Median IC50 Value Research Articles

Antimalarial activities of medicinal plants and herbal formulations used in Thai traditional medicine

Malaria is one of the world's leading killer infectious diseases with high incidence and morbidity. The problem of multidrug-resistant Plasmodium falciparum has been aggravating particularly in Southeast Asia. Therefore, development of new potential antimalarial drugs is urgently required. The present study aimed to investigate antimalarial activities of a total of 27 medicinal plants and 5 herbal formulations used in Thai traditional medicine against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) P. falciparum clones. Antimalarial activity of the ethanolic extracts of all plants/herbal formulations against K1 and 3D7 P. falciparum clones was assessed using SYBR Green I-based assay. All plants were initially screened at the concentration of 50 μg/ml to select the candidate plants that inhibited malaria growth by ≥50%. Each candidate plant was further assessed for the IC50 value (concentration that inhibits malaria growth by 50%) to select the potential plants. Selectivity index (SI) of each extract was determined from the IC50 ratio obtained from human renal epithelial cell and K1 or 3D7 P. falciparum clone. The ethanolic extracts from 19 medicinal plants/herbal formulation exhibited promising activity against both K1 and 3D7 clones of P. falciparum with survival of less than 50% at the concentration of 50 μg/ml. Among these, the extracts from the eight medicinal plants (Plumbago indica Linn., Garcinia mangostana Linn., Dracaena loureiri Gagnep., Dioscorea membranacea Pierre., Artemisia annua Linn., Piper chaba Hunt., Myristica fragrans Houtt., Kaempferia galanga Linn.) and two herbal formulations (Benjakul Formulation 1 and Pra-Sa-Prao-Yhai Formulation) showed potent antimalarial activity with median range IC50 values of less than 10 μg/ml against K1 or 3D7 P. falciparum clone or both. All except G. mangostana Linn. and A. annua Linn. showed high selective antimalarial activity against both clones with SI>10. Further studies on antimalarial activities in an animal model including molecular mechanisms of action of the isolated active moieties are required.

Initial testing (stage 1) of temozolomide by the pediatric preclinical testing program

The DNA methylating agent temozolomide was developed primarily for treatment of glioblastoma. However, preclinical data have suggested a broader application for treatment of childhood cancer. Temozolomide was tested against the PPTP solid tumor and ALL models. Temozolomide was tested against the PPTP in vitro panel at concentrations ranging from 0.1 to 1,000 µM and was tested against the PPTP in vivo panels at doses from 22 to 100 mg/kg administered orally daily for 5 days, repeated at day 21. In vitro temozolomide showed cytotoxicity with a median relative IC50 (rIC50 ) value of 380 µM against the PPTP cell lines (range 1 to > 1,000 µM). The three lines with rIC50 values lesser than 10 µM had low MGMT expression compared to the remaining cell lines. In vivo temozolomide demonstrated significant toxicity at 100 mg/kg, but induced tumor regressions in 15 of 23 evaluable solid tumor models (13 maintained CR [MCR], 2 CR) and 5 of 8 ALL models (3 MCR, 2 CR). There was a steep dose response curve, with lower activity at 66 mg/kg temozolomide and with tumor regressions at 22 and 44 mg/kg restricted to models with low MGMT expression. Temozolomide demonstrated high level antitumor activity against both solid tumor and leukemia models, but also elicited significant toxicity at the highest dose level. Lowering the dose of TMZ to more closely match clinical exposures markedly reduced the antitumor activity for many xenograft lines with responsiveness at lower doses closely related to low MGMT expression.

Combining Dose-Response Slope with in Vitro Sensitivity to ABL Tyrosine Kinase Inhibitors As an Improved Correlative Metric for Predicting Clinical Response in Chronic Myeloid Leukemia

