WO2009021551A1

WO2009021551A1 - A method for assessing the risk of toxicity in a chemotherapy

Info

Publication number: WO2009021551A1
Application number: PCT/EP2007/058349
Authority: WO
Inventors: Joseph Ciccolini; Cédric Mercier
Original assignee: Universite De La Mediterranee; Assistance Publique Hôpitaux De Marseille
Priority date: 2007-08-13
Filing date: 2007-08-13
Publication date: 2009-02-19

Abstract

The invention relates to an in vitro method for determining the risk of toxicity upon a chemotherapeutic agent intake in a patient afflicted with cancer, wherein metabolism of the chemotherapeutic agent involves cytidine deaminase (CDA), which method comprises determining the CDA activity in a biological sample of the patient, wherein an alteration in CDA activity compared to a control value is indicative of a risk of toxicity upon the chemotherapeutic agent intake.

Description

A method for assessing the risk of toxicity in a chemotherapy

The present invention relates to the field of cancer treatments. More particularly, it provides a method for determining the risk of toxicity upon a chemotherapeutic agent intake in patients afflicted with a cancer.

BACKGROUND :

Gemcitabine is an antimetabolite drug used in the treatment of various solid tumors, including lung, pancreatic, or gynaecological cancers. Innovative combinational strategies (e.g. gemcitabine + capacitabine (Gemcap), or gemcitabine + oxaliplatin (Gemox)) make gemcitabine an extensively prescribed drug now. Gemcitabine is characterized by a narrow therapeutic index and its liver elimination depends upon a key enzymatic step, driven by cytidine deaminase (CDA). CDA is prone to gene polymorphism [1 , 2, 3, 4, 5]. Recently, clinical evidences showed that patients bearing the 208A>G mutation on the CDA gene were at risk of developing haematological toxicities after gemcitabine exposure, thus highlighting the fact that the toxic profile of this drug could be dramatically worsen when administered to patients affected by gene polymorphism [3, I]. Overall, about 20 polymorphisms have been reported so far, with mixed, when not contradictory, impacts on resulting activities [1 , 4, 6]. Anticipating gemcitabine-induced toxicities solely on the basis of screening for genetic mutations can sometimes be uncertain, especially since epigenetic regulations of the CDA gene are still largely unknown.

SUMMARY OF THE INVENTION : The inventors now propose to detect patients with abnormal CDA activity by phenotyping them prior to the administration of a chemotherapeutic agent such as gemcitabin.

For that purpose it is herein provided a simple, rapid and inexpensive method, based upon the assay of residual CDA activity in serum. An object of the invention is thus an in vitro method for determining the risk of toxicity upon a chemotherapeutic agent intake in a patient afflicted with a cancer, wherein metabolism of the chemotherapeutic agent involves cytidine deaminase (CDA), which method comprises determining the CDA activity in a biological sample of the patient, wherein an alteration in CDA activity compared to a control value is indicative of a risk of toxicity upon the chemotherapeutic agent intake. In the present application, metabolism of the chemotherapeutic agent means in particular liver elimination of the chemotherapeutic agent or in vivo activation of the chemotherapeutic agent, i.e., in vivo conversion of the pro-drug into a pharmacologically active drug. In a particular embodiment, the chemotherapeutic agent is gemcitabine. Similarly, this method can be fully used for example either to detect patients with impaired CDA activity, at risk of severe toxicities with Ara-C, CNDAC, decitabine and/or tezacitabine intake, or, conversely, to detect patients displaying an abnormal increase of the CDA activity ("extensive activity") compared to a control value, at risk of severe toxicities with capecitabine and/or DMDC intake.

The CDA activity may be determined by radioactive High Performance Liquid Chromatography (HPLC), or by visible spectrophotometry. The CDA activity is advantageously determined by visible spectrophotometry.

