Chromatin conformation changes in peripheral blood can detect prostate cancer and stratify disease risk groups.

BACKGROUND: Current diagnostic blood tests for prostate cancer (PCa) are unreliable for the early stage disease, resulting in numerous unnecessary prostate biopsies in men with benign disease and false reassurance of negative biopsies in men with PCa. Predicting the risk of PCa is pivotal for making an informed decision on treatment options as the 5-year survival rate in the low-risk group is more than 95% and most men would benefit from surveillance rather than active treatment. Three-dimensional genome architecture and chromosome structures undergo early changes during tumourigenesis both in tumour and in circulating cells and can serve as a disease biomarker. METHODS: In this prospective study we screened whole blood of newly diagnosed, treatment naïve PCa patients (n = 140) and cancer-free controls (n = 96) for the presence of 14,241 chromosomal loops in the loci of 425 genes. RESULTS: We have detected specific chromosome conformation changes in the loci of ETS1, MAP3K14, SLC22A3 and CASP2 genes in peripheral blood from PCa patients yielding PCa detection with 80% sensitivity and 80% specificity. Further analysis between PCa risk groups yielded prognostic validation sets consisting of HSD3B2, VEGFC, APAF1, BMP6, ERG, MSR1, MUC1, ACAT1 and DAPK1 genes that achieved 80% sensitivity and 93% specificity stratifying high-risk category 3 vs low risk category 1 and 84% sensitivity and 89% specificity stratifying high risk category 3 vs intermediate risk category 2 disease. CONCLUSIONS: Our results demonstrate specific chromosome conformations in the blood of PCa patients that allow PCa diagnosis and risk stratification with high sensitivity and specificity.

Chromosome conformation signatures define predictive markers of inadequate response to methotrexate in early rheumatoid arthritis.

BACKGROUND: There is a pressing need in rheumatoid arthritis (RA) to identify patients who will not respond to first-line disease-modifying anti-rheumatic drugs (DMARD). We explored whether differences in genomic architecture represented by a chromosome conformation signature (CCS) in blood taken from early RA patients before methotrexate (MTX) treatment could assist in identifying non-response to DMARD and, whether there is an association between such a signature and RA specific expression quantitative trait loci (eQTL). METHODS: We looked for the presence of a CCS in blood from early RA patients commencing MTX using chromosome conformation capture by EpiSwitch™. Using blood samples from MTX responders, non-responders and healthy controls, a custom designed biomarker discovery array was refined to a 5-marker CCS that could discriminate between responders and non-responders to MTX. We cross-validated the predictive power of the CCS by generating 150 randomized groups of 59 early RA patients (30 responders and 29 non-responders) before MTX treatment. The CCS was validated using a blinded, independent cohort of 19 early RA patients (9 responders and 10 non-responders). Last, the loci of the CCS markers were mapped to RA-specific eQTL. RESULTS: We identified a 5-marker CCS that could identify, at baseline, responders and non-responders to MTX. The CCS consisted of binary chromosome conformations in the genomic regions of IFNAR1, IL-21R, IL-23, CXCL13 and IL-17A. When tested on a cohort of 59 RA patients, the CCS provided a negative predictive value of 90.0% for MTX response. When tested on a blinded independent validation cohort of 19 early RA patients, the signature demonstrated a true negative response rate of 86 and a 90% sensitivity for detection of non-responders to MTX. Only conformations in responders mapped to RA-specific eQTL. CONCLUSIONS: Here we demonstrate that detection of a CCS in blood in early RA is able to predict inadequate response to MTX with a high degree of accuracy. Our results provide a proof of principle that a priori stratification of response to MTX is possible, offering a mechanism to provide alternative treatments for non-responders to MTX earlier in the course of the disease.

Circulating Chromosome Conformation Signatures Significantly Enhance PSA Positive Predicting Value and Overall Accuracy for Prostate Cancer Detection.

