherapy or radiotherapy. It has been also demonstrated that this protective effect is selective to normal tissues, as it requires functional p53. We have developed a preclinical model to assess the efficacy of low dose arsenic trioxide (LDA) as a cytoprotective agent against bone marrow toxicity induced by radioimmunotherapy using Y-90 ibritumomab tiuxetan as a model. MethodsTo test the hypothesis that LDA protects bone marrow against Y-90 ibritumomab tiuxetan induced damage, sex-matched BAL/c mice (4-6 weeks of age) were randomized into four groups: control, LAD only, Y-90 ibritumomab tiuxetan only, LAD pretreatment followed by Y-90 ibritumomab tiuxetan. LDA pretreatment was carried out by feeding mice with water containing 1 mg/L arsenic trioxide for three days. Y-90 ibritumomab tiuxetan was then injected into mice at the dose of 200uCi via tail vein. Tissue samples were collected at different time points (3 hours to 5 weeks) after treatment. Bone marrow damage was analyzed histologically with H&E staining, and DNA damage was assessed with pH2AX staining. To test the hypothesis that LDA does not protect malignant cells, a mouse xenograft model was generated using a CD20 expressing lymphoma cell line, Karpas 422. Treatments were initiated 1 week after implantation when tumors became palpable. Tumor volumes were measured with a caliper periodically. Tumor volume was calculated using the equation: volume = length × width × depth × 0.5236 mm3. Two independent experiments were done and the tumor volumes are expressed as means ± SE. ResultsY-90 ibritumomab tiuxetan treatments were associated with severe damages to bone marrow cells, and such damages were significantly reduced by LDA pretreatment (Fig 1). Consistent with this observation, much more DNA damage was accumulated in mice treated with Y-90 ibritumomab tiuxetan from as early as 3 hours to a week after treatment, as compared to mice pretreated with LDA (Fig 2). Remarkably, while DNA damage was eliminated in LDA-pretreated mice by 2wk to 5wk after treatment, damage was still observed in mice without LAD pretreatment. [Display omitted] [Display omitted] In tumor xenograft models, the tumor volume of the control group continued to increase with time. LDA pretreatment did not have any detectable effect on the growth of the implanted tumors. As expected, treatment with a single dose of Y-90 ibritumomab tiuxetan resulted in marked tumor growth suppression. LDA pretreatment showed little effect on radiation-induced tumor growth suppression (Fig 3). [Display omitted] ConclusionOur results demonstrate that a brief pretreatment with LDA is associated with a marked protection of bone marrow without compromising the ability of irradiation to kill lymphoma cells. A clinical trial is being developed based on our findings. Disclosures:No relevant conflicts of interest to declare.

Cavernous sinus hemangioma: A fourteen year single institution experience

Cavernous sinus hemangioma (CSH) is a rare extra-axial vascular neoplasm that accounts for 2% to 3% of all cavernous sinus tumors. Their location, propensity for profuse bleeding during surgery, and relationship to complex neurovascular structures are factors which present difficulty in excising these lesions. The authors describe their experience of 22 patients with CSH over 14years at a tertiary care center. Patients were managed with microsurgical resection using a purely extradural transcavernous approach (13 patients) and with Gamma Knife radiosurgery (GKRS; Elekta AB, Stockholm, Sweden) (nine patients). Retrospective data analysis found headache and visual impairment were the most common presenting complaints, followed by facial hypesthesia and diplopia. All but one patient had complete tumor excision in the surgical series. Transient ophthalmoparesis (complete resolution in 6–8weeks) was the most common surgical complication. In the GKRS group, marked tumor shrinkage (>50% tumor volume reduction) was achieved in two patients, slight shrinkage in five and no change in two patients, with symptom improvement in the majority of patients. To our knowledge, we describe one of the largest series of CSH managed at a single center. Although microsurgical resection using an extradural transcavernous approach is considered the treatment of choice in CSH and allows complete excision with minimal mortality and long-term morbidity, GKRS is an additional tool for treating residual symptomatic lesions or in patients with associated comorbidities making surgical resection unsuitable.

Abstract PL04-04: Synthetic lethal approaches to targeting Ras mutant cancers.

