NEONATAL SCREENING FOR CYSTIC FIBROSIS. PAST AND FUTURE. Pilot Study and RECENT INFORMATION EVA PCMA vzw Antwerp-Belgium.

F. Eyskens, MD, PhD 1,2 1 UZA, 2 PCMA vzw, Antwerp, Belgium

Neonatal Mass Screening in Flanders from 01/01/2012 Ministry of Health of the Flemish Community PCMA vzw VCBMA 40.000-50.000 newborns screened/lab/year 40.000-50.000 newborns screened/lab/year

Phenylketonuria/Hyperphenylalaninemia (1968) Congenital Hypothyroidism (1981) Congenital adrenal hyperplasia (2003) Biotinidase deficiency (2007) MCADD, MADD, MMA, PA, GA1, IVA, MSUD (2007) Proposed selective screening Galactosemia New pilot screening programs: Lysosomal Storage Diseases Proposed mass screening program Duchenne Muscular Dystrophy (stop 2012; approval needed) Doctoral thesis university of Antwerp 1997 * Pilot study from 1 may 2002-30 april 2004, implemented in Flanders Screening Program from January 2007 Cystic Fibrosis IRT (stop: 2003)

CRITERIA for Screening Programs (WHO: Wilson & Jungner) Important health problem Asymptomatic stage in natural evolution Accepted treatment for patients Parents are in favour to screen for «untreatable disease(s)» Secondary prevention by genetic counseling and prenatal diagnosis Facilities for further diagnosis Suitable test available: simple, reasonably priced, repeatable, sensitive, specific, acceptable Natural history of the disease is well known Screening should be a continuous process

Verdeling van de verschillende diagnoses gesteld met de neonatale MASSASCREENING in de provincie Antwerpen (percentueel jaarlijks aandeel). PKU BIOT CAH PKU CH CAH Duchenne Biot.def. DUCHENNE CH

Congress Caen Prof. G. Travert Results: Brittany, France East Anglia, UK Antwerp, Belgium

IRT SCREENING Crossley et al, 1979 Pancreas enzyme precursor; spill over in blood due to obstruction of pancreatic ductuli Increased in blood in case of: CF Perinatal stress Renal insufficiency Trisomie 13 Methods: (1979-1988: RIA Gnost Trypsin test) 1988-1990: AGEN trypsinogen Neoscreen 1990-1997 : Delfia-IRT

METHODOLOGY ImmunoReactiveTrypsin(ogen) test: Delfia IRT: Best screening test available in blood spots Intra-assay VC%: 3.9; Interassay VC%= 7.1* Best reproducibility *validity for values of 70 ng/ml (cut-off value).

METHODOLOGY: PROBLEMS Calibration watery solutions delivered with the kit Every kit (different lot numbers) had to be evaluated while the cut-off value changed so that 97th centile values for age should be used instead of absolute values as cut-off IRT calibrators in blood spots are instable, even when stored at 20 C IRT levels in blood spots are influenced by storage

CUT-OFF VALUES FOR THE INTERPRETATION OF IRT LEVELS The decline or IRT values with age was studied in 102 infants with an initial IRT value above the 97th centile of age and in whom repeat samples were obtained after 14-20, 21-30 and 31-90 days By plotting the log of the geometric mean values again the log of the mean ages of repeat testing, a straight line was obtained with the following regression equation: y= -69.84 logx + 131.6 (r-0.98)

IRT SCREENING VOOR CF: COMPARISON OF INITIAL AND FOLLOW-UP VALUES IN CF PATIENTS AND FALSE POSITIVES.

IRT SCREENING: RESULTS Total number screened (1987-1999): 372,674 CF patients found: 92 Prevalence: 1:3,900 (95th confidence interval: 1:2,900-1:4,800) False positives: 2;445 (0.7-1.25%) False negatives: 8 (Delfia: 6) Sensitivity (%): 92 (Delfia: 87.2) 87% in Wisconsin using the IRT/IRT screening approach Specificity (%): 99.3 Positive predictive value: 3.6 % Negative predictive value: 99.99%

IRT SCREENING: PROBLEMS Only serum calibrators available Stability of IRT (storage at +4 C)- Thermolabile IRT component High false positive rate-cut-off? Relative high false negatives Not all patients are found!

