It may be worthwhile continuing aggressive resuscitation efforts even for prolonged periods before attaining ROSC for OHCA in patients found hypothermic from metabolic illnesses as compared to only from environmental exposures  as has been traditionally reported in the medical literature.Despite the decreased survival associated with diabetes with out-of-hospital cardiac arrest and the overall low survival to hospital discharge, we would like to present two cases of OHCA in diabetics who despite prolonged resuscitation efforts had complete neurological recovery likely due to concomitant hypothermia. There is a steady decreasing rate of ROSC with longer durations of CPR so that outcomes are best when <20 minutes compared to prolonged resuscitation efforts (>30-40 minutes). It has been previously recognized that hypothermia prior to cardiac arrest can be neurologically protective even with up to 9 hours of cardiopulmonary resuscitation. Hypothermia has been associated with DKA and although often indicates sepsis with mortality rates of 30-60%, it may indeed be protective if occurring prior to cardiac arrest. The critical factor for neuroprotection may be a slow drop to a temperature <25 Despite the decreased survival associated with diabetes with out-of-hospital cardiac arrest  and the 0C so an intravenous femoral warming catheter was placed for a rapid goal of 36 oC and warmed blankets were placed over her including wrapping around her head; warmed IV fluids and hydrocortisone 100mg were also given. She received CPR for over 40 minutes before we achieved return of spontaneous circulation (ROSC) as she was now back in sinus bradycardia with heart rate 40-50, and EKG performed 2 hours later demonstrating Osborne Waves with shivering artifact  as EMS brought her stretcher into the ED. After defibrillation x1 with 200J, she remained in a pulseless wide-complex slow rhythm so CPR was initiated and continued while receiving intravenous epinephrine and crystalloid boluses; Intravenous regular insulin, calcium chloride, and bicarbonate were given for presumed hyperkalemia with DKA. Her trachea was intubated and a right internal jugular intravenous line was placed. Her skin was cold to the touch and her rectal temperature was 22artifact . A Dopam0C so that the intravascular warming/cooling device thermostat was reset to 340C for our goal of post-arrest targeted temperature management (TTM). In the ICU five hours later, she was awake following commands and mouthing words. The TTM was discontinued and her temperature normalized over the next three hours. The next morning she was extubated and transferred to the floor later in the day. She was discharged home 2 days later completely normal neurologically.Laboratory studies confirmed DKA with a serum HCO3 of <5 meq/L, Anion Gap of 36, and Glucose of 967 mg/dL. She remained unresponsive on mechanical ventilation and approximately three hours after arrival her core temperature reached 320C, and glucose was 1,200 mg/dL with a serum HCO3 of <5 meq/L, Anion Gap of 42 and base deficit of -28. An esophageal warming/cooling device was placed set to a temperature of 360C, and an external warm-air circulating blanket was placed over him. An insulin infusion and warmed fluids were administered.A 27 year-old man was found by his mother lying face down unresponsive on his apartment floor. He was diagnosed with type I diabetes at age 11 and had multiple admissions for DKA due to noncompliance with insulin therapy. His past medical history also included asthma, and gastroparesis. She turned him over and began CPR, and he was asystolic when EMS arrived so they transported him with CPR in progress for 10-15 minutes while giving ACLS medications via an intraosseous (IO) line. CPR was continued for approximately 60 additional minutes in the ED after his trachea was intubated and a right femoral venous line was placed. He received intravenous epinephrine every 3-5 minutes , crystalloid boluses, calcium chloride and bicarbonate, as well as defibrillation once for VF before achieving return of spontaneous circulation (ROSC). His rectal temperature was 240C. He was extubated 4 days later and discharged home completely normal neurologically with normal cardiac and hepatic function but still requiring intermittent dialysis for several weeks. He has had numerous repeat admissions for DKA in subsequent years despite endocrinology changing his insulin regimen to a pump, case management involvement, psychiatry evaluation, and intense parental interventions including attempts to gain legal guardianship. Tragically four years after this index presentation, he was found dead at his home at age 31.Despite having anuric acute kidney injury (creatinine >5 mg/dL), rhabdomyolysis , shock liver , severe ARDS (pO2/FiO2 ratio <100) and acute myocardial stunning (EF 30%) requiring CRRT and epinephrine infusion he was able to write notes using his cellphone 8 hours later when his temperature was now 360C prior to out of hospital cardiac arrest (OHCA) as is achieved in deep hypothermic circulatory arrest for operative procedures of the aortic arch and great vessels


TAPBP, the top gene, is critically involved in immune function and encompasses several UV-altered methylation sites that were validated by targeted sequencing, providing cost-effective opportunities for clinical application. The DNA methylome also reveals non UV-related aberrations underlying pathological differences between the cutaneous and 17 acral melanomas. Unsupervised epigenomic mapping demonstrated that non UV-mutant cutaneous melanoma more closely resembles acral rather than UV-exposed cutaneous melanoma, with the latter showing better patient prognosis than the other two forms. These gene-environment interactions reveal translationally impactful mechanisms in melanomagenesis.Ultraviolet radiation (UV) is causally linked to cutaneous melanoma, yet the underlying epigenetic mechanisms, known as molecular sensors of exposure, have not been characterized in clinical biospecimens. Here, we integrate clinical, epigenome (DNA methylome), genome and transcriptome profiling of 112 cutaneous melanoma from two multi-ethnic cohorts. We identify UV-related alterations in regulatory regions and immunological pathways, with multi-OMICs cancer driver potential affecting patient survival.  While cutaneous melanoma is linked to UV radiation, acral melanoma is not. Epigenetic mechanisms function as sensors to exposures and determinants of cell identity. Here, the authors use DNA methylation data to identify dysregulated pathways associated with UV radiation and pathobiology in cutaneous and acral melanomas. It accounts for more than 75% of skin cancer-related deaths though it represents <5% of all cutaneous malignancies2. The incidence of melanoma has been increasing worldwide3 and this trend has been observed for decades in some populations 4.Melanoma is a neoplasm arising from melanocytes in the skin, mucosa, or uvea5. However, there are types of melanoma that arise in body parts protected from direct UV light, and these are acral, mucosal and uveal melanomas. These types represent uncommon cancers, among which the most frequent is the acral melanoma, which occurs on the glabrous skin  and the subungual area7. Even though it is rare in the general population, acral melanoma is the most common melanoma among people with darker skin8.Epidemiological studies have highlighted that the strongest risk factors for cutaneous melanoma development are severe sunburns during childhood and intense intermittent ultraviolet (UV) exposure, which consists of UVC (100–280 nm), UVB (290–320 nm), and UVA (320–400 nm)10. Recently, ten mutated UV signature genes were identified in both clinical samples and animal models, and patients harboring the UV mutation signature presented longer disease-free and overall survival11. Although associations between genetic changes and UV exposure have been well characterized, the role of epigenetic modifications induced by UV exposure has never been investigated directly in human melanoma tissues , including the Blue Books by the International Agency for Research on Cancer (IARC), presented evidence based on epidemiologic, clinical, histopathologic and genomic featuresWe hypothesize that epigenetic alterations, interplaying with transcriptional and mutational events, constitute critical biological mechanisms underpinning intrinsic pathological differences and extrinsic responses to UV exposure in cutaneous and acral melanomas. We perform differential DNA methylome-wide analysis in cutaneous melanoma patients comparing UV-exposed and non UV-exposed melanomas in two independent clinical cohorts, including a sample population from Brazil which encompasses the white and pigmented phenotypes Fig. . UV expoBRAF mutations were relatively more enriched in BCH than in TCGA patients .UV mutation status was inferred in cutaneous melanoma patients using WGS and WES data from Barretos Cancer Hospital (BCH) in the context of International Cancer Genome Consortium (ICGC)-Brazil project and The Cancer Genome Atlas (TCGA) study, respectively Fig. . Similarversus non UV-mutant cutaneous melanoma patients, DNA methylome-wide analysis based on Infinium HumanMethylation450 (450 K) array was performed in BCH samples and compared with that in the TCGA cohort   , 1541 (58.8%) were hypermethylated and 1,079 (41.2%) were hypomethylated  Fig. . In CpG 01) Fig. .p = 1.95e−03), which now encompasses almost all non UV-mutants analyzed. A similar pattern was observed in the TCGA cohort. Cluster C1 was fully occupied by non UV-mutant samples and was visually distinct, exhibiting again an upper hypermethylation (red) stretch and a lower hypomethylation (blue) stretch, relative to the other clusters. Even if C1 is merged with the adjacent cluster C2, the non UV-mutant patients remain statistically enriched in this combined cluster (p = 3.00e−10), which now encompasses almost all non UV-mutants analyzed , which is wild-type for BRAF, RAS, and NF-1, lacks the UV mutational signature and has higher copy number and complex rearrangements9. Indeed, we observed that the TN molecular subgroup is significantly enriched in non UV-mutant patients in both BCH and TCGA cohorts  in BCH and TCGA, respectively. Among them, a large proportion (10 pathways) were identical between BCH and TCGA, 8 and 6 of which remained significant after adjustment for the number of CpGs per gene and FDR, respectively , vitiligo (BCH and TCGA), melanoma (BCH), and skin cancer (BCH), particularly among the top and false discovery rate (FDR)-adjusted ontologies (FDR < 0.05) . Complementary analysis using differentially expressed genes comparing non UV-mutant and UV-mutant cutaneous melanoma patients , demonstrating that immune cell composition was indeed different between UV-mutant and non UV-mutant cutaneous melanoma patients Fig. . Specifip = 2.3e−109 and p = 3.71e−29, respectively) between BCH and TCGA cohorts  were hypermethylated and 30 CpGs  were hypomethylated in both BCH and TCGA, considering CpGs with absolute effect sizes ≥10% and with no significant enrichment in SNPs   were significantly correlated with expression, among which 7 CpGs were indeed located in the TAPBP gene, including its cg18930100  analysis applied to DNA methylome and transcriptome data profiled on the same samples Fig. . We firssis Fig.  and founges Fig. . We thenges Fig.  in order100 Fig. . All thents Fig. . Notablyion Fig.  and incrion Fig.  in UV-mu17. This was performed using data on copy number variation, point mutations, RNA expression and DNA methylation profiled in cutaneous melanoma patients. We found that the top half of the CpGs with the highest cancer driver potential were largely predominated by CpGs of the TAPBP gene  TAPBP methylation, which was correlated with melanoma-specific survival and RNA expression and concurred with other genome-wide deregulations that led to its high multi-OMICs driver potential, we performed technical validation of TAPBP methylation using bisulfite pyrosequencing in the BCH cohort (Supplementary Data TAPBP hypomethylation (including similar effect sizes and baseline methylation levels) in UV-mutant relative to non UV-mutant cutaneous melanoma  had the UV mutation signature as expected. The majority (47.6%) of the acral melanoma patients did not exhibit mutations in BRAF, NRAS or NF1, and a substantial portion  presented a pigmented skin phenotype.We further investigated whether the DNA methylome could also underlie differences between pathologically different melanomas, with interaction by UV mutation status, namely between melanoma types predominantly associated with UV exposure (cutaneous melanoma) and those not UV-associated . Table p < 1.00e−04).Based on PLS-DA modeling in BCH, we observed that the DNA methylome of the non UV-mutant cutaneous melanomas resembles more that of the pathologically different acral melanomas than the pathologically related UV-mutant cutaneous melanomas Fig. . This wap < 0.05) of the differentially methylated genes in skin disorders , we observed consistent findings in both at the CpG, gene and biological pathway levels. Moreover, UV-related DNA methylation alterations showed similar distributions between BCH and TCGA in hypo- and hypermethylated regions as well as similar enrichments in regulatory and CpG density regions, in skin disorders and in immunological pathways. Among the CpGs and genes differentially methylated in both BCH and TCGA, methylation levels of TAPBP  were significantly associated with RNA expression of this gene, concurred with other genome-wide deregulations to yield a high multi-OMICs driver potential and were significantly correlated with melanoma-specific survival. The array-based methylation results of TAPBP were independently validated by bisulfite pyrosequencing, further reinforcing the robustness of the findings and providing promising opportunities for clinical application via pyrosequencing as a cost-effective technique.The only available melanoma dataset with methylome and genome data for replication of our BCH findings was from TCGA. The number of detectable signals was higher in the BCH relative to the TCGA cohort, and this is probably not due to statistical power differences as both datasets had similar sample sizes. This could be rather due in the BCH dataset to 1) the better quality of samples and/or their processing using our in-house optimized automated workflow to generate DNA methylome data, coupled to a priori designed sample distribution on the array that minimizes confounding with batch effects based on statistical semi-randomization, and (2) more accurate technology, using WGS rather than WES, to assess UV exposure. Even though there were some clinicopathological and ethnic dissimilarities  and the transporter associated with antigen processing21. Downregulation of TAPBP (tapasin) protein expression has been observed in multiple cancers as an immune escape mechanism of human tumors, which is restored after cytokine administration, indicating that deficient TAPBP expression might be due to dysregulation than to structural alterations22. Our findings show that TAPBP transcription is significantly inversely associated with its DNA methylation levels, and the latter are altered in relation to UV exposure rather than to melanoma pathological identity since this gene was not found to be differentially methylated in non UV-mutant cutaneous versus acral melanomas  expression, as demonstrated in vitro, highlighting the possibility that patients with defects in MHC-I antigen-processing machinery may benefit from combining immunotherapeutic strategies with demethylating agents (such as those that could restore TAPBP expression)25. In complement with TAPBP, several HLA genes were also differentially methylated in UV-mutant versus non UV-mutant cutaneous melanoma in both BCH and TCGA, and these genes were centrally involved in the multiple immunological pathways identified  they were reproduced in two independent populations, including different ethnicities, which offer a natural means of effect randomization , (2) they showed a dose-response , and (3) they yielded a cancer driver potential for TAPBP that was comparable to that of known cancer driver genes. Still, more datasets will be needed to better reinforce the causality of the associations, for example, by using in larger sample sizes germline data as proxies for TAPBP methylation through Mendelian Randomization.The relation between TAPBP, we reported that the UV mutational signature is associated with a high load (thousands) of epigenetic alterations affecting the methylome landscape of cutaneous melanoma. This highlights that, even though the UV-mutant and non UV-mutant cutaneous melanomas are supposed to share the same pathological/cellular origin, they may need to be classified separately, at least based on their underlying epigenomic landscape, which has the potential to capture markers of both exposures and cell identity. Moreover, the non UV-mutant cutaneous melanoma, by resembling in its epigenome the acral melanoma, may have a poorer prognosis and require a different therapeutic approach than the UV-exposed cutaneous melanoma. This is in line with our data showing that patient survival is worse for the non UV-mutant cutaneous and acral melanomas relative to the UV-mutant cutaneous melanomas. Our findings also corroborate those of another study showing that cutaneous melanoma patients harboring the UV mutation signature had higher disease-free and overall survival11. The consistency between findings is notable especially that our dataset included a different ethnic group and a more accurate methodology (WGS rather than WES) to infer the UV signature.In addition to the findings focused on 11. Among them, PKHD1L1, LRP1B, ADGRV1 and DNAH10 were hypomethylated in UV-mutant compared with non UV-mutant cutaneous melanoma patients in BCH    uncovering potentially powerful exposure and cancer epigenetic biomarkers that can be exploited in risk stratification; (2) enhancing tumor classification within and across melanoma types; (3) revealing molecular drivers in melanomagenesis that could be at the origins of this cancer, hence, suitable for targeted therapy; and (4) diminishing population disparities and knowledge inequalities in melanoma pathobiology. The translational impact of the work covers common and less frequent melanomas and offers a roadmap guiding similar gene-environment investigations of other melanoma types.In this work, we applied multiple powerful technologies, encompassing WGS, WES, RNA sequencing and DNA methylome-wide profiling, coupled to state-of-the-art bioinformatics tools onto a unique series of cutaneous and acral melanoma samples. Specifically, we leveraged publicly available data and complemented that with the generation of new datasets, with larger sample sizes, higher genomic coverage, more detailed phenotypic assessment, high-quality frozen tissue samples, and the inclusion of melanomas other than cutaneous and of ethnicities besides European-descent. In fact, less than 5% of genetic studies worldwide include participants with multiple ethnicity34 and tumor tissues by 450 K DNA methylation array. We selected two subsets of patients from ICGC-Brazil (BCH cohort): first, Discovery cohort 1, encompassing 54 cutaneous melanomas patients harboring or not the UV mutation signature; second, Discovery cohort 