Abstract The RAS oncogenes are very frequently activated by mutation in human tumors, which very often show continued dependency on the signaling functions of RAS. In addition to controlling the RAF/MEK/ERK pathway, RAS proteins are established to be important direct regulators of type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino terminal RAS binding domain (RBD). Disruption of PI3K p110alpha activation by oncogenic KRAS has been shown to prevent tumor development in a mouse model of lung cancer. Disruption of RAS-PI3K p110alpha interaction in established lung tumors prevents tumor progression and results in long-term stabilization of lung tumors. Coordinate inhibition of the MAPK pathway at the same time promotes tumor regression and dramatically reduces tumor burden. However, as yet it is unclear whether the combined targeting of pathways that RAS controls, such as RAF/ERK and PI 3-kinase/AKT, will prove effective in the treatment of RAS mutant cancers in the clinic, or whether this approach will prove unacceptably toxic. It is therefore important that other means of targeting this critical oncogene are considered. One such approach is the development of agents that directly inhibit the function of RAS proteins, for example by blocking interaction with effectors. Another approach, which will be explored here, is the identification of unique dependencies of RAS mutant tumor cells through the use of functional genomic screens. We have used RNA interference libraries on a panel of lung cancer cell lines to determine genes that are required for the survival of cells with oncogenic KRAS mutations, but not wild type KRAS. This has led to the identification of signaling networks that are essential for RAS mutant cells, including a transcriptional programme controlled by the GATA2 transcription factor. In mouse models of lung cancer, deletion of GATA2 leads to dramatic regression of RAS induced tumors. Although GATA2 itself is likely to be undruggable, investigation of the transcriptional targets of GATA2 has revealed three key pathways that are much more tractable: the proteasome, NF-kB and Rho signaling. Combined suppression of GATA2 regulated pathways with clinically approved inhibitors of the proteasome and Rho kinase causes marked tumor clearance. Discovery of the non-oncogene addiction of KRAS mutant lung cancers to GATA2 function presents a network of druggable pathways for therapeutic exploitation. We are currently testing combinations of GATA2 functional targeting with other approaches that show potential, such as MEK plus PI 3-kinase inhibition, and immune checkpoint modulators. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL04-04. Citation Format: Julian Downward. Synthetic lethal approaches to targeting Ras mutant cancers. [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 PL04-04.

Abstract B198: Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC).

Abstract Selinexor (KPT-330) is a novel small molecule inhibitor of nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IκB, resulting in selective death of cancer cells. Preclinical studies have shown that oral Selinexor is well tolerated, even with prolonged (4-8 month) administration. Human Phase 1 clinical studies with Selinexor in hematological and solid cancer patients are ongoing (Clinicaltrials.gov NCT01607892 and NCT01607905). In this study we evaluated the mechanisms involved in Selinexor-induced growth inhibition of NSCLC in vitro and in vivo. Methods: A panel of NSCLC cells with various genetic backgrounds were treated with Selinexor and tested in proliferation (MTT), clonogenic survival, and flow cytometric assays. The cellular localization of TSPs were evaluated by immunofluorescence (IF). Western blotting was used to study levels of cell cycle regulating proteins and quantify markers of cell survival. Functional inhibition of NF-κB was measured using a transcription factor assay kit. A SCID mouse xenograft model of NSCLC, adenocarcinoma A549 (wt p53, NF-κB IC50 of 1.19 uM), was used to evaluate Selinexor effects in vivo and tumors were evaluated by histology and immunohistochemistry (IHC). Results: Selinexor induced cell cycle arrest, inhibited tumor cell growth and clonogenic formation and induced apoptosis in lung cancer cell lines regardless of genetic background (IC50 25-700 nM). Treated normal human dermal fibroblasts entered into cell cycle arrest but resisted apoptosis induction (IC50 4.0 uM). IF of the TSPs p53, p21, IκB, E2F4, and survivin demonstrated strong nuclear localization after 4 to 24 hour treatment with Selinexor followed by apoptosis as determined by cleavage of PARP, caspases 3 and 8 by immunoblot analysis. Functional evaluation of NF-kB in A549 and H1299 revealed transcriptional repression with IC50 values similar to MTT assays. Finally, A549 xenografts treated with Selinexor (10 - 20 mg/kg QoDx3) showed marked tumor suppression when compared to vehicle and cisplatin-treated mice (growth inhibition was 78% at 20 mg/kg, p<0.0001). Histological and IHC analysis of treated tumors indicated increased nuclear accumulation of p53, p21, FOXO1, survivin, NF-κB and IκB, reduced cell proliferation, and tumor cell death. Conclusions: In NSCLC, Selinexor forces nuclear retention of TSPs, inhibits NF-κB transcriptional activity, inhibits tumor growth and induces cell death regardless of p53 status. Selinexor thus has therapeutic potential for the treatment of NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B198. Citation Format: William Senapedis, Marsha Crochiere, Tami Rashal, Trinayan Kashyap, Boris Klebanov, Jean-Richard Saint-Martin, Ori Kalid, Sharon Shechter, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Eran Shacham, Sharon Tamir, Dilara McCauley, Erkan Baloglu, Michael Kauffman, Sharon Shacham, Yosef Landesman. Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC). [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 B198.

