Clinical applications of NIPT; Our experience in an NHS Regional Genetics Lab Fiona Togneri Principal Clinical Scientist West Midlands Regional Genetics Laboratory

As many of you will know, at the start of 2016, the UKNSC recommended evaluative implementation of NIPT into the FASP for NHS patients with high screen risk results from standard ANS

WMRGL Implementation In anticipation of this recommendation, in Dec 2014 we released a tender for selection of the most appropriate NIPT technology before the tender was awarded to Illumina for their Verifi test and our service was launched in September 2015

1000 sample experience To date we’ve processed more than 1000 patients through our service with a technical failure rate of just 0.24%, this represents 2 patients, one of whom failed due to biological reasons of trisomy for chromosome 7 and therefore only one patient sample to date has failed due to technical processing issues, highlighting this as an extremely robust assay 0.24% technical failure rate from clinical service

11/ 31 (35%) continued without any confirmations studies Performance 11/ 31 (35%) continued without any confirmations studies One False positive T18 patient (CPM) Trisomy for 18p was observed in placental villi from this patient following birth Two false positive T13 patients Both double aneuploidies Indication for lower confidence result and follow-up by AF Our service shows extremely competitive performance statistics but this is a screening test as opposed to diagnostic and therefore false positive and false negative results are observed To date we’ve observed 1 FP trisomy 18 result with the cause confirmed as being due to CPM and trisomy for 18p observers in placental Villi following birth 2 FP trisomy 13 results have also been observed and although placental material was not available for these pregnancies, CPM is also our hypothesised cause for these false results. Interestingly, both of these were double aneuploid results and this supports double aneuplodies as indicators for lower confidence NIPT results that should be followed up by AF. Although no true FN results have been observed, unfortunately one patient reported as being unlikely to be affected with trisomy following NIPT through our service was later diagnosed at birth with mosaicism for trisomy 21. This unfortunately is a limitation of this testing with the hypothesised cause being that the trophoblast cells of the placenta (where the cffDNA originates) are non-trisomic. To date we have correctly identified 31 pregnancies with trisomy 21 and what is interesting is that in 35%, families have chosen to continue their pregnancy without invasive confirmations, with instead diagnoses being confirmed following birth. This percentage is consistent with recent literature and historic figures and supports to disprove the perception in the popular media that introduction of NIPT will lead to a decrease in patients being born with Down syndrome. *One euploid patient was diagnosed following birth as having mosaicism for trisomy 21 (limitation of the test)

Metrics I’d like to go back to this key metrics table and concentrate on the samples referred to our clinical service...

Robertsonian translocation Referral Indications Robertsonian translocation in parent 1% Missed screening 1% To highlight one thing which is that although our service was launched in anticipation of UKNSC recommendations for provision of NIPT for high screen risk patients, these actually represent just half of the samples referred for this testing currently

(49.4% of referrals) (48.4% of referrals) (2.2% of referrals) Screening risk And the pattern of sample referrals varies according to gestational age. Many patients will chose to access NIPT in the first trimester as a first line screen, as an alternative to waiting on current NHS ANS Also many known robertsonian translocation carriers and patients with a priori risk from a previous trisomic pregnancy will access this testing via our clinical genetics service as soon as possible after the 10 weeks minimum gestation threshold The majority of patients with high screen risk results will then access testing in the 2nd trimester as well as an increasing number of patients with abnormal ultrasound scan findings And these patients then represent by far the most common indication for NIPT in the third trimester

Robertsonian translocation Referral Indications Other applications Robertsonian translocation in parent 1% Missed screening 1% So as mentioned previously, just 50% of samples referred to our NIPT service are referred due to high risk from ANS results And what I’m most interested in for this presentation are the other 50% not covered by current recommendations and I’ll spend the rest of this presentation discussing these other current and future possible applications of NIPT + potential future applications

Ultrasonographic findings 10% of referrals to NIPT Clinical Service Patients who decline invasive sampling (gold standard) Maternity Hospital with specialised fetal medicine centre Late gestation Pregnancies at 28 weeks gestation with MCA Patient 1- trisomy 18 confirmed at birth Patient 2- trisomy 13 confirmed following stillbirth at 31+3 weeks Testing to inform management at delivery and in neonatal period I particular I’m interested in the patients with abnormal scan findings who currently represent 10% of referrals to our service These are crucially all patients who have declined invasive sampling which of course represents gold standard testing for these patients Many of these patients are referred for NIPT at extremely late gestations, such as these 2 patients referred with MCA at 28 weeks gestation identified with Edwards and Patau syndromes respectively. Such prenatal testing in these patients, who have previously declined invasive sampling has been acknowledged as greatly informative by both clinicians and patients. To inform the most appropriate management of these patients during delivery and also most appropriate palliative care for their children following birth. With the Patau syndrome child mentioned for example, this prenatal diagnosis allowed things to be in place for the family to take the child home in a cold cot and spent some precious time together as a family.

Ultrasonographic findings Patients unlikely to opt for TOP Patient 3 Increased NT at 11 weeks gestation (>1 in 5 screen risk) Trisomy 13 by NIPT continuation of pregnancy Result confirmed following IUD 18/40 Truncus arteriosus, VSD, cleft palate, polydactyly 146 patients (PAGE and clinical service) 19 with isolated markers only 127 fetal structural anomalies Something that many of these patients have in common is that they are extremely committed to their pregnancies irrespective of outcome. And a patient who highlights this is patient 4. Identified with an increased NT at 11 weeks gestation and reported as being highly likely to be affected by trisomy 13 following NIPT. Despite this result, the family opted to continue this pregnancy without invasive sampling to confirm and this result was then confirmed following IUD at 18 weeks with complex scan findings In total 146 patients with abnormal scan findings have been analysed by NIPT at our laboratory, these are both patients recruited as part of the Page fetal exome sequencing project for NIPT validation studies and also samples receieved as part of our clinical service and analysis of these patient indications against results allows for determination of high and low risk scan findings

NIPT results following abnormal ultrasound scan findings (n=146) 75% trisomy 50% trisomy 21 (duodenal atresia) 29% trisomy 46% trisomy This graph shows the number of pregnancies along the bottom referred with each of the scan indications noted down the left hand side. Pink lines indicate negative NIPT results and blue lines, positive. Ant abd wall defects, gastrointestinal defects, mca and increased NT represent high risk scan findings for aneuploidly while isolated markers, skeletal defects and babies small for gestational age represent weaker indications

Beyond aneuploidy screening Patient 4 Invasive sampling at 13+1 weeks gestation with NT 6.7mm Consented to PAGE project and sample taken for NIPT verification studies (2015) CVS result by microarray and reflex cytogenetics 46,XX,der(6)t(3;6)(q21;q25)mat 71Mb deletion at 3qter and 10.5Mb deletion at 6qter Of course when studying fetal structural anomalies, aneuploid screening is a bit of a backwards step, just because patients with skeletal defects are not then being diagnosed with trisomy via NIPT, it doesn’t mean that there’s no genetic cause,really we need to be looking further into the genome and that is where we would like to go. The fourth patient Id like to present who shows you some of the possibilities here and was recruited to the PAGE study and a maternal blood sample taken for NIPT validation studies. Analysis of CVS material by microarray and reflex cytogenetic studies showed a derivative chromosome 6 from a balanced maternally derived translocation between one chromosome 3 and one chromosome 6. With significant imbalance of 3qter and 6qter

WIthin-SamplE COpy Number aberration DetectOR (WISECONDOR) These images show the array data from this patient. NIPT analysis in this instance was by the freely available whole genome analysis pipeline wisecondor that conducts segmental copy number aberration detection. Ana as you can see both imbalances were clearly detected. This work was done by Emily Colley who’s a PhD student in our lab and the reason Emily was interested in identifying unbalanced translocations by NIPT is that she’s currently looking into another potential future application of NIPT which Id like to move onto now and that is for couples experiencing miscarriage Work by Emily Colley

Application of NIPT in Miscarriage One in 5 pregnancies 1% recurrent miscarriage POC analysis Not always successful ?? NIPT What are the levels of cffDNA in early miscarriages? Can cffDNA be used to reliably detect fetal aneuploidies and other chromosome abnormalities? 1 in 5 pregnancies will unfortunately end in miscarriage and one percent of couples experience miscarriage will go on to have recurrent miscarriages. Currently green top guidelines are for poc analysis but as many of you will be aware, sadly this process is not always successful and we wondered therefore could be use NIPT here? Work by Emily Colley

Trisomy 15 POC microarray Miscarriage at 7 weeks POC microarray NIPT from mat blood taken at diagnosis of miscarriage Emily has very kindly agreed for me to show you an example from her provisional results. This was a lady who miscarried at 7 weeks gestation. POC analysis was successful and this showed trisomy 15, a blood sample for POP analysis was also taken at diagnosis of miscarriage and this excitingly correctly identifies the imbalance. Work by Emily Colley

Conclusions Initial NHS funding for the evaluative roll out of NIPT is for patients with high screen-risk results. ~50% of referrals to the current service Data presented here highlights groups of patients with other clinical indications for NIPT that will not be covered by this clinical pathway. Translocation carriers, patients with structural defects on scan, miscarriage families…. Beyond aneuploidy detection (?exome)

Acknowledgements Tommy’s Project Team Emily Colley Stephanie Allen WMRGL Prenatal and RM Team Stephanie Allen Elizabeth Young Mike Griffiths NGS Team BWH Fetal Medicine Team Mark Kilby Clinical Genetics Service Denise Williams Illumina Inc. Marion Burdin Tommy’s Project Team Emily Colley Stephanie Allen Arri Coomarasamy Data recently submitted for publication