Abstract
Gastric cancer (GC) is histologically divided into intestinal (INT) and diffuse (DIF) clinical subtypes. Even though these two GC groups are structurally and biologically different, this ...classification is not used to inform choice of treatment.
The taxanes (i.e., docetaxel and cabazitaxel) are widely used for the treatment of cancer and for GC specifically the TAX-325 study revealed a significantly improved survival when docetaxel (DTX) was added to cisplatin and fluorouracil. Despite this advancement, patients still exhibit intrinsic or acquired resistance to DTX. Yet, the molecular basis of drug resistance remains unknown.
We performed a post-hoc analysis of the TAX-325 study to examine the potential influence of GC subtypes in clinical response to DTX. We classified randomized patients as diffuse or non-diffuse histology and correlated histology with clinical outcomes using a Cox proportional hazards model. Non-diffuse GC showed a significant improvement in overall survival with the addition of DTX (12.1 v 8.8 mo, p=0.002), whereas diffuse histology was not associated with an improvement in survival (8.3 v 8.5 mo, p=0.66). To investigate the molecular mechanism of GC DIF subtype resistance to taxanes, we used a panel of 12 GC cell lines representative of both subtypes (4 intestinal subtype, 8 diffuse subtype). DTX cytotoxicity assays revealed that similarly to what we observed clinically, 5/8 (63%) DIF GC cell lines were resistant (IC50 > 600 nM) to DTX compared to 1/4 (25%) INT GC cell lines. Further functional studies revealed that there was minimal DTX drug-target engagement in the DIF GC cells, as, assessed by confocal microscopy of the microtubule network and tubulin polymerization assays. These results suggested that DTX interaction with its target, microtubule, was impaired in the DIF GC cell lines. To rule out multi-drug resistance (MDR) as potential cause of intrinsic DTX resistance in these cells we performed flow cytometric evaluation of P-glycoprotein and found that all of the DIF GC cell lines were negative. Additionally, drug accumulation studies with C-14 radiolabeled DTX revealed that the drug accumulated intracellularly in all of GC cell lines in our panel.
Next generation sequencing of our panel of untreated or DTX-treated GC cell lines revealed a number of genes/pathways that are differentially expressed in the drug-sensitive versus the resistant cell lines. Gene set enrichment analysis is currently ongoing in order to identify a subset of genes/pathways implicated in intrinsic DTX resistance in GC. These studies will provide novel insights into mechanism of intrinsic drug resistance and will ultimately allow us to design more effective targeted therapies to overcome chemo-resistance and eventually prolong patient survival.
Citation Format: Giuseppe Galletti, Kyle Cleveland, Chao Zhang, Ada Gjyrezi, Alexandre Matov, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Elucidating the molecular basis of intrinsic taxane resistance in gastric cancer. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2014-897
Abstract
The taxanes (i.e., paclitaxel, docetaxel (DTX) and cabazitaxel) are microtubule (MT)-stabilizing drugs widely used to treat solid tumor malignancies. Their success is limited by the presence ...of intrinsic or acquired drug resistance. Understanding the molecular mechanisms of taxane resistance is a key to significantly improve clinical outcomes of taxane-based chemotherapy. In gastric cancer (GC), retrospective analysis of the TAX-325 trial revealed that the addition of docetaxel (DTX) to standard cisplatin/fluorouracil increased progression-free and overall survival, primarily only in intestinal subtype (INT) GC, suggesting that diffuse (DIF) GC may be intrinsically taxane resistant. In fact, our previous data supported this hypothesis; DIF GC cell lines had higher incidence (63%) of DTX resistance than INT GC cell lines (25%). Flow cytometric analyses showed negligible P-glycoprotein expression on the cell surface of all cell types, and accumulation of C14-labeled DTX in cells was observed in both sensitive and resistant GC cell lines. These results ruled out drug efflux as a possible mechanism that confers taxane resistance in DIF GC cells.
Next, we used fluorescein-conjugated paclitaxel (Flutax) as a probe to investigate the affinity of taxanes to MTs. Live cell imaging showed strong binding of Flutax to MTs in a sensitive cell line at least for 60 minutes. In contrast, Flutax failed to stay bound to MTs in resistant GC cell lines,. In addition, discontinuous decoration of MTs with Flutax was seen in resistant but not in sensitive cell lines. Our observation suggests that MTs of resistant GC cell lines have lower affinity for taxanes, and there might be subtle structural changes of MTs. We are currently conducting the competition of Flutax/DTX to quantify the affinity of DTX in both types of GC cell lines.
