In November 2020, a public announcement was made of a partnership between NHS England and GRAIL, an American healthcare company that has been developing a plasma DNA-based test called ‘Galleri’ for many cancer sites. Sir Simon Stevens, NHS Chief Executive, has called the blood test potentially game-changing for the early detection of cancers. The announcement states that “a pilot” “will involve 140,000 participants aged 50 to 79 who have no symptoms but will have annual blood tests for three years”. If this is correct, the application of the test would effectively be considered as a new screening method. The announcement also states that “another 25,000 people with possible cancer symptoms will also be offered testing to speed up their diagnosis after being referred to hospital in the normal way”, implying the introduction of the test into routine medical practice. It is unfortunately unclear precisely what is planned and what are the scientific objectives. The GRAIL test depends upon finding DNA markers of cancer in peripheral blood. It exploits the fact that tumour formation depends upon DNA mutations acquired during life (somatic mutations), in some cases in combination with inherited mutations. At the cellular level, gain-of-function mutations in proto-oncogenes such as C-MYC in many tumour types or BRAF in some colon cancers, together with loss-of-function mutations in tumour suppressor genes such as TP53 and BRCA1 in breast cancer, combine to disrupt a few key cellular pathways for cell-cycle regulation, cell signalling and DNA metabolism. As malignancy evolves through genomic instability and selection for cell proliferation, these socalled driver mutations accumulate together with less functionally relevant ‘passenger’ mutations. Remarkably, driver mutations in as few as about 160 genes out of the whole genomic complement account for nearly all tumorigenesis. In addition to mutations in particular genes, genomic instability results in changes in the number of copies of DNA regions, either their amplification or deletion, and hypermethylation of the DNA in many regions of the genome. As tumours develop, some altered DNA is released in the form of cell-free fragments, presumably through cell death, into blood. Early in the disease process, the altered DNA is present in plasma in very small amounts. The GRAIL test uses the most sensitive DNA analytical methods to detect these fragments and characterise them. These methods have sometimes, perhaps optimistically, been called a ‘liquid biopsy’ for a range of tumours. The use of cell-free DNA analysis in screening has great potential, but unlike its use in antenatal screening for Down’s syndrome, trisomy 18 and trisomy 13, its use in cancer screening remains uncertain. Four questions need to be answered before service pilot studies are launched. First, what is the screening performance of the GRAIL blood test for specific cancer sites expressed as detection rates (sensitivity) for specified false-positive rates for given intervals before clinical presentation? In results on DNA methylation in a case-control study, the reported detection rate was only 18% of stage 1 cancers for a 0.7% false-positive rate. Screening performance can be estimated with the data from a large prospective study adopting a nested case-control design using stored plasma samples. Second, what is the accuracy in identifying the site of the cancer in people with screen positive results among asymptomatic people? Third, what is the probability that the source of the cancer in people with screen positive results cannot be found in diagnostic investigations (e.g. magnetic resonance imaging or endoscopy) or the probability they will find unexpected abnormalities of little or no clinical significance? Fourth, what, in a large randomised trial, is the reduction in the prespecified site-specific cancer mortality rate (not survival because of lead time and length of time bias, and not cancer incidence which increases in screening due to early detection) following the treatment of people with screen positive results? The design of the study involving 140,000 people without symptoms (i.e. screening) is unclear from what is publicly available. Once there is clarity over what is planned and the above questions are addressed and published, a pilot study could be considered on many fewer people. A starting point before considering the test for screening would be to assess the test performance in patients with symptomatic stage I tumours for the individual cancer sites the test would be used to screen for. If that shows poor performance, it is likely to be even poorer in asymptomatic patients and may not therefore be worth taking further. We are concerned that the study design and aims described in the public announcement are not clear, and that the NHS may be taking on a major screening initiative without obtaining the necessary evidence on efficacy, J Med Screen 2021, Vol. 28(1) 1–2 ! The Author(s) 2020

A problem at the interface of genomic medicine and medical screening is that genetic associations of etiological significance are often interpreted as having predictive significance. Genome-wide association studies (GWAS) have identified many thousands of associations between common DNA variants and hundreds of diseases and benign traits. This knowledge has generated many publications with the understandable expectation that it can be used to derive polygenic risk scores for predicting disease to identify those at sufficiently high risk to benefit from preventive intervention. However, the expectation rests on the incorrect assumption that odds ratios derived from polygenic risk scores that are important etiologically are also directly useful in risk prediction and population screening.