Another subject of the invention is a kit for determining the risk of toxicity upon a chemotherapeutic agent intake in a patient afflicted with a cancer, wherein liver elimination of the chemotherapeutic agent or in vivo activation of the chemotherapeutic agent involves cytidine deaminase (CDA), which kit comprises : a container comprising cytidine; a container comprising ammonium; - a leaflet which describes a method for determining the CDA activity in a biological sample, by measuring the amount of ammonium released through conversion of cytidine into uridine by spectrophotometry, wherein the leaflet indicates that a CDA activity inferior to about 1 U/mg is indicative of a risk of a severe toxicity upon intake of a chemotherapeutic agent the liver elimination of which involves CDA, such as gemcitabine, Ara-C, CNDAC, decitabine and/or tezacitabine, whereas a CDA activity superior to 8 U/mg is indicative of a risk of a severe toxicity upon intake of a chemotherapeutic agent the in vivo activation (conversion of a pro-drug into a drug) of which involves CDA, such as capecitabine and/or DMDC.

LEGENDS TO THE FIGURES :

Figure 1 shows distribution of seric CDA activities in 45 reference patients without toxicities upon gemcitabine exposure, and comparison with the CDA activity measured in the patient with fatal outcome. Plain line: mean, dotted lines: +/- SD, dashed line: threshold.

Figure 2 shows High-resolution melting analysis of CDA single nucleotide polymorphisms 79A>C, 208G>A and 435C>T. Genomic DNA from reference patients were used for PCR and post-PCR melting curve analysis carried out on a LightCycler®480 instrument. Wild-type DNA was used as reference for each allele.

DETAILED DESCRIPTION OF THE INVENTION : The inventors have developed a method to determine phenotypically CDA status in cancer patients, more particularly as an attempt to detect those at risk upon gemcitabine, Ara-C, CNDAC, decitabine, tezacitabine, capecitabine and/or DMDC intake. Conjointly to genotypic investigations, this method was used to phenotype, in a retrospective setting, a female patient displaying extremely severe, and eventually lethal, toxicities after administration of a standard gemcitabine/carboplatin protocol. The phenotypic investigation showed a marked CDA deficiency (-75%) in this patient when compared with a reference, non-toxic population. Genetic studies undertaken next to screen mutations possibly at the origin of this deficiency showed heterozygosity for the 79A>C single point mutation, whereas the canonical CDA 208A>G polymorphism was not found. Taken together, this case-report demonstrates for the first time that CDA down-regulation can lead to toxic-death in patients exposed to gemcitabine. Besides, the inventors showed here that their cost-effective and simple phenotypic approach enables the detection of patients displaying an abnormal CDA phenotype. Patients displaying an abnormal CDA phenotype may be patients displaying a deficient CDA activity at risk upon administration of gemcitabine, and/or of chemotherapeutic agents the metabolism and liver detoxification of which are similar to those of gemcitabine, such as Ara-C, CNDAC, decitabine and tezacitabine. Other patients displaying an abnormal CDA phenotype may be patients displaying an extensive CDA activity at risk upon administration of capecitabine, and/or of chemotherapeutic agents the metabolism and in vivo activation (conversation of a pro-drug into a drug) of which are similar to those of capecitabine, such as DMDC.

The method of the invention comprises determining the level of CDA activity in a cancer patient who is to receive a chemotherapy, and comparing it to a control value. An alteration in the CDA activity compared to the control value is indicative of a toxicity upon administration of the chemotherapeutic agent, the metabolism of which involves CDA.

In the context of the invention, the term "alteration" means that CDA does not show a normal activity, compared to a control value. The "control value" may be the CDA activity of a reference non-toxic population. Preferably the reference population does not show any mutation in the CDA gene that is known to modify the activity of the gene product.

The control value may be a cut-off value as indicated below. The cut-off value is expressed in CDA activity unit per milligram of sample total protein (U/mg).

In a particular embodiment, the alteration is a deficiency in CDA activity, or in other words, an abnormally decreased CDA activity compared to a control value.

In another particular embodiment, the alteration is a CDA extensive activity, or in other words, an abnormally increased (or extensive) CDA activity compared to a control value.

Gemcitabine (marketed as Gemzar®) is 2'-deoxy-2',2'-difluorocytidine. A CDA activity, in a serum sample, inferior to a control value of about 1 U/mg is abnormal and indicative of a risk of severe toxicity upon gemcitabine uptake.