AbstractAbstract 1670BCR-ABL mutations result in clinical resistance to ABL tyrosine kinase inhibitors (TKIs) in CML. Although in vitro IC50 values for specific mutations have been suggested to guide TKI choice in the clinic, quantitative relationship between IC50 and clinical response has never been demonstrated. Notably, IC50 value constitutes only one point on the dose-response curve for a given drug. Most dose-response curves can be described by Hill's equation (equation 1), which incorporates both IC50 and slope (m) parameters: [Display omitted] Here, fa is cell fraction affected by treatment and D is drug dose. We report estimation of the slope of in vitro dose-response curves for wild-type and kinase domain-mutant BCR-ABL against clinical ABL TKIs for CML and examine the value of this incorporated parameter for predicting clinical response. Dose response curves for imatinib, nilotinib and dasatinib were determined by methanethiosulfonate-based cell viability assay in Ba/F3 cells expressing wild-type BCR-ABL or each of 15 specific kinase domain point mutations (O’Hare T. et al Cancer Res 65:4500-5). The parameters m and IC50 were determined for each mutation and drug by fitting the log-transformed equation 1 to the respective dose-response curve. Excellent goodness of fit (r2 range 0.94–0.99) was observed for all drug-mutation pairings, confirming the adequacy of Hill’s equation to describe the effect of ABL TKIs on cellular viability in vitro. Inhibitory potential at peak concentration (IPP) was subsequently calculated as: [Display omitted] Here, fu is cell fraction unaffected by treatment, and D is mean peak concentration (Cmax) reported in plasma (Laneuville P. et al J Clin Oncol 28:e169-71). IPP and IC50 values for each Ba/F3 BCR-ABL mutant were compared with previously reported CCyR rates for nilotinib (Kantarjian H. et al Blood 110:3540-6) and dasatinib (Muller M.C. et al Blood 114:4944-53). Consistent with the particularly negative effect of P-loop mutations on drug binding and clinical outcome with imatinib, we found that 4 of 7 these mutations tested (G250E, Y253H, E255K, E255V) showed lower dose-response slope relative to wild-type BCR-ABL in addition to high IC50, while all other mutations showed variably increased slopes. Furthermore, the range of IPPs of imatinib for these mutations was lower than (and not overlapping with) all other mutations (0.084–1.66 vs 2.93–5.59; p=6*10−6). Slope variability provided particular additional interpretive value in cases where in vitro IC50 and clinical response are disparate. For example, G250E and V379I mutants feature comparable cellular IC50 values for imatinib (1184 and 1140 nM, respectively), but only G250E harbors worse clinical prognosis, arguably due to a lower dose-response slope of G250E, as also reflected in a lower IPP value compared to V379I (1.66 vs 3.04). Similarly, the M244V mutant does not confer substantial clinical resistance to imatinib despite increased IC50, possibly due to an exceptionally high slope value (m=5 vs. m=1.87 for wild-type BCR-ABL) reflecting a very steep dose-response curve which may render patients with this mutation particularly vulnerable to consequences of unfavorable imatinib pharmacokinetic profile or reduced compliance. Lastly, we examined whether higher IPP values were predictive of better clinical response to nilotinib and dasatinib in second-line clinical setting. Dasatinib-treated patients with mutations resulting in high IPP (IPP>7) had a significantly higher mean CCyR rate than those patients with lower IPPs (53% vs 31%; p=0.038). In contrast, this relationship was not evident when IC50 alone was used. In nilotinib-treated patients the difference in mean CCyR rate between patients with values above or below the median IPP or IC50 value approached but did not reach statistical significance (p=0.055 for both cases), potentially due to the lower number of patients for which response data by mutation has been reported (7 vs 11 mutations, 65 vs 295 patients with mutations, for nilotinib and dasatinib respectively). Taken together, our data suggest an integrated metric such as IPP may have both further relevance in conjunction with individual pharmacokinetic measurements and application to improved interpretation of mutationally-guided TKI treatment selection in CML and other malignancies. Disclosures:Vainstein:Neumedicines Inc: Employment. Druker:MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Dr Druker is a scientific founder, consultant, and stock holder in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD., OHSU and Dr. Druker have a financial interest in MolecularMD. Dr Druker is a scientific founder, consultant, and stock holder in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Other; Novartis, Bristol-Myers Squibb: Dr Druker's institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr Druker does not derive salary, nor does his lab receive funds from these contracts. Other.

Genome Wide Studies in Multiple Myeloma Identify XPO1/CRM-1 As a Critical Target Validated Using the Selective Inhibitor of Nuclear Export (SINE) KPT-276

AbstractAbstract 573Using high throughput RNA interference screening on 6,722 druggable genes we previously identified XPO1/CRM1 as one of the 50 most vulnerable targets in Multiple Myeloma (MM)1. XPO1 knockdown proved lethal in MM cell lines, but had no effect on human embryonic kidney (293) cells or lung cancer (A549) cells, showing that XPO1 is a specific myeloma vulnerability, and that myeloma cell survival is dependent upon XPO1 expression. XPO1 encodes the protein exportin 1, a nuclear transport protein that exports tumor suppressor proteins from the nucleus, where they are active, to the cytoplasm, where they become inactive. We next analyzed XPO1 in MM via gene expression profiling (GEP). XPO1 expression is up-regulated as the disease progresses: patients with active MM have a higher level of XPO1 compared to normal plasma cells (p<0.04) and to patients with monoclonal gammopathy of undetermined significance or smoldering MM (p<0.0001). The highest levels were in human MM cell lines. TC classification revealed highest levels in t(11;14) and lowest levels in t(4;14) disease.Selective inhibitors of nuclear export (SINE) compounds have recently been developed that irreversibly inhibit XPO1/CRM1 and its nuclear export function. One such inhibitor, KPT-276, decreased the viability of all 12 MM cell lines tested in vitro, as shown by MTT assay. After 72 hours of drug treatment, a median IC50 value of approximately 175 nM (range 30–1000 nM) was observed. No synergy with other commonly used anti-MM therapeutics was observed in vitro. In contrast, the drug had little effect in 8 solid tumor cell lines with the exception of the B cell lymphoma line Ramos. KPT-276 was also consistently active in inducing apoptosis against MM primary patient samples. Using an IC80 dose of KPT-276, drug-treated samples had a reduced population of cells in S phase (8%) compared to cells treated with DMSO (21%). Using the vkappa*myc transgenic MM model, KPT-276 reduced monoclonal spikes (by a mean of 56%) in all mice treated orally with 150 mg/kg dose three times per week for 4 weeks. Furthermore, KPT-276 significantly reduced tumor growth in a xenograft MM1.S mouse model. GEP was performed in the presence or absence of drug in two different MM cell lines. Two genes of probable relevance, cell division cycle 25 homolog A (CDC25A) and Bromodomain-containing protein 4 (BRD4), were dysregulated by SINE treatment. Both are involved in cell cycle control and have been linked to MYC. RT-PCR and western blotting confirm that MYC, CDC25A and BRD4 are down-regulated, as soon as six hours, after treatment with KPT-276. KPT-276 has shown marked anticancer activities against B cell malignancies in vitro and is active and tolerated in Phase I canine studies. KPT-330, a close analog of KPT-276, is currently in Phase 1 studies in human with advanced hematological and solid tumors. Disclosures:Schmidt:Karyopharm: Research Funding. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