Background: A source of error in prenatal screening for trisomies is PCR amplification error associated with guanine-cytosine (GC) content of DNA fragments in maternal plasma. We describe a simple method of allowing for this. Methods: Data from a Reflex DNA screening programme (67 trisomy 18 and 83 unaffected pregnancies) were used to compare the ratio of chromosome 18 DNA fragment counts to chromosome 8 DNA fragment counts (because chromosome 8 has a similar GC content to chromosome 18) with the percentage of chromosome 18 DNA counts using counts from all autosomes in the denominator, with and without an all autosome correction for the GC content of the DNA fragments. Results: A chromosome 18 to 8 ratio of DNA fragment counts was more discriminatory than the percentage of all autosome counts arising from chromosome 18 without, or with an all autosome correction for GC content bias. It achieves a high screening performance, eg. for a 0.25% false-positive rate, a 97% detection rate instead of 49% without a correction for GC content, and 91% with an all autosome correction for GC content. Conclusion: Consideration can be given to using the ratio of chromosome 18 DNA fragment counts to chromosome 8 DNA fragment counts in cell-free DNA prenatal screening for trisomy 18, avoiding the need for more complex methods of making a correction for the GC content currently used.

Background Treatments that generate T cell-mediated immunity to a patient’s unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galβ1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity. Methods Various immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT −/− ) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel–Cox test, C5a data by Mann–Whitney test, and single tumor regression by repeated measures analysis. Results In vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT −/− mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth. Conclusions We have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.