Major Cancer Regressions in Mesothelioma After Treatment with an Anti-Mesothelin Immunotoxin and Immune Suppression

Immunotoxins are potent anticancer agents with an unusual mechanism of action: inhibition of protein synthesis resulting in apoptotic cell death. Immunotoxins have produced many durable complete responses in refractory hairy cell leukemia, where patients rarely form antibodies to the bacterial toxin component of the immunotoxin. Patients with mesothelioma, however, have normal immune systems and form antibodies after one cycle, and tumor responses to the immunotoxin have not been observed in this disease. We describe the results of a trial in which major antitumor responses were seen in patients with advanced mesothelioma who received the anti-mesothelin immunotoxin SS1P, together with pentostatin and cyclophosphamide, to deplete T and B cells. Of 10 patients with chemotherapy-refractory mesothelioma, 3 have had major tumor regressions with 2 ongoing at 15 months, and 2 others responded to chemotherapy after discontinuing immunotoxin therapy. Antibody formation was markedly delayed, allowing more SS1P cycles to be given, but this alone does not appear to account for the marked antitumor activity observed.

Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations

Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. Eighty percent of UMs harbor mutations in the Gαq family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQ(Q209L)-transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a UM xenograft model. Our data identify PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and demonstrate that combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone.

Bronchoscopic findings for bevacizumab-related pulmonary hemorrhage in advanced non–small cell lung cancer

We report contemporaneous bronchoscopic findings for a case of bevacizumab-related pulmonary hemorrhage in a patient with advanced non-small cell lung cancer (NSCLC). Flexible bronchoscopy at diagnosis revealed abnormal capillary dilation that was suggestive of endobronchial involvement at the primary tumor location. The patient developed massive hemoptysis despite of marked tumor shrinkage achieved by bevacizumab-containing chemotherapy. Emergency flexible bronchoscopy for hemoptysis suggested that the location of the primary tumor was the source of bleeding. Subsequent follow-up flexible bronchoscopy revealed an ulcerative mucosal-like lesion associated with a white necrotic substance as well as attenuation of the dilation of submucosal vessels compared with that apparent at diagnosis. Our case report highlights the potential mechanistic insights into bevacizumab-related bleeding and importance of performing bronchoscopy at diagnosis in NSCLC patients, given that abnormal bronchoscopic findings may be a risk factor for bleeding.

Intratumoral Gas in Hepatocellular Carcinoma following Transarterial Chemoembolization: Associated Factors and Clinical Impact

PurposeTo determine the frequency and factors associated with the presence of intratumoral gas-containing areas in hepatocellular carcinoma (HCC) on computed tomography (CT) scans obtained 4–6 weeks after transarterial chemoembolization. Materials and MethodsFrom June 2010 to December 2011, 201 patients underwent 286 chemoembolization procedures for HCC (n = 497 tumors) and were retrospectively included. The presence of intratumoral gas was assessed on CT 4–6 weeks after chemoembolization. Clinical and biologic data and tumoral and chemoembolization procedure characteristics were noted. Factors associated with the presence of intratumoral gas were evaluated. Tumor response was assessed by using European Society for the Study of the Liver criteria. Tumors containing gas or not containing gas were compared by univariate and multivariate analysis. ResultsIntratumoral gas was found in 26 tumors (5%) after 26 chemoembolization procedures (9.1%) in 26 patients (13%). Gas was related to abscess formation in three patients (11.5%). On multivariate analysis, a large mean tumor diameter at baseline (72.4 mm vs 40.2 mm; P = .003), chemoembolization with drug-eluting beads (P = .033), and superselective approach (P = .024) were independently associated with the presence of gas. Tumors that exhibited gas-containing areas at 1 month had a significantly higher objective response rate than those that did not (P < .0001). ConclusionsIntratumoral gas-containing areas after chemoembolization are rarely related to the formation of abscesses. The presence of intratumoral gas on CT 4–6 weeks after chemoembolization could be a surrogate marker for marked tumor necrosis.

