Levels Of Cytidine Deaminase Research Articles

Cytidine deaminase-dependent mitochondrial biogenesis as a potential vulnerability in pancreatic cancer cells

Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine as part of the pyrimidine salvage pathway. Elevated levels of CDA are found in pancreatic tumors and associated with chemoresistance. Recent evidence suggests that CDA has additional functions in cancer cell biology. In this work, we uncover a novel role of CDA in pancreatic cancer cell metabolism. CDA silencing impairs mitochondrial metabolite production, respiration, and ATP production in pancreatic cancer cells, leading to a so-called Pasteur effect metabolic shift towards glycolysis. Conversely, we find that CDA expression promotes mitochondrial biogenesis and oxidative phosphorylation, independently of CDA deaminase activity. Furthermore, we observe that patient primary cells overexpressing CDA are more sensitive to mitochondria-targeting drugs. Collectively, this work shows that CDA plays a non-canonical role in pancreatic cancer biology by promoting mitochondrial function, which could be translated into novel therapeutic vulnerabilities.

Nucleotide metabolism in cancer cells fuels a UDP-driven macrophage cross-talk, promoting immunosuppression and immunotherapy resistance

Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y6. Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDAhigh cancer cells match with T cell cytotoxicity dysfunction and P2RY6high TAMs. Overall, we suggest CDA and P2Y6 as potential targets for cancer immunotherapy.

The MitoNEET Ligand NL-1 Mediates Antileukemic Activity in Drug-Resistant B-Cell Acute Lymphoblastic Leukemia.

Disease relapse in B-cell acute lymphoblastic leukemia (ALL), either due to development of acquired resistance after therapy or because of de novo resistance, remains a therapeutic challenge. In the present study, we have developed a cytarabine (Ara-C)-resistant REH cell line (REH/Ara-C) as a chemoresistance model. REH/Ara-C 1) was not crossresistant to vincristine or methotrexate; 2) showed a similar proliferation rate and cell surface marker expression as parental REH; 3) demonstrated decreased chemotaxis toward bone marrow stromal cells; and 4) expressed higher transcript levels of cytidine deaminase (CDA) and mitoNEET (CISD1) than the parental REH cell line. Based on these findings, we tested NL-1, a mitoNEET inhibitor, which induced a concentration-dependent decrease in cell viability with a comparable IC50 value in REH and REH/Ara-C. Furthermore, NL-1 decreased cell viability in six different ALL cell lines and showed inhibitory activity in a hemosphere assay. NL-1 also impaired the migratory ability of leukemic cells, irrespective of the chemoattractant used, in a chemotaxis assay. More importantly, NL-1 showed specific activity in inducing death in a drug-resistant population of leukemic cells within a coculture model that mimicked the acquired resistance and de novo resistance observed in the bone marrow of relapsed patients. Subsequent studies indicated that NL-1 mediates autophagy, and inhibition of autophagy partially decreased NL-1-induced tumor cell death. Finally, NL-1 showed antileukemic activity in an in vivo mouse ALL model. Taken together, our study demonstrates that mitoNEET has potential as a novel antileukemic drug target in treatment refractory or relapsed ALL.

The augmented expression of the cytidine deaminase gene by 5-azacytidine predicts therapeutic efficacy in myelodysplastic syndromes.

5-Azacytidine (5AC), a hypomethylating agent, is clinically used for the treatment of patients with myelodysplastic syndromes (MDS). Cytidine deaminase (CDA) is a key enzyme in the detoxification of 5AC. We investigated whether the CDA expression could predict response to 5AC in MDS. Among leukemia-derived cell lines, MDS-L, an MDS-derived cell line with a relatively low CDA expression level, was found to be the most sensitive to 5AC. Combination with tetrahydrouridine, an inhibitor of CDA, synergistically potentiated the cytotoxic effect of 5AC. Treatment with 5AC markedly enhanced the expression level of CDA mRNA and showed demethylation at CpG sites in the 5′-flanking region of the CDA gene. We further compared the protein expression levels of CDA in matched clinical samples before and after treatment with 5AC in bone marrow cells from 8 MDS patients by an immunohistochemical analysis. The CDA expression level showed an approximately 2- to 3-fold increase after 5AC treatment in 3 of these cases, and these three patients with relatively higher CDA expression levels after 5AC treatment all showed better clinical responses to 5AC. In contrast, the 5 remaining patients, whose CDA expression showed no augmentation, observed no clinical benefit. Taken together, the optimized determination of the CDA expression levels before and after 5AC treatment, and the methylation status at CpG sites of 5′-flanking region of the CDA gene, may contribute to the development of precise 5AC therapy for MDS.