In vitroantimalarial susceptibility and molecular markers of drug resistance in Franceville, Gabon

BackgroundMalaria remains a major public health problem, due largely to emergence and widespread P. falciparum drug resistance. WHO recommends artemisinine combination based therapy (ACT) to overcome P. falciparum drug resistance, but reports of declining ACT efficacy have been published. A thorough understanding of the molecular bases of P. falciparum resistance to existing drugs is therefore needed. The aims of this study were to analyze the in vitro sensitivity of P. falciparum field isolates from Franceville, Gabon, to chloroquine (CQ), mefloquine (MF), dihydroartemisinine (DHA) and monodesethylamodiaquine (MDAQ), and to investigate polymorphisms associated with drug resistance.MethodsWe conducted a cross-sectional study of 53 field isolates. Field isolates sensitivity to CQ, MF, DHA and MDAQ was assessed using the colorimetric DELI test. The Pfmdr1 codons 86 and 1246, Pfcrt (haplotype codon 72 to 76) and the PfATPAse6 codons 110 and 2694 were analysed by PCR-RFLP. Associations between drug sensitivity and parasite gene polymorphisms were evaluated with the Chi square test, and routine hematological parameters were analyzed with Fisher’s exact test implemented with Epinfo software. In all statistical tests, significance was assumed at p<0.05.ResultsA total of 46 P. falciparum isolates were successfully cultured in vitro and their sensitivity was tested. The proportions of isolates resistant to CQ, MF and MDAQ were 43.5%, 23.4% and 56.5%, respectively. Some isolates (23.9%) had DHA IC50 values higher than 10 nM. The median IC50 values were 71.67 (interquartile range (IQR, 1–438.2), 6.59 (IQR, 0.08-96), 64.79 (IQR, 0.09-448) and 6.45 nM (IQR, 0.09-23) for CQ, MF, MDAQ and DHA, respectively. The strongest correlation between diminished DHA sensitivity and MF resistance was observed (r2=0.73), followed by correlation between diminished DHA sensitivity and CQ resistance. Cross-resistance between CQ and MF was also observed. The prevalence of the 86Y and 1246Y mutations in Pfmdr1, 76T in Pfcrt, and 110A and 2694T in PfATPase6 was respectively 42% and 17.1%, 97.8%, and 0% and 22.2%.ConclusionThese high levels of antimalarial drug resistance in Franceville, Gabon, call for reinforced surveillance of drug efficacy.

Cytotoxicity, toxicity, and anticancer activity of Zingiber officinale Roscoe against cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line (CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3) was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/ DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median IC50 (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and 27.86 μg/ml, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.

Initial testing (stage 1) of the phosphatidylinositol 3′ kinase inhibitor, SAR245408 (XL147) by the pediatric preclinical testing program

Activation of the PI3 kinase pathway occurs frequently in many adult cancers and is implicated in tumor cell proliferation, survival, and resistance to chemotherapy and radiotherapy. However, less is known regarding the relevance of this pathway in pediatric cancers. Here we have evaluated SAR245408, a novel small molecule PI3K inhibitor, against childhood cancer cell lines and xenografts. SAR245408 was tested against the PPTP in vitro cell line panel at concentrations from 10 to 100 µM and against the PPTP in vivo xenograft panels at a dose of 100 mg/kg administered orally daily × 14. In vitro SAR245408 demonstrated cytotoxic activity, with a median relative IC50 value of 10.9 µM (range 2.7-24.5 µM). SAR245408 was well tolerated in vivo, and all 44 tested xenograft models were evaluable for efficacy. SAR245408 induced significant differences in EFS distribution compared to control in 29 of 37 (79%) of solid tumor xenografts and in two of seven (29%) ALL xenografts. SAR245408 induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C > 2) in 4 of 37 (11%) solid tumor xenografts. Intermediate EFS T/C activity was also observed for two of seven (29%) evaluable ALL xenografts. Objective responses were not observed for solid tumor or for ALL xenografts. Under the conditions evaluated in this study, SAR245408 achieved modest single-agent activity against most PPTP preclinical models. Further exploration of SAR245408 in combination with standard agents or with other signaling inhibitors could be considered.

Antitumor activity of zoledronic acid in primary breast cancer cells determined by the ATP tumor chemosensitivity assay.

BackgroundThe NeoAzure study has demonstrated that the use of the bisphosphonate zoledronic acid (Zol) in the neoadjuvant setting increases the rate of complete response in primary breast cancer and therefore indicates direct antitumor activity. The purpose of this study was to compare the antitumor effect of Zol with standard chemotherapy in primary breast cancer cells using ATP-tumor chemosensitivity assay (ATP-TCA).MethodsBreast cancer specimens were obtained from patients with breast cancer who underwent primary breast cancer surgery at the Department of Obstetrics and Gynecology, Tübingen, Germany, between 2006 through 2009. Antitumor effects of Zol, TAC (Docetaxel, Adriamycin, Cyclophosphamide) and FEC (5-Fluorouracil, Epirubicin, Cyclophosphamide) were tested in 116 fresh human primary breast cancer specimens using ATP-TCA. ATP-TCA results were analyzed with different cut-off levels for the half maximal inhibitory concentration (IC50), for IC90 and for the sensitivity index (IndexSUM). Each single agent or combination was tested at six doubling dilutions from 6.25, 12.5, 25, 50, 100, and 200% of test drug concentrations (TDC) derived from the plasma peak concentrations determined by pharmacokinetic data. The assay was carried out in duplicate wells with positive and negative controls.ResultsThe median IndexSUM value was lower for Zol than for the combined regimen FEC (36.8%) and TAC (12.9%), respectively, indicating increased antitumor activity of Zol in primary breast cancer cells. The difference regarding Zol and FEC was significant (p < 0.05). The median IC50 value for Zol (8.03% TDC) was significantly lower than the IC50 values for FEC (33.5% TDC) and TAC (19.3% TDC) treatment (p < 0.05). However, the median IC90 value for Zol (152.5% TDC) was significantly higher than the IC90 value obtained with TAC (49.5% TDC; p < 0.05), but similar to the IC90 value for FEC (180.9% TDC). In addition a significant positive correlation was observed for the IndexSum of Zol and the ER status (p < 0.01).ConclusionZoledronic acid has a strong antitumor effect on primary breast cancer cells in vitro which is equal or superior to commonly used chemotherapeutic regimens for treating breast cancer.