THE RATE OF FALSE NEGATIVE RESULTS WOULD NOT BE ACCEPTED IF CF SHOULD BECOME A TREATABLE DISEASE. WHAT ABOUT CARRIER DETECTION?

RESULTS OF DNA ANALYSIS Homozygote for F 508: 61% Heterozygote for F 508 and another known mutation: 12% Heterozygote for F 508 and an unknown mutation: 12% Heterozygote for another known mutation: 3% No mutation found: 12%

WISCONSIN CF SCREENING PROGRAM IRT testing alone: sensitivity: 87% IRT/ F 508: sensitivity: 92% IRT/ multiple (25)mutation analysis: sensitivity: 99%

CF screening USA

CF screening USA Two-tier screening strategy: screen for several different mutations: 15?-30? LESS or MORE??

CF screening Switzerland 2011-20 Dtsch Arztebl Int 2013; 110(20): 356-363

FAILSAFE

INFLUENCE OF STORAGE AT 20 C DURING 1 MONTH ON IRT LEVELS Age Nr Before After P value 3-9 47 µ: 82 (68-133) 14-20 11 µ: 47 (28-131) 21-30 25 µ: 36.5 (11-70) 31-90 11 µ: 30 (17-58) µ: 80 (53-127) µ: 49 (26-99) µ: 36 (11-71) µ: 31 (11-71) < 0.005 0.6<P<0.7 0.6<P<0.7 0.4<P<0.5

THERMOLABILE IRT COMPONENT Literature: IRT immunoasay detects several forms of trypsinogen isoenzymes and trypsin Dhondt et al: the decline of IRT with age is different in CF patients compared to patients with persistent hypertrypsinemia Our finding: Thermolabile IRT component present in blood spots taken the first 10 days of life seems to be specific for CF

Switzerland IRT/DNA (7 mutations) Results of one-year evalutation

Comparison: Switzerland IRT/DNA)- Antwerp IRT/IRT Total number screened (2011): 83,198 Cut off IRT: P99.2 CF patients found: 30 Prevalence: 1:3,600 False positives: 54 (617 IRT only)(0.65%) False negatives: 1 Sensitivity (%): 96.8 Specificity (%): 99.9 Positive predictive value: 36 % Total number screened (1987-1999): 372.674 Cut off IRT: P97 CF patients found: 92 Prevalence: 1:3,900 (95th confidence interval: 1:2,900-1:4,800) False positives: 2.445 (0.7-1.25%) False negatives: 8 (Delfia: 6) Sensitivity (%): 92 (Delfia: 87.2) 87% in Wisconsin using the IRT/IRT screening approach Specificity (%): 99.3 Positive predictive value: 3.6 %

Commonest CF gene mutations; Belgium-Switzerland F508del 3905insT G542X R553X W1282X 1717-1G>A N1303K F508del G542X R553X W1282X 1717-1G>A N1303K 3272-26G>A (R117H) S1251N 2789+5G>A

Information given to parents! GEINFORMEERD KIEZEN/ INFORMED DISSENT Explicit parental consent is not necessary for mandated public health newborn screening, if the asssay used meets the following criteria: Analytically and clinically validated Clinical utility is documented Treatment is available to prevent or reduce adverse outcome of condition (ref.: Good Laboratory Practices for Biochemical Genetic Testing and Newborn Screening for Heritable Diseases. CLIAC Meeting February 9-10, 2010,USA; gepubliceerd document door B. Chen, CDC, Atlanta, USA ).

CF screening in Switzerland: opting-out screening strategy Even when DNA analysis is performed: Explicit parental consent is not necessary DNA results obtained in the sceening center are NOT communicated to the CF reference centers

Improving early diagnostics, diagnosis and start of treatment by CF screening (Switzerland): Before NBS: From birth to genetically confirmed diagnosis of CF: 198 days (range: 13-1307) After NBS was implemented: From birth to genetically confirmed diagnosis of CF: 34 days (range: 13-135)

CF diagnosis in Belgium (without newborn screening) 59%

KCE report 132B (2010): Should we screen for cystic fibrosis in Belgium?

IRT D 3-5 120.000 Report: CF not suspected IRT < 99.5th centile IRT >99.5th centile 600 * No consent for DNA analysis DNA analysis (commercial kit) * 60 (10%) ± 600 15 45 540 (90%) Two CF mutations * One CF mutation * No mutation detected * F508del/ F508del Other IRT D3 < 99.9th centile Report: CF not suspected IRT D3 >99.9th centile 120 15 IRT D21 > 40 µg/l IRT D21 ** IRT D21 < 40 µg/l ± 90%? ± 10%? [Cl-] (mmol/l) 60 60 30-59 60 30-59 <30 60 30-59 <30 SWEAT TEST CFTR gene sequencing * 10 5 5 2 0-1 * i) Results will have to be available within 3 weeks. ii) In order to limit the number of equivocal cases, R117H and several other mutations will not be reported, depending on what will be known from the CFTR2 project on their impact on CFTR function (cut-off percentage of activity to be determined). iii) In vitro characterization of the impact of the very rare mutations that are not described in the the CFTR2 database to be considered. ** if not controlled at D30 (recall failure), perform a sweat test.

In order to be efficient and fluid, most information between labs, CF centres and the supervising structure will be transmitted in electronic form. M a t e r n i t y Midwife - blood sampling - consent for DNA testing Paediatrician Refer for sweat testing Supervising structure NN Screening labs (6) «CF suspected» Contact the pediatrician within 1 month and provide him (her): - the list of hospitals accredited for the sweat test - the list of the 7 accredited CF centres Department of Genetics * Complete - very simple screening cards when «CF not suspected» - detailed screening cards in other cases Send them to the supervising structure Confirm to the competent Community Institution that payment is due to the NN screening lab for «n» analyses Competent Community Institutions 2 mutations 0-1 mutation Belgian CF Registry Other lab accredited for the sweat test Sweat Chloride < 30 mmol/l Sweat Chloride 30 mmol/l - confirm (or not) the diagnosis a(within a short delay) - proceed to further investigation as needed - start treatment (if indicated) - provide genetic counseling (as indicated) Accredited CF centres (7) * The link between each NN screening lab and a Department of Genetics shoud be fluid and tight. This is straightforward in the French Community (3 Universities each including in one place a NN screening lab, a Department of Genetics and an accredited CF centre). Things will be more complicated in the other Communities.

Supervising structure Pulmonologists ABLM-BVSM 1) Coordinate information about the program Paediatricians Medical literature, congresses 2) Continuous process towards algorithm s improvement Acredited CF centres Others (GP, fertility clinics ) Complete screening cards and transmit them to the supervising structure: Include detailed feedback if indicated: - sweat test (where? Results?) - final diagnosis (which? where?) - other NN Screening lab In case of «CF suspected», contact the CF centre to make sure that extensive information is available 3) Collect and manage the full sets of data (database) Confirm to the competent Community Institution that payment is due to the NN screening lab for «n» analyses Competent Community Institution Payment for: - x IRT dosages - y DNA analysis Competent Community Institutions Medical Committtee of ABLM-BVSM NN Screening labs Accredited CF Centres Belgian CF Registry 4) Publish annual results Accredited CF centres 5) Collect information about false negative Other Sweat test 6) Ensure external quality control IRT DNA testing