To the Editor: The prenatal reflex DNA test described in our paper1.Wald NJ, Huttly WJ, Bestwick JP et al. Prenatal reflex DNA screeningfor trisomies 21, 18, and 13. Genet Med; e-pub ahead of print 9 November 2017.Google Scholar is a method of screening that has advantages over the recall method.2.Chitty L.S. Wright D. Hill M. et al.Uptake, outcome and costs of implementing non-invasive prenatal testing for Down’s syndrome into NHS maternity care: prospective cohort study in eight diverse maternity units.10.1136/bmj.i3426BMJ. 2016; 354: i3426Google Scholar,3.Public Health England. NHS public health functions agreement 2017-18. Service specification no.16. NHS Fetal Anomaly Screening Programme—screening for Down’s, Edwards’ and Patau’s Syndromes (Trisomy 21, 18 & 13). https://www.england.nhs.uk/wp-content/uploads/2017/05/serv-spec-16.pdf. Accessed 29 November 2017.Google Scholar In the recall method as described,3.Public Health England. NHS public health functions agreement 2017-18. Service specification no.16. NHS Fetal Anomaly Screening Programme—screening for Down’s, Edwards’ and Patau’s Syndromes (Trisomy 21, 18 & 13). https://www.england.nhs.uk/wp-content/uploads/2017/05/serv-spec-16.pdf. Accessed 29 November 2017.Google Scholar a combined test is carried out and women with a risk ≥1 in 150 receive a positive result; they are invited to return for counseling and offered either a second screening test based on a DNA analysis or an invasive diagnostic test—of course, without an obligation to accept either. Walker, in her letter,4.Walker C.L. Response to Wald et al.1:CAS:528:DC%2BC1cXhtVamsb7I10.1038/gim.2017.237Genet Med. 2018; 20: 1Google Scholar does not appear to fully accept the advantages of the reflex method. Reflex DNA screening results in a higher rate of detection (95% vs. 81%) of pregnancies with trisomy 21, 18, and 13 and a false-positive rate (0.02% vs. 2.4%) about a hundred times lower than the recall method. Consequently, the reflex method means fewer women with an unaffected pregnancy are given a positive screening result, while a high detection rate is maintained. A quantitative comparison of the two methods in respect of screening for trisomy 21 is given at http://www.wolfson.qmul.ac.uk/ReflexDNAversusRecallDNA. This shows that in 10,000 pregnancies screened by either the reflex or recall methods, respectively, the following numbers apply: (i) women recalled, 0 vs. 259; (ii) invasive diagnostic tests in unaffected pregnancies, 1.8 vs. 30.3; (iii) trisomy 21 pregnancies detected, 30.3 vs. 25.9; and (iv) odds of being affected given a positive result, 17:1 vs. 1:1. These advantages of the reflex method over the recall method are clear and, contrary to Walker’s opinion, involve no extrapolation. It is untenable to suggest that worrying women and their partners with the news of a positive screening result when this is completely unnecessary is good medical practice, or even ethical. Calling women back for another test when this can be avoided causes unnecessary harm and this is always wrong. It is not an issue that is relevant to patient autonomy or choice, which is, of course, important.Our responses to Walker’s four points are as follows. (i) The decision to be screened is an option that women can, of course, discuss with their partners; obtaining consent to reflex DNA screening is no different from obtaining consent to other screening tests. (ii) Notifying women that they have a positive screening result is obviously distressing. If this can be avoided without loss of efficacy it should be done. It is not a legitimate or ethical matter for research. (iii) The duty of health professionals is to offer the most effective and safe tests or interventions that are affordable. This is not paternalistic; it is the expected duty of care. There is no withholding of information, as Walker states in relation to reflex DNA screening, because consent is obtained for the reflex test incorporating all its components. The combined test itself has several components and no one argues that separate consent should be sought for each component (nuchal translucency measurement and two blood measurements). The concept underlying the reflex DNA test is to capture the advantages of a single screening test with several components instead of performing two separate sequential tests. (iv) Avoiding unnecessary harm without loss of efficacy is always a benefit and therefore not a valid research issue.While there is agreement about using the combined test followed by a DNA test in some women, there is disagreement on how this is done. Walker holds the view that there is merit in women being told that they have a positive combined test result, and then being recalled for counseling. Given that reflex DNA screening can avoid this step entirely, and deliver a test with an improved screening performance, we see no advantage in the recall strategy.We are puzzled by Walker’s view that avoiding the unnecessary reporting of false-positive results is not a self-evident benefit, and puzzled by her questioning the consequential reduced use of clinical resources, which was unambiguously clear to the midwives and clinicians involved. We are also at a loss to understand how Walker seems to lean toward the recall method, when the evidence so strongly favors the reflex method with the added benefit of avoiding needless worry among the women screened.Ethical declarationDisclosureN.J.W. is Director of Logical Medical Systems, which produces software for the interpretation of Down syndrome screening tests. The other authors declare no conflict of interest. To the Editor: The prenatal reflex DNA test described in our paper1.Wald NJ, Huttly WJ, Bestwick JP et al. Prenatal reflex DNA screeningfor trisomies 21, 18, and 13. Genet Med; e-pub ahead of print 9 November 2017.Google Scholar is a method of screening that has advantages over the recall method.2.Chitty L.S. Wright D. Hill M. et al.Uptake, outcome and costs of implementing non-invasive prenatal testing for Down’s syndrome into NHS maternity care: prospective cohort study in eight diverse maternity units.10.1136/bmj.i3426BMJ. 2016; 354: i3426Google Scholar,3.Public Health England. NHS public health functions agreement 2017-18. Service specification no.16. NHS Fetal Anomaly Screening Programme—screening for Down’s, Edwards’ and Patau’s Syndromes (Trisomy 21, 18 & 13). https://www.england.nhs.uk/wp-content/uploads/2017/05/serv-spec-16.pdf. Accessed 29 November 2017.Google Scholar In the recall method as described,3.Public Health England. NHS public health functions agreement 2017-18. Service specification no.16. NHS Fetal Anomaly Screening Programme—screening for Down’s, Edwards’ and Patau’s Syndromes (Trisomy 21, 18 & 13). https://www.england.nhs.uk/wp-content/uploads/2017/05/serv-spec-16.pdf. Accessed 29 November 2017.Google Scholar a combined test is carried out and women with a risk ≥1 in 150 receive a positive result; they are invited to return for counseling and offered either a second screening test based on a DNA analysis or an invasive diagnostic test—of course, without an obligation to accept either. Walker, in her letter,4.Walker C.L. Response to Wald et al.1:CAS:528:DC%2BC1cXhtVamsb7I10.1038/gim.2017.237Genet Med. 2018; 20: 1Google Scholar does not appear to fully accept the advantages of the reflex method. Reflex DNA screening results in a higher rate of detection (95% vs. 81%) of pregnancies with trisomy 21, 18, and 13 and a false-positive rate (0.02% vs. 2.4%) about a hundred times lower than the recall method. Consequently, the reflex method means fewer women with an unaffected pregnancy are given a positive screening result, while a high detection rate is maintained. A quantitative comparison of the two methods in respect of screening for trisomy 21 is given at http://www.wolfson.qmul.ac.uk/ReflexDNAversusRecallDNA. This shows that in 10,000 pregnancies screened by either the reflex or recall methods, respectively, the following numbers apply: (i) women recalled, 0 vs. 259; (ii) invasive diagnostic tests in unaffected pregnancies, 1.8 vs. 30.3; (iii) trisomy 21 pregnancies detected, 30.3 vs. 25.9; and (iv) odds of being affected given a positive result, 17:1 vs. 1:1. These advantages of the reflex method over the recall method are clear and, contrary to Walker’s opinion, involve no extrapolation. It is untenable to suggest that worrying women and their partners with the news of a positive screening result when this is completely unnecessary is good medical practice, or even ethical. Calling women back for another test when this can be avoided causes unnecessary harm and this is always wrong. It is not an issue that is relevant to patient autonomy or choice, which is, of course, important. Our responses to Walker’s four points are as follows. (i) The decision to be screened is an option that women can, of course, discuss with their partners; obtaining consent to reflex DNA screening is no different from obtaining consent to other screening tests. (ii) Notifying women that they have a positive screening result is obviously distressing. If this can be avoided without loss of efficacy it should be done. It is not a legitimate or ethical matter for research. (iii) The duty of health professionals is to offer the most effective and safe tests or interventions that are affordable. This is not paternalistic; it is the expected duty of care. There is no withholding of information, as Walker states in relation to reflex DNA screening, because consent is obtained for the reflex test incorporating all its components. The combined test itself has several components and no one argues that separate consent should be sought for each component (nuchal translucency measurement and two blood measurements). The concept underlying the reflex DNA test is to capture the advantages of a single screening test with several components instead of performing two separate sequential tests. (iv) Avoiding unnecessary harm without loss of efficacy is always a benefit and therefore not a valid research issue. While there is agreement about using the combined test followed by a DNA test in some women, there is disagreement on how this is done. Walker holds the view that there is merit in women being told that they have a positive combined test result, and then being recalled for counseling. Given that reflex DNA screening can avoid this step entirely, and deliver a test with an improved screening performance, we see no advantage in the recall strategy. We are puzzled by Walker’s view that avoiding the unnecessary reporting of false-positive results is not a self-evident benefit, and puzzled by her questioning the consequential reduced use of clinical resources, which was unambiguously clear to the midwives and clinicians involved. We are also at a loss to understand how Walker seems to lean toward the recall method, when the evidence so strongly favors the reflex method with the added benefit of avoiding needless worry among the women screened. Ethical declarationDisclosureN.J.W. is Director of Logical Medical Systems, which produces software for the interpretation of Down syndrome screening tests. The other authors declare no conflict of interest. DisclosureN.J.W. is Director of Logical Medical Systems, which produces software for the interpretation of Down syndrome screening tests. The other authors declare no conflict of interest. N.J.W. is Director of Logical Medical Systems, which produces software for the interpretation of Down syndrome screening tests. The other authors declare no conflict of interest.

