Cytidine Deaminase Deficiency Research Articles

Cytidine deaminase deficiency in mice enhances genetic instability but limits the number of chemically induced colon tumors

Cytidine deaminase (CDA) catalyzes the deamination of cytidine (C) and deoxycytidine (dC) to uridine and deoxyuridine, respectively. We recently showed that CDA deficiency leads to genomic instability, a hallmark of cancers. We therefore investigated whether constitutive CDA inactivation conferred a predisposition to cancer development. We developed a novel mouse model of Cda deficiency by generating Cda-knockout mice. Cda+/+ and Cda−/− mice did not differ in lifetime phenotypic or behavioral characteristics, or in the frequency or type of spontaneous cancers. However, the frequency of chemically induced tumors in the colon was significantly lower in Cda−/− mice. An analysis of primary kidney cells from Cda−/− mice revealed an excess of C and dC associated with significantly higher frequencies of sister chromatid exchange and ultrafine anaphase bridges and lower Parp-1 activity than in Cda+/+ cells. Our results suggest that, despite inducing genetic instability, an absence of Cda limits the number of chemically induced tumors. These results raise questions about whether a decrease in basal Parp-1 activity can protect against inflammation-driven tumorigenesis; we discuss our findings in light of published data for the Parp-1-deficient mouse model.

Cytidine deaminase activity increases in the blood of breast cancer patients

Cytidine deaminase (CDA), an enzyme of the pyrimidine salvage pathway, deaminates cytidine, deoxycytidine and analogs, such as gemcitabine. Constitutive low levels of CDA activity have been reported in the blood of patients with hematological malignancies or suffering from gemcitabine toxicity. We previously reported that cellular CDA deficiency leads to genetic instability. We therefore hypothesized that constitutive CDA deficiency might confer a predisposition to cancer. We analyzed CDA activity and expression in blood samples from breast cancer (BC) patients with a suspected predisposition to the disease, and in healthy controls. Contrary to our hypothesis, we found that both CDA activity and mRNA levels were higher in blood samples from BC patients than in those from controls, and that this difference was not due to excess neutrophils. CDA activity levels were significantly higher in the serum samples of BC patients treated by radiotherapy (RT) than in those of untreated healthy controls, and hormone therapy in RT-treated BC patients was associated with significantly lower levels of CDA activity. A preliminary analysis of CDA activity in the serum of the very few BC patients who had undergone no treatment other than surgery suggested that the increase in CDA activity might be due to the breast cancer itself. Our findings raise important questions, which should lead to studies to elucidate the origin and significance of the increase in CDA activity in the serum of BC patients, and the impact of hormone therapy.

Cytidine deaminase deficiency in tumor cells is associated with sensitivity to a naphthol derivative and a decrease in oncometabolite levels

Identifying new molecular targets for novel anticancer treatments is a major challenge in clinical cancer research. We have shown that cytidine deaminase (CDA) expression is downregulated in about 60% of cancer cells and tissues. In this study, we aimed to develop a new anticancer treatment specifically inhibiting the growth of CDA-deficient tumor cells. High-throughput screening of a chemical library led to the identification of a naphthol derivative, X55, targeting CDA-deficient tumor cells preferentially, without affecting the growth of non-tumoral cells regardless of CDA expression status. Metabolomic profiling revealed that CDA-deficient HeLa cells differed markedly from control HeLa cells. X55 treatment had a moderate effect on control cells, but greatly disturbed the metabolome of CDA-deficient HeLa cells, worsening the deregulation of many metabolites. In particular, the levels of the three oncometabolites, fumarate, succinate and 2-hydroxyglutarate, were significantly lower in CDA-depleted cells, and this decrease in levels was exacerbated by X55 treatment, revealing an unexpected link between CDA deficiency, mitochondrial function and X55 response. Finally, we identified strong downregulation of MAPT (encoding Tau, a microtubule associated protein) expression as a reliable predictive marker for tumor cell X55 sensitivity.