The Related Roles of a Ubiquitin Processing Protease, Nutrient Sensor, and Cytidine Deaminase in the Growth‐to‐Development Transition of Dictyostelium Development

The ubiquitin processing protease, UbpA (a homolog of yeast Ubp14 and human IsoT/USP5), is required for the growth‐to‐development transition of Dictyostelium. To understand the role of UbpA in regulating this transition, ubpA− cells were used to elucidate UbpA‐dependent pathways. ubpA− cells were hypersensitive to oxidative and nitrosative stresses, and miss‐regulated genes at the growth‐to‐development transition, including lmcB. ubpA is required for growth‐stage expression of lmcB which is involved in sensing nutrient levels and signaling cells to develop upon starvation. Exogenous expression of ubpA in ubpA− cells restored lmcB transcript levels, while exogenous expression of lmcB partially restored the developmental defect in ubpA− cells. Interestingly, lmcB is downregulated in wild‐type cells once the growth‐to‐development transition has occurred. Pull down experiments utilizing His‐tagged LmcB and TAP‐tagged LmcB expressed in Dictyostelium identified putative interacting proteins that included LmcA, PsmC5, CdaA, and a cAMP‐inducible protein. Direct binding experiments confirmed the interaction between LmcB and LmcA, and between LmcB and CdaA, a cytidine deaminase. To investigate cytidine deaminase (CDA) activity levels at the growth‐to‐development transition, a CDA assay was adapted for Dictyostelium. Wild‐type cells showed relatively low levels of CDA activity when growing and substantially increased levels upon starvation. Both growing and starving ubpA− cells have high CDA levels similar to the starving wild type cells. It is possible that LmcB binds CdaA to repress CDA activity during growth; then, upon starvation, LmcB is downregulated releasing CdaA. These results will lead to a better understanding of the mechanisms cells use to sense starvation stress and respond by making the transition from growth to development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A randomized phase II trial comparing different schedules of nab-paclitaxel (nabP) combined with gemcitabine (GEM) as first line treatment for metastatic pancreatic adenocarcinoma (mPDAC).

4100 Background: NabP+GEM chemotherapy improves survival compared with GEM monotherapy as treatment for mPDAC. A PDAC mouse model suggested that nabP potentiates GEM activity by reducing cytidine deaminase levels and scheduling may be critical to optimise clinical benefit. Methods: Patients (pts) were randomised to receive standard concomitant (CON) nabP+GEM or sequential (SEQ) administration, with nabP given 24 hours before GEM. After 6 cycles, pts benefiting from treatment could continue the same regimen until disease progression. The primary endpoint was progression-free survival (PFS) by RECIST v1.1; secondary endpoints included safety, objective response rate (ORR), overall survival (OS) and quality of life (QoL). Serial blood and baseline tumour samples were collected for exploratory biomarkers. Results: Between March 2014 and 2016, 146 pts (71 SEQ, 75 CON) were recruited. Median age (range) was 66 (45-82) years; Karnofsky performance status was 70 (in 12% pts), 80 (27%), 90 (38%) or 100 (24%); 47% had pancreatic head primaries; 84% had liver metastases. Median no. cycles received was 4 SEQ, 3 CON; 51 pts (35%) received ≥6 cycles of treatment (42% SEQ, 28% CON). A 24+2hr interval was achieved in > 90% SEQ admin. Grade ≥3 adverse events experienced by ≥10% pts (SEQ, CON) were neutropaenia (54%, 30%; p = 0.003), febrile neutropaenia (12%, 12%), fatigue (22%, 15%), vomiting (7%, 11%) and anaemia (10%, 5%). G-CSF was administered at local investigator's discretion to 35 pts (23 SEQ, 12 CON; p = 0.015). To date, 112 pts have died. 6 month (m) PFS by SEQ and CON arms were 47% and 33%; median PFS were 5.8 and 4.0m; hazard ratio (HR) = 0.66, 95% CI = 0.46-0.95; 12m OS by SEQ and CON arms were 29% and 26%; median OS were 10.1 and 7.9m; HR = 0.88, 95% CI = 0.61-1.29. ORR was 50% SEQ and 33% CON (p = 0.065). Mean baseline QoL Global health status score was 60.6 SEQ and 63.4 CON. The mean change in QoL score from baseline at 24 weeks was -2.1 SEQ and -12.1 CON. Conclusions: Sequential delivery of nabP combined with GEM trended towards improving all clinically relevant efficacy end points: PFS, OS, and ORR. Translational correlates will be reported in due course. Clinical trial information: ISRCTN71070888.

Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma

ABSTRACTHepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.

Acquired resistance to decitabine and cross-resistance to gemcitabine during the long-term treatment of human HCT116 colorectal cancer cells with decitabine.

The aim of the present study was to determine the effects of long-term exposure of decitabine (DAC) to HCT116 colorectal cancer (CRC) cells on the acquisition of resistance to DAC as well as cross-resistance to anticancer drugs used for CRC or other epigenetic modifiers. In the present study, DAC-resistant HCT116 CRC cells were established through long-term treatment with increasing concentrations of DAC (10 to 540 nM); and the cross-resistance to other drugs was subsequently examined. DAC-resistant HCT116 cells were obtained following a 104-day treatment with DAC, including DAC-free intervals. The results demonstrated that the IC50 value of DAC was increased ~100-fold in DAC-resistant HCT116 cells. Messenger (m)RNA expression of secreted frizzed-related protein 1 (SFRP1), which is regulated by DNA methylation, was not detected in DAC-resistant cells; however, SFRP1 mRNA was present in HCT116 cells treated with DAC for 52 days. DNA methyltransferase 1 (DNMT1) protein levels were slightly decreased until day 81 and then returned to control levels in DAC-resistant cells. Further experiments using DAC-resistant HCT116 cells revealed that these cells exhibited cross-resistance to gemcitabine (Gem); however, cross-resistance was not observed for other DNMT inhibitors (azacitidine and zebularine), histone deacetylase inhibitors (trichostatin A, vorinostat and valproic acid) or anticancer drugs for CRC (5-fluorouracil, irinotecan and oxaliplatin). Furthermore, the protein expression levels of cytidine deaminase (CDA) were increased, while those of deoxycytidine kinase (dCK) were decreased in DAC-resistant HCT116 cells; by contrast, the mRNA expression levels for these proteins were not significantly altered. In conclusion, the results of the present study indicated that the long-term treatment of HCT116 cells with DAC led to the acquisition of resistance to both DAC and Gem. In addition, these results may be partly attributed to changes in CDA and/or dCK, which are involved in metabolic pathways common to these two drugs.

CP-029 Evaluation of nab-paclitaxel plus gemcitabine treatment in patients with metastatic pancreatic cancer

BackgroundPancreatic cancer remains a fatal and difficult to-treat disease. Gemcitabine (G) is a cytotoxic agent that is potent against pancreatic adenocarcinoma. Nab-paclitaxel (nab-P), an albumin-bound formulation of paclitaxel, appears to...

DI-017 Safety of nab-paclitaxel plus gemcitabine in patients with metastatic pancreatic cancer

BackgroundPancreatic cancer remains a highly fatal and difficult-to-treat disease. Nab-paclitaxel (nab-P), an albumin-bound formulation of paclitaxel, appears to decrease levels of cytidine deaminase, which is the primary gemcitabine catabolic enzyme;...

Cytidine Deaminase Status As a Predictive Marker Of Response and Toxicity In Patients With Haematological Malignancies Treated With Azacytidine Or Cytarabine