BACKGROUNDAn estimate of fetal fraction (FF) is needed for DNA-based screening for trisomy 21 and other aneuploidies, but there is no gold standard to validate FF measurement methods. We specify a gold standard and use it to validate a method of measuring FF (SeqFF) in singleton pregnancies.METHODSThe gold standard was a formula derived from 2 elements: (a) an estimate of the percentage of DNA fragments in maternal plasma from chromosome 21 (%Ch21) in pregnancies without trisomy 21, 18, or 13 (PU) and (b) calculation of %Ch21 with increasing FF in trisomy 21 pregnancies (P21). The SeqFF method was evaluated by plotting regression lines of %Ch21 and SeqFF estimates of FF in 31 singleton male and 31 female trisomy 21 pregnancies and comparing the regressions with the reference line derived from the gold standard formula.RESULTSThe gold standard formula was P21 = (1/2)PUFF + PU, with FF expressed as a proportion, or converting %Ch21 to multiples of the median (MoM), P21(MoM) = (1/2)FF + 1. Based on 3865 pregnancies, the PU was 1.2935%. The regression lines for trisomy 21 pregnancies with male and female fetuses were almost identical to the gold standard reference line (regression slopes in MoMs 0.52 and 0.50, respectively, compared with 0.50 for the gold standard reference line).CONCLUSIONSThe proposed gold standard can be used to validate different methods of estimating FF in singleton pregnancies. SeqFF is an accurate method of estimating FF.