BACKGROUND: Prostate cancer (PCa) has a high lifetime prevalence (one out of six men), but currently there is no widely accepted screening programme. Widely used prostate specific antigen (PSA) test at cut-off of 3.0 ng/mL does not have sufficient accuracy for detection of any prostate cancer, resulting in numerous unnecessary prostate biopsies in men with benign disease and false reassurance in some men with PCa. We have recently identified circulating chromosome conformation signatures (CCSs, Episwitch® PCa test) allowing PCa detection and risk stratification in line with standards of clinical PCa staging. The purpose of this study was to determine whether combining the Episwitch PCa test with the PSA test will increase its diagnostic accuracy. METHODS: n = 109 whole blood samples of men enrolled in the PROSTAGRAM screening pilot study and n = 38 samples of patients with established PCa diagnosis and cancer-negative controls from Imperial College NHS Trust were used. Samples were tested for PSA, and the presence of CCSs in the loci encoding for of DAPK1, HSD3B2, SRD5A3, MMP1, and miRNA98 associated with high-risk PCa identified in our previous work. RESULTS: PSA > 3 ng/mL alone showed a low positive predicted value (PPV) of 0.14 and a high negative predicted value (NPV) of 0.93. EpiSwitch alone showed a PPV of 0.91 and a NPV of 0.32. Combining PSA and Episwitch tests has significantly increased the PPV to 0.81 although reducing the NPV to 0.78. Furthermore, integrating PSA, as a continuous variable (rather than a dichotomised 3 ng/mL cut-off), with EpiSwitch in a new multivariant stratification model, Prostate Screening EpiSwitch (PSE) test, has yielded a remarkable combined PPV of 0.92 and NPV of 0.94 when tested on the independent prospective cohort. CONCLUSIONS: Our results demonstrate that combining the standard PSA readout with circulating chromosome conformations (PSE test) allows for significantly enhanced PSA PPV and overall accuracy for PCa detection. The PSE test is accurate, rapid, minimally invasive, and inexpensive, suggesting significant screening diagnostic potential to minimise unnecessary referrals for expensive and invasive MRI and/or biopsy testing. Further extended prospective blinded validation of the new combined signature in a screening cohort with low cancer prevalence would be the recommended step for PSE adoption in PCa screening.

Development and Validation of Blood-Based Predictive Biomarkers for Response to PD-1/PD-L1 Checkpoint Inhibitors: Evidence of a Universal Systemic Core of 3D Immunogenetic Profiling across Multiple Oncological Indications.

BACKGROUND: Unprecedented advantages in cancer treatment with immune checkpoint inhibitors (ICIs) remain limited to only a subset of patients. Systemic analyses of the regulatory 3D genome architecture linked to individual epigenetic and immunogenetic controls associated with tumour immune evasion mechanisms and immune checkpoint pathways reveal a highly prevalent molecular profile predictive of response to PD-1/PD-L1 ICIs. A clinical blood test based on a set of eight (8) 3D genomic biomarkers has been developed and validated on the basis of an observational trial to predict response to ICI therapy. METHODS: The predictive eight biomarker set is derived from prospective observational clinical trials, representing 280 treatments with Pembrolizumab, Atezolizumab, Durvalumab, Nivolumab, and Avelumab in a broad range of indications: melanoma, lung, hepatocellular, renal, breast, bladder, colon, head and neck, bone, brain, lymphoma, prostate, vulvar, and cervical cancers. RESULTS: The 3D genomic eight biomarker panel for response to immune checkpoint therapy achieved a high accuracy of 85%, sensitivity of 93%, and specificity of 82%. CONCLUSIONS: This study demonstrates that a 3D genomic approach can be used to develop a predictive clinical assay for response to PD-1/PD-L1 checkpoint inhibition in cancer patients.

Monocytes acquire prostate cancer specific chromatin conformations upon indirect co-culture with prostate cancer cells.