Total en Bloc Spondylectomy for Spinal Metastasis of Differentiated Thyroid Cancers

A retrospective case series. To evaluate the long-term effectiveness and problems of total en bloc spondylectomy (TES) for treating metastatic thyroid cancer of the spine. Although TES is indicated for low-grade malignant spinal tumors, its efficacy in treating thyroid cancer metastases in the spine is uncertain. This study followed 8 patients who underwent TES for spinal metastasis of thyroid cancer (5 males, 3 females; mean age, 55.3 y; mean follow-up period, 6.4 y), 6 with follicular carcinomas and 2 with papillary carcinomas. In 6 patients without a marked paravertebral tumor extension, TES was performed using a posterior approach. Two patients had prominent anterior or anterolateral tumor extension into the thoracic and retroperitoneal cavities, and a combined anterior and posterior approach was used. We evaluated the patients' clinical symptoms, operative procedures, complications, and prognoses. Before surgery, the patients' neurological status was evaluated by the Frankel classification (grade C, 4 patients; grade D, 1 patient; grade E, 3 patients) and Tokuhashi scores (9, 1 patient; 11, 2 patients; 13 and 14, 1 patient each; 15, 3 patients). One year after surgery, all the patients had improved by at least a Frankel grade, and 4 patients classified as grade C before surgery had become ambulatory. At the final follow-up (average of 6.4 y), 5 patients had a grade E Frankel classification and 1 had grade D, but 2 patients had suffered neurological deterioration, because of tumor recurrence, at the same level as the TES in one patient and at an adjacent level in the other. At follow-up, all patients were alive; 5 showed no evidence of disease. Two patients experienced rod breakage, at 2.8 and at 3.3 years after TES, and 1 patient suffered an endplate fracture 6 months after TES; this patient developed a late infection, 7 years after TES. TES was effective in controlling spinal thyroid cancer metastasis locally and in preserving the ability to walk. However, instrumentation failure and other late complications arose during the patients' long survival times after TES.

A phase Ib dose-escalation study of TRC105 (anti-endoglin antibody) in combination with bevacizumab (BEV) for advanced solid tumors.

3059 Background: CD105 (endoglin) is an endothelial cell membrane receptor highly expressed on angiogenic tumor vessels that is essential for angiogenesis and upregulated by hypoxia and VEGF inhibition. TRC105 is an anti-CD105 monoclonal antibody that potentiates VEGF inhibitors in preclinical models. This study assessed safety, PK and preliminary efficacy of TRC105 in combination with BEV. Methods: Pts with advanced solid tumors, ECOG PS 0-1, and normal organ function were treated with escalating doses of IV TRC105 (3, 6, 8 or 10 mg/kg/wk) plus bevacizumab (BEV) at 15 mg/kg q3wk or 10 mg/kg q2wk. Results: Thirty one pts (median age = 62; M:F 14:17; median 4 prior regimens; primarily metastatic colorectal or ovarian cancer) were treated with TRC105 wkly + BEV. TRC105 3 mg/kg wkly + 15 mg/kg q3wk BEV was well tolerated. Concurrent administration of TRC105 6 mg/kg wkly + 15 mg/kg BEV q3wk resulted in headaches in 4 of 5 pts on cycle 1 day 1 (two grade 3). Dose escalation to the recommended single-agent phase II dose of 10 mg/kg TRC105 weekly + BEV (10 mg/kg q2wk) was tolerated when the initial TRC105 dose was introduced one week after BEV dosing and administered over 2 days. Headache was the only serious adverse drug reaction observed. Adverse events characteristic of each individual drug were not increased in frequency or severity. Target TRC105 serum concentrations were achieved at 6 mg/kg. Mucocutaneous telangiectasia, a marker of TRC105 target modulation, was observed beginning at 6 mg/kg and was dose proportional. Five of 19 heavily pretreated, BEV or VEGF receptor tyrosine kinase inhibitor (TKI) refractory pts with colorectal and ovarian cancer, each with marked tumor burden, experienced radiographic reductions in tumor volume (10-17%). Three of these patients remained on study longer than the prior VEGF inhibitor treatment and two are ongoing. Seven ongoing patients have been treated for 2-8 months. Conclusions: TRC105 10 mg/kg wkly was well tolerated with BEV 10 mg/kg q2wk. The combination demonstrated activity in BEV and VEGF TKI refractory pts. Randomized phase II trials of BEV +/- TRC105 have commenced in renal cell cancer and glioblastoma. Clinical trial information: NCT01332721.