OBJECTIVE:To compare costs and efficacy of reflex and recall prenatal DNA screening for trisomy 21, 18 and 13 (affected pregnancies). In both methods women have Combined test markers measured. With recall screening, women with a high Combined test risk are recalled for counselling and offered a DNA blood test or invasive diagnostic testing. With reflex screening, a DNA analysis is automatically performed on plasma collected when blood was collected for measurement of the Combined test markers.METHODS:Published data were used to estimate, for each method, using various unit costs and risk cut-offs, the cost per woman screened, cost per affected pregnancy diagnosed, and for a given number of women screened, numbers of affected pregnancies diagnosed, unaffected pregnancies with positive results, and women with unaffected pregnancies having invasive diagnostic testing.RESULTS:Cost per woman screened is lower with reflex v recall screening: £37 v £38, and £11,043 v £11,178 per affected pregnancy diagnosed (DNA £250, Combined test markers risk cut-off 1 in 150). Reflex screening results in similar numbers of affected pregnancies diagnosed, with 100-fold fewer false-positives and 20-fold fewer women with unaffected pregnancies having invasive diagnostic testing.CONCLUSIONS:Reflex DNA screening is less expensive, more cost-effective, and safer than recall screening.

Objective: To develop a screening test for fetal trisomy 13, 18, and 21 using cell-free DNA from maternal blood with an automated workflow using the Ion Proton sequencing platform. Methods: An automated next-generation sequencing workflow was developed using the Ion Proton sequencing platform and software developed for straightforward bioinformatic analysis. An algorithm was developed using 239 samples to determine the likelihood of trisomy, using DNA fragment counts and a fetal fraction validity check; the results were compared with those from invasive diagnostic procedures. A further 111 samples were used to assess the tests' sensitivity (detection rate) and specificity (1 minus false-positive rate). Results: The 110 of a possible 111 valid samples used to verify the IONA® test gave 100% sensitivity and specificity, compared with invasive diagnostic procedures; one failed the fetal fraction validity check giving a sample failure rate of 0.29% across all 350 analysed samples. Conclusion: The data indicate that the IONA test provides a robust, accurate automated workflow suitable for use on maternal blood samples to screen for trisomies 13, 18, and 21. The test has the potential to reduce the number of unnecessary invasive procedures performed and facilitate testing by screening laboratories.

Background: Prenatal cystic fibrosis (CF) screening is currently based on determining the carrier status of both parents. We propose a new method based only on the analysis of DNA in maternal plasma. Methods: The method relies on the quantitative amplification of the CF gene to determine the percentage of DNA fragments in maternal plasma at targeted CF mutation sites that carry a CF mutation. Computer modelling was carried out to estimate the distributions of these percentages in pregnancies with and without a fetus affected with CF. This was done according to the number of DNA fragments counted and fetal fraction, using the 23 CF mutations recommended by the American College of Medical Genetics for parental carrier testing. Results: The estimated detection rate (sensitivity) is 70% (100% of those detected using the 23 mutations), the false-positive rate 0.002%, and the odds of being affected given a positive screening result 14:1, compared with 70%, 0.12%, and 1:3, respectively, with current prenatal screening based on parental carrier testing. Conclusions: Compared with current screening practice based on parental carrier testing, the proposed method would substantially reduce the number of invasive diagnostic procedures (amniocentesis or chorionic villus sampling) without reducing the CF detection rate. The expected advantages of the proposed method justify carrying out the necessary test development for use in a clinical validation study.