HIV-specific antibodies capable of ADCC are common in breastmilk and are associated with reduced risk of transmission in women with high viral loads.

There are limited data describing the functional characteristics of HIV-1 specific antibodies in breast milk (BM) and their role in breastfeeding transmission. The ability of BM antibodies to bind HIV-1 envelope, neutralize heterologous and autologous viruses and direct antibody-dependent cell cytotoxicity (ADCC) were analyzed in BM and plasma obtained soon after delivery from 10 non-transmitting and 9 transmitting women with high systemic viral loads and plasma neutralizing antibodies (NAbs). Because subtype A is the dominant subtype in this cohort, a subtype A envelope variant that was sensitive to plasma NAbs was used to assess the different antibody activities. We found that NAbs against the subtype A heterologous virus and/or the woman's autologous viruses were rare in IgG and IgA purified from breast milk supernatant (BMS) – only 4 of 19 women had any detectable NAb activity against either virus. Detected NAbs were of low potency (median IC50 value of 10 versus 647 for the corresponding plasma) and were not associated with infant infection (p = 0.58). The low NAb activity in BMS versus plasma was reflected in binding antibody levels: HIV-1 envelope specific IgG titers were 2.2 log10 lower (compared to 0.59 log10 lower for IgA) in BMS versus plasma. In contrast, antibodies capable of ADCC were common and could be detected in the BMS from all 19 women. BMS envelope-specific IgG titers were associated with both detection of IgG NAbs (p = 0.0001)and BMS ADCC activity (p = 0.014). Importantly, BMS ADCC capacity was inversely associated with infant infection risk (p = 0.039). Our findings indicate that BMS has low levels of envelope specific IgG and IgA with limited neutralizing activity. However, this small study of women with high plasma viral loads suggests that breastmilk ADCC activity is a correlate of transmission that may impact infant infection risk.

Abstract LB-317: Pediatric Preclinical Testing Program (PPTP) evaluation of volasertib (BI 6727), a Polo-like kinase (PLK) inhibitor

Abstract Background: Volasertib is a first in class, selective and potent cell cycle kinase inhibitor that induces mitotic arrest and apoptosis by targeting PLK. Genomic screens have identified PLK1 as a potential therapeutic target for several pediatric cancers, including rhabdomyosarcoma and neuroblastoma. Methods: Volasertib (provided by Boehringer Ingelheim) was tested in vitro at concentrations from 0.1 nM to 1.0 µM. Volasertib was tested against the PPTP solid tumor xenografts using a dose of 30 mg/kg administered intravenously weekly x 3. For the ALL panel (using NOD-SCID mice), the MTD was 15 mg/kg, and this dose was used for efficacy testing. The total planned treatment period was 3 weeks with an additional 3 weeks observation. Two measures of antitumor activity were primarily used: 1) an objective response measure modeled after the clinical setting; and 2) a time to event (4-fold increase in tumor volume) measure based on the median event-free survival (EFS) of treated (T) and control (C) animals for each xenograft. Intermediate activity requires EFS T/C &amp;gt; 2, with high activity additionally requiring regression at the end of the observation period. Results: The median relative IC50 (rIC50) value for the PPTP cell lines was 14.1 nM, with a range from 6.0 nM to 135 nM. The median rIC50 values were lowest for the ALL cell line panel compared to the remaining cell lines (11.9 versus 16.0 nM, respectively), but this difference was not significant, and overall there were no differences in rIC50 by histotype. Against the PPTP in vivo panels volasertib induced significant differences in EFS distribution compared to control in 19 of 32 (59%) evaluable solid tumor xenografts and in 2 of 4 (50%) evaluable ALL xenografts. Volasertib induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity in 11 of 30 (37%) evaluable solid tumor xenografts. Intermediate activity for the EFS T/C metric was most consistently observed in the neuroblastoma (4 of 6) and glioblastoma (2 of 3) panels. For the ALL panel, 2 of 4 (50%) xenografts met criteria for intermediate activity. Objective responses were observed for 4 of 32 solid tumor and 1 of 4 ALL xenografts. Two of 6 neuroblastoma xenografts demonstrated CRs, as did 1 of 3 glioblastoma and 1 of 5 rhabdomyosarcoma xenografts evaluable for this response measure. Conclusions: Volasertib showed low nanomolar in vitro potency against the PPTP cell lines with no histotype selectivity. Volasertib induced regressions in 5 of 36 evaluable PPTP xenografts with the neuroblastoma panel showing the most consistent pattern of responsiveness to volasertib. Given available pharmacokinetic data showing that mice tolerate higher systemic exposure to volasertib than humans, it is unlikely that the PPTP in vivo results are under-estimating the potential clinical activity of volasertib against the childhood cancer types evaluated here. (Supported by NCI NO1CM42216) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-317. doi:1538-7445.AM2012-LB-317

Evaluation of arsenic trioxide by the pediatric preclinical testing program with a focus on Ewing sarcoma

Arsenic trioxide was tested against the PPTP in vitro panel (1.0 nM to 10 µM) and against the PPTP Ewing sarcoma in vivo panel administered intraperitoneally at a dose of 2.5 mg/kg daily × 5 per week for a planned treatment duration of 3 weeks. Arsenic trioxide showed a median relative IC(50) value of 0.9 µM, with Ewing sarcoma cell lines having IC(50) values similar to those of the remaining PPTP cell lines. Arsenic trioxide did not induce significant differences in EFS distribution compared to control in any of the Ewing sarcoma xenografts studied, and no objective responses were observed.