BackgroundCytarabine and azacytidine are mainstays for treating haematological malignancies. As most nucleosidic analogs, both azacytidine and cytarabine are metabolized in the liver by an exclusive enzymatic step driven by cytidine deaminase (CDA). CDA expression polymorphism has been associated with clinical outcome in patients treated with gemcitabine. MethodsWe determined CDA levels in 39 adult patients (16F, 23M, mean age 77 years), treated mainly for AML and myelodysplastic syndromes with either aza-cytidine or a cytarabine-containing regimen. Response and treatment-related toxicities were monitored following current standards. In addition, impact of CDA status on azacytidine tolerance was evaluated in mice with or without CDA deficiency, as a proof of concept for the actual implication of metabolic deregulations in the toxicities observed in patients. ResultsIn patients, mean CDA activity was 3.7 +/-2.8 U/mg (min: 1, max: 14.8 U/mg). Ten out of 39 patients (i.e., 25%) had low CDA activities and were considered as poor metabolizers (PM). Conversely, 8 patients (i.e., 20%) displayed high CDA activities (i.e., > 6U/mg) and were considered as high metabolizers (HM). PM showed severe toxicities, including two toxic-deaths. Conversely, HM showed little efficacy when treated with either azacytidine or cytarabine. In mice with CDA-deficiency, standard dose azacytidine led to profound and long-lasting neutropenia, as compared with normal mice. Drug monitoring confirmed that individuals with low CDA activity and toxicities showed higher concentrations of azacytidine as compared with normal individuals. Conclusionsthis study suggests that CDA status could be a relevant marker for predicting clinical outcome in patients treated with either azacytidine or cytarabine. CDA status could be further used as a covariate to tailor drug dosage so as to ensure an optimal efficacy/toxicity balance in patients with haematological malignancies. Disclosures:No relevant conflicts of interest to declare.

Gender, Cytidine Deaminase, and 5-Aza/Decitabine—Letter

We have read with interest the observational study by Mahfouz and colleagues ([1][1]), who examined the role of cytidine deaminase (CDA) on outcomes of patients treated with 5-azacytidine or decitabine. Surprisingly, gender emerged as the main determinant of CDA levels, and consequently, male

High cytidine deaminase expression in the liver provides sanctuary for cancer cells from decitabine treatment effects

We document for the first time that sanctuary in an organ which expresses high levels of the enzyme cytidine deaminase (CDA) is a mechanism of cancer cell resistance to cytidine analogues. This mechanism could explain why historically, cytidine analogues have not been successful chemotherapeutics against hepatotropic cancers, despite efficacy in vitro. Importantly, this mechanism of resistance can be readily reversed, without increasing toxicity to sensitive organs, by combining a cytidine analogue with an inhibitor of cytidine deaminase (tetrahydrouridine). Specifically, CDA rapidly metabolizes cytidine analogues into inactive uridine counterparts. Hence, to determine if sheltering/protection of cancer cells in organs which express high levels of CDA (e.g., liver) is a mechanism of resistance, we utilized a murine xenotransplant model of myeloid cancer that is sensitive to epigenetic therapeutic effects of the cytidine analogue decitabine in vitro and hepato-tropic in vivo. Treatment of tumor-bearing mice with decitabine (subcutaneous 0.2mg/kg 2X/week) doubled median survival and significantly decreased extra-hepatic tumor burden, but hepatic tumor burden remained substantial, to which the animals eventually succumbed. Combining a clinically-relevant inhibitor of CDA (tetrahydrouridine) with a lower dose of decitabine (subcutaneous 0.1mg/kg 2X/week) markedly decreased liver tumor burden without blood count or bone marrow evidence of myelotoxicity, and with further improvement in survival. In conclusion, sanctuary in a CDA-rich organ is a mechanism by which otherwise susceptible cancer cells can resist the effects of decitabine epigenetic therapy. This protection can be reversed without increasing myelotoxicity by combining tetrahydrouridine with a lower dose of decitabine.

Abstract 1869: Severe toxicities in patients undergoing gemcitabine, ARA-C, capecitabine, or azacytidine treatments: Is deregulated cytidine deaminase the bad guy