3, constituting of 17 acral melanomas that are non UV-mutant . No compensation was provided to the participants in this study and informed consent was obtained by all participants included in BCH cohort. Patients were recruited at BCH in the context of the ICGC-Brazil project, which encompassed 100 melanoma patients prior to any systemic treatment and from whom paired tumor/blood tissues were profiled by WGS6/2013. N9.The second cohort comprised 58 cutaneous melanoma samples from TCGA-SKCM for which information about UV signature was available Fig.  based on39. Briefly, ~25 mg tissue in 180 μL ATL Buffer was homogenized  at 6500 1 × 10/10 s for three times. After samples were centrifuged for 1 min at 2867 × g, supernatants were transferred for another tube, 25 μL of Proteinase K were added per sample, and samples were incubated at 56 °C, 134 × g for 3 h. Then 4 μL RNase were added per sample and DNA was isolated using QIAsymphony .DNA from fresh frozen BCH-cohort samples were isolated using the DNA Mini Qiasymphony kit  following BCH Biobank procedures and the manufacturer’s instructions9. Molecular subgroups were defined by investigating somatic single-nucleotide mutations in BRAF hotspot, RAS hotspot and NF1 throughout Mutect40 algorithm and further annotated using Annovar41. The TN molecular subgroup denoted melanoma patients who did not harbor mutations in any of the three genes34.The WGS library construction and sequencing of BCH samples were performed at Mendelics . A total amount of one μg of each matched normal and tumor DNA was submitted to sonication fragmentation and further library preparation by Illumina TruSeq DNA PCR-Free Library Preparation kit  using the 350 bp protocol. Libraries were quantified by Qubit Fluorometer  and qualified by 2100 Bioanalyzer . The sequencing was carried out using Illumina HiSeq 2500 by paired-end strategy at a minimum of 30X coverage. WES data of TCGA samples was available from GDC Legacy Portal42. We used the Non-negative Matrix Factorization (NMF) algorithm43 to determine the consensus signatures among the 71 patients. At the moment of the analyses, we used the 21 signatures10 that were available and identified a consensus signature with more than 0.8 cosine similarity. For both BCH and TCGA cohorts, we classified samples as harboring an UV mutation signature (Cosmic Signature 7) based on the recommended criteria in which C > T transitions at dipyrimidine sites accounted for more than 60% or CC > TT mutations more than 5% of the total mutation burden9.The UV mutational signature identification was performed using the SomaticSignatures Bioconductor packageThe isolated DNA (500 ng) from BCH-cohort was bisulfite-modified using the EZ DNA Methylation Kit  following the manufacturer’s instructions for Illumina Infinium 450 K beadchip assay. Modified DNA was stored at −20 °C when short intervals were required between bisulfite conversion and further processing, and at −80 °C for long-term storage.9. For BCH, bisulfite converted DNA samples were profiled using 450K  and a well-established workflow optimized at IARC for high-throughput analyses through an automated robotic system (Freedom EVO 150 by Teca) that can process the chips with minimal human error. Chips are scanned using Illumina iScan to produce two-color raw data files (.idat format). Sample allocation to the arrays was based on a semi-randomization design that ensures minimum confounding by technical variation and minimizes the masking of biological covariates of interest by batch effects.The 450K data of BCH were generated in-house, and those of TCGA were downloaded from the GDC Legacy Portal44  to  as it is more suitable for running regressions. Surrogate variable analysis (SVA) was performed on the methylome data to correct for potential batch effects, to adjust for differences in cell type composition as a reference-free method46, and to adjust for latent variables, a choice validated by the findings of our benchmarking47. SVA also increases statistical power by removing (unwanted) variability through aggregating information at the data level and constraining the data’s variability to the phenotype of interest48.For the bioinformatics pre-processing, IDAT files from both cohorts were imported and processed using R software. Quality-control graphs and bimodal distributions for each dataset are shown in Supplementary Figs. versus non UV-mutant cutaneous melanoma patients . At least 90% of DMP-based genes overlapped with those derived from DMRs across all models  to test four statistical models in the discovery cohort 1 (BCH), including one crude and three adjusted models, comparing the DNA methylome of UV-mutant versus non UV-mutant cutaneous melanoma patients. We used the Metal tool51 and the Dmrff package in R to perform DMR fixed effects inverse variance-weighted meta-analysis52, using the crude model as prioritized in the cohort-specific analysis. The meta-analysis lambda value was 1.16, showing low inflation. Statistically significant DMRs were defined as those with FDR-adjusted P value < 0.05. Due to the larger number of hits expected with the increased statistical power afforded by the meta-analyses, we also reported the more stringent Bonferroni-adjusted p values, especially considering the higher likelihood of false positivity due to clinicopathological, ethnic and methodological differences between meta-analysed BCH and TCGA.We complemented the cohort-specific analysis by a meta-analysis across the BCH and TCGA cohorts comparing UV-mutant 53. This approach performs classification of samples using partial least squares regression of the categorical outcome Y (cancer subtype) on the predictor variables (DNA methylation). PLS-DA is a clustering technique that allows the quantification of the discrimination relevance of a given variable (CpG) and to predict the phenotype of new samples (independent of DMPs or DMRs). This method is especially suited to deal with a much larger number of variables than samples, as in next-generation microarray and sequencing data, and we aided this method further by a filtering step using median absolute deviation (MAD)54. We selected the 100 most variable CpGs and applied PLS-DA on the methylation matrix on this subset of sites to assess the discriminative potential of the DNA methylome between UV-mutant and non UV-mutant cutaneous melanomas .In addition to generating DMPs and DMRs, methylation data from BCH and TCGA samples were further investigated using Partial Least Squares Discriminant Analysis (PLS-DA)TAPBP (7 CpGs) gene , were downloaded from TCGA-SKCM project58 (https://maayanlab.cloud/Enrichr). Given that genes with larger numbers of probes are more likely to have significantly differentially methylated CpGs, potentially biasing gene set analysis, we implemented the gometh function of the missMethyl package59 in R to adjust for the number of CpGs per gene, which ranges on the 450K array from 1 to 1299 CpGs.Gene ontology and pathway analysis were performed using the Jensen Disease ontology and KEGG pathway databases available on Enrichr websiteRegarding eQTMs, we applied Pearson correlation between RNA expression and DNA methylation data of the 169 genes that are common between BCH and TCGA DMRs, while limiting the analysis of a given gene to its constituent CpGs.60 that uses LASSO61 penalization technique to select the 25 most informative CpGs and transcript probes in each dataset  or a deep deletion (≤ −2), and this number was divided by the maximum CNV value obtained across the analysed genes in order to generate the CNV score; [MUT: Mutation], being the number of tumors affected by at least one single-nucleotide alteration, and this number was divided by the maximum MUT value obtained across the analysed genes in order to generate the MUT score; [EXP: Expression], being the number of UV-mutant cases presenting variations in RNA expression (|logFC| > 2) relative to non UV-mutant patients, and this number was divided by the maximum EXP value obtained across the analysed genes in order to generate the EXP score; and [METH: Methylation], being the number of UV-mutant cases presenting variations in methylation (|delta betas| > 0.1) relative to non UV-mutant patients, and this number was divided by the maximum METH value obtained across the analysed genes in order to generate the METH score. The driver score for each gene was then calculated as the sum of these four proportions, representing a derivation of our recently reported cancer driver score17 by additionally including DNA methylation data on top of genomic and transcriptomic data.For the prediction of multi-OMICs driver score, genome, transcriptome and DNA methylome data were downloaded from TCGA-SKCM (473 cases)64; however, we preferred to estimate the power based on the OMICs with the largest dimension as a more conservative approach. Statistical power was further enhanced by implementing dimension reduction and SVA approaches , and the false positive likelihood was reduced by monitory and correcting for potential inflation, by adjusting for multiple-testing, and by replication of findings in two independent cohorts as well as by two different techniques (array- and pyrosequencing-based).The OMIC with the largest dimension  would require the largest number of samples analysed to maintain a high statistical power. In this work, it would be the DNA methylome  followed by the transcriptome . Although WGS has a larger dimension than either, it is not being used agnostically in this work, but rather to screen for specific mutational signatures or mutated genes known to be genetically altered in melanoma. Accordingly, statistical power is estimated based on the methylome as such: the overall mean standard deviation (SD) of methylation probes in the BCH or TCGA data is 0.11 . Given an effect size ≥10% methylation difference  OR (melanomas)) AND  OR (methylome) OR (methylome-wide) OR (DNA methylation)). In total, we found 867 studies of which 20 (Supplementary Data Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesReporting SummarySupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Supplementary Data 15Supplementary Data 16Supplementary Data 17Supplementary Data 18Supplementary Data 19Supplementary Data 20Supplementary Data 21Supplementary Data 22Supplementary Data 23Supplementary Data 24Supplementary Data 25Supplementary Data 26Supplementary Data 27Supplementary Data 28Supplementary Data 29