In another embodiment, the chemotherapeutic agent is selected from the group consisting of cytarabine (also known as Ara-C), CNDAC, decitabine and tezacitabine.

Cytarabine is 4-amino-1-beta-D-arabinofuranosyl-2(1 H)-pyrimidinone. CNDAC is 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosylcytosine.

Decitabine is 5-aza-2'-deoxycytidine.

Tezacitabine is (E)-2'-deoxy-2'-(fluoromethylene)cytidine.

Ara-C, CNDAC, decitabine and tezacitabine are anti-metabolite agents.

A CDA activity, in a serum sample, inferior to a control value of about 1 U/mg is abnormal and indicative of a risk of severe toxicity upon Ara-C, CNDAC, decitabine and/or tezacitabine uptake.

In still another embodiment, the chemotherapeutic agent is selected from the group consisting of capecitabine and/or DMDC. Capecitabine (marketed as Xeloda®) is pentyl[1-(3,4-dihydroxy-5-methyl- tetrahydrofuran-2-yl)- 5-fluoro-2-oxo-1 H-pyrimidin- 4-yl]aminomethanoate.

The activation of capecitabine follows a pathway with three enzymatic steps and two intermediary metabolites, 5'-deoxy-5-fluorocytidine (5'-DFCR) and 5'-deoxy-5- fluorouridine (5'-DFUR), to form 5-fluorouracil. Capecitabine can trigger toxicity due to its liver transformation into 5-fluorouracil in patients displaying an abnormal increase of the CDA activity compared to a control value (extensive CDA activity). A CDA activity, in a serum sample, superior to a control value of about 8U/mg is abnormal and indicative of a risk of severe toxicity upon capecitabine uptake.

DMDC is a chemotherapeutic agent the metabolism and in vivo activation (conversation of a pro-drug into a drug) of which is similar to those of capecitabine. DMDC is 2'-deoxy-2'-methylidenecytidine.

A CDA activity, in a serum sample, superior to a control value of about 8U/mg is abnormal and indicative of a risk of severe toxicity upon DMDC uptake.

In the context of the invention, the term « severe toxicity » refers to a grade 3 toxicity or higher according to standard WHO grading (see Reporting Guidelines of the National Cancer Institute - http://ctep.cancer.gov/reporting/ctc.html). Toxicity is of a grade 3 when said toxicity causes haematological disorders (e.g., leucopoenia, thrompopenia, anaemia, pancytopenia) and subsequent infectious diseases (such as fever, sepsis) and digestive diseases (such as diarrhoea, mucitis, nausea or vomiting). Toxicity of the highest grade (grade 5) causes death of the patient.

Such a severe toxicity means that the chemotherapeutic agent is not suitable for the tested patient and may be life-threatening. Therefore, most preferably, the method of the invention should be performed in a patient before the chemotherapeutic agent is prescribed. The dosage regimen should then be adapted to the risk of severe toxicity herein determined or another chemotherapeutic agent which does not involve CDA for kidney elimination, should be chosen instead.

In a preferred embodiment, the CDA activity is determined by spectrophotometry, preferably by visible spectrophotometry, most preferably at 630nm.

More preferably, the CDA activity may be determined by measuring the amount of ammonium released through conversion of cytidine into uridine, by spectrophotometry.

In a particular method of the invention, the CDA activity is determined by: a. incubating the biological sample with cytidine; b. setting up a calibration curve of ammonium to be incubated similarly with the sample; c. precipitating proteins so as to stop the reaction ; d. centrifuging and recovering the upper layer; e. incubating the recovered upper layer of step d) with a mixture of phenol and sodium hypochlorite and recovering the upper layer comprising the ammonium; f. detecting ammonium in the recovered upper layer of step e) with a spectrophotometer, preferably at 630nm; g. calculating the CDA activity, in regard with the signahactivity relationship generated by the calibration curve and the amount of proteins in the sample.

The protein amount of the sample is measured before incubating the sample with cytidine. For each patient, one additional sample is incubated without the substrate (blank).

The biological sample may be a body fluid, such as serum, plasma, and blood. It may also be a tissue biopsy, in particular a liver biopsy. Preferably, the biological sample is serum.