Initial testing (stage 1) of the cyclin dependent kinase inhibitor SCH 727965 (dinaciclib) by the pediatric preclinical testing program

SCH 727965 is a novel drug in clinical development that potently and selectively inhibits CDK1, CDK2, CDK5, and CDK9. The activity of SCH 727965 was evaluated against the PPTP's in vitro and in vivo panels. SCH 727965 was tested against the PPTP in vitro panel using 96 hours exposure at concentrations ranging from 0.1 nM to 1.0 µM. It was tested against the PPTP in vivo panels at a dose of 40 mg/kg administered intraperitoneally twice weekly for 2 weeks and repeated at Day 21 with a total observation period of 6 weeks. The median IC(50) value for the cell lines was 7.5 nM, with less than fourfold range between the minimum (3.4 nM) and maximum (11.2 nM) IC(50) values. SCH 727965 demonstrated an activity pattern consistent with cytotoxicity for most of the cell lines. Forty-three xenograft models were studied and SCH 727965 induced significant delays in event free survival distribution compared to control in 23 of 36 (64%) evaluable solid tumor xenografts and in 3 of 7 ALL xenografts. SCH 727965 did not induce objective responses in the solid tumor panels and the best response observed was stable disease for one osteosarcoma xenograft. In the leukemia panel, there were two objective responses with a complete response observed in a single xenograft. SCH 727965 shows an interesting pattern of activity suggesting its potential applicability against selected childhood cancers, particularly leukemias.

Abstract C106: Pediatric Preclinical Testing Program (PPTP) Stage 1 evaluation of the PI3K inhibitor XL147 (SAR245408).

Abstract Introduction: XL147 is a potent, orally bioavailable inhibitor of the class I PI3K family of lipid kinases with IC50 values in the nanomolar range in biochemical assays. XL147 inhibits phosphorylation of downstream effectors of PI3K in preclinical models as well as in surrogate tissues and tumors in adults with cancer. XL147 is under clinical evaluation for adults with cancer in combination with certain cytotoxic agents as well as with other cell signaling pathway inhibitors (e.g., the MEK inhibitor MSC1936369B). The activity of XL147 was evaluated against the in vitro and in vivo panels of the Pediatric Preclinical testing Program (PPTP). Methods: XL147 was tested against the PPTP's in vitro cell line panel at concentrations ranging from 10.0 nM to 100 μM using the PPTP's standard 96 hour exposure period. For in vivo testing, an XL147 dose of 100 mg/kg administered orally daily for 14 days with a total planned observation period of 6 weeks. Results: XL147 demonstrated cytotoxic activity, with Ymin values approaching 0% for all of the cell lines at the highest concentration tested (100 μM). The median relative IC50 value for the PPTP cell lines was 10.9 μM, with a range from 2.7 μM (CHLA-10) to 24.5 μM (TC-71). There were no significant differences by histotype in median relative IC50 values, though there was a trend for lower values for the rhabdomyosarcoma panel (median IC50 5.6 μM) and higher values for the neuroblastoma panel (median IC50 19.5 μM). XL147 was tested against 31 solid tumor xenografts and 7 acute lymphoblastic leukemia (ALL) xenografts. A dose of 100 mg/kg administered orally daily for 14 days for a total planned observation period of 6 weeks was utilized. XL147 was generally well tolerated, with a &amp;lt;1% toxicity rate in the treated groups, similar to that observed for control animals. XL147 induced significant differences in EFS distribution compared to control in 26 of 30 (87%) of the evaluable solid tumor xenografts and in 2 of 7 (29%) of the evaluable ALL xenografts. XL147 induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C &amp;gt; 2) in 3 of 29 (10%) evaluable solid tumor xenografts (2 of 6 rhabdomyosarcoma and 1 of 5 neuroblastoma), and intermediate or high EFS T/C activity was observed for 2 of 7 (29%) evaluable ALL xenografts. Basal levels of activation of PI3K signaling, as assessed by phospho-AKT, levels did not correlate with in vivo response to XL147. Expression of PI3K isoforms at the RNA level was evaluated using Affymetrix U133 Plus 2.0 arrays, with the most striking observation being the pattern of expression of PIK3CD, which was virtually restricted to the ALL and lymphoma cell lines and xenografts, as would be expected from the hematopoietic cell specificity of this isoform in normal tissues. Conclusions: Under the conditions evaluated in this study, XL147 achieved modest single-agent activity against the PPTP preclinical models. Available data suggests that genomic activation of PI3K signaling (e.g., through PIK3CA mutation or PTEN deletion) is less common in the pediatric solid tumors setting compared to the adult cancer setting, which may explain the limited single agent activity. Preclinical evaluations of XL147 in combination with particular standard agents and with other pathway signaling inhibitors are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C106.