The interplay between active biological processes and DNA repair is central to mutagenesis. Here, we show that the ubiquitous process of replication initiation is mutagenic, leaving a specific mutational footprint at thousands of early and efficient replication origins. The observed mutational pattern is consistent with two distinct mechanisms, reflecting the two-step process of origin activation, triggering the formation of DNA breaks at the center of origins and local error-prone DNA synthesis in their immediate vicinity. We demonstrate that these replication initiation–dependent mutational processes exert an influence on phenotypic diversity in humans that is disproportionate to the origins’ genomic size: By increasing mutational loads at gene promoters and splice junctions, the presence of an origin significantly influences both gene expression and mRNA isoform usage. Last, we show that mutagenesis at origins not only drives the evolution of origin sequences but also contributes to sculpting regulatory domains of the human genome. Activation of DNA replication origins triggers DNA breaks and mutagenesis driving phenotypic diversity in human populations. Mutations are the building blocks of genome evolution and the driving force for both Mendelian disorders and cancer. Detailed catalogs of human genetic variation in normal cells and tumors have begun to reveal some of the determinants of mutation distribution in human cells. Large-scale genomic features, including replication timing , from 19 human cell samples . We refiWe first quantified mutation rates by assessing the distribution of single-nucleotide variants from de novo mutations (DNMs) in the vicinity of the three replication origin classes. Because DNMs occur within one generation, i.e., are present in an offspring but not in either parent, and are subject to minimal selective pressure, their distribution reflects the germline mutation rate. We observed an increased frequency of DNMs at constitutive origins, but not at stochastic and core origins, compared with their flanking regions . We deteTo characterize the mutational processes associated with origins, we identified the mutational signatures operating at constitutive origins using an established nonnegative matrix factorization (NMF) approach  and analWe next identified three small common indel signatures, hereafter referred to ID A, B, and C, operating in concert with SBS A, B, and C, respectively . We found that ID A and C signatures are mainly composed of indels at long (≥5 bp) homopolymers, indicative of DNA polymerase slippage and incomplete DNA mismatch repair (MMR) , which displays high affinity for negatively supercoiled DNA , with ~80% of constitutive origins lying within gene bodies. Constitutive origins locally accumulate at gene element boundaries such as transcription start sites (TSSs) and splice junctions . This suggests that origin activation, rather than origin environment, is the cause of DSB accumulation. We observed the same trends when considering the ATAC-seq signal  and found that the number of DSBs at all origins or intergenic origins correlates with TOP2B . These observations suggest that the sites of efficient replication initiation in accessible chromatin are associated with accumulation of topoisomerases and are hotspots for DSBs. Further, the number of DSBs detected at constitutive origins correlates with the number of DNMs detected in human populations at the same sites . However, we also found that the density of DSBs at TSSs and splice sites depends on the expression of the associated genes (fig. S4I). To further understand the relationship between origins and transcription in generating DSBs at origins, we assessed the impact of origin usage, efficiency, and expression levels of the associated genes on DSB density at origins found at either TSSs or splice sites  independently of gene expression, meaning that the contribution of transcription to DSB density at origins is minimal. When comparing origins at TSSs or splice sites of genes expressed at similar levels, increased origin usage/efficiency  leads to a ~5.2-fold increase in DSBs, while increased expression  leads to a ~1.9-fold increase in DSBs. These observations show that the presence of efficient origins triggers a local accumulation of DSB independently from transcription. We note that the presence of constitutive origins in a gene does not impede its overall transcriptional activity (fig. S4J).Because constitutive origins are enriched within gene bodies , we assece sites . We founP = 7.49 × 10−206, χ2 test of independence; fig. S5A). We then investigated whether the presence of a constitutive origin within a gene feature increases the mutational load in the associated gene. We first categorized genes according to the presence or absence of a constitutive origin at their TSSs or at splicing sites and computed the density of functional variants around their TSSs and splice sites . Similarly, splice sites marked by an origin display a ~1.34-fold increase in mutational load . We then investigated how transcription contributes to this observed increase in mutational loads. To do this, we computed mean levels of gene expression in adult male germ cells from the spermatogonia, spermatocyte, and spermatid lineages . We extracted the sequence context of all functional variants found within 2.5 kb around TSSs and created lists of predicted TFBSs associated with the functional variants. By comparing the enrichment of TF motifs around TSSs marked by an origin compared with those that are origin free , we obseTo investigate the long-term consequences of mutagenesis at constitutive origins on the evolution of the human genome, we devised an in silico model in which a library of DNA sequences, calibrated on noncoding human genomic sequences, was evolved according to rules defined by the observed mutation prevalence, pattern, and replication strand bias at constitutive origins . We compP = 8.26 × 10−4, χ2 test of independence; P < 2.2 × 10−16, χ2 test of independence; We next analyzed variation in the Shannon diversity index, a measure of sequence diversity, around our modeled in silico origins . SequencHere, we establish that a class of highly efficient replication origins allows the observation of specific mutational patterns associated with human replication initiation that substantially affect genome evolution and expression. The mutational processes we report are distinct, by both their nature and consequences, from the known processes that have been previously associated with replication . Our datThe accumulation of DNMs at constitutive origins provides direct evidence for increased germline mutation rates at early and efficient origins, supporting the mutagenic properties of origin activation. However, we note that stochastic and core origins exhibit a similar mutation rate to their flanking regions . This coA direct relationship between constitutive origin activation and mutagenesis is supported by the significant correlations between origin efficiency and mutation frequency. We identified two replication-dependent mutational processes operating at constitutive origins that may result from the two-step process of origin activation . SBS B iUsing the H9 experimental cell system, we confirmed the mutagenic properties of constitutive origins by demonstrating the impact of origin activation on genetic instability. We found that, while transcription contributes to the accumulation of DSBs at TSSs and splice sites, origin efficiency is the main determinant of DSB accumulation at origins . It is nWe demonstrated that origin usage and activation is the driving force for origin sequence evolution and impose the positioning of local nucleotide skews  and splice sites of GENCODE basic transcripts  to prioritize full-length coding transcripts over partial or noncoding transcripts. Intron coordinates were recovered by subtracting the coordinates of exons, 5′ untranslated regions (5′UTRs), and 3′UTRs from transcript coordinates using the “subtract” command of bedtools  to late-replicating (S4 + G2) DNA values as a function of genomic positions in 50-kb sliding windows at 1-kb intervals. We averaged the obtained timing values from experiments using the GM12812, BG02, IMR-90, and MCF7 cell lines. Enrichment of replication origins within replication timing domains was assessed by computing the fraction of origins and associated replication timing values in 100-kb windows of the human genome.Replication origins, mapped using SNS-seq, from 19 human cell samples were recovered from Akerman n = 34,082,281) and short insertions/deletions  were recovered using VCFtools  for each window were then calculated as The total number of mutations, Evaluation and visualization of mutational patterns at origins were performed using the MutationalPatterns  and available from To determine the contribution of polymerase ε and δ to DNA synthesis in the vicinity of constitutive origins, we analyzed the contribution