Because tubulin mutations have been reported in many taxane-resistant cell lines, we sequenced the different tubulin isotypes in GC cell lines using both Sanger and next generation sequencing. No mutations were identified. In addition, we did not see any significant changes in the expression of βIII-tubulin or tubulin post-translational modifications between the sensitive and resistant cell lines. Pathway analyses on the RNA-Seq datasets derived from the panels of sensitive and resistant cell lines before and after taxane treatment is ongoing to identify the key molecular events underlying taxane resistance.Taken together, our data indicate that new molecular pathway(s) or change of overall MT structure and/or function may contribute to the impaired taxane binding to MTs in resistant GC cells. Our study will lead us to novel mechanistic insights in intrinsic drug resistance, and will ultimately allow us to target key molecule(s) to overcome chemo-resistance, the key determinant to improve overall survival of patients.
Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Prashant V. Thakkar, Ada Gjyrezi, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2015-3600
Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on ...tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on β-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the βI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a β-tubulin-binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the β-tubulin isotype composition of the cells was examined. Increased expression of βII- and βIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action.
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Background: A better understanding of taxane sensitivity/resistance in mCRPC is needed to optimize treatment. Preclinically, taxane efficacy has been linked to the ability of ...microtubules (MT) to inhibit AR nuclear trafficking. In this prospective biomarker study, we used circulating tumor cells (CTCs) from patients (pts) in TAXYNERGY to perform real-time analysis of AR nuclear localization (ARNL) and MT stabilization (bundling; MTB) in order to predict taxane sensitivity. Methods: TAXYNERGY (NCT01718353) is a phase 2 trial randomizing chemo-naïve mCRPC pts 2:1 to docetaxel or cabazitaxel, with a switch to the alternative taxane in the absence of a ≥ 30% PSA drop by C4. Here we present the co-primary biomarker endpoints. CTCs at baseline (C1D1) were compared to CTCs after 1 week of taxane treatment (C1D8), and were analyzed by multiplex confocal microscopy for %ARNL (integrated AR intensity in the cell and nuclear areas) and MTB (assessed for increase compared to C1D1 on a scale from 0–3 from no to most MTB increase). Associations between %ARNL and MTB with clinical outcomes were sought. Results: Of 63 randomized pts, 26 had evaluable CTCs both at C1D1 and C1D8. At C1D8, mean %ARNL was significantly lower in pts achieving a ≥ 50% PSA drop by C4 vs those without (44% vs 64%; p = 0.004). A taxane-induced decrease in mean %ARNL (C1D8 vs C1D1) was associated with a higher rate of ≥ 50% PSA response (73% vs 13%; p = 0.009); mean %ARNL decreased by 18% in responders and increased by 2% in non-responders (p = 0.02). Finally, a taxane-induced increase in mean MTB trended higher in pts achieving a ≥ 30% PSA drop by C4 vs those without (0.69 vs 0.09; p = 0.09); increase in mean MTB score was indicative of response and observed in pts who did not require a taxane switch after C4 (0.75 vs 0.09; p = 0.06). Conclusions: We provide the first prospective data suggesting that taxane-induced shifts in ARNL and MTB (measured in CTCs) may serve as an early biomarker of taxane sensitivity. Consistent with preclinical data, AR nuclear exclusion caused by microtubule bundling may be a clinically-actionable marker of taxane efficacy. Funding: Sanofi Genzyme. Clinical trial information: NCT01718353.
Abstract
Androgen receptor signaling inhibitors (ARSi) and taxanes are mainstays for patients with metastatic castration-resistant prostate cancer (mCRPC). However, patient response is heterogeneous, ...and the molecular underpinnings of treatment resistance are not well elucidated.
To identify clinically meaningful mechanisms of treatment resistance, we performed transcriptome analysis of circulating tumor cells (CTCs) isolated from mCRPC patients enrolled in two independent prospective clinical trials: PROPHECY, a clinical study of patients (n=118) treated with abiraterone or enzalutamide followed by docetaxel; and TAXYNERGY where patients were randomized to docetaxel or cabazitaxel treatment. CTCs were obtained at baseline (before treatment), on treatment and at progression and their comprehensive transcriptomic analysis was correlated with clinical outcomes. To uncover potential involvement of the circulating immune macroenvironment (CIME) in treatment resistance, we performed transcriptomic analysis of matching peripheral blood mononuclear cells (PBMCs) using an established, rigorous, blood-derived transcriptional modular framework.