Patterns of Immune Dysregulation in Primary Immunodeficiencies: A Systematic Review

Immune dysregulation is as important as susceptibility to infection in defining primary immunodeficiencies (PIDs). Because of the variability and nonspecificity of the symptoms of PIDs, diagnosis can be delayed-especially if a patient presents with immune dysregulation. Diagnosis is then based on certain combinations of symptoms and relies on the clinician's ability to recognize a pattern. So far there is no large report linking patterns of immune dysregulations to the underlying genetic defects. To identify immune dysregulatory patterns associated with PIDs and to help clinicians to detect an underlying PID in certain patients with noninfectious inflammatory diseases. A systematic literature review was performed. We included 186 articles that reported on n= 745 patients. The most common immune dysregulation category was "autoimmunity" (62%, n= 463), followed by "intestinal disease" (38%, n= 283) and "lymphoproliferation" (36%, n=268). Most patients (67%) had 1 or more symptoms of immune dysregulation. Autoimmune hemolytic anemia, the most common autoimmune phenotype, was most frequently reported in patients with LPS responsive beige-like anchor protein deficiency (when combined with hypogammaglobulinemia or gastrointestinal symptoms), activation-induced cytidine deaminase deficiency (when combined with autoimmune hepatitis), or RAG1 deficiency (when it was the only symptom of immune dysregulation). Eczema, allergies, and asthma were reported in 34%, 4%, and 4% of the patients, respectively. Patterns of immune dysregulation may help the physician to recognize specific PIDs. This systematic review provides clinicians with an overview to better assess patients with immune dysregulation.

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

The Emerging Role of Cytidine Deaminase in Human Diseases: A New Opportunity for Therapy?

The recycling activity of cytidine deaminase (CDA) within the pyrimidine salvage pathway is essential to DNA and RNA synthesis. As such, CDA deficiency can lead to replicative stress, notably in Bloom syndrome. Alternatively, CDA also can deaminate cytidine and deoxycytidine analog-based therapies, such as gemcitabine. Thus, CDA overexpression is often associated with lower systemic, chemotherapy-related, adverse effects but also with resistance to treatment. Considering the increasing interest of CDA in cancer chemoresistance, the aims of this review are to describe CDA structure, regulation of expression, and activity, and to report the therapeutic strategies based on CDA expression that recently emerged for tumor treatment.

Abstract 3898: CDA predicts life-threatening toxicities in AML patients treated with cytarabine

Abstract Cytarabine (Ara-C) is the backbone for treating accute myeloid leukemia (AML). As most nucleoside analogs, Ara-C is metabolized in the liver by an exclusive enzymatic step driven by cytidine deaminase (CDA). CDA is a ubiquitous enzyme that proved to be highly polymorphic, with activities in patients ranging from poor metabolizer (PM) to ultra metabolizer (UM). To date, deregulated CDA has been evidenced as a culprit for life-threatening toxicities with gemcitabine. Little evidences have been collected thus far about the possible role erratic CDA could further play with Ara-C. Here, we investigated whether deregulations affecting CDA could be at the origin of unexpectedly severe, and sometimes lethal, toxicities with Ara-C in patients with AML. In this clinical observational study we determined CDA activity levels and searched for CDA genetic polymorphisms in 58 adult patients (25F, 33M, mean age 63 ±15 years) hospitalized for AML and scheduled for induction therapy. All patients were treated following current standard care by Ara-C-containing regimen (i.e., 7+3 protocol) and monitored for toxicity (CTCAEv4 grading) and response. Efficacy was evaluated one-month after starting the induction protocol. Mean CDA activity in the studied population was 3.14 ± 3 U/mg. No relationship was found between CDA phenotype and genotype (i.e., 79A>C polymorphism). A total of 20 patients (43%) displayed severe toxicities (i.e. grade-3 and above) upon administration, including 3 unrecoverable grade-5 toxicities (5%). Relationship between CDA activity and severe toxicity was investigated using ROC analysis. We observed that the incidence of CDA deficiency was much higher in AML patients (40%) than what we previously observed in patients with solid tumors. A cut-off value in CDA ≤ 2 U/mg was associated with increased risk of experiencing severe/lethal toxicities with 74% sensitivity and 65% specificity. Among the 20 patients with severe toxicities, 14 (72 %) were categorized as PM. Importantly, patients who experienced lethal toxicities were all profoundly PM patients. CR was achieved in 35 patients (60%), CRI achieved in 4 patients (7%) whereas 16 patients (27%) failed to respond (PD). No relationship was evidenced between CDA status and response, however regardless of the karyotype we observed that PM patients tended to have longer PFS (278 vs. 517 days) and OS (570 days vs. not reached) as compared with EM patients. Overall this proof-of-concept study strongly suggests that CDA status could be a relevant marker for predicting clinical outcome in patients treated with Ara-C, especially treatment-induced toxicities. A prospective study including full PK and pharmacogenetics support is currently ongoing in our institute to confirm these observations. Should the role CDA plays be confirmed, this marker could be used as a covariate to customize Ara-C dosing in AML patients. Citation Format: Raphaelle Fanciullino, Laure Farnault, Mélanie Donnette, Vadim Ivanov, Geoffroy Venton, Joseph Ciccolini, Yael Berda-Haddad, Catherine Roche, L'Houcine Ouafik, Bruno Lacarelle, Regis Costello. CDA predicts life-threatening toxicities in AML patients treated with cytarabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3898.

Cytidine Deaminase Deficiency Reveals New Therapeutic Opportunities against Cancer.

Purpose: One of the main challenges in cancer therapy is the identification of molecular mechanisms mediating resistance or sensitivity to treatment. Cytidine deaminase (CDA) was reported to be downregulated in cells derived from patients with Bloom syndrome, a genetic disease associated with a strong predisposition to a wide range of cancers. The purpose of this study was to determine whether CDA deficiency could be associated with tumors from the general population and could constitute a predictive marker of susceptibility to antitumor drugs.Experimental Design: We analyzed CDA expression in silico, in large datasets for cancer cell lines and tumors and in various cancer cell lines and primary tumor tissues using IHC, PDXs, qRT-PCR, and Western blotting. We also studied the mechanism underlying CDA silencing and searched for molecules that might target specifically CDA-deficient tumor cells using in silico analysis coupled to classical cellular experimental approaches.Results: We found that CDA expression is downregulated in about 60% of cancer cells and tissues. We demonstrate that DNA methylation is a prevalent mechanism of CDA silencing in tumors. Finally, we show that CDA-deficient tumor cells can be specifically targeted with epigenetic treatments and with the anticancer drug aminoflavone.Conclusions: CDA expression status identifies new subgroups of cancers, and CDA deficiency appears to be a novel and relevant predictive marker of susceptibility to antitumor drugs, opening up new possibilities for treating cancer. Clin Cancer Res; 23(8); 2116-26. ©2016 AACR.

Highlights of This Issue

Salt-inducible kinase 2 (SIK2) is overexpressed in 30% of ovarian cancers and regulates centrosome splitting, mitotic progression, and activation of PI3K. A small-molecule inhibitor of SIK2 (ARN-3236) blocks the activity of SIK2 kinase, inhibits growth of ovarian cancer cell lines, and enhances sensitivity to paclitaxel in cell culture and in xenografts. ARN-3236 produces nuclear-centrosome dissociation, inhibits centrosome splitting, blocks mitotic progression, induces polyploidy, reduces AKT/survivin signaling, and triggers apoptotic cell death. Thus, ARN-3236 appears to be a drug with promise for enhancing primary treatment of ovarian cancers.Mameri and colleagues report that cytidine deaminase (CDA) expression is downregulated in about 60% of tumors, mostly due to DNA methylation. Immunohistochemistry assessments of CDA expression status defined two new subgroups of tumors: CDA-deficient tumors and CDA-proficient tumors. CDA-deficient tumor cells, but not CDA-proficient tumor cells, present a marked susceptibility to aminoflavone, indicating that CDA expression status could be used to guide anticancer therapy. These results constitute a proof-of-concept that CDA deficiency may turn out to be a new predictive marker of susceptibility to antitumor drugs, thus opening up new possibilities for treating cancer.Patients with glioblastoma (GBM) have a median survival of <15 months despite surgical resection, high-dose radiation and chemotherapy with temozolomide (TMZ). Cytomegalovirus (CMV) proteins are expressed in over 90% of sampled GBM and expression is restricted to glioma cells not surrounding normal brain tissue. Batich and colleagues targeted the CMV antigen pp65 using dendritic cells (DCs) in combination with dose-intensified temozolomide (TMZ) and evaluated patient antitumor immune responses and survival. Despite increases in regulatory T cells after TMZ, patients treated with pp65-DCs showed increased pp65 immunity and long-term survival extending beyond predicted rates. Randomized studies on the prevention of generated regulatory T cells in the context of TMZ treatment and CMV targeting are under way.The lncRNA PVT1 is an important oncogene in human tumors; however, the mechanisms underlying the PVT1 functions remain elusive. Xu and colleagues demonstrate a reciprocal link between PVT1 and FOXM1 in gastric cancer (GC). PVT1 binds to the FOXM1 protein and enhances its stability, fulfills its functions in a FOXM1-mediated manner. Moreover, FOXM1 binds directly to and constitutively transactivates the PVT1 promoter. The authors report a novel mechanism by which transcript-induced lncRNA facilitate mRNA function by stabilizing a transcript-coding protein posttranscriptionally. PVT1 expression may be a useful biomarker for GC prognosis, and the PVT1-FOXM1 loop could be a therapeutic target.