Background: Three-dimensional chromosome loop conformations are powerful regulators of gene expression. These chromosome conformations can be detected both in tumour and in circulating cells and have significant disease biomarker potential. We have recently detected specific chromosome conformations in circulating cells of patients with prostate cancer (PCa) which were similar to ones found in their primary tumours, however, the possibility of horizontal transfer of chromosome conformations was not studied previously. Methods: Human monocytes (U937) were co-cultured in Boyden chambers through 0.4 uM membrane with or without PC-3 human PCa cells or their conditioned media and a custom DNA microarray for 900,000 chromosomal loops covering all coding loci and non-coding RNA genes was performed on each part of the co-culture system. Results: We have detected 684 PC-3 cell-specific chromosome conformations across the whole genome that were absent in naïve monocytes but appeared in monocytes co-cultured with PC-3 cells or with PC-3-conditioned media. Comparing PC3-specific conformations to the ones we have previously detected in systemic circulation of high-risk PCa patients revealed 9 positive loops present in both settings. Conclusions: Our results demonstrate for the first time a proof of concept for horizontal transfer of chromosome conformations without direct cell-cell contact. This carries high clinical relevance as we have previously observed chromatin conformations in circulating cells of patients with melanoma and PCa similar to ones in their primary tumours. These changes can be used as highly specific biomarkers for diagnosis and prognosis. Further studies are required to elucidate the specific mechanism of chromosome conformations transfer and its clinical significance in particular diseases.

Environmental biosafety and transgenic potato in a centre of diversity for this crop.

The Nuffield Council on Bioethics suggests that introgression of genetic material into related species in centres of crop biodiversity is an insufficient justification to bar the use of genetically modified crops in the developing world. They consider that a precautionary approach to forgo the possible benefits invokes the fallacy of thinking that doing nothing is itself without risk to the poor. Here we report findings relevant to this and other aspects of environmental biosafety for genetically modified potato in its main centre of biodiversity, the central Andes. We studied genetically modified potato clones that provide resistance to nematodes, principal pests of Andean potato crops. We show that there is no harm to many non-target organisms, but gene flow occurs to wild relatives growing near potato crops. If stable introgression were to result, the fitness of these wild species could be altered. We therefore transformed the male sterile cultivar Revolucion to provide a genetically modified nematode-resistant potato to evaluate the benefits that this provides until the possibility of stable introgression to wild relatives is determined. Thus, scientific progress is possible without compromise to the precautionary principle.

Initial Identification of a Blood-Based Chromosome Conformation Signature for Aiding in the Diagnosis of Amyotrophic Lateral Sclerosis.

BACKGROUND: The identification of blood-based biomarkers specific to the diagnosis of amyotrophic lateral sclerosis (ALS) is an active field of academic and clinical research. While inheritance studies have advanced the field, a majority of patients do not have a known genetic link to the disease, making direct sequence-based genetic testing for ALS difficult. The ability to detect biofluid-based epigenetic changes in ALS would expand the relevance of using genomic information for disease diagnosis. METHODS: Assessing differences in chromosomal conformations (i.e. how they are positioned in 3-dimensions) represents one approach for assessing epigenetic changes. In this study, we used an industrial platform, EpiSwitch™, to compare the genomic architecture of healthy and diseased patient samples (blood and tissue) to discover a chromosomal conformation signature (CCS) with diagnostic potential in ALS. A three-step biomarker selection process yielded a distinct CCS for ALS, comprised of conformation changes in eight genomic loci and detectable in blood. FINDINGS: We applied the ALS CCS to determine a diagnosis for 74 unblinded patient samples and subsequently conducted a blinded diagnostic study of 16 samples. Sensitivity and specificity for ALS detection in the 74 unblinded patient samples were 83∙33% (CI 51∙59 to 97∙91%) and 76∙92% (46∙19 to 94∙96%), respectively. In the blinded cohort, sensitivity reached 87∙50% (CI 47∙35 to 99∙68%) and specificity was 75∙0% (34∙91 to 96∙81%). INTERPRETATIONS: The sensitivity and specificity values achieved using the ALS CCS identified and validated in this study provide an indication that the detection of chromosome conformation signatures is a promising approach to disease diagnosis and can potentially augment current strategies for diagnosing ALS. FUND: This research was funded by Oxford BioDynamics and Innovate UK. Work in the Oxford MND Care and Research Centre is supported by grants from the Motor Neurone Disease Association and the Medical Research Council. Additional support was provided by the Northeast ALS Consortium (NEALS).