The patient is any human adult or child afflicted with a cancer, for the treatment of which chemotherapy is needed. Solid tumors that may benefit from gemcitabine include lung, pancreatic, bladder and breast cancers. Cancers that may benefit from Ara-C include acute myeloid leukemia, chronic myeloid leukemia, acute lymphoid leukemia, lymphomas. Cancers that may benefit from capecitabine include digestive cancers, in particular colorectal cancer, and breast cancer. Solid tumors may also benefit from CNDAC, decitabine, tezacitabine and/or DMDC.

In order to perform the method of the invention routinely, It is herein provided a kit for determining the risk of toxicity upon a chemotherapeutic agent intake in a patient afflicted with cancer, the metabolism of the chemotherapeutic agent involving cytidine deaminase (CDA), which kit comprises : a container comprising cytidine; a container comprising ammonium; - a leaflet which describes a method for determining the CDA activity in a biological sample, by measuring the amount of ammonium released through conversion of cytidine into uridine by spectrophotometry, wherein the leaflet indicates that a CDA activity inferior to about 1 U/mg, in a serum sample, is indicative of a risk of a severe toxicity upon intake of a chemotherapeutic agent the liver elimination of which involves CDA, such as gemcitabine, Ara-C, CNDAC, decitabine and/or tezacitabine, whereas a CDA activity superior to 8 U/mg, in a serum sample, is indicative of a risk of a severe toxicity upon intake of a chemotherapeutic agent the in vivo activation (conversation of a pro-drug into a drug) of which involves CDA, such as capecitabine and/or DMDC.

Preferably, the kit is a ready-to-use kit which, besides the leaflet, comprises seven containers: a container comprising cytidine; a container comprising ammonium; a container comprising sodium and phosphate buffers; a container comprising phenol and nitroprusside; - a container comprising tungstate; a container comprising hypochlorite; and a container comprising sulphuric acid.

In a preferred embodiment, the kit and the protocol may be as follows:

• Buffers in the kit:

- Solution A : KH2PO4 0.07M + Na2H2PO4, 0.07M PH7

- Solution B : sodium tungstate (1g/10 ml H2O)

- Solution C : Ammonium (102 mg in 500 μl_ H2O) - Solution D : Phenol (12 g phenol + 60 mg sodium nitroprusside in 900 ml H2O)

- Solution E : Hypochlorite (1 g hypochlorite + 8.4 g sodium hydroxyde in 1 I H2O)

- Solution F : cytidine 2 mM in solution A

- Solution G : sulfuric acid 1 N.

Solutions are aliquoted as working solutions (vol : 1 ml, except for solution A : 20 ml and solution D : 15 ml) and stored at -80⁰C.

• Protocol:

A blood sample is obtained when the patient is not undergoing chemotherapy and stored at 4°C. The blood tube is centrifuged at 2500 rpm, during 20 min and at 4°C, then the serum fraction is isolated.

An aliquote of 50 μl_ is isolated and serum proteins are assayed according to a standard method, such as the Bradford method.

Three aliquotes of 100 μl_ serum are isolated. The ammonium control is prepared by mixing 10 μl_ solution C in 10 ml solution A = 40

CDA Activity Units.

Dilution is performed (20/10/5/2.5 /1.25/0 A U.) in solution A. The control and the sample are incubated as follows:

400 μl of solution F, 100 μl_ sample (n=2/patients), 100 μl_ sample (n=1/blank patient) or 400 μl_ of solution F + 100 μl_ of each control curve point at 37°C during 16h. The incubation is stopped by adding 400 μl_ of solution F into the blank samples. The reaction is stopped by precipitating proteins with 200μl solution B + 200μl_ solution G. After centrifugation (5000 rpm, room temperature,-5 min) 500 μl_ of supernatant are recovered.

Coupling is achieved by adding 1.5 ml solution D and 2 ml solution E. The mixture is vortexed and incubated during 30 min at 37°C, before being read at 630 nm. The means of the two activities per patient is calculated after subtracting the blank value.

The CDA activity is expressed as Activity Units (U) taking into account the level of serum proteins.

The below examples illustrate the invention without limiting its scope.