Molecular analysis of pfatp6 and pfmdr1 polymorphisms and their association with in vitro sensitivity in Plasmodium falciparum isolates from the Thai-Myanmar border

The association between pfatp6, pfmdr1 polymorphisms (gene mutation and amplification) and in vitro susceptibility to mefloquine (MQ), artesunate (AS), quinine (QN), and chloroquine (CQ) was investigated in a total of sixty-three Plasmodium falciparum isolates collected from the Thai-Myanmar border. The mutations of pfatp6 at codons R37K, G639D, S769N, and I898I and of pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Pfatp6 and pfmdr1 gene copy numbers were analyzed by quantitative real time-polymerase chain reaction (qRT-PCR). In vitro susceptibility test was successful in 58 culture-adapted isolates. Median (range) IC50 values for MQ, AS, QN, and CQ were 28.96 (3.4–100.5), 1.74 (0.8–5.57), 223.9 (14.99–845.47), and 69.93 (9.6–183.18) nM, respectively. There was a significant positive correlation (R2=0.58) of parasite susceptibility to MQ, AS, and QN. Twelve isolates showed marked decline in susceptibility to AS [median (range) IC50=3.78 (3.07–5.57) nM]. Almost all isolates carried wild-type pfatp6 and pfmdr1 alleles at the investigated codons, while only three isolates (5%) carried pfmdr1 mutation alleles at codon 86. Mutation at codon 86 was associated with a significant increase in the susceptibility of parasite isolates to MQ and QN. All of the sixty-three isolates carried only one pfatp6 copy number. Thirty-three out of the 58 isolates showed increase in pfmdr1 gene copies, which was associated with reduced in vitro susceptibility to MQ, AS, and QN. No association between mutation or amplification of pfatp6 gene and in vitro susceptibility of P. falciparum isolates was found.

Abstract 5356: Pediatric preclinical testing program (PPTP) evaluation of the DNA methylating agent temozolomide

Abstract Background: Temozolomide is DNA methylating agent that has been approved in the United States for treatment of astrocytoma. This drug in many ways resembles more established compounds, such as dacarbazine and procarbazine, in that it gives rise to a methyl diazonium ion that attacks nucleophilic sites including the O6-guanine position in DNA. Temozolomide, however, differs from these drugs, which have to be activated by enzymatic oxidation, in that it degrades spontaneously via base-catalyzed hydrolysis to the final active methylating species. Methods: The PPTP includes a molecularly characterized in vitro panel of cell lines (n=27) and in vivo panel of xenografts (n=61) representing most of the common types of childhood solid tumors and childhood acute lymphoblastic leukemia (ALL). Temozolomide (provided by the NCI Drug Repository) was tested in vitro at concentrations from 100 nM to 1 mM. Temozolomide was administered PO using a daily × 5 schedule repeated at 21 days at a dose of 100 mg/kg, or 66 mg/kg. Three measures of antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a treated to control (T/C) tumor volume measure; and 3) a time to event (4-fold increase in tumor volume) measure based on the median event-free survival (EFS) of treated and control animals for each xenograft. Biomarkers of temozolomide sensitivity, MGMT, MLH1, MSH2 and p53 genotype were determined. Results: The median temozolomide IC50 value for the PPTP cell lines was 380 μM (range 2 to &amp;gt; 1000 μM), with the neuroblastoma cell line NB-1643 having the lowest IC50 value. There were no significant differences in IC50 values between the rhabdomyosarcoma, neuroblastoma, Ewing sarcoma, and ALL cell lines. In vivo temozolomide induced significant toxicity at 100 mg/kg resulting in exclusion of 10/42 lines from analysis. For the 32 lines evaluable there were 13 maintained complete responses (MCR) and 2 CR in the solid tumor panels and 3 MCR and 2 CR in the ALL panel. Retesting the excluded lines at 66 mg/kg resulted in excessive toxicity leading to exclusion of 2/9 lines. At this dose there were 2 MCR and progressive disease in the remaining 5 lines. Overall 17/30 tumor models demonstrated objective responses (≥PR). Of these 7 are MGMT-negative, 3 are MLH1-negative, and 11 have wild type p53. All 7/7 MGMT-negative tumors responded, irrespective of p53 genotype (5/7 wild type). Conclusions: In vitro temozolomide induced cytotoxicity with no apparent cell type specificity. In vivo temozolomide demonstrated high activity, but with a steep dose response curve, consistent with other DNA damaging agents. Responses poorly correlated with MGMT, MLH1, MSH2 levels, or p53 genotype. However, all MGMT-negative tumors were responsive, irrespective of MLH1 or p53 status. The results suggest that temozolomide may have potential for treatment of a range of pediatric malignancies. (Supported by NO1-CM91003-03) 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 5356. doi:10.1158/1538-7445.AM2011-5356

Abstract 740: Pediatric Preclinical Testing Program (PPTP) evaluation of the NEDD8-activating enzyme (NAE) inhibitor MLN4924