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions.

Stretched histone regions, such as super-enhancers and broad H3K4me3 domains, are associated with maintenance of cell identity and cancer. We connected super-enhancers and broad H3K4me3 domains in the K562 chronic myelogenous leukemia cell line as well as the MCF-7 breast cancer cell line with chromatin interactions. Super-enhancers and broad H3K4me3 domains showed higher association with chromatin interactions than their typical counterparts. Interestingly, we identified a subset of super-enhancers that overlap with broad H3K4me3 domains and show high association with cancer-associated genes including tumor suppressor genes. Besides cell lines, we could observe chromatin interactions by a Chromosome Conformation Capture (3C)-based method, in primary human samples. Several chromatin interactions involving super-enhancers and broad H3K4me3 domains are constitutive and can be found in both cancer and normal samples. Taken together, these results reveal a new layer of complexity in gene regulation by super-enhancers and broad H3K4me3 domains.

A recurring rollercoaster ride: a qualitative study of the emotional experiences of parents of children with juvenile idiopathic arthritis.

BACKGROUND: Despite the wealth of clinical research carried out in children with juvenile idiopathic arthritis (JIA), little is known about the emotional experiences of their parents. This article describes the predominant emotional experiences reported by parents of children with JIA in two Canadian cities. METHODS: Research participants included 15 experienced parents and 8 novice parents (<6 months since children's JIA diagnosis). Their children were 2 to 16 years old with various JIA categories. A qualitative dataset including audio recordings and verbatim transcripts of three focus groups, and written reports of 59 reciprocal interviews (parents interviewing each other) were examined by a multidisciplinary research team following a four-step qualitative analytical process. RESULTS: Parents of children with JIA experienced recurrent mixed negative and positive emotions that varied over time. Between disease onset and diagnosis, mounting anxiety, fear and confusion were the predominant emotions. Shortly after diagnosis there were shock, disbelief, and fear, with a sense of having being blindsided by the disease. At times of disease quiescence there was hope and gratitude, but also fatigue and frustration with ongoing treatment and fear of flares. During periods of increasing or ongoing symptoms there was admiration and sympathy for the courageous way children coped with JIA, as well as sorrow and frustration for ongoing pain and limitations. There were at times, frustration and indignation with peers and teachers unable to understand the child's fluctuations in physical activity and schoolwork. Throughout the disease, parents felt an underlying anxiety and powerlessness. CONCLUSIONS: Parents of children with JIA described complex emotional journeys akin to the recurring ups and downs of rollercoaster rides, instead of ordered emotional phases ending in resolution. This has implications for healthcare providers who need to be aware of the complexity of these emotional journeys to support parents more effectively, thereby helping improve patient outcomes.

Transgenic potatoes for potato cyst nematode control can replace pesticide use without impact on soil quality.

Current and future global crop yields depend upon soil quality to which soil organisms make an important contribution. The European Union seeks to protect European soils and their biodiversity for instance by amending its Directive on pesticide usage. This poses a challenge for control of Globodera pallida (a potato cyst nematode) for which both natural resistance and rotational control are inadequate. One approach of high potential is transgenically based resistance. This work demonstrates the potential in the field of a new transgenic trait for control of G. pallida that suppresses root invasion. It also investigates its impact and that of a second transgenic trait on the non-target soil nematode community. We establish that a peptide that disrupts chemoreception of nematodes without a lethal effect provides resistance to G. pallida in both a containment and a field trial when precisely targeted under control of a root tip-specific promoter. In addition we combine DNA barcoding and quantitative PCR to recognise nematode genera from soil samples without microscope-based observation and use the method for nematode faunal analysis. This approach establishes that the peptide and a cysteine proteinase inhibitor that offer distinct bases for transgenic plant resistance to G. pallida do so without impact on the non-target nematode soil community.

Prototype demonstration of transgenic resistance to the nematode Radopholus similis conferred on banana by a cystatin.

Cavendish banana was transformed using Agrobacterium tumefaciens to express a protein engineered rice cystatin (OcIdeltaD86) of value for control of plant parasitic nematodes. Expression for each line was under control of a constitutive promoter from the maize ubiquitin gene (UBI-1), a constitutive, chimeric promoter from the octopine and mannopine synthase genes of A. tumefaciens or a promoter from a root-preferentially expressed tubulin gene Arabidopsis (TUB-1). Lines were selected as of potential interest after 8 weeks challenge in containment if their mean R. similis/25 g roots for three sibling plants were more than 1 standard normal variate below the grand mean for all plants in c7-15 lines challenged concurrently. A total of 16 lines were selected on this basis as putative positives. Western blots confirmed that eight of these lines expressed cystatin with a mean of 0.08 +/- 0.04% tsp. All but two of 19 negatively selected lines from bioassays did not express cystatin. The mean resistance level of the confirmed positive lines was 69 +/- 17%. ELISA established the positive lines under control of UBI provided significantly higher expression levels of OcIdeltaD86 than recorded for the other two promoters. Lines of interest were confirmed as producing a transcript for OcIdeltaD86 by RT-PCR. Eight plants of one UBI promoter line expressing only 0.1 +/- 0.004% tsp as cystatin were re-challenged with R. similis and achieved a resistance of 70 +/- 10%. Subsequent repeat western blotting confirmed that this line still produced the cystatin after the trial. This is the first report of transgenic resistance against a major pest or disease of banana.

Guidelines for blood test monitoring of methotrexate toxicity in juvenile idiopathic arthritis.

OBJECTIVE: To assess the utility of the American College of Rheumatology guidelines for monitoring methotrexate (MTX)-related toxicity in a cohort of children with juvenile idiopathic arthritis (JIA). METHODS: Eighty-nine patients with JIA treated with MTX were monitored prospectively: aspartate aminotransferase (AST), alanine aminotransferase (ALT), complete blood count (CBC), and differential blood count were measured prior to starting MTX, and then monthly. Significantly abnormal blood tests (SABT) were prospectively defined as (1) significantly elevated liver enzymes (SELE) greater than twice the upper limit of normal; (2) granulocyte count < 1.5 109/l; (3) lymphocyte count < 0.9 109/l; or (4) hemoglobin decreased by > 2 g/l from previous level. Clinical interventions, current and cumulative MTX dose, duration of treatment, comorbidity, and concurrent medications at the time of the first SABT identification were recorded. Independent t tests and chi-squared tests were used for comparisons, and the probability of developing a SABT was calculated by Kaplan-Meier survival analysis. RESULTS: Forty percent of patients had a SABT: 26% had hematological abnormalities and 14% had SELE. Ninety-five percent of patients with SABT had symptoms consistent with a viral infection when the SABT was drawn and MTX dose was withheld until results had normalized on repeat testing. SABT persisting beyond one month occurred in only 2 patients, and their abnormalities resolved by 6 months with no specific identified cause; they resumed MTX at a later time without recurrence of SABT. There were no differences between patients with and without SABT with respect to current or cumulative MTX dose, duration of treatment, and concurrent medications at the time of the SABT. The probability of developing a SABT was estimated to be 11% at 3 months, compared to 10% probability of having an abnormal blood test by chance alone. CONCLUSION: Routine blood tests every 4 to 8 weeks in children with JIA are unnecessarily frequent.