Magnetic dipole interaction of endogenous magnetic nanoparticles with magnetoliposomes for targeted drug delivery

Dynamics of magnetoliposomes binding to the tumor cells and the efficiency of their recognition for targeted drug delivery is largely determined by physical interaction. In this paper we assess the strength of magnetic dipole interaction that occurs between endogenous magnetic nanoparticles in tumor cells and exogenous magnetic nanoparticles as a component of magnetoliposomes, and compare it with the forces of specific binding of the antigen-antibody complex. To assess the strength of magnetic dipole interaction the model of chains of identical particles was used, and an order of magnitude, 9(-10) N, was obtained. Thus, the indicated force has an order of magnitude close to the forces of specific binding, and even more. The force of magnetic dipole interaction between a magnetically marked dosage form and tumor cells is virtually the additional specific binding force--"passive targeting" for targeted drug delivery in consequence of the fact that tumor cells tend to contain the number of biogenic nanoparticles of magnetite (Fe3O4) by an order of magnitude greater than normal.

Abstract SY17-03: Targeting BRAF and PI3′-kinase signaling for therapy of melanoma.

Abstract Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with greater than 50% of tumors expressing the BRAF(V600E) oncoprotein. BRAF(V600E) is a constitutively active protein kinase that promotes sustained activation of BRAF→MEK→ERK signaling in the melanoma cell. Importantly, the marked tumor regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF(V600E) in melanoma maintenance. However, since most patients relapse with lethal vemurafenib resistant disease, understanding and preventing mechanism(s) of drug resistance will be critical to extending the durability of patients' responses. Although mutational activation of BRAF is often the initiating event, progression of BRAF mutated melanomas is frequently associated with silencing of the PTEN tumor suppressor, a negative regulator of PI3′-kinase signaling. Whilst PTEN silencing is common in human melanoma, mutational activation of PIK3CA (encoding the alpha catalytic subunit of PI3′-kinase) is extremely rare, even though PIK3CA mutations are common in other human cancers. Since PTEN is reported to have tumor suppressor functions independent of its PI3′-lipid phosphatase activity, PTEN silencing may have more profound tumor promoting activity than mutational activation of PIK3CA. To test this, we generated mice carrying a conditional knock-in allele of Pik3ca that permits Cre-induced expression of PIK3CA(H1047R), a mutationally activated form of PIK3CA detected in many cancer types. Expression of PIK3CA(H0147R) in melanocytes elicited no obvious phenotype, consistent with the absence of phenotype in PTEN(Null) melanocytes. As observed previously, melanocyte-specific expression of BRAF(V600E) combined with PTEN silencing led to metastatic melanoma. Interestingly, metastatic melanoma also arose from melanocytes expressing both BRAF(V600E) and PIK3CA(H1047R), although these tumors grew more slowly than their BRAF(V600E)/PTEN(Null) counterparts. Both BRAF(V600E)/PTEN(Null) and BRAF(V600E)/PIK3CA(H1047R) melanomas were sensitive to the tumor prevention effects of pharmacological blockade of class 1 PI3′-kinases. However, the clinically relevant subset of BRAF(V600E)/PTEN(Null) melanoma was resistant to pharmacological blockade of AKT. Using genetically engineered mouse models and melanoma-derived cell lines, we demonstrate cooperative anti-tumor effects of combining RAF and PI3′-kinase inhibitors, suggesting that combined pathway-targeted chemotherapy may maximize melanoma patients' therapeutic response while delaying the onset of lethal drug resistant disease. Using patient derived xenografts propagated in immunocompromised mice we explored the cause and consequences of drug resistance, which was selected by continuous vemurafenib administration. As observed previously, resistant tumors displayed continued dependence on BRAF(V600E)→MEK→ERK signaling due to elevated BRAF(V600E) expression. However, remarkably, we observed that vemurafenib-resistant melanomas displayed a striking drug dependency for their continued proliferation, such that cessation of drug administration led to regression of established drug-resistant tumors. We further demonstrated that a discontinuous dosing strategy, which exploits the fitness deficit displayed by drug-resistant cells in the absence of the vemurafenib, forestalls the onset of lethal drug resistant disease. These data highlight the concept that drug-resistant cells may also show dependency, such that altered drug dosing may prevent the emergence of lethal drug resistance. Citation Format: Martin McMahon, Meghna Das Thakur, Victoria Marsh, Jillian Silva, Allison Landman, Marian Deuker, Fernando Salangsang, Nancy Pryer, Wayne Phillips, Mitchell Levesque, Reinhard Dummer, Marcus Bosenberg, William R. Sellers, Darrin Stuart. Targeting BRAF and PI3′-kinase signaling for therapy of melanoma. [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 SY17-03. doi:10.1158/1538-7445.AM2013-SY17-03