In PROPHECY, CTC RNA-seq identified that RB loss concurrently with enhanced E2F signaling networks were associated with intrinsic ARSi resistance. Using single sample GSEA (ssGSEA) score, we identified that the RB/E2F common signature at baseline was associated with short PFS (median PFS=6.5 months) and OS (median OS=24.5 months) (hazard ratio (HR) = 3.5; 95% CI 1.5-8.2) in men with mCRPC. We further developed a BRCA-loss transcriptional signature, and validated it in the SU2C mCRPC patient cohort, expanding the identification of patients with BRCA-loss phenotypes beyond genomic loss. Applying this signature to PROPHECY baseline samples, we showed that men with high BRCA-loss scores experienced shorter OS (HR=2.42; 95% CI=1-5.9). Through the comparison of CTC transcriptomic profiles at progression with baseline, we identified an inflammatory response signature in CTCs which was significantly associated with acquired ARSi resistance. Transcriptomic PBMC analysis further identified enrichment of inflammasome gene signatures at progression, with concurrent downregulation of CD8+ T and NK cells.
Furthermore, preliminary data from both clinical trials, showed a significant upregulation of TGF-β1 and corresponding TGFβ-Receptor signaling pathway in CTCs from patients at progression following taxane treatment, suggesting a role for TGFβ pathway in clinical response to taxane chemotherapy.
Taken together, these data demonstrate that liquid biopsy transcriptomics of both tumor cells and immune cells can identify molecular pathways associated with treatment resistance paving the way for treatment optimization and the development of novel precision therapies in patients with mCRPC.
Citation Format: Jiaren Zhang, Bob Zimmermann, Giuseppe Galletti, Susan Halabi, Ada Gjyrezi, Qian Yang, Santosh Gupta, Akanksha Verma, Andrea Sboner, Monika Anand, Daniel J. George, Simon G. Gregory, Prerna Mahtani, Seunghee Hong, Virginia Pascual, Clio P. Mavragani, Emmanuel S. Antonarakis, David M. Nanus, Scott T. Tagawa, Olivier Elemento, Andrew J. Armstrong, Paraskevi Giannakakou. Liquid biopsy transcriptomics identify pathways associated with poor outcomes and immune phenotypes in men with mCRPC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 646.
Abstract
Farnesyltransferase inhibitors (FTIs) are a class of anticancer drugs that have exhibited modest activity in the clinic as single agents, but were found to have promising activity in ...combination with the microtubule-stabilizing drug Taxol (TX), both in vitro and in vivo. Clinical trials with an FTI/TX combination have demonstrated a clinical benefit for a subset of patients refractory to previous taxane-treatment. However, the precise cellular machinery underlying the FTI/TX synergistic drug combination still needs to be elucidated. To this end, we undertook an unbiased experimental/computational approach designed to identify the key cellular components that either contribute to or drive the therapeutic synergy of this drug combination. The approach combined (i) measurements of drug-mediated changes on tubulin biomarkers and overall survival; (ii) whole-genome transcriptome analysis using the combination index drug synergy algorithm; (iii) utilization of interaction and functional databases to identify pathway connections and (iv) a reverse-engineering forward simulation (REFS) approach to identify genes that drive synergy.
Drug-induced transcriptome changes were examined using whole-genome Affymetrix HGU133 Plus 2 microarrays. Gene changes deemed significant using a fold change criterion, identified as synergistic with the combination index analysis, and their biological relevance was assessed by experimental validation by qPCR and siRNA studies. Also, the transcriptome changes unique to the LNF/TX combination were evaluated in the context of detailed molecular interaction maps and groups of biological function. Our analyses identified several major network hubs that include: (i) overexpression of the class II β-tubulin isotype TUBB2A and of several heat shock protein such as HSP40 and HSP70 and (ii) synergistic overexpression of cell cycle genes CDKN1A and MDM2 (iii) through REFS modeling, prosaposin and granulin were identified as drivers of drug synergy (iv) induction of the focal adhesion molecule paxillin (PXN). Interestingly, stable knockdown of protein farnesyltransferase mimicked the effects of FTI treatment and resulted in strong substratum attachment and migration defects suggesting that PXN could be regulated by farnesyltransferase and its pharmacologic inhibitors. Ultimately, we plan to check the expression levels of the key genes in tumor biopsies from patients enrolled in the Phase 1 FTI/Taxotere clinical trial and correlate their expression in pre- and post-treatment biopsies with clinical response to the combination chemotherapy.