Abstract Background: Nucleoside analogs are anticancer drugs widely used both in adults and in children. Genetic polymorphisms leading to impaired ability to detoxify drugs in the liver are a major issue in clinical oncology. Cytidine deaminase (CDA) is a ubiquituous enzyme responsible for the metabolism of several pyrimidine derivatives. CDA is involved in the activation pattern of capecitabine, but conversely is responsible for the liver catabolism of most other nucleosidic analogs. So far, downregulated CDA is considered as an issue with gemcitabine, but little is known about the impact of deregulated CDA on the clinical outcome with other pyrimidine analogs. Methods: A retrospective study was perfomed as a proof-of-concept for the implication of CDA in severe toxicities with several pyrimidine analogs. Phenotypic and genotypic investigations were performed in a subset of patients presenting with severe toxicities soon after being treated for the first time with a pyrimidine derivative. CDA status was evaluated on a phenotypic basis using a surrogate enzymatic test. Additionally, polymorphisms associated with loss of CDA activity (i.e. 79A>C, 435 T>C, 208 G>A mutations) or conversely with increased expression (i.e. rs532545 deletion) were screened. Toxicities were monitored following the standard CTC 4.0 grading. Results & Discussion: We followed five patients (4 adults, 1 child), all presenting with extremely severe toxicities soon after being treated with a nucleoside analog (i.e. capecitabine, gemcitabine, azacytidine and ara-C, respectively). Two patients treated with capecitabine underwent severe toxicities, including a lethal one. Both patients were phenotyped as CDA ultra-metabolizer (UM: CDA > 6 U/mg). The rs532545 deletion in the CDA promoter region was found in the patient with lethal outcome. This suggests that CDA UM status triggers toxicities through an increased activation of capecitabine towards its toxics metabolites. Conversely, poor metabolizer (PM) profile (i.e. CDA < 2 U/mg) was evidenced in the 3 other patients with severe toxicities upon gemcitabine, azacytidine and Ara-C intake. Of note, one of the patient exhibited the double 79A>C and rs532545 polymorphisms, which have opposite impacts on CDA levels eventually. In this case, phenotypic determination proved to be critical to picture the CDA status, and confirmed her PM phenotype. Our observations suggest that CDA deficiency makes patients administered with gemcitabine, azacytidine and ara-C at risk of early severe toxicities alike. Conclusions: Deregulated CDA (i.e. PM or UM patients) proved to be associated with a serial of severe/lethal toxicities in patients treated with nucleoside analogs frequently administered in clinical oncology. Overall, this POC study warrants the identification of CDA status in patients prior to starting pyrimidine-based treatments. 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 1869. doi:1538-7445.AM2012-1869

Nab-Paclitaxel Potentiates Gemcitabine Activity by Reducing Cytidine Deaminase Levels in a Mouse Model of Pancreatic Cancer

Nanoparticle albumin-bound (nab)-paclitaxel, an albumin-stabilized paclitaxel formulation, demonstrates clinical activity when administered in combination with gemcitabine in patients with metastatic pancreatic ductal adenocarcinoma (PDA). The limited availability of patient tissue and exquisite sensitivity of xenografts to chemotherapeutics have limited our ability to address the mechanistic basis of this treatment regimen. Here, we used a mouse model of PDA to show that the coadministration of nab-paclitaxel and gemcitabine uniquely demonstrates evidence of tumor regression. Combination treatment increases intratumoral gemcitabine levels attributable to a marked decrease in the primary gemcitabine metabolizing enzyme, cytidine deaminase. Correspondingly, paclitaxel reduced the levels of cytidine deaminase protein in cultured cells through reactive oxygen species-mediated degradation, resulting in the increased stabilization of gemcitabine. Our findings support the concept that suboptimal intratumoral concentrations of gemcitabine represent a crucial mechanism of therapeutic resistance in PDA and highlight the advantages of genetically engineered mouse models in preclinical therapeutic trials. This study provides mechanistic insight into the clinical cooperation observed between gemcitabine and nab-paclitaxel in the treatment of pancreatic cancer.

Combining Tetrahydrouridine with Decitabine Addresses Malignant Cell Sanctuary in the Liver, a Tissue That Expresses High Levels of Cytidine Deaminase,