NN Screening Labs (n:6) M a t e r n i t y Midwife - blood sampling - consent for DNA testing 2) - Organize ASAP DNA testing if indicated 3) - Classify the results of the screening process and - Notify before Day 30 CF not suspected Internal quality control 1) - Perform IRT dosages (Day 3-5 & 20 if indicated) Very simple negative screening card Internal quality control DNA testing Department of Genetics No sample received Detail Failure to recall Detail IRT2 > Day 20 Detail + data about the sweat test (where? Results?) Supervising structure Negative screening card CF suspected Contact the pediatrician within 1 month and provide him (her): - the list of hospitals accredited for the sweat test - the list of the 7 accredited CF centres Include detailed feedback about sweat testing (where? results?) and referral (where? Conclusions?) Positive screening card Paediatrician M a t e r n i t y 4) Forward payment for DNA testing from competent Community Institution to the Department of Genetics

NN Screening lab Accredited centres (n:7) CF suspected 1) Provide expertise in sweat testing Internal quality control Sweat Chloride < 30 mmol/l Other lab accredited for the sweat test Sweat Chloride 30 mmol/l 2) - confirm (or not) the diagnosis (within a short delay) - proceed to further investigation as needed - start treatment (if indicated) - provide genetic counselling (as indicated) Belgian CF Registry M a t e r n i t y 2 mutations Paediatrician Refer for sweat testing 0-1 mutation 3) Provide any additional information requested by the - NN screening lab or - the supervising structure

Pancreatitis-Associated Protein (PAP) has been reported to be elevated in newborn infants with CF Sarles et al I Pediatr. 147, 302-305 2005 Pancreatitis-associated protein (PAP)- a screening marker for CF? A lectin-related secretory protein present in small amounts in normal pancreas and over expressed during the acute phase of pancreatitis. In animal models PAP is constitutively expressed in the intestinal tract, but not in other tissues. PAP mrna could not be evidenced in liver, stomach, salivary glands, brain, kidney or testis. Its pattern of expression during severe pancreatic aggression suggests that it might be a stress protein involved in the control of bacterial proliferation. PAP has been suggested to be a marker of pancreatic sufficiency in individuals with CF

J Pediatr. 2005 Sep;147(3):302-5. Combining immunoreactive trypsinogen and pancreatitis-associated protein assays, a method of newborn screening for cystic fibrosis that avoids DNA analysis. Sarles J, Berthézène P, Le Louarn C, Somma C, Perini JM, Catheline M, Mirallié S, Luzet K, Roussey M, Farriaux JP, Berthelot J, Dagorn JC.

The benefit of the integration of the PAP test in the algorithm is still under study. This test might have the potential to further reduce the number of DNAanalyses that are required, and thus to reduce the number of detected healthy carriers and equivocal mutations.

The Addition of Pancreatitis Associated Protein (PAP) in a Two-Tier IRT/DNA Screening Strategy for Cystic Fibrosis is Less Effective in Programs that Screen at 48 hours of Age. Enzo Ranieri Biochemical Genetics Head South Australian Neonatal Screening Centre Directorate of Genetic and Molecular Pathology Women s and Children s Hospital Campus, Adelaide SA Pathology, South Australia, Australia DANGER: false-negatives increase! http://www.aphl.org/conferences/proceedings2013-newborn- Screening-Symposium

KCE report 132B: Should we screen for cystic fibrosis in Belgium? Yearly: 15,000 DNA mutation analysis of CFTR gene Carrier detection; prenatal diagnosis Costs: around 5,000,000 euro 6500 sweat tests We can screen the entire newborn population for 5 years with that budget.

Conclusion 1 Proposed Screening strategy: IRT/DNA Swiss model: second tier DNA: non-diagnostic; limited number of mutations (panel of 6-7): Failsafe measurement? Recall IRT? Highest IRT (>99.9%) without mutation

Conclusion 2 False-negatives remain! False-positives-carriers: Reference centers CF: Good psychosocial support INFORMATION to the parents of the screening perinatal-prenatal Oral information > Written information Good communication between the different actors: screening labs, reference centers CF, dept of medical genetics, pediatricians (caring physicians)

Conclusion 3 Screening strategy and process should be evaluated periodically and adjusted when and where necessary