Novel and recurrent AID mutations underlie prevalent autosomal recessive form of HIGM in consanguineous patients.

Immunoglobulin class switch recombination deficiencies (Ig-CSR-D) are characterized by normal or elevated serum IgM level and absence of IgG, IgA, and IgE. Most reported cases are due to X-linked CD40L deficiency. Activation-induced cytidine deaminase deficiency is the most frequent autosomal recessive form, whereas CD40 deficiency is more rare. Herein, we present the first North African study on hyper IgM (HIGM) syndrome including 16 Tunisian patients. Phenotypic and genetic studies allowed us to determine their molecular basis. Three CD40LG mutations have been identified including two novels (c.348_351dup and c.782_*2del) and one already reported mutation (g.6182G>A). No mutation has been found in another patient despite the lack of CD40L expression. Interestingly, three AICDA mutations have been identified in 11 patients. Two mutations were novel (c.91T>C and c.389A>C found in one and five patients respectively), and one previously reported splicing mutation (c.156+1T>G) was found in five patients. Only one CD40-deficient patient, bearing a novel mutation (c.109T>G), has been identified. Thus, unlike previous reports, AID deficiency is the most frequent underlying molecular basis (68%) of Ig-CSR-D in Tunisian patients. This finding and the presence of specific recurrent mutations are probably due to the critical role played by inbreeding in North African populations.

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

Abstract 5549: Recipient-donor contradictory genotype with impact on anticancer drug pharmacogenetics after liver transplant: A deadly gift

Abstract A 5-year old girl with liver transplant was scheduled for a therapy including high-dose cytarabine for her Burkitt lymphoma. Cytarabine pharmacogenetics focus on cytidine deaminase (CDA) , the polymorphic liver enzyme responsible for its detoxification. Preliminary double testing for her CDA status on DNA lymphocytes showed none of the germline polymorphisms usually associated with CDA deficiency (ie, 79A>C (K27Q), 208G>A), but surprisingly with a functional mild deficiency syndrom. Despite 30% reduction in cytarabine dosing, life-threatening toxicities showed quickly and treatment was discontinued. Genetic HRM-based investigations performed in the patient on liver biopsy failed in generating a clear genotype. Further HRM+sequencing retrospective investigations on liver biopsy collected from the donor showed that he was actually bearing the homozygous CDA*2 allelic variant (ie, 79AA), a genotype usually associated with severe CDA deficiency. Based upon the donor liver genotype, and not the recipient genotype, treatement was resumed with further dose reduction, close monitoring and better tolerance eventually. The patient is now in complete remission more than a year after completion of the treatment. This case report illustrates the limits and risks of searching germline polymorphisms in patients with liver transplant when the story plays in the liver. Citation Format: Cindy Serdjebi, Raphaelle Fanciullino, Frederic Fina, Arnaud Verschuur, Bertrand Roquelaure, Bruno Lacarelle, L'Houcine Ouafik, Joseph Ciccolini, Nicolas André. Recipient-donor contradictory genotype with impact on anticancer drug pharmacogenetics after liver transplant: A deadly gift. [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 5549. doi:10.1158/1538-7445.AM2014-5549

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.