Abstract 3509Non-cytotoxic DNA methyl-transferase 1 (DNMT1) depletion by decitabine induces p53-independent differentiation mediated cell cycle exit in acute myeloid leukemia (AML) cells and spares normal hematopoietic stem cells, providing a potentially important alternative to conventional apoptosis-based therapy. However, there are pharmacologic barriers that impede optimal clinical translation of pre-clinical observations. One major barrier is the enzyme cytidine deaminase (CDA), which drastically reduces the half-life of decitabine from many hours in vitro to a few minutes in vivo. This problem is significant since decitabine is S-phase specific in its mechanism of action, and for effective DNMT1 depletion, low peak levels but extended half-life are optimal. Furthermore, CDA is highly expressed in the liver and intestines, generating potential sanctuary sites from the effects of decitabine. Finally, upregulation of CDA expression is a potential mechanism by which AML cells may resist decitabine at the cellular level. To overcome the multiple CDA-mediated barriers to effective therapy, we evaluated the combination of very low dose decitabine with the CDA inhibitor tetrahydrouridine (THU) in a murine xenotransplantation model of aggressive human AML (THP1 AML cells – p53 and p16-null). Treatment groups (n=5 per group) received equal volume vehicle (PBS), low dose cytarabine (0.1 mg/kg subcutaneous [SC]) with (THU) (4mg/kg intraperitoneal [IP]), decitabine (0.1 mg/kg SC) with THU (4mg/kg IP), and decitabine alone (0.2 mg/kg SC). Median survival in decitabine (61 days) and THU-decitabine (70 days) treated mice was significantly extended compared to the PBS (38 days) and THU-cytarabine (50 days) treatment groups (Log Rank p=0.00156 (figure 1). In decitabine treated mice, eventual emergence of disease was concentrated in the liver, consistent with a sanctuary function of this organ. Combination of THU with decitabine largely eliminated liver infiltration by AML, reducing liver tumor load from average 5.2g in PBS treated mice to 0.8g in THU-Decitabine treated mice (t-test p<0.001, livers were harvested at different time-points corresponding to the Kaplan-Meier curve). In separate experiments without AML cell xenotransplantation, the non-cytotoxic, DNMT1 depleting character of the decitabine therapy was confirmed by hematologic and bone marrow evaluation for DNA damage by phospho-H2AX staining. In conclusion, combining THU with decitabine can address malignant cell sanctuary in the liver, an organ that expresses high levels of cytidine deaminase. The ability of this combination to address other aspects of CDA-mediated resistance to therapy is also being evaluated. Disclosures:No relevant conflicts of interest to declare.

Hybridoma Passage In Vitro May Result in Reduced Ability of Antimannan Antibody To Protect against Disseminated Candidiasis

We previously reported the enhanced resistance of monoclonal antibodies B6.1 (an immunoglobulin M [IgM]) and C3.1 (an IgG3) against experimental candidiasis. Both MAbs recognize the same fungal epitope. We have since found that a highly passaged B6.1 hybridoma (hp-B6.1) resulted in antibody that has little protective potential. The potential clinical applicability of the antibody and our interest in understanding antibody protection against candidiasis led us to investigate an explanation for this phenomenon. Antibody genetic structure of hp-B6.1, the original hybridoma clone (ori-B6.1) stored frozen since 1995, a subclone of hp-B6.1 that produces protective antibody, the IgG3-producing hybridoma, and a nonprotective IgG1-producing hybridoma were compared. Variable region gene sequences of heavy (V(H)) and light chains showed genetic instability of V(H) chains with only the hp-B6.1; the V(H) sequences from ori-B6.1 and the subclone were, however, identical. Activation-induced cytidine deaminase levels were greatest in the B6.1 hybridomas, which may explain the instability. The constant region CH3 domain remained unchanged, implying normal N-glycation and complement-fixing potential, and antibody binding affinities appeared unchanged. Complement fixation assays surprisingly showed that ori-B6.1 antibody fixes C3 more rapidly than does hp-B6.1 antibody. The V(H) region primary structure may affect complement activation, which could explain our result. Indeed, antibody from the hp-B6.1 subclone fixed complement like antibody from ori-B6.1. These results show that the greatest protection occurs when antimannan antibodies possess the dual abilities of recognizing the appropriate carbohydrate epitope and rapidly fixing complement; loss of the latter property results in the loss of protective potential by the antibody.