Abstract 2713: The Pten and p53 tumor suppressors cooperate to constrain the PI3K/mTOR pathway activation.

Abstract Loss of Pten expression and mutational inactivation of p53 are frequent molecular events in liver cancer associated with advanced disease stage and poor prognosis. The tumor suppressor PTEN is a phosphoinositide phosphatase that antagonizes the class IA PI3K-engaged oncogenic signaling by dephosphorylating PtdIns(3,4,5)P(3); however, the specific roles of class IA PI3K isoforms, p110a and p110b, have not been assessed in genetically engineered mouse models of liver cancer with Pten inactivation. Furthermore, effective treatment strategies for Pten and p53 deficient liver malignancy remain to be defined. In this study, we showed that combined deletion of Pten and p53 in liver led to rapid development of hepatocellular carcinoma and cholangiocellular carcinoma, the two most common types of liver cancer, with complete penetrance. While a single ablation of p110a or p110b markedly reduced the tumor burden, inactivation of both p110a and p110b completely blocked the liver tumorigenesis. Although liver tumors driven by inactivation of Pten and p53 showed a good response to GDC-0941, a pan-PI3K inhibitor, the combined treatment with both GDC-0941 and Rad001, an mTOR inhibitor or single-agent BEZ235, a dual pan-PI3K and mTOR inhibitor, led to marked tumor regression. These studies suggest that Pten and p53 cooperate to constrain the PI3K/mTOR pathway activation and inhibitors targeting both PI3K and mTOR may effectively treat Pten and p53 deficient liver cancers. Citation Format: Pixu Liu, Hailing Cheng, Lynn Symonds, Erbo Xu, Sauveur-Michel Maira, Emmanuelle di Tomaso, Thomas Roberts, Jean Zhao. The Pten and p53 tumor suppressors cooperate to constrain the PI3K/mTOR pathway activation. [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 2713. doi:10.1158/1538-7445.AM2013-2713

Abstract SY25-02: Synthetic lethal approaches to targeting RAS mutant tumors: Is there an Achilles' heel.