Herein, we demonstrate a combined experimental and computational approach that includes functional analysis, data-driven simulation and experimental validation, and its power to identify key cellular components, both known and novel, in the anticancer activity of drug combinations exhibiting therapeutic synergy.
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 4911. doi:10.1158/1538-7445.AM2011-4911
Abstract
Androgen receptor (AR) signaling drives castration resistant prostate cancer (CRPC) disease progression. The taxanes (TXN) represent the only class of chemotherapy agents that improve ...survival in CRPC. Our recent work revealed that TXNs work by blocking AR nuclear accumulation and signaling downstream of microtubule (MT) stabilization. Moreover, we showed that AR cytoplasmic sequestration in CRPC patients’ circulating tumor cells (CTCs) significantly correlated with clinical response to TXN therapy. Our data, suggest that TXN-induced blockade of AR nuclear accumulation partly underlies the clinical activity of TXNs in CRPC. Recently several AR splice variants, lacking the ligand binding domain, have been identified in CRPC. These variants are constitutively active in the nucleus and likely to be insensitive to current therapies targeting either ligand biosynthesis or ligand-receptor interactions. Currently, the role of AR splice variants on TXN chemotherapy response in CRPC is unknown. Our preliminary data in preclinical PC models suggest that certain AR variants depend on MTs for nuclear accumulation and are TXN sensitive while others are MT-independent and TXN-insensitive. In this study we are prospectively evaluating whether the presence of AR splice variants and the integrity of the MT-AR axis in CTCs can predict clinical response to TXN chemotherapy in CRPC. CTCs are captured by the geometrically enhanced differential immunocapture (GEDI) microfluidic device that uses PSMA-based immunocapture and size-selective cell transport for maximum specificity of CRPC-derived CTCs. We captured viable CTCs from CRPC patients (median 54 CTC/ml) and performed molecular and functional analyses. Comparison of CTCs captured by CellSearch or GEDI, using same day blood draw from 30 CRPC patients, revealed a 2-400 fold increase in CTC numbers with the GEDI. GEDI-capture of live CTCs allowed us to evaluate ex-vivo TXN sensitivity and determine drug-target-engagement (DTE) by MT bundling. CTCs isolated from docetaxel (DTX)-resistant CRPC patients did not show any evidence of DTX activity in this assay suggesting that it can be used to monitor patient's response in real time. In addition, we have miniaturized RNA sequencing (RNA-Seq) to enable molecular analyses of GEDI-captured CTCs. RNA-Seq of 50 GEDI-captured LNCap 86.2 cells spiked into 1 ml healthy donor blood, detected both the T868A AR point mutation and ARv567 splice variant. In an ongoing prospective clinical study, we are capturing CTCs from CRPC patients treated with TXNs and are evaluating the integrity of the MT-AR signaling axis, in relation to clinical response to therapy. Our CTC analyses include RNA-Seq for the detection of AR splice variants, alterations in tubulin isotypes and MT related proteins, and multiplex confocal microscopy of DTE and AR localization. These analyses are performed longitudinally and may identify biomarkers predictive of clinical TXN efficacy.