Molecular determinants of capecitabine efficacy in colorectal cancer

3606 Background: Capecitabine offers physicians a more convenient treatment for advanced colorectal cancer (CRC), with manageable toxicity and antitumor activity comparable to that of continuous-infusion therapies with 5-fluorouracil (5-FU). However, there are no validated and established predictive factors for clinical outcome of capecitabine efficacy in CRC. Therefore we investigated whether intratumoral mRNA expression levels of genes involved in the capecitabine/5-FU metabolism (cytidine deaminase (CDA), dihydropyrimidine dehydrogenase (DPD), folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH), thymidine phosphorylase (TP), thymidylate synthase (TS)) and in angiogenesis (cyclooxygenase 2 (Cox-2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF)) are associated with the clinical outcome of patients with metastatic CRC treated with first-line capecitabine. Methods: Thirty-seven patients with metastatic CRC were enrolled in this study and treated with single agent capecitabine.The intratumoral mRNA levels of CDA, COX-2, DPD, EGFR, FPGS; GGH, TP, TS, and VEGF were assessed from paraffin-embedded tissue samples using laser-capture-microdissection methods and quantitative real-time PCR. Results: There were20 women and 17 men with a median age of 61 years (range 49–74). The median progression-free survival was 6.7 months (95% CI, 4.8–11.6 months), with a median follow up of 14.4 months (range: 1.3 to 18.7 months). Complete response was observed in 1 (3%), partial response in 6 (20%), stable disease in 14 (47%) and progressive disease in 9 (30%) patients (response was inevaluable in 7 patients). Higher gene expression levels of DPD were associated with resistance to capecitabine (P= 0.032; Kruskal-Wallis test). Patients with a lower mRNA amount of DPD (≤0.46) had a longer progression-free survival compared with patients that had a higher mRNA amount (8.0 vs. 3.3 months; adjusted P=0.048; log-rank test). Conclusions: This pilot study suggests that intratumoral gene expression levels of DPD may be useful to predict the clinical outcome of patients with metastatic CRC with first-line single agent capecitabine treatment. Our data are hypothesis generating and should be validated in larger and prospective clinical trials. [Table: see text]

Cytidine deaminase in polymyalgia rheumatica and elderly onset rheumatoid arthritis

Serum cytidine deaminase (CD) as a marker of inflammatory disease was assessed in 44 patients and 47 controls to differentiate polymyalgia rheumatica (PMR) from elderly onset rheumatoid arthritis (EORA). The patients were divided into four groups: PMR with and without synovitis and seropositive and seronegative EORA. No statistically significant differences were found when serum CD levels of seropositive EORA patients were compared with serum CD of PMR patients without synovitis, neither when serum CD levels of all PMR patients were compared with a seronegative EORA group, nor when serum CD levels of PMR patients with synovitis were compared with those with EORA. Nevertheless, statistically significant differences were detected between EORA's serum CD levels and the control group (p=0.023). This difference was 10% when comparing CD levels of PMR patients with the control group (p=0.070). We did not demonstrate that serum CD levels could be a useful tool to differentiate PMR from EORA, but these findings could nevertheless reflect the presence of an inflammatory disease.

Pharmacogenetic correlates of clinical benefit from gemcitabine and platinum in patients with advanced bladder cancer

3202 Background: The expression level of the DNA excision repair gene ERCC1 has been correlated with outcome in pts receiving platinum agents for a variety of malignancies. While ribonucleotide reductase (RRM1) levels have been associated with responsiveness to gemcitabine (G), deoxycytidine kinase (DOCK), cytidine deaminase (CD), and other enzymes may also be useful to predict response to G. Therefore, pharmacogenetic correlations were carried out in pts with transitional cell bladder cancer (BC). Methods: mRNA enzyme levels (MEL) of ERCC1, RRM1, DOCK, and CD were determined using quantitative realtime PCR (Taqman) in microdissected paraffin-embedded tumor from treatment-naïve patients with locally advanced or metastatic BC who received G and a platinum agent. The result was expressed as a ratio of the PCR product of each enzyme compared to the β-actin gene. Thresholds for chemotherapy (CT) resistance were determined using the bootstrap technique. Statistical correlations between MEL and clinical endpoints were performed using the Chi square method. Results: 50 consecutive pts with locally advanced or metastatic BC (41 M, 9 F) received G and cisplatin or carboplatin. Among 18 pts with metastatic disease, there were 6 PD, 3 SD, 5 PR, and 2 CR. The median survival for pts with SD, PR, and CR was 13.4 m (range 6.5–25.0); and for pts with PD, 7.6 m (range 3.3–13.5). Among 25 pts who received adjuvant post-operative therapy, 7 died of disease an average of 10.6 m from the start of CT. An additional 4 pts relapsed and are receiving treatment, while 14 remain disease free with a median F/U of 19 m. Among 4 pts with bladder sparing CT-XRT, 3 are alive and disease free and 1 has succumbed to disease with a median F/U of 23.6 m. Among 2 pts with neoadjuvant CT, downstaging occurred in both cases, and both are alive with a median F/U of 12.3 m. Correlations with MEL will be presented at the meeting. Conclusions: MEL may permit the identification pts with a high probability of benefit before CT and avoid ineffective CT when the enzyme profile indicates resistance. Prospective validation of these results would establish MEL for treatment selection in patients with BC. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics Response Genetics Response Genetics Eli Lilly