EXAMPLES :

Toxic death case in a patient undergoing gemcitabine-based chemotherapy in relation with cytidine deaminase down regulation.

PATIENTS AND METHODS Reference Subset:

Forty-five adult Caucasian patients (26 males and 19 females), mean age 67 +/- 7 years, were included in this work for studying the reference distribution of CDA activities in serum. All these reference patients had received previously gemcitabine without notable toxicities as part of standard treatment of their cancer. Patients were hospitalized at La Timone University Hospital, Marseille, France, from May to December 2006. All the samples used in this study were the same than the ones withdrawn routinely for standard biological monitoring of patients with cancer at La Timone University Hospital, and no extra-sampling was therefore necessary. Written informed consents and local ethic committee approval were obtained prior to perform both phenotypic and genotypic investigations. Toxic-Death Case: A 74-year old Caucasian female patient underwent gemcitabine/carboplatin therapy for her metastatic vesical cancer, as following: gemcitabine D1 + D8 (1250 mg/m²/day) combined with adapted carboplatin (AUC5) on D2 [8]. Soon after the end of the D8 infusion of gemcitabine (e.g., D 17), this patient showed extremely severe haematological toxicities (G4 neutropenia, G4 thrombopenia, G3 anemia, sepsis, WHO grading). Despite appropriate and heavy supportive treatment (transfer to intensive care unit, administration of broad-spectrum antibiotherapy, daily platelet-concentrate infusions, growth factor administration), her condition quickly deteriorated and fatal outcome was finally observed on D22. CDA phenotypic assay:

Residual serum CDA activity was assayed as a surrogate marker for the overall functionality of this enzyme. To avoid any risk of drug-induced CDA inhibition, a wash- out period of minimum 15-day (e.g., 1500 half-lives of gemcitabine) after the last administration was observed before sampling. Once whole blood samples had been withdrawn, they could be maintained at 4°C for a maximum of 6 hours at 4°C, and then centrifuged at 4°C for 20 min at 20Og for serum isolation. Serum fractions were stored at -80⁰C until analyzed. Protein assay was performed using the standard Bradford method. CDA activity was assayed following a simplified spectrophotometric method modified from the literature, based upon the release and detection of ammonium from cytidine [9]. The test protocol was as described above. Standard solutions were composed of ammonium chloride in water, ranging from 0 to 40 U of enzymatic activity, with 1 U = 4.10^"3 μMol of ammonium released per min and per ml of serum. Final results were expressed as U/mg proteins.

CDA genotypic assay:

Genomic DNA were obtained after standard extraction from whole blood with the QIAamp DNA blood extraction kit (Qiagen, France). Genomic DNA were quantified spectrophometrically at 260nm, and 10 ng were used for each PCR run. The three most relevant polymorphisms, i.e. 79A>C, 208G>A, 435T>C, were checked by high resolution melting (HRM) analysis [10]. Primers were designed in order to yield small amplicons containing putative sequence variation. PCR and post-PCR melting of double-stranded amplicons were carried out on the Lightcycler™ 480 instrument (Roche-Diagnostics, Meylan, France) using the HRM mastermix™ according to the manufacturer procedure.

RESULTS

Performance of the phenotypic method: Mean equation describing the signal/activity relationship was o.d (630 nm) = 0.025^*activity + 0.000823, with a mean r² of 0.9991 (n=5). lntraday and interday precisions at LOQ (1.25U) were 9.9 and 12.1%, respectively, lntraday and interday accuracies were -2.4 and -5.1%, respectively (n=5). CDA activity in whole blood proved to be stable at least 6 hours at 4°C. CDA activity in serum proved to remain stable for at least 4 month at -80⁰C. Moderate haemolysis did not affect CDA activity in the samples. Reference subset:

Distribution of the CDA activities recorded in the reference group is displayed in Figure 1. Mean of the recorded values was 3.6 +/- 1.6 U/mg protein. Kolmogorov-Smirnov test showed a normal distribution (K-S=O.115, p=0.17). Conjointly, genotypic studies were undertaken, and the three most relevant polymorphisms (79A>C, 208G>A, 435T>C), were checked by HRM (Figure 2). The non synonymous single nucleotide polymorphism (SNP) 79A>C resulting in a Lys27Gln amino acid change was found in 48% of the reference patients. The synonymous SNP 435T>C was found in 63% of the patients (50% heterozygotes and 13% homozygotes, respectively). Overall, 14 reference patients displayed both 79 A>C and 435T>C mutations. Conversely, none of the reference patients was bearing the 208 G>A mutation. No significant relation was observed between the presence/absence of these three mutations and final CDA activities (p=0.941 , One-Way Anova). Toxic-Death case: The female patient displaying severe toxicities was sampled soon before she died (D21 , 13 days after her last gemcitabine administration) while receiving supportive care in the ICU unit. Evaluation of her CDA activity in serum showed a value of 0.9 U/mg, about 75% lower than the mean value of the reference, non-toxic subset (Figure 1 ). Screening for genetic mutations on her CDA gene revealed a heterozygoty for the 79A>C SNP. Further search for the other SNPs (e.g., 208G>A, 435T>C) in this very patient was negative.

Taken together, these data demonstrate for the first time that CDA deficiency could be at the origin of a toxic-death case upon gemcitabine administration. So far, only the 208G>A SNP was associated with increase of treatment-related toxicities. Here, no such mutation was found, despite dramatic loss of CDA activity and subsequent lethal toxicities after gemcitabine exposure. This discrepancy suggests that the 208G>A mutation may not be as clinically relevant as once thought to detect patients at risk, at least in non-Japanese populations. Finally, the inventors showed that this simple, cheap and rapid phenotypic method is a convenient and unambiguous approach for anticipating CDA-deficiency-driven severe/lethal toxicities with gemcitabine, a major anticancer drug extensively used in clinical oncology. REFERENCES

1. Gilbert JA, Salavaggione OE, Ji Y, Pelleymounter LL, Eckloff BW, Wieben ED, et al. Gemcitabine pharmacogenomics: cytidine deaminase and deoxycytidylate deaminase gene resequencing and functional genomics. Clin Cancer Res. 2006; 12(6): 1794-803.

2. Fitzgerald SM, Goyal RK, Osborne WR, Roy JD, Wilson JW, Ferrell RE. Identification of functional single nucleotide polymorphism haplotypes in the cytidine deaminase promoter. Hum Genet. 2006;119(3):276-83.

3. Sugiyama E, Kaniwa N, Kim SR, Kikura-Hanajiri R, Hasegawa R, Maekawa K, at al. Pharmacokinetics of gemcitabine in Japanese cancer patients: the impact of a cytidine deaminase polymorphism. J Clin Oncol. 2007;25(1 ):32-42.

4. Kirch HC, Schroder J, Hoppe H, Esche H, Seeber S, Schutte J. Recombinant gene products of two natural variants of the human cytidine deaminase gene confer different deamination rates of cytarabine in vitro. Exp Hematol. 1998;26(5):421-5.

5. Schroder JK, Kirch C, Seeber S, Schutte J. Structural and functional analysis of the cytidine deaminase gene in patients with acute myeloid leukaemia. Br J Haematol.

1998; 103(4):1096-103.

6. Yue L, Saikawa Y, Ota K, Tanaka M, Nishimura R, Uehara T, et al. A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity. Pharmacogenetics. 2003; 13(1 ):29-38.

7. Yonemori K, Ueno H, Okusaka T, Yamamoto N, lkeda M, Saijo N, et al. Severe drug toxicity associated with a single-nucleotide polymorphism of the cytidine deaminase gene in a Japanese cancer patient treated with gemcitabine plus cisplatin. Clin Cancer Res. 2005;1 1 (7):2620-4.

8. Mercier C, Ciccolini J, Pourroy B, Fanciullino R, Duffaud F, Digue L, et al. Dose individualization of carboplatin after a 120-hour infusion schedule: higher dose intensity but fewer toxicities. Ther Drug Monit. 2006;28(2):212-8.