Abstract Background: MLN4924 is a potent and selective small molecule NAE inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. The activity of MLN4924 was evaluated against the PPTP's in vitro and in vivo panels. Methods: The PPTP includes a molecularly characterized in vitro panel of cell lines (n=27) and in vivo panel of xenografts (n=61) representing most of the common types of childhood solid tumors and childhood acute lymphoblastic leukemia (ALL). MLN4294 (provided by Millennium Pharmaceuticals) was tested in vitro at concentrations from 1.0 nM to 10.0 µM and was tested against the PPTP in vivo panel using a 100 mg/kg dose (66mg/kg for ALL panel) BID administered SQ daily × 5 for 3 weeks, with observation for 6 weeks. Three measures of antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a treated to control (T/C) tumor volume measure; and 3) a time to event (4-fold increase in tumor volume) measure based on the median event-free survival (EFS) of treated and control animals for each xenograft. Results: The median relative IC50 for MLN4924 against the PPTP cell lines was 143 nM, with a range from 15 nM to 678 nM. The median relative IC50 value for the Ewing panel (31 nM) was significantly lower than that of the remaining PPTP cell lines. MLN4924 demonstrated an activity pattern consistent with cytotoxic activity. MLN4924 was well tolerated, with a toxicity rate of 4.6%. 42 of 45 xenograft models were considered evaluable for efficacy. MLN4924 induced significant differences in EFS distribution compared to control in 20 of 34 (59%) solid tumor xenografts. MLN4924 induced intermediate activity (EFS T/C values &amp;gt; 2) in 9 of the 33 evaluable xenografts (27%), including 4 of 4 glioblastoma xenografts, 2 of 3 Wilms tumor xenografts, 2 of 5 rhabdomyosarcoma xenografts, and 1 of 4 neuroblastoma xenografts. For the ALL panel, 5 of 8 evaluable xenografts showed intermediate activity for the EFS T/C measure. The maximum shift in time to event for treated animals was 3.8-fold (ALL-3), and two other xenografts had shifts in time to event of 3.0-fold (ALL-2 and ALL-17). MLN4924 did not induce objective responses in the PPTP solid tumor or ALL panels, with the best response being stable disease for 1 ALL xenograft. Conclusions: MLN4924 potently inhibits the cell lines of the PPTP in vitro panel and shows an interesting pattern of in vivo activity. Pharmacokinetic and pharmacodynamic studies are ongoing. (Supported by NCI NO1CM42216) 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 740. doi:10.1158/1538-7445.AM2011-740

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1×CD3 BiTE Antibody

AbstractAbstract 2890Response rates of ±80% in Acute Myeloid Leukemia (AML) are observed after conventional therapy but ±30% of patients experience a relapse. In the elderly the outcome is even worse. A small population of therapy resistant leukemia cells, minimal residual disease (MRD), are thought to be responsible for relapse of AML. The leukemic stem cells (LSC) herein have self renewal potential and reside in the CD34+CD38- stem cell compartment and side population (SP) compartment and can be identified via aberrant marker expression and scatter properties. Several markers are identified that show differential expression on AML (stem) cells versus normal hematopoietic stem cells (HSC). Previously we showed that CLEC12A (CLL-1, MICL, KLRL1, DCAL-2) is expressed on blasts of 90% of AML patients with varying expression. Importantly, CLEC12A is expressed on LSC but not on normal HSC (van Rhenen, Blood 110(7), 2007). This unique expression pattern paves the way to develop therapies that potentially eliminate CLEC12A-positive LSC and preserves CLEC12A-negative HSC. Drug-conjugated antibodies (ADCs) targeting CLEC12A and Bispecific T cell Engager (BiTE) scFv-antibodies targeting T-cells to CLEC12A positive cells could be instrumental to achieve this goal.We evaluated the response of AML cells to ADCs conjugated via cleavable and non-cleavable linkers to the maytansine derivates DM1 and DM4 and to the BiTE antibody CLL-1×CD3. ADC activity was assessed by colony formation capacity after 24 hours exposure to 0.1–100 nM ADC in 29 freshly obtained AML samples. The response to the BiTE antibody was tested by flow cytometry in 9 AML samples via induction of apoptosis (Annexin V/7AAD) after 24 hours exposure. To determine the effect of ADC on self-renewal in normal bone marrow (NBM), colony formation capacity was asseses during long term liquid culture after 24 hours exposure to 1–100 nM ADC. Furthermore internalisation of CLEC12A in AML progenitor and stem cells was tested. Several splice variants of CLEC12A are reported (CLL-1, MICLα, MICLβ, MICLγ) that have different intra-cellular signalling motifs or lack the transmembrane motif or the extra-cellular c-type lectin-like domain. Since these variants could not all be distinguished or detected by extra-cellular antibody binding, we evaluated these splice variants by Q-RT-PCR.After 24 hours exposure, a median IC50 value of >100 nM was observed for the unconjugated antibody CR2357. The median IC50 values for ADCs with non-cleavable linkers were 10 nM for CR2357-SMCC-DM1 (4,3 DM1/mAb), 2 nM for CR2357-PEG4-MAL-DM1 (5.9 DM1/mAb) and 0.8 nM for CR2357-PEG4-MAL-DM1 (10 DM1/mAb). For CR2357-SPDB-DM4 (4 DM4/mAb) which has a cleavable linker the median IC50 was 4 nM. The median IC50 of ADCs with non-cleavable linkers were significantly correlated to each other (r=0.730-0.784, p<0.01). CR2357-PEG4-MAL-DM1 (10 DM1/mAb) was significantly correlated to CLEC12A membrane expression (r=0.649, p<0.05). Prelimanary data of colony formation capacity during long term liquid culture of NBM showed that at >5 weeks after exposure, this was reduced to 15–50% for CR2357 and CR2357-PEG4-MAL-DM1 (10 DM1/mAb) relative to the untreated control. Exposure of AML cells to the CLL-1×CD3 BiTE antibody with donor T-cells (E:T=10:1 and 1:1) showed a dose dependent activation of T-cells as measured by increased CD25 and CD69 expression on CD4+ and CD8+ T-cells. Importantly, besides T-cell activation, Annexin V/7AAD staining of AML cells showed a specific decrease of CLEC12A-positive viable cells while in CLEC12A-negative cells viability remained constant. Internalisation of CR2357 antibody in CD34+/CD38+ progenitor cells and in CD34+/CD38- LSC was clearly demonstrated. Q-RT-PCR of CLEC12A splice variant expression showed highest expression for MICLα > MICLβ ∼F MICLγ > CLL-1 indicating that MICLα is the main variant expressed on the cellular membrane. Downstream signalling will therefore mainly be mediated by SHP-1/2 phosphatases. Although expression levels in AML, NBM, and sorted sub-populations varied, the ratio between the splice variants remained almost similar suggesting that the individual splice variants play a similar role in the different cell populations.In conclusion: these result show that targeting of CLEC12A-positive AML cells by ADCs and BiTE antibodies results in specific cell kill and might be a promising approach for the eradication of LSC that survive conventional therapy. Disclosures:No relevant conflicts of interest to declare.

