Lisa Simi Scienze Biomediche Sperimentali e Cliniche

1. SNPs analysis in DPYD gene for primary prevention of severe toxicity induced by 5 fluorouracil treatment in cancer patients Lisa Simi Scienze Biomediche Sperimentali e Cliniche

2. DPYD Dihydropyrimidinedehydrogenase 5-FU (5-FLUOROURACIL) DPD is the rate-limitingenzymefor fluoropyrimidinecatabolism <80% Enzymaticlevels show high inter and intraindividualvariationinfluencingresponseofpatientsto 5-FU in termsoftoxicity, resistance and efficacy. • 5-FU treatment in individualdeficient in DPYD expression can cause: • Myelosuppression • Neurotoxicity • Mucositis • Hand-footsyndrome • Dyarrhea 10-30% toxicity 0,5-1% mortality Single NucleotidesPolymorphisms (SNPs) in DPYD gene predictiveriskalleles for developing severe toxicity to the commonlyprescribedanticancerdrug 5-fluorouracil

3. DPYD gene: 23 codingexons on chromosome 1p22 DPYD DPYD*2A (IVS14+1G>A; c.1905+1G>A) is the mostwellstudiedvariant: SNPs at the intronboundaryofexon 14 thatresults in a splicingdefect–skippingof the entireexon- non functional DPD. c.1679T>G and c.2846A>T are relatively rare butresult in low DPD activity Allelicfrequency of eachvariantisstrictlyrelated to the population, the 3 variants IVS14+1G>A, c.1679T>G and c.2846A>T are mostlikely to be detected in individuals of europeansancestrybut are far lesspenetrant in otherracialgroups. Vol 94 NO6- DEC2013-Clinical Pharmacology and Therapeutics • Up tonow more than 100 variantshavebeendescribed on this gene.

4. 128 missensevariantsreported 3 variantshaveconsistentlybeenreported to be associated with 5FU toxicity and impaired DPD enzymeactivity More than 100 additionalvariantshavebeenreportedthatmaycontribute to DPD deficiencyespecially in populationsnot of Europeanancestrythathavebeen under represented in large case-control clinicalassociationstudies of 5FU toxicity *2A like *13 like D949V like

5. QUASAR2 Quick and Simple and Reliable trial Importantto note: data fromthis trial are relative to a largenumberofindividualssubmittedexactlyto the sametherapy Formallysignificantassociation of *2A and c.2846A with global toxicity for capecitabine Exclusionofseveralunwarranted SNP from the currentlyavailable FU toxicity test Thereisstill the need to identify and characteriseadditional 5-FU variants to closely monitor patientswho are at increasedrisk of toxicityor to increase 5-FU dosage in thosewho are at low risk of toxicity

6. https://www.pharmgkb.org/

8. In ourlaboratory an optimised HRMA-basedprotocolhavebeendefined in the last twoyears to detectpresence of SNP in the exon 14 and relative flankingregions Primary prevention of toxicity in patients that will begin the treatment with 5-FU

9. High ResolutionMelting Analysis provide a reliableapproach to screening DNA variants with high specificity and reducedcostthanothermethodsallowing the detection of differentvariants Actually the insertion of the detection of the variants c.2846A>T and c.1679T>G in the primarypreventionprotocolisstill in progress

10. RESULTS Total 611 samples Total of 35 variantsdetected in thisstudy Secondarily to the screening of *2A, the detection of 4 variants (c.2846A>T, c.1679T>G, c.1627A>G and c.1601G>A) havebeenperformed in order to include in the analysisalso the recentlyrecommendedvariants to be detected in primaryprevention of toxicity (CPIC guidelines)

11. After the analysis of the intronicvariant, a deepercharacterization of SNPs in DPYD gene isstill in progress with a twiceaims: 2 Analyzeindividualsheterozygous for *2A thatdeveloped a differentresponse to the sametherapy to identifypossibleassociationbetween more thanoneSNPs and treatment response (data from a retrospectivestudy ) Analyzeindividuals wild type for the *2A variant(576/611) and thatunderwenttoxicities, to assess the relevanceof otherSNPs. Evaluation in progress, preliminary data relative to 125/576. Listofvariantsanalysed

12. Up to now 8%,10 individuals relative to 125/576 wild type for *2A (follow up data still under collection) experiencedtoxicity. Amongthem: Variantsreportedas “low level of evidence”: variant-drug combination evaluated in multiple studies but lacking clear evidence of an association with toxicity

13. Sample #2 Phenotypicrelevance of «lowlevel of evidence» variantsmayincreasewhen in association with the SNPs with an high impact on DPD functions IVS14+1G>A c.1627A>G Forwardsequence Reverse sequence

14. Conclusion • The relevanceof *2A detection in individualsselectedfor 5-FU therapyisconfirmed and actually the detection of on c.1679 and c.2846 isstronglyrecommended • To date predictivetestsof 5-FU toxicityhavehadlimitedvalueascarriersofknownDPD-deficiency-associatedalleles (*2A, I560S and D949V), althoughrepresents the mostadversereactionto 5-FU, constitute a relativelysmallpercentageoftoxicitycases. • More efforts should be performed for the definition of a more specific and informative panel of SNPs in DPYD gene by using a time-cost effective methodological approach suitable to detect variants for the primary prevention of toxicity • It should be reminded that the final aims of DPYD genetic test is to prevent the most severe and fatal instances of toxicity, but also to give a preliminary information for the best selection of the adequate therapy , suggesting a reduction of dose in carriers individuals but also the possibility to use personalised protocol in those at low risk of toxicity, in order to obtain a maximum effectiveness of cancer therapy

15. SCIENZE BIOMEDICHE SPERIMENTALI E CLINICHE University of Florence Prof. Mario Pazzagli Prof. Claudio Orlando Dr. Pamela Pinzani Dr. StefaniaGelmini Dr. Irene Mancini Dr. Francesca Malentacchi Dr. Francesca Salvianti Thankyouverymuch for yourattention!!!