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 3634. doi:1538-7445.AM2012-3634
Abstract
Prostate cancer progression into castration-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR) signaling despite surgical and chemical androgen ablation. The ...taxanes represent the only class of chemotherapy that improves overall survival in CRPC patients. Despite their success, CRPC patients do progress on taxane treatment rendering taxane-resistant tumors. The molecular mechanisms underlying clinical taxane resistance in CRPC have not been well elucidated due to the lack of available tumor tissue to study. Circulating tumor cells (CTCs) represent a liquid biopsy of the original tumor and isolation of them can lead to their molecular characterization potentially revealing predictive biomarkers for taxane sensitivity or resistance. Here, we use a geometrically enhanced differential immunocapture (GEDI) microfluidic device that couples an anti-prostate specific membrane antigen (PSMA) antibody with optimized 3D geometry to capture and isolate live CTCs from whole blood of CRPC patients. The GEDI-microfluidic device was shown to have a 2-400 fold higher sensitivity for CTC capture than the FDA-approved CellSearch® system. We have previously shown that CRPC patient CTCs can be used to derive functional information that correlates to clinical response to taxane chemotherapy, namely AR subcellular localization status. We have developed a suite of other functional assays that can be performed on live GEDI-captured CTCs that enable their molecular characterization and allow us to test specific mechanistic hypotheses based on our extensive preclinical data. Included, and herein described, are the determination of AR subcellular localization, extent of effective drug-target engagement assessed by microtubule bundling, identification of RNA species relevant to the mechanism of taxane resistance and computer vision algorithms that will allow for enriched and automated analysis of high-volume image sets of GEDI-captured CTCs. In addition, we will be testing the hypothesis that distinct AR splice variants may affect patient sensitivity to taxane-based chemotherapy. This suite of assays are being rigorously applied in a phase II clinical trial in which chemotherapy-naïve CRPC patients will be initially treated with either docetaxel or cabazitaxel and clinically evaluated for an early switch to the other taxane following disease progression. This prospective, randomized, multi-site clinical trial will enroll 100 CRPC patients within one year. Patients will be followed until relapse and each patient will have 15 independent GEDI assays performed across five time points from baseline to chemotherapy crossover to relapse. The depth of coverage this suite of assays provides will offer unique insights for potential mechanisms of clinical taxane resistance and predictive biomarkers for taxane sensitivity in CRPC patient CTCs.
Citation Format: Matthew S. Sung, Ada Gjyrezi, Guang Yu Lee, Alexandre Matov, Giuseppe Galletti, Matthew Loftus, Yusef Syed, Timothy Lannin, Atanas Hristov, Christopher Mason, Scott Tagawa, Brian Kirby, David Nanus, Paraskevi Giannakakou. Using CTCs to interrogate mechanisms of taxane resistance in the prospective TAXYNERGY clinical trial in prostate cancer. 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 3492. doi:10.1158/1538-7445.AM2013-3492
Abstract
Resistance to chemotherapy is a major obstacle in cancer therapy. Intrinsic or acquired drug resistance occurs frequently in most cancers. In this report, we developed a novel nanoparticle ...formulation with the intent of significantly increasing the bioavailability and enhancing the efficacy of chemotherapeutic agent, paclitaxel in the experimental model of resistant human squamous cell carcinama. The targeted nanoparticle (HFT-T) was loaded with paclitaxel in its hydrophobic core, which is composed of Heparin-Folate-Paclitaxel (HFT) conjugates (Wang X., et al. ACS Nano 2009, 3, 3165–3174). In vitro cytotoxicity of nanoparticle HFT-T was assessed on a resistant human squamous cancer cell line KB-8–5 (ten time resistant to paclitaxel compare to the sensitive cell line KB-3–1). The targeted nanoparticle HFT-T was more effective in inhibiting cell growth, when compared with non-targeted nanoparticle (HT-T) or free paclitaxel in multidrug resistance cells. In vitro cellular uptake, retention, and efflux of HFT-T nanoparticles studies suggested that the HFT-T formulation may counteract the transporter-mediated drug resistance in KB-8–5 cells, thereby increasing intracellular concentrations of paclitaxel and enhancing its cytotoxicity. Compared to free paclitaxel, HFT-T was found to be capable of effectively delivering and maintaining markedly higher concentrations (by 2∼3-fold) of paclitaxel in KB-8–5 cells when added to the cultures at various times. To test the in vivo efficacy of HFT-T particles, nude mice bearing subcutaneous human KB-8–5 tumors were randomly divided into four groups (n=8 per group), and treated with HFT-T, free paclitaxel, HT-T or PBS (control), respectively. The drugs (at a dose of 40 mg/kg paclitaxel or equivalent) were administrated intravenously via tail vein once a week for 3 weeks (3 injections in total). At the end of the experiment, the tumor volume in the HFT-T-treated group were significantly reduced compared with all other treatment groups including the control (p=0.0075), free paclitaxel (p=0.0155), and HT-T (p=0.0381) treated groups. In contrast, tumor volumes in paclitaxel (p=0.2287) or HT-T-treated (P=0.1423) groups were not significantly different from the control group. The results indicated that the therapeutic efficacy of targeting HFT-T nanoparticle is significantly superior to free paclitaxel and non-targeted nanoparticle in drug-resistant xenograft models. In conclusion, this study provides a novel strategy using targeted nanoparticle as drug deliver system to minimize the drug resistance in human cancers. (Supported by NCI P50 CA128613 and U54CA119338–04)
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 LB-196. doi:10.1158/1538-7445.AM2011-LB-196
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