Abstract The RAS oncogene is frequently activated by mutation in human tumors, including most pancreatic cancers and many lung and colon cancers, which very often show continued dependency on its signaling functions. RAS proteins are established to be important direct regulators of p110alpha and p110gamma type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino terminal RAS binding domain (RBD). Disruption of PI3K p110alpha activation by oncogenic KRAS has been shown to prevent tumor development in a mouse model of lung cancer. Disruption of Ras-PI3K p110alpha interaction in established lung tumors prevents tumor progression and results in long-term stabilization of lung tumors. Coordinate inhibition of the MAPK pathway at the same time promotes tumor regression and dramatically reduces tumor burden. However, as yet it is unclear whether the combined targeting of pathways that RAS controls, such as RAF/ERK and PI 3-kinase/Akt, will prove effective in the treatment of RAS mutant cancers in the clinic. It is therefore important that other means of targeting this critical oncogene are considered. One such approach is the development of agents that directly inhibit the function of RAS proteins, for example by blocking interaction with effectors. Another approach, which will be explored here, is the identification of unique dependencies of RAS mutant tumor cells through the use of functional genomic screens. We have used RNA interference libraries on a panel of lung cancer cell lines to determine genes that are required for the survival of cells with oncogenic KRAS mutations, but not wild type KRAS. This has led to the identification of signaling networks that are essential for RAS mutant cells, including a transcriptional programme controlled by the GATA2 transcription factor. In mouse models of lung cancer, deletion of GATA2 leads to dramatic regression of RAS induced tumors. Although GATA2 itself is likely to be undruggable, investigation of the transcriptional targets of GATA2 has revealed three key pathways that are much more tractable: the proteasome, NF-kB and Rho signaling. Combined suppression of GATA2 regulated pathways with clinically approved inhibitors of the proteasome and Rho kinase causes marked tumor clearance. Discovery of the non-oncogene addiction of KRAS mutant lung cancers to GATA2 function presents a network of druggable pathways for therapeutic exploitation. We are currently investigating the ability of these targeting strategies to impact on RAS induced cancer formation in other common RAS driven cancer models. The ability of targeting GATA2 function to cause regression of KRAS mutant lung cancers with loss of p53 function has also been established in mouse models, suggesting that this approach may be viable in more advance tumors. Future challenges will include how to convert the major regressions that are seen when targeting GATA2 in mouse models of lung cancer into lasting clearance of the disease. In order to do this we are testing combinations of GATA2 functional targeting with other approaches that show potential, such as MEK plus PI 3-kinase inhibition. A major limitation to the efficacy of these approaches is likely to be the evolution of drug resistance, so mechanisms whereby this can occur are being actively investigated, both for the synthetic lethal hits and also the RAS signaling pathway inhibitors. Citation Format: Julian Downward. Synthetic lethal approaches to targeting RAS mutant tumors: Is there an Achilles' heel. [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 SY25-02. doi:10.1158/1538-7445.AM2013-SY25-02

Poly(β-amino ester) Nanoparticle Delivery of TP53 Has Activity against Small Cell Lung Cancer In Vitro and In Vivo

Small cell lung cancer (SCLC) is an aggressive disease with one of the highest case-fatality rates among cancer. The recommended therapy for SCLCs has not changed significantly over the past 30 years; new therapeutic approaches are a critical need. TP53 is mutated in the majority of SCLC cases and its loss is required in transgenic mouse models of the disease. We synthesized an array of biodegradable poly(β-amino ester) (PBAE) polymers that self-assemble with DNA and assayed for transfection efficiency in the p53-mutant H446 SCLC cell line using high-throughput methodologies. Two of the top candidates were selected for further characterization and TP53 delivery in vitro and in vivo. Nanoparticle delivery of TP53 resulted in expression of exogenous p53, induction of p21, induction of apoptosis, and accumulation of cells in sub-G1 consistent with functional p53 activity. Intratumoral injection of subcutaneous H446 xenografts with polymers carrying TP53 caused marked tumor growth inhibition. This is the first demonstration of TP53 gene therapy in SCLC using nonviral polymeric nanoparticles. This technology may have general applicability as a novel anticancer strategy based on restoration of tumor suppressor gene function.

Therapeutic efficacy of a systemically delivered oncolytic adenovirus – Biodegradable polymer complex

Despite great efforts to develop a more effective oncolytic adenovirus (Ad) for eradicating tumors, in vivo application via systemic administration is strictly limited to local injection due to host immune responses by Ad surface proteins and liver accumulation by the inherent nature of the Ad. In the last decade, numerous techniques using synthetic polymers have widely emerged to shield the exterior of therapeutic Ad vectors for systemic delivery. We developed a cationic polymer linked with polyethylene glycol for systemically delivering oncolytic Ad. The increased transduction efficiency and oncolytic effect of the Ad vectors physically coated with the polymer were evaluated, showing the optimal size (130 nm) of the Ad/polymer complex for systemic administration and prolonged stability of the Ad/polymer complex. Marked tumor growth suppression of the oncolytic Ad delivered by the polymer through systemic injection was observed in HT1080 and A549 xenograft models. The masking effect of the Ad surface by the polymer elicited evasion of innate adaptive immune responses and the tumor-to-liver ratio of the complex was significantly elevated 1229-fold greater than that of a naked Ad. These results demonstrate that the potential system of oncolytic Ad complexed with the biodegradable polymer may be useful for developing therapeutic vector systems via systemic delivery.