Abstract Background: AGI-134 is a fully synthetic glycolipid, composed of an alpha-Gal (Galá1-3Galâ1-4GlcNAc-R) sugar epitope attached via a linker to a lipid tail. Natural antibodies to the alpha-Gal epitope are responsible for the hyperacute rejection of xenografts in humans. It is proposed that intratumorally administered AGI-134 will incorporate into the cell membranes of the tumor cells, presenting the alpha-Gal epitope for binding of anti-Gal antibodies to the tumor cells. This will initiate an immune response that attacks the injected tumor and, through uptake of immune-complexed tumor antigens by antigen presenting cells, will create a patient-specific, systemic anti-tumor response against distant metastases. Results: We demonstrate that AGI-134 incorporates into tumor cell membranes in vitro and that the exposed alpha-Gal epitope binds anti-Gal IgG and IgM antibodies from human serum to the tumor cell surface. Using flow cytometry and a complement-dependent cytotoxicity assay we show that tumor cell opsonization with anti-Gal antibodies leads to deposition of complement proteins C3b and C5b-9, which ultimately leads to tumor cell lysis. Furthermore, we demonstrate that AGI-134-labeled tumor cells opsonized with human serum proteins are phagocytosed by professional APCs. Using the B16-F10 melanoma model in anti-Gal producing á1,3-galactosyltransferase knockout (GT KO) mice we present data to demonstrate that AGI-134 injection into a primary tumor provides significant dose-dependent protection from the development of established distant lesions. Using GT KO mouse serum we demonstrate in vitro that deposition of complement on AGI-134-labeled mouse tumor cells is both alpha-Gal and anti-Gal dependent. In vivo, we demonstrate that the effect of AGI-134 is due to the alpha-Gal moiety by replacing it with human blood group antigens. The protection from secondary lesions conferred by AGI-134 is long lasting in the GT KO mouse melanoma model (monitored up to 90 days). Importantly, when sub-optimal concentrations of AGI-134 were tested in vivo in combination with an anti-PD-1 antibody (RMP1-14), a significant enhancement in efficacy over either of the agents administered alone was observed. Citation Format: Stephen Shaw, Sascha Kristian, Kim Wigglesworth, Jenny Middleton, Mel Glossop, Giles Whalen, Robert Old, Mike Westby, Chris Pickford. AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4862.

3083 Background: Unlike virtually all non-primate mammals, humans lack α-Gal (Gal-α-1,3-Gal-β-1,4-GlcNAc) epitopes and produce antibodies against α-Gal-positive antigens. Anti-α-Gal (anti-Gal) antibodies are among the most abundant antibodies in humans and responsible for hyper-acute rejection of α-Gal-positive xenotransplants. Studies using rabbit-derived α-Gal glycolipids provided a preclinical proof of principle for α-Gal glycolipids as cancer immunotherapy, inducing a CD8+ T cell response to tumor associated antigens (TAAs) that correlated with protection from distal tumor growth in mice. In the current study we show that a fully synthetic, α-Gal glycolipid-like small molecule, AGI-134, displays potent anti-tumor activity by engaging the cellular and humoral immune system against TAAs. Methods: in vitro and in vivo studies. Results: AGI-134 inserts into the plasma membranes of mouse and human cancer cells in vitro. Anti-Gal IgM and IgG bind to the α-Gal labeled cancer cells and mediate an anti-cancer cell response via complement dependent cytotoxicity, antibody dependent cellular cytotoxicity, and cancer cell phagocytosis by antigen presenting cells in vitro. In anti-Gal producing α-1,3-galactosyltransferase knockout (GT KO) mice, intratumorally (IT) administered AGI-134 greatly inhibits the growth of untreated distal B16-F10 melanoma lesions in an α-Gal- and anti-Gal-dependent fashion. Moreover, we show evidence that AGI-134 acts synergistically with an anti-programmed cell death 1 receptor (PD-1) antibody. In a PK study in GT KO mice, AGI-134 shows low systemic exposure following IT and subcutaneous (SC) administration. In a non-GLP toxicity study in cynomolgus monkeys, 5 SC or intravenous AGI-134 doses ranging from 2-50 mg/kg/dose were well tolerated. The no-observed-adverse-effect level was considered to be 50 mg/kg SC. Conclusions: We propose that AGI-134 has potential as a new immunotherapy for the treatment of solid tumors. The data suggest that AGI-134 may be an excellent combination partner for checkpoint inhibitors in treating cancer. Currently, the drug is being prepared for a Phase I/II clinical trial.

The ever-increasing threat of multi-drug resistant bacterial infections has spurred renewed interest in alternative approaches to classical antibiotic therapy. In contrast to other mammals, humans do not express the galactose-α-1,3-galactosyl-β-1,4-N-acetyl-glucosamine (α-Gal) epitope. As a result of exposure of humans to α-Gal in the environment, a large proportion of circulating antibodies are specific for the trisaccharide. In this study, we examine whether these anti-Gal antibodies can be recruited and redirected to exert anti-bacterial activity. We show that a specific DNA aptamer conjugated to an α-Gal epitope at its 5′ end, herein termed an alphamer, can bind to group A Streptococcus (GAS) bacteria by recognition of a conserved region of the surface-anchored M protein. The anti-GAS alphamer was shown to recruit anti-Gal antibodies to the streptococcal surface in an α-Gal-specific manner, elicit uptake and killing of the bacteria by human phagocytes, and slow growth of invasive GAS in human whole blood. These studies provide a first in vitro proof of concept that alphamers have the potential to redirect pre-existing antibodies to bacteria in a specific manner and trigger an immediate antibacterial immune response. Further validation of this novel therapeutic approach of applying α-Gal technology in in vivo models of bacterial infection is warranted.

Pregnancy is a normal physiological condition in which the maternal β-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of β-cells to analyse the origin of new β-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, β-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HPAP). First, we conclude that the lineage tracing system was highly specific for β-cells. Secondly, we scored the proportion of the β-cells marked with HPAP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labelling index in pregnant animal pancreata, compared to non-pregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labelling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-β-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only β-cell duplication, but also the activation of a non-β-cell progenitor population. Further, there was no transdifferentiation of β-cells to other cell types in a two and half month period following labelling, including the period of pregnancy.

Background: Genomic DNA sequences in cell-free plasma are biomarkers of cancer prognosis, where characteristic changes in methylation of turnout suppressor or oncogene DNA regions are indicative of changes in gene activity. Also, cell-free fetal DNA can be distinguished, by its methylation status, from the maternal DNA in the plasma of pregnant women, hence providing DNA biomarkers for the proposed minimally-invasive diagnosis of fetal aneuploidies, including Down's syndrome. However, the production and clearance of cell-free DNA from plasma in relation to its methylation status, are poorly understood processes.Methods: We studied the methylation status of DNA derived from the imprinted GNAS1 locus, in cell-free plasma DNA of healthy adults. Heterozygotes were identified that carried the SNP rs1800905 in the imprinted region. The parent-of-origin-dependent DNA methylation was analysed by bisulfite conversion, followed by cloning and sequencing.Results: Genomic DNA molecules derived from both the methylated, maternal, allele and the unmethylated, paternal, allele were found in plasma. Methylated and unmethylated DNA molecules were present in equal numbers.Conclusions: Our data indicate that the methylation status of a DNA sequence has no effect on its steady state concentration in the cell-free DNA component of plasma, in healthy adults. (C) 2008 Elsevier B.V. All rights reserved.

The discovery of cell-free fetal (cff) DNA and RNA in the maternal circulation has driven developments in noninvasive prenatal diagnosis (NIPD) for the past decade. Detection of paternally derived alleles in cff DNA is becoming well established. Now much interest is focussing on NIPD of fetal chromosomal abnormalities, such as trisomy 2 1, which is a considerable challenge because this demands accurate quantitative measurements of the amounts of specific cff DNA or cff RNA sequences in maternal blood samples. Emerging strategies for distinguishing and quantifying the fetal nucleic acids in the maternal circulation promise continued development of the field, and pose a number of unanswered questions. Copyright (c) 2007 John Wiley & Sons, Ltd.

Objectives: To assess whether different genomic cell-free DNAs are equally abundant in the plasma of individual donors, and any relationship between DNA methylation and representation in plasma.Design and methods: The concentrations of DNA in plasma were determined by real-time PCR.Results: Different DNA sequences were not equally represented. The relative abundances were similar in different donors.Conclusions: Different DNA sequences are not equally abundant in plasma, with no relationship between DNA methylation and abundance. (C) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

The discovery of cell-free fetal (cff) DNA and RNA in the maternal circulation has driven developments in noninvasive prenatal diagnosis (NIPD) for the past decade. Detection of paternally derived alleles in cff DNA is becoming well established. Now much interest is focussing on NIPD of fetal chromosomal abnormalities, such as trisomy 21, which is a considerable challenge because this demands accurate quantitative measurements of the amounts of specific cff DNA or cff RNA sequences in maternal blood samples. Emerging strategies for distinguishing and quantifying the fetal nucleic acids in the maternal circulation promise continued development of the field, and pose a number of unanswered questions.