Cytidine deaminase status as a predictive marker in patients with hematologic malignancies treated with azacytidine or cytarabine.

7117 Background: Cytarabine 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 is highly polymorphic and dysregulations have been repeatedly associated with poor clinical outcome with gemcitabine. Methods: We have used a test to determine, on a phenotype basis, CDA status in patients. This test was used prospectively in a subset of 39 adult patients (16F, 23M, mean age 77 years), all treated for various haematological malignancies (i.e., CML, AML, lymphomas, 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. Results: In 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%) showed low CDA activities and were considered as PM. Conversely, 8 patients (i.e., 20%) displayed CDA activities particurlary elevated (i.e., > 6U/mg) and were considered as UM patients. Of note, PM patients all showed severe toxicities, including two toxic-deaths. Conversely, UM patients showed little efficacy when treated with either azacytidine or cytarabine. In mice with CDA-deficiency, standard 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. Conclusions: Overall this pilot study strongly 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.

Abstract 2208: Cytidine deaminase deficiency and severe toxicities upon aza-cytidine intake: Clinical observations and experimental evidences.

Abstract Azacytidine is a nucleosidic analog used for treating a variety of haematological malignancies. Azacytidine pharmacokinetics is dependent upon a liver detoxification step driven by cytidine deaminase (CDA). CDA is affected by several genetic polymorphisms leading to a wide inter-individual variability in resulting enzymatic activities. In particurlar, dysregulated CDA has been associated with life-threatening toxicities with several nucleosidic analogs such as after cytarabine, capecitabine and gemcitabine intake. However, little is known about the impact of CDA deficiency on the clinical outcome of patients treated with azacytidine. In our institute, a lethal toxicity after pancytopenia has been observed with a 82-years old female patient upon aza-cytidine intake. CDA deficiency was evidenced after retrospective phenotyping (functional activity was found to be reduced by 65% as compared with standard CDA values: 1.2 VS. 3.5 U/mg), thus strongly suggesting that impaired ability to detoxify azacytidine could be the culprit indeed for this case. To further establish the exact role CDA-deficiency could have played in this toxic-death, a non-clinical study was undertaken in rodents. To mimic CDA deficiency, 8 mice were pre-treated with 100 mg/kg of tetrahydrouridine I.P. before being administered with 24 mg/kg of azacytidine. Another group of 8 mice received 24 mg/kg azacytidine only, and were therefore considered as control-CDA. Four mice were used as untreated controls when evaluating tolerance to the treatment. Neutrophils were monitored by flow-cytometry before and after treatment over 10 days as a surrogate marker for global azacytidine-related toxicities. CDA was measured using the same test than the one used at bedside and showed that the two groups differed markedly in their CDA phenotypes (ie, 7.7 VS. 0.98 U/mg) and that deep CDA deficiency (Poor Metabolizer: PM status) was achieved indeed in THU-treated animals. Pharmacokinetics study was carried out on a satellite group (T10, T20, T40 and T60 min) by revered-phase HPLC-UV analysis and showed a marked overexposure in azacytidine plasma levels in PM animals displaying CDA-deficiency. In full line with this observation, longer and deeper neutropenia was observed in this PM subset, as compared with the mice with normal CDA activity treated with the same dosage of azacytidine. Overall, our experimental data strongly support the hypothesis that CDA deficiency is a condition associated with increased risk to undergo life-threatening toxicities after azacytidine treatment. Detecting PM patients should help to secure the use of azacytidine at bedside. Citation Format: Raphaelle Fanciullino, Cindy Serdjebi, Severine Mollard, Regis Costello, L'Houcine Ouafik, Joseph Ciccolini, Cedric Mercier. Cytidine deaminase deficiency and severe toxicities upon aza-cytidine intake: Clinical observations and experimental evidences. [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 2208. doi:10.1158/1538-7445.AM2013-2208

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

Case of a 10-Year-Old Boy with Hyper–Immunoglobulin M Immunodeficiency Caused by Activation-Induced Cytidine Deaminase Deficiency

We report the case of a 10-year-old boy, with a history of recurrent sinopulmonary infections, presenting with pneumococcal meningitis. Immune workup found low immunoglobulin G (IgG), IgA, and IgE and elevated IgM. Specific antibodies to pneumococcus, tetanus, and mumps were low. Genetic testing identified CD40L and uracil-DNA glycosylase as normal. Sequence analysis for mutations in activation-induced cytidine deaminase (AID) gene on chromosome 12p13 revealed a single missense mutation in exon 3 caused by a point mutation. AID is an enzyme that is specifically present in activated B cells and necessary for isotype switching. This case highlights the significance of AID deficiency in hyper-IgM immunodeficiency.

High-Resolution Melting Analysis of Sequence Variations in the Cytidine Deaminase Gene (CDA) in Patients With Cancer Treated With Gemcitabine

Gemcitabine (2',2'-difluorodeoxycytidine) is a major antimetabolite cytotoxic drug with a wide spectrum of activity against solid tumors. Hepatic elimination of gemcitabine depends on a catabolic pathway through a deamination step driven by the enzyme cytidine deaminase (CDA). Severe hematologic toxicity to gemcitabine was reported in patients harboring genetic polymorphisms in CDA gene. High-resolution melting (HRM) analysis of polymerase chain reaction amplicon emerges today as a powerful technique for both genotyping and gene scanning strategies. In this study, 46 DNA samples from gemcitabine-treated patients were subjected to HRM analysis on a LightCycler 480 platform. Residual serum CDA activity was assayed as a surrogate marker for the overall functionality of this enzyme. Genotyping of three well-described single nucleotide polymorphisms in coding region (c.79A>C, c.208G>A and c.435C>T) was successfully achieved by HRM analysis of small polymerase chain reaction fragments, whereas unknown single nucleotide polymorphisms were searched by a gene scanning strategy with longer amplicons (up to 622 bp). The gene scanning strategy allowed us to find a new intronic mutation c.246+37G>A in a female patient displaying marked CDA deficiency and who had an extreme toxic reaction with a fatal outcome to gemcitabine treatment. Our work demonstrates that HRM-based methods, owing to their simplicity, reliability, and speed, are useful tools for diagnosis of CDA deficiency and could be of interest for personalized medicine.

Cytidine Deaminase Residual Activity in Serum Is a Predictive Marker of Early Severe Toxicities in Adults After Gemcitabine-Based Chemotherapies

PURPOSE Anticipating toxicities with gemcitabine is an ongoing story, and deregulation in cytidine deaminase (CDA) could be associated with increased risk of developing early severe toxicities on drug exposure. PATIENTS AND METHODS A simple test to evaluate CDA phenotypic status was first validated in an animal model investigating relationships between CDA activity and gemcitabine-related toxicities. Next, relevance of this test as a marker for toxicities was retrospectively tested in a first subset of 64 adult patients treated with gemcitabine alone, then it was tested in a larger group of 130 patients who received gemcitabine either alone or combined with other drugs and in 20 children. Additionally, search for the 435 T>C, 208 G>A and 79 A>C mutations on the CDA gene was performed. Results In mice, CDA deficiency impacted on gemcitabine pharmacokinetics and had subsequent lethal toxicities. In human, 12% of adult patients experienced early severe toxicities after gemcitabine administration. A significant difference in CDA activities was observed between patients with and without toxicities (1.2 +/- 0.8 U/mg v 4 +/- 2.6 U/mg; P < .01). Conversely, no genotype-to-phenotype relationships were found. Of note, the patients who displayed particularly reduced CDA activity all experienced strong toxicities. Gemcitabine was well tolerated in children, and no CDA deficiency was evidenced. CONCLUSION Our data suggest that CDA functional testing could be a simple and easy marker to discriminate adult patients at risk of developing severe toxicities with gemcitabine. Particularly, this study demonstrates that CDA deficiency, found in 7% of adult patients, is associated with a maximum risk of developing early severe toxicities with gemcitabine.

Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease.

Activation-induced cytidine deaminase (AID) expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM) and class switch recombination (CSR). Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2) associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID−/− mice spontaneously develop tertiary lymphoid organs (TLOs) in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID−/− mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF) conditions. Gastric autoantigen -specific serum IgM was elevated in AID−/− mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.