9. Okamura T, Kigasawa K, Takeuchi T, Ohta C. Cytidine deaminase activity in abnormal pregnancy, lnt J Gynaecol Obstet. 1993 Apr;41 (1 ):53-60. 10. Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, Voelkerding KV. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem. 2006;52(3):494-503

Claims

1. An in vitro method for determining the risk of toxicity upon a chemotherapeutic agent intake in a patient afflicted with a cancer, wherein metabolism of the chemotherapeutic agent involves cytidine deaminase (CDA), which method comprises determining the CDA activity in a biological sample of the patient, wherein an alteration in CDA activity compared to a control value is indicative of a risk of toxicity upon the chemotherapeutic agent intake.

2. The method of claim 1 , wherein the alteration in CDA activity is a deficiency.

3. The method of claim 1 or 2, wherein the chemotherapeutic agent is selected from gemcitabine, Ara-C, CNDAC, decitabine and tezacitabine.

4. The method of claim 3, wherein the chemotherapeutic agent is gemcitabine.

5. The method of claims 3 or 4, wherein the chemotherapeutic agent is gemcitabine , Ara-C, CNDAC, decitabine or tezacitabine, and a CDA activity inferior to about 1 CDA activity unit per milligram of serum sample total protein (1 U/mg) is indicative of a risk of severe toxicity upon gemcitabine, Ara-C, CNDAC, decitabine or tezacitabine intake.

6. The method of claim 1 , wherein the alteration in CDA activity is an extensive CDA activity.

7. The method of claim 1 or 6, wherein the chemotherapeutic agent is selected from capecitabine and DMDC.

8. The method of claim 7, wherein the chemotherapeutic agent is capecitabine or DMDC, and a CDA activity superior to about 8 CDA activity units per milligram of serum sample total protein (8U/mg) is indicative of a risk of severe toxicity upon capecitabine or DMDC intake.

9. The method of any of claims 1 to 8, wherein the CDA activity is determined by spectrophotometry.

10. The method of claim 9, wherein the CDA activity is determined by visible spectrophotometry.

1 1. The method of claim 9 or 10, wherein the CDA activity is determined by measuring the amount of ammonium released through conversion of cytidine into uridine, by spectrophotometry.

12. The method of claim 1 1 , which comprises the step consisting of: a. incubating the biological sample with cytidine; b. setting up a calibration curve of ammonium to be incubated similarly with the sample; c. precipitating proteins so as to stop the reaction ; d. centrifuging and recovering the upper layer; e. incubating the recovered upper layer of step d) with a mixture of phenol and sodium hypochlorite and recovering the upper layer comprising the ammonium; f. detecting ammonium in the recovered upper layer of step e) with a spectrophotometer; g. calculating the CDA activity, in regard with the signahactivity relationship generated by the calibration curve and the amount of proteins in the sample.

13. A kit for determining the risk of toxicity upon a chemotherapeutic agent intake in a patient afflicted with cancer, the metabolism of the chemotherapeutic agent involving cytidine deaminase (CDA), which kit comprises : i. a container comprising cytidine; ii. a container comprising ammonium; iii. a leaflet which describes a method for determining the cytidine deaminase (CDA) activity in a biological sample, by measuring the amount of ammonium released through conversion of cytidine into uridine by spectrophotometry, wherein the leaflet indicates that a CDA activity inferior to about 1 U/mg, in a serum sample, is indicative of a risk of a severe toxicity upon intake of a chemotherapeutic agent the liver elimination of which involves CDA, whereas a CDA activity superior to 8 U/mg, in a serum sample, is indicative of a risk of a severe toxicity upon intake of a chemotherapeutic agent the in vivo activation of which involves CDA.

14. The kit of claim 13, wherein the chemotherapeutic agent the liver elimination of which involves CDA is selected from the group consisting of gemcitabine, Ara-C, CNDAC, decitabine and tezacitabine.

15. The kit of claim 13, wherein the chemotherapeutic agent the in vivo activation of which involves CDA is selected from the group consisting of capecitabine and DMDC.

16. The kit of anyone of claims 14 to 16, further comprising: i. a container comprising sodium and phosphate buffers; ii. a container comprising phenol and nitroprusside; iii. a container comprising tungstate; iv. a container comprising hypochlorite; and v. a container comprising sulphuric acid.