Initial testing (stage 1) of the multi-targeted kinase inhibitor sorafenib by the pediatric preclinical testing program

Sorafenib is an inhibitor of multiple kinases (e.g., VEGF receptors, PDGFR, FLT3, RET, BRAF, KIT) and is approved by FDA for treatment of two adult cancers. The activity of sorafenib was evaluated against the PPTP's in vitro and in vivo panels. Sorafenib was evaluated against the PPTP in vitro panel using 96-hr exposure at concentrations ranging from 1.0 nM to 10.0 µM. It was tested against the PPTP in vivo panels at a dose of 60 mg/kg administered by oral gavage daily for 5 days per week, repeated for 6 weeks. In vitro sorafenib demonstrated cytotoxic activity, with a median IC(50) value of 4.3 µM. Twenty of 23 cell lines had IC(50) values between 1.0 and 10.0 µM. A single cell line (Kasumi-1) with an activating KIT mutation had an IC(50) value < 1.0 µM (IC(50) = 0.02 µM). In vivo sorafenib induced significant differences in event-free survival (EFS) distribution compared to control in 27 of 36 (75%) of the evaluable solid tumor xenografts and in 1 of 8 (12.5%) of the evaluable ALL xenografts. Sorafenib induced tumor growth inhibition meeting criteria for intermediate activity (EFS T/C) in 15 of 34 (44%) evaluable solid tumor xenografts. No xenografts achieved an objective response. The primary in vitro activity of sorafenib was noted at concentrations above 1 µM, with the exception of a more sensitive cell line with an activating KIT mutation. The primary in vivo effect for sorafenib was tumor growth inhibition, which was observed across multiple histotypes.

Abstract 5266: Pediatric Preclinical Testing Program (PPTP) evaluation of the CDK inhibitor SCH 727965

Abstract Background: SCH 727965 is a potent inhibitor of CDK1, CDK2 and CDK9 activities in vitro and has been shown to be effective as a single agent against a wide-spectrum of adult tumor xenograft models including lung, prostate, breast, ovarian and multiple myeloma, with tumor growth inhibition from 12% to 150% (regression) noted. The activity of SCH 727965 was evaluated against the PPTP's in vitro and in vivo panels. Methods: The PPTP includes a molecularly characterized in vitro panel of cell lines (n=27) and in vivo panel of xenografts (n=61) representing most of the common types of childhood solid tumors and childhood acute lymphoblastic leukemia (ALL). SCH 727965 (provided by Schering-Plough) was tested in vitro at concentrations from 0.1 nM to 1.0 μM and was tested against the PPTP in vivo panel using a 40 mg/kg dose administered IP twice-weekly for 2 weeks, repeated at Day 21 with a total planned treatment and observation period of 6 weeks. Three measures of antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a treated to control (T/C) tumor volume measure; and 3) a time to event (4-fold increase in tumor volume) measure based on the median event-free survival (EFS) of treated and control animals for each xenograft. Results: The median IC50 value for the PPTP cell lines was 7.5 nM, with a less than 4-fold range between the minimum (3.4 nM) and maximum (11.2 nM) IC50 values. SCH 727965 demonstrated an activity pattern consistent with cytotoxic activity for most of the PPTP cell lines with minimum (Ymin) T/C% values approaching 0% for most cell lines. SCH 727965 was well tolerated in vivo, with a toxicity rate &amp;lt; 5%. All 43 (36 solid tumor and 7 ALL) xenograft models studied were evaluable for efficacy. SCH 727965 induced significant differences in EFS distribution compared to control in 23 of 36 (64%) evaluable solid tumor xenografts. Seven of 33 solid tumor xenografts evaluable for the EFS T/C activity measure showed a 2-fold or greater increase in time to event for the SCH 727965 treated group. SCH 727965 did not induce objective responses in the PPTP solid tumor panels. The best response in these panels was stable disease (SD) which was observed for the osteosarcoma xenograft OS-1. PD2 (progressive disease with growth delay) was observed in 15 of 36 xenografts, being most commonly observed in the rhabdomyosarcoma (4 of 6), rhabdoid tumor (2 of 2) and the Ewing sarcoma (3 of 5) tumor panels. In the ALL panel, the best response was a complete response (CR), which was observed in a single xenograft (ALL-17). A T-cell ALL xenograft (ALL-16) attained a partial response (PR). The remaining 5 ALL xenografts showed progressive disease. Conclusions: SCH 727965 potently inhibits the cell lines of the PPTP in vitro panel and shows an interesting pattern of in vivo activity suggesting its potential applicability against selected childhood cancer subtypes. (Supported by NCI NO1CM42216) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5266.