of mutational signatures attributed to defective proofreading due to acquired mutations in the exonuclease domains of POLE or POLD1. To do this, we used an iterative fitting approach, as described previously, but using only the polymerase-associated signatures SBS10a, SBS10b, SBS10c, and SBS10d (COSMIC v3.2) and found that SBS10b (defective POLE proofreading) and SBS10c (defective POLD1 proofreading) contribute to mutagenesis at constitutive origins. Calculating the absolute number of mutations attributed to both signatures in 100-bp windows spanning the 20-kb origin domains allows the demonstration that polymerase δ, but not polymerase ε, is active in a small 4-kb domain centered at the origins.n = 2), TOP2B (n = 2), DSBs , BRCA1 (n = 4), XRCC5 (n = 2), RAD51 (n = 1), TOP1 (n = 3), PARP1 (n = 3), RPA , and phospho-RPA2-S33 (n = 1). We then computed coverage plots using the “map” function of bedtools in windows of 100 bp covering not only 20-kb domains centered on constitutive origins but also domains covering low-, medium-, and high-efficiency constitutive origins. Enrichments over background were computed by normalizing the local enrichment values by the average values obtained for the first and last 20 windows of origin domains. We finally averaged the coverage plots over the different analyzed cell lines and conditions when available.The distribution of DNA repair factors and DSBs around constitutive origins was assessed using available ChIP-seq and other datasets. The references and associated Gene Expression Omnibus (GEO) accession numbers for the datasets used in these analyses are reported in table S1. We selected datasets assessing the chromatin distribution of ORC  plates. Cells were passaged every 3 to 4 days based on cell density. Cells tested negative for mycoplasma contamination throughout the study. All DNA libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit for Illumina Platforms (Roche) and were tested for quality and primer and adapter contamination using Agilent 2100 Bioanalyzer High Sensitivity DNA chip before proceeding to high-throughput sequencing.Human H9 embryonic stem cells (WiCell WA09) were cultured at 37°C, 5% COd-lysine–coated 96-well plate (Greiner Bio-One). Once adhered by centrifugation, cells were cross-linked in 4% paraformaldehyde (Thermo Fisher Scientific) for 10 min followed by two washes with 1× PBS. Plates were tightly sealed, and cells were kept in 1× PBS at 4°C until downstream library preparation. INDUCE-seq was performed as previously described (Cells were harvested using Accutase (Thermo Fisher Scientific) to obtain a single-cell suspension and counted, followed by two washes in Dulbecco’s phosphate-buffered saline (PBS). Cells  were deposited into a poly-ATAC-seq was performed as previously described  and following the manufacturer’s instructions. Primary antibodies were incubated overnight at 4°C [Anti-Topoisomerase II alpha  and Anti-Topoisomerase II beta ]. DNA was purified from input and enriched chromatin samples using DNA spin columns . CUT&RUN libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina following the CUT&RUN assay kit recommendations to avoid denaturation and loss of small DNA fragments. CUT&RUN assays were performed in biological triplicate, and each was sequenced three times to ensure high-quality signals. After assessing reproducibility by comparing the genome-wide CUT&RUN signal intensity in 10 kb windows, replicates were combined.RNA was isolated using the RNeasy Kit (Qiagen) following the manufacturer’s instructions and quantified before running on an Agilent RNA Pico 6000 chip using an Agilent 2100 Bioanalyzer. RNA with a RIN (RNA integrity score) score above 7 was used to generate RNA libraries. Libraries were prepared using a NEBNext Ultra II RNA library preparation kit and a NEBNext human rRNA depletion kit (New England Biolabs). Complementary DNA libraries were generated from 750 ng of RNA, amplified for eight PCR cycles, and barcoded with NEBNext Multiplex Oligos for Illumina (New England Biolabs). RNA-seq analysis was performed in triplicate.INDUCE-seq reads were processed as previously described  of all functional variants, as previously defined, found within 5 kb of TSSs and identified overlapping TFBSs using the TFBSTools  and sQTL  for 49 tissues were recovered from the GTEx project website at rsat.sb-roscoff.fr/random-seq_form.cgi). PDF-1 was constructed by fitting a multipeak Gaussian profile to the observed mutation rates reported in To determine the effect of mutagenesis at constitutive origins on their sequences, we devised an in silico model of evolution in which a library of synthetic DNA sequences was evolved according to rules defined by observed mutation processes operating at constitutive origins. We defined three PDFs that describe (i) the probability of a base to mutate according to its distance from origin centers , (ii) the probability of the resulting mutation according to its trinucleotide context and the nature of the known mutational signatures operating at its position , and (iii) the probability for a mutation to be fixed on the Watson or Crick strand . The library of 20-kb DNA sequences contained 1000 sequences calibrated on noncoding upstream sequences from the hg38 version of the human genome and using a Markov model with an oligonucleotide size of 6  and the following equation: ip is the proportional abundance of one of the 4096 6-mers. As SDI values depend on the number of sequences analyzed, computed values were corrected to account for different sample size when appropriate.DNA sequence analysis was performed using the seqinr (gerp_conservation_scores.homo_sapiens.GRCh38.bw), reporting substitution rates computed from the multiple alignment of 111 mammalian genomes, was obtained from ftp://ftp.ensembl.org/pub/release101/compara/conservation_scores/111_mammals.gerp_conservation_score/. GERP scores were computed as averages from rolling windows of 100 nt spanning 20 kb centered on origin midpoints. For consistency of presentation with plots of mutation rates and sequence diversity, the y axes in plots showing GERP scores have been inverted so that greater constraint is low and greater diversity is high.GERP scores .


Avian influenza (AI) is an important disease that has significant implications for animal and human health. High pathogenicity AI (HPAI) has emerged in consecutive seasons within the UK to cause the largest outbreaks recorded. Statutory measures to control outbreaks of AI virus (AIV) at poultry farms involve disposal of all birds on infected premises. Understanding of the timing of incursions into the UK could facilitate decisions on improved responses. During the autumnal migration and wintering period (autumn 2019– spring 2020), three active sampling approaches were trialled for wild bird species considered likely to be involved in captive AI outbreaks with retrospective laboratory testing undertaken to define the presence of AIV.n = 594) caught during routine and responsive mist net sampling failed to detect AIV. Cloacal sampling of hunter-harvested waterfowl (n = 146) detected seven positive samples from three species with the earliest detection on the 17 October 2020. Statutory sampling first detected AIV in wild and captive birds on 3 November 2020. We conclude that hunter sourced sampling of waterfowl presents an opportunity to detect AI within the UK in advance of outbreaks on poultry farms and allow for early intervention measures to protect the national poultry flock.Faecal sampling of birds ( Avian influenza (AI) is caused by a zoonotic viral pathogen  hosted predominantly by wild birds but with the ability to jump to other taxonomic groups , includiUK poultry farmers are legally obliged to report suspicion of AIV infection within their flocks, which is followed by testing and implementation of control measures if notifiable AIV is confirmed . DetectiAnatidae; ducks, geese and swans [Anatidae bird families has been found between studies [Anatidae into poultry is unclear but transfer via intermediate bridge species, which may include passerines, has been proposed [The primary wild host for both LP and HPAVI is thought to be the nd swans  with annnd swans . Howevernd swans . Substan studies –18. The proposed . ConfirmHundreds of thousands of individual passerines (and other birds) are ringed (fitted with uniquely coded rings upon the tarsus or tibia) across the UK as part of voluntary national monitoring of bird populations overseen by the British Trust for Ornithology . FurtherThree approaches were tested to evaluate their ability to detect AIV in wild birds between Autumn 2019 and Spring 2021: (1) sampling of hunter-harvested waterfowl, (2) volunteer sampling of migrating birds and (3) responsive sampling of birds caught close to a poultry farm AI outbreak.post mortem from hunter-harvested waterfowl at a private site on the northern side of the outer Humber Estuary in northeast England . The site was chosen because of its position on the east coast of the UK, where many migratory birds enter the country during their autumn migration [Cloacal swab samples were collected igration .Faeces passed by migratory birds upon capture were collected in the same region at three sites with ongoing bird banding/ringing projects during autumn 2019 and 2020 .Fig. 1.Faecal samples were collected from passerines caught in mist nets from one hour before dawn until catches became minimal during daylight hours. Mist nets were used under licence from the British Trust for Ornithology (BTO) and birds were extracted and placed singly into a cloth bag from where faecal samples were collected into 1.5 ml screw cap microcentrifuge tubes. Tubes were labelled with a unique sample code, the date, species of origin and the number of any ring present or fitted during the bird's capture.Each cloth bag was only used once per sampling session to avoid cross contamination of samples, and bags were soaked in a weak bleach solution  and washWading birds were caught monthly in mist nets over autumn and winter 2019/2020 and 2020/2021 during nights with a waning or new moon to limit the ability of waders to see the nets. Mixed species vocalisation play backs were used to attract waders to the catching areas. Upon capture, waders were placed into single species holding crates, which were lined with plain paper. Once all birds were processed (biometrics taken and ringed), individual faecal samples were collected from the boxes and placed into 1.5 ml microcentrifuge tubes labelled by batch number (for which the ring numbers of birds in each batch were recorded), species and date. Lining paper was replaced between each batch to avoid cross contamination.A single duck trap was placed on Kilnsea Wetlands (Spurn peninsula) and baited with grain for ducks during the winter 2020/2021. Further, dead birds found in the wider Spurn area were collected, and sampled for AIV via a cloacal swab.On 7th December 2019, low pathogenicity H5N3 was confirmed on a commercial poultry farm in mid-Suffolk as part of the UK notifiable disease investigation process. In response to this outbreak, passerines were captured at bird ringing sites see  using miThe Animal and Plant Health Agency (APHA) publishes data on AIV detections in wild birds  in additAll swabs and faecal samples were stored dry in 35 ml centrifuge tubes and placed into a chest freezer at −20 °C within 24 h and then stored within a −80 °C freezer within 4 days of sample collection. Samples were transported on dry ice within 24 h of removal from −80 °C storage for virological investigation at the APHA in Weybridge, Surrey. Samples were retrospectively tested with the Nagy matrix (M)-gene detection real-time reverse transcription polymerase chain reaction (RRT-PCR) for generic detection of AIV RNA . PositivTo compare the timing of AIV incursion detected by each of the sampling approaches with bird migration trends, data on relative abundance (% of sites in the UK where a species was present in any given week) were downloaded from eBird's Basic Dataset (EBD), a downloadable citizen science repository for bird sightings .Between 18 October 2020 and 13 January 2021, cloacal swabs were collected from 146 shot birds from 7 different species of waterfowl. A total of 7 shot birds tested positive for AIV .Table 1Low pathogenicity H5 was detected in Teal (1) and highly pathogenicity H5 was detected in Eurasian Teal (1) and Eurasian Wigeon (1) .Table 2Among teal, one shot on 19 October 2020 had been ringed on the 10 April 2017 at Nidingen, Halland, Sweden, one shot on 28 November 2020 had been ringed at Ottenby, Öland, Sweden on 4 August 2019, and another shot on 28th November 2020 had been ringed in Murmansk Oblast, Russia on 21 July 2016 see .Fig. 3.n = 382) were collected at Spurn peninsula and 66 were collected from Filey Brigg  were also collected from 3 species of wader: bar-tailed godwit , redshank  and knot . The duck trap caught a small sample of waterfowl: mallard  and shoveler . Finally, four birds were sampled when discovered dead or weakened at Spurn: one each of cormorant , whooper swan (Cygnus cygnus), mute swan (Cygnus olor) and common scoter (Melanitta nigra). None of the active faecal migration samples tested positive for AIV.Faecal samples (n = 119) were collected from 16 species at 4 sites between 7.5 and 38.8 km of the outbreak site  had been found dead on the 3 November 2020. Over the outbreak season (November 2020–April 2021), 311 of 1345 different wild bird carcasses from 22 species tested positive for AIV from locations across the UK [During autumn/winter of 2020/2021, HPAI H5N8 AIV was first confirmed in the UK by passive surveillance of wild birds on the 9 November 2020. The birds (a greylag s the UK .Waterfowl migration into the UK during autumn 2020  varied bn = 152), 7 ducks tested positive for AIV in 2020 in comparison with no positive results obtained from 474 samples collected from passerines during 2019 and 2020. Moreover, none of 119 samples collected within the locality of an AI infected poultry farm tested positive for AIV. These results further support priority surveillance for AIV in Anseriformes. The percentage of hunter-harvested samples testing positive for HPAIV was surprisingly consistent with the results of a study involving 4729 hunter-harvested birds in the USA during 2014-15; 1.3%, with Eurasian teal and Eurasian wigeon prevalent within the sample [During the autumn/winter of 2020/21 LPAI was retrospectively confirmed in a Eurasian teal that had been shot and sampled on 19 October, a full 11 days before LPAI was first detected as part of a statutory notifiable avian disease investigation following a non-negative result from active serological surveillance within poultry. Further, HPAI was retrospectively detected in a hunter-harvested Eurasian wigeon that was shot on 31 October, three days before HPAI was first detected on a poultry farm and three days before HPAI was detected in wild birds as part of the UK AIV passive surveillance scheme (which was confirmed 6 days later). Despite a small sample size  so, whist week of January, 5 hunter harvested birds tested positive in the last week of the study (week 4 of the year) indicating that a longer study period may reveal more about changing AIV prevalence in different locations [A similar study was conducted in wild waterfowl at two sites in northern Italy between November 2020 and January 2021 where 823 hunted and 521 live captured ducks were sampled . Results demonstrated higher AIV prevalence than was detected on the Humber, with 6.7% of sample positive for AIV in hunter-harvested birds and 9.7% in samples from birds that were captured and released, compared with 4.8% on the Humber. Whilst AIV detection was most frequent in northern Italy during November and January, different peaks were evident between the two sets of samples. Week 49 and 50 (of the year) showed the largest number of positive samples for live captured birds and week 47 was highest for hunter harvested birds (though no birds were sampled by this method in week 49). Whilst no live captured birds tested positive after the 1ocations .The active sampling methods in the current study were highly spatially focussed in comparison with the UK's current passive surveillance of found-dead birds, but these approaches offered the additional advantage of sampling clinically healthy individuals as well as those that had yet to develop symptoms of disease. Asymptomatic but transmissible infections of AIV have been detected in waterfowl and other avian species during challenge experiments and it is plausible that the same findings may be seen in wild birds . The impAnatidae species are predominantly responsible for the seasonal re-emergence of AIV [Anatidae to reliably indicate the seasonal re-emergence of AIV within the UK. Furthermore, similar methods used along the migration pathways of these species would further aid in tracking international AIV dynamics.Passive monitoring did detect infection among wild birds across the UK, but no spatio-temporal pattern was discernible. Most migratory bird species enter the UK from breeding grounds to the east , but the first passive detection within British wild birds was recorded in Gloucestershire, in the southwest of the UK. If e of AIV , then ite of AIV  and to aDetection of the same strain of AIV among wild birds that occupy poultry farms could help identify those species that pose the greatest risks to poultry . HoweverAnatidae. However, this is not universal, with Gronesova et al. [et al., [Passer montanus) but 94/800 seropositive samples from the same species indicating that although 94 individuals had been immunologically challenged by AIV, none were actively excreting at the time of sampling. An extensive US study [n = 13 046). Whilst there will likely be differences in the epidemiological network between the new and old world (different species and families), a UK or flyway-wide study of similar magnitude may be required to clarify the potential roles of passerines in AIV epidemiology in Eurasia.Previous studies investigating AIV prevalence in passerines have mostly detected low levels or no AIV within their samples, though the numbers of studies focussing on wild passerines is heavily outweighed by those focussing on a et al.  detectin[et al.,  reportedUS study  involvinst to January 31st under all devolved administrations except the Isle of Man. This extends to February 20th in England, Wales and Scotland when hunting below the high-water mark. Legal quarry also limits what can be sampled. Gadwall (Mareca strepera), common goldeneye , mallard, northern pintail (Anas acuta), common pochard (Aythya ferina), northern shoveler, Eurasian teal, tufted duck (Aythya fuligula) and Eurasian wigeon are legal quarry for ducks and Canada goose, greylag goose, pink-footed goose (Anser brachyrhynchus) and European white-fronted goose  can all be lawfully shot, but other duck and goose species and all swans cannot [Anatidae in the UK with the possible exception of barnacle geese (Branta leucopsis) [Active sampling of hunter-harvested waterfowl is limited to certain species that can be lawfully harvested (see below) and by the UK open season which covers the period from the September 1s cannot . These aucopsis) . The resUK autumn migration in dabbling ducks rose in mid-late August varying by species, with most wintering birds present by mid to late November , but thet al. [et al. [Bevin et al.  and Gobb [et al. , have al


Gastric cancer is a heterogenous group of tumours, and a better understanding of the carcinogenesis and cellular origin of the various sub-types could affect prevention and future treatment. Gastric neuroendocrine tumours (NETs) and adenocarcinomas that develop in the gastric corpus and fundus of patients with chronic atrophic gastritis have atrophic gastritis, hypoacidity, and hypergastrinemia as common risk factors and a shared cellular origin has been suggested. In particular, signet ring cell carcinomas have previously been suggested to be of neuroendocrine origin. We present a case of a combined gastric NET and signet ring cell carcinoma in a patient with hypergastrinemia due to autoimmune chronic atrophic gastritis. The occurrence of such a combined tumour strengthens the evidence that gastric NETs and signet ring cell carcinomas develop from a common origin. The proliferation of enterochromaffin-like (ECL) cells of the gastric corpus and fundus is stimulated by gastrin and most gastric neuroendocrine tumours (NETs) develop due to long-term hypergastrinemia. Gastric NETs develop in patients with autoimmune chronic atrophic gastritis (CAG) type 1), as well as in patients with gastrinomas type 2), and type 1 gastric NETs regress after treatment with a gastrin receptor antagonist [, as well, and typWe here present a case of a combined type 1 gastric NET and signet ring cell carcinoma which offers novel information concerning the aetiology and origin of signet ring cell carcinomas.An 80-year-old woman with a pervious history of coronary heart disease and pulmonary embolism, using acetylsalicylic acid and warfarin, was referred to our department due to iron deficiency anaemia. After a colonoscopy without relevant pathology, she underwent upper endoscopy. A polyp of 8 mm (Paris Is/Isp) with reddish appearance  located Immunohistochemical methods: 4 µm sections from formalin fixed and paraffin embedded tissue blocks were incubated with antibodies against neuroendocrine markers CgA  and synaptophysin . Proliferation rate was assessed in sections labelled with an antibody against Ki-67 . The immunoreaction against synaptophysin was amplified using Mouse Link ; and, finally, all immunoreactions were visualized using an EnVision-HRP kit with DAB+ . Hyperplasia and dysplasia of neuroendocrine cells in the oxyntic mucosa were described according to Solcia .Blood samples included serum gastrin 1226 pM (<60 pM), chromogranin A 243 µg/L <101.9 µg/L), anti-intrinsic factor > 480 kU/L (<10 kU/L), anti-parietal cell IgG 3 kU/L (<10 kU/L), suggestive of marked hypergastrinemia due to autoimmune chronic atrophic gastritis. Biopsies from the antrum and corpus were H pylori PCR negative and the flat corpus mucosa had changes consistent with autoimmune chronic atrophic gastritis with linear and micronodular neuroendocrine cell hyperplasia [1.9 µg/L,+K+ATPase, where a combined NET G2 and adenocarcinoma also was found [The case of a combined gastric NET and signet ring cell carcinoma is a rare occurrence, which we consider an illustrative argument of a common origin of neoplastic lesions with a different morphology. It has been reported that chronic atrophic gastritis and hypergastrinemia predispose to gastric NETs and adenocarcinoma to a similar extent  and otheas found ,16. Simias found .According to the 2019 WHO classification, a mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) has components  that each constitute at least 30% , a limitAltogether, the current case report describes a rare event but supports the hypothesis that gastric ECL cell NETs and signet ring cell carcinomas share a common origin.


To assess the effect of filgotinib, which preferentially inhibits Janus kinase 1 (JAK1), on MRI measures of structural change in the SI joint in patients with active AS in the TORTUGA trial.n = 58) or placebo (n = 58) once daily for 12 weeks. In this post hoc analysis, T1-weighted MRI scans of the SI joint were evaluated by two independent readers using Spondyloarthritis Research Consortium of Canada (SPARCC) Sacroiliac Joint Structural Score (SSS) definitions for erosion, backfill, fat metaplasia and ankylosis. Correlations between SPARCC SSS and improvement in clinical outcomes were also assessed.Adults with active AS and inadequate response/intolerance to two or more NSAIDs were randomized 1:1 to filgotinib 200 mg (P = 0.02) and an increase in backfill score (P = 0.005) vs placebo, with no significant between-group differences for ankylosis (P = 0.46) or fat metaplasia (P = 0.17). At week 12, the change in SPARCC MRI SI joint inflammation scores correlated positively with erosion scores but negatively with backfill scores.MRI scans from 87 patients  were evaluated. At baseline there were no notable differences between filgotinib and placebo for any MRI structural lesion types. From baseline to week 12, filgotinib was associated with a significant reduction in SI joint erosion score (The significant changes in MRI structural lesions induced by filgotinib in the SI joint by week 12 demonstrate that tissue repair can be observed very soon after starting treatment with a JAK1 preferential inhibitor. This could have prognostic implications for development of ankylosis.http://clinicaltrials.gov, NCT03117270ClinicalTrials.gov,  Rheumatology key messagesThe JAK inhibitor filgotinib was associated with MRI structural benefits in patients with AS.vs placebo.SPARCC SI joint erosion and backfill significantly decreased and increased, respectively, with filgotinib Structural changes occurred within the first 12 weeks of treatment, correlating with a reduction in inflammation.AS is a chronic immune-mediated condition characterized by inflammation in the axial skeleton, with early involvement of the sacroiliac (SI) joint and spine , 2. DespMRI is the only imaging modality that can directly detect both inflammatory and structural changes in the SI joint and spine and thus is useful for the early assessment of AS . Fat-supAS is sometimes referred to as radiographic or axial spondyloarthritis . Although the terms are based on different criteria , they represent largely overlapping populations .The ASAS recently updated definitions for SI joint structural MRI lesions, which included erosion, fat lesions, fat metaplasia in an erosion cavity , ankylosis and non-bridging bone bud . ErosionUntil recently, treatment for patients whose AS persisted despite physiotherapy and continuous NSAIDs comprised TNF inhibitors . These aWe present a post hoc analysis of the effects of filgotinib on MRI measures of structural changes in the SI joint of patients from the TORTUGA trial. MRIs were reassessed using the Spondyloarthritis Research Consortium of Canada (SPARCC) Sacroiliac Joint Structural Score (SSS), a validated method to assess the structural lesions of erosion, backfill, fat metaplasia and ankylosis on T1W MRI scans .n = 58) or placebo (n = 58) once daily for 12 weeks. Previous use of one TNF inhibitor was permitted (limited to 30% of enrolled patients). The study protocol was reviewed and approved by the central or individual independent ethics committee in each participating country (Rheumatology online).TORTUGA (NCT03117270) was a multicentre, placebo-controlled, double-blind, randomized trial. The study design has been reported previously . BrieflyMRI scans were taken at baseline and week 12 (or at an early discontinuation visit). Changes in BMO from baseline were assessed in an independent reading campaign using SPARCC MRI SI joint and spine inflammation scoring methods applied to SI joint and spine MRI STIR scans , 27. MRIwww.carearthritis.com/mriportal/sss/index/) for the SPARCC SSS score before assessment of the TORTUGA MRI scans [In this post hoc analysis, T1W MRI scans of the SI joint from the TORTUGA trial that were originally evaluated for SPARCC SI joint inflammation were re-evaluated using standardized SPARCC SSS definitions . While tRI scans .Endpoints included the change from baseline in SPARCC SSS total erosion, total backfill, total fat metaplasia and total ankylosis scores at week 12; the proportion of patients with a decrease, increase or no change in structural lesion score  at week 12, according to the baseline SPARCC BMO; correlation between changes in structural lesions and the change in clinical parameters; and the number of patients with erosion who developed backfill at week 12.All patients with MRI scans with one or more measurement at both baseline and week 12 or an early discontinuation visit during the TORTUGA trial were included. Observed changes from baseline were evaluated using analysis of covariance with factors for treatment, baseline value and randomization stratification by prior TNF inhibitor use. Least squares mean changes from baseline and between-group differences with 95% CIs were calculated. Cumulative probability plots were generated to compare the change from baseline to week 12 for each structural lesion for the treatment groups.The mean of the two reader scores was used to compare the change in structural lesion scores between treatment groups. Interreader intraclass correlation coefficients (ICCs) were calculated to assess the consistency and reliability of scoring between the two MRI readers using the ICC 2.1 model. As pre-specified, interreader discrepancies were resolved by a third independent adjudicator if the change from baseline in SPARCC SSS for structural change measurements for any domain differed by ≥4 points in different directions  or ≥6 points in the same direction (both detected either improvement or worsening). For cases requiring adjudication, the final score was derived from the mean of the adjudicator’s score and the closest score of the two primary readers.An ordered logistic regression model tested for treatment differences among categories of change from baseline to week 12  for each structural lesion type, according to baseline SPARCC BMO ≥2 or <2. A SPARCC MRI SI joint score ≥2 is an established indicator of SI joint inflammation on MRI . PearsonP-values should be considered nominal.The TORTUGA trial was not powered for comparison of these post hoc endpoints; all Rheumatology online). In most cases, MRI scans were missing because patients were unavailable or unwilling to undergo the MRI scan at the week 12/early discontinuation visit. Of patients included in the current analysis, there were no notable differences in demographics or baseline disease characteristics, including SPARCC SSS scores, between the filgotinib and placebo groups  with an evaluable MRI at both baseline and week 12 (or an early discontinuation visit) were re-evaluated in this post hoc analysis. Disease activity was similar between these patients and the 29 patients from the TORTUGA trial who were excluded because of missing MRI scans (o groups . The totRheumatology online). Erosion scores decreased in the filgotinib group and increased in the placebo group (P = 0.02 for between-group difference). Backfill scores increased in the filgotinib group but not in the placebo group (P = 0.005). An example of backfill development during 12 weeks of filgotinib treatment is shown in The mean change from baseline to week 12 in SPARCC SSS for erosion and backfill was significantly different for filgotinib and placebo (P = 0.46) or fat metaplasia (P = 0.17) (Rheumatology online). These findings were supported by cumulative probability plots showing changes from baseline to week 12 in erosion, backfill, ankylosis and fat metaplasia  and decreased in 1/22 patients (4.5%), whereas in the placebo group, backfill did not increase in any patients but decreased in 3/22 patients . Treatment differences for erosion, ankylosis and fat metaplasia were not significant. There were no significant differences for any of the structural lesions in the subgroup of patients with a SPARCC BMO score <2 at baseline (Rheumatology online).r = 0.359, P = 0.013) and placebo group . A negative correlation was observed for backfill in both the filgotinib  and placebo  groups. No significant correlations were observed between erosion or backfill scores and other clinical endpoints. No significant correlations were observed between ankylosis or fat lesion scores with any of the clinical endpoints assessed.Changes in erosion score at week 12 correlated positively with changes in the SPARCC MRI SI joint inflammation score in both the filgotinib group (Rheumatology online).According to individual reader data, erosion resolved in 8 patients (reader 1) and 11 patients (reader 2) in the filgotinib group and in no patients (reader 1) and 5 patients (reader 2) in the placebo group (resolution was defined as being present at baseline but not at week 12/early discontinuation at a particular location within the joint). Backfill developed in 54.2% (reader 1) and 57.3% (reader 2) of locations (SI joint quadrants) with resolved erosion in the filgotinib group and in 20.0% of the locations with resolved erosion reported by reader 2 in the placebo group (there were no cases of resolved erosion reported by reader 1)  vs placebo over 12 weeks [TORTUGA was the first clinical study to investigate a selective JAK1 inhibitor for the treatment of adult patients with active AS. As previously reported, the initial study met its primary endpoint, with filgotinib-treated patients showing decreased disease activity, including MRI-documented inflammation. SPARCC SI joint and spine inflammation scores decreased significantly (12 weeks . The redP = 0.017) and an increase in SPARCC SSS backfill  with etanercept after 12 weeks vs placebo [Similar effects have been observed with anti-TNF therapy , 18. A p placebo , so it wThe mechanisms by which JAK inhibitors might ameliorate inflammation and induce erosion repair (and how this influences new bone formation) remain to be elucidated. Many cytokines implicated in innate and adaptive immune responses underlie the pathogenesis of AS signal through JAK pathways . InterfeOur study had some limitations. It was a post hoc analysis with small patient numbers. Half the patients demonstrated substantial ankylosis in the SI joint, limiting the capacity to demonstrate change in other structural features. No changes in total ankylosis score were expected during the relatively short timeframe (12 weeks) and given that ankylosis was generally at an advanced state at baseline. The impact of filgotinib, and JAK inhibitors in general, on structural lesions should be explored in longer-term studies.Although MRI scans were unavailable for 29 patients, the population in the MRI substudy was generally similar to the overall population. In addition, missing MRIs were mainly due to patient-related factors (e.g. claustrophobia or deformation of the spine preventing the MRI from being performed) or logistical issues leading to MRI scans being obtained outside the protocol-defined time window, all of which occurred prior to unblinding of the study. In addition, an unpublished sensitivity analysis in the main TORTUGA trial, which included a broader set of out-of-window MRIs, revealed a similar outcome to the main analysis for the SPARCC SI joint endpoint. We therefore believe that, other than reducing the number of evaluable patients, the missing MRIs did not have a relevant impact on the interpretation of the data. Patients with MRI structural lesions, especially fat lesions and backfill, represent a subset with higher prognostic risk of progression—not just in the SI joint, but also the spine . They arIn conclusion, in addition to previously reported decreases in SPARCC inflammation observed with filgotinib in AS during the TORTUGA trial, filgotinib was associated with a significant reduction in SPARCC SSS erosion and an increase in backfill as early as 12 weeks. Research is needed to confirm these findings in larger clinical trials, and the prognostic implications of the structural changes observed in this analysis for disease/ankylosis progression warrant further research. In particular, investigation is needed into how the early effects of treatment on structural lesions might influence future development of ankylosis of the SI joint, new bone formation in the spine and the impact on clinical and mobility outcomes and disability.keab543_Supplementary_DataClick here for additional data file.


Most of the patient-specific medical 3D printing begins with volume based medical images of the patient. Several digital manipulations are typically performed to prescribe a final anatomic representation that is then 3D printed. Among these are image segmentation where a volume of interest such as an organ or a set of tissues is digitally extracted from the volumetric imaging data. Image segmentation requires medical expertise, training, software, and effort. The theme of image segmentation has a broad intersection with medical 3D printing. The purpose of this editorial is to highlight different points of that intersection in a recent thematic series within  Medical 3D printing uses specialized segmentation and computer-aided design software , 2, and Visual feedback during stent-deployment is impossible to obtain as deployment is performed under fluoroscopic imaging. Using 3D printed models, De Backer et al. fabricated patient-specific anatomies Fig.  for stenIn small children, both CT and MRI imaging is rare since minimization of radiation and sedation is important. Hopfner et al. used image processing and computer-aided design software to allow unlimited variations of 3D heart models based on single patient scans . The aduMedical training in retrograde intrarenal surgery for treating renal stone disease is arduous owing to the complexity of the procedure. A series of six 3D printed models of upper urinary tract and stones (Fig. Full color and realistic joint models can be valuable for studying complex cases. A new method for developing multi-color and multi-material life-like knee joint anatomical models Fig.  was deveSingle field orthovoltage radiation has dosimetric pitalls and unnecessarily excessive exposure of radiation to organs at risk. Cheng et al. present a novel technique incorporating an optical scanner and 3D printing to deliver treatments using parallel opposed fields . A retroLimited access to key diagnostic tools is detrimental to priority health needs of populations. In situations where an otoscope is unavailable due to financial constraints, a self-fabricated low-cost otoscope might represent a feasible opportunity. Capobussi et al. share the design and development of an open-source 3D printed otoscope and the prototype is compared to a commercial solution Fig.  demonstrVisualizing patient-specific three-dimensional imaging data in augmented reality may improve the surgeon’s understanding of anatomy and surgical pathology, thereby allowing for improved surgical planning, superior intra-operative guidance, and ultimately improved patient care. Wake et al. developed a workflow using the Microsoft Hololens device to visualize prostate and renal cancer models Fig.  to guideSterilization of a 3D printed model could negatively impact its geometric fidelity. The sterility, biocompatibility, mechanical properties, and geometric fidelity of anatomic models must be carefully considered. Toro et al. investigated the geometric fidelity of material extrusion 3D printed acrylonitrile butadiene styrene polymer using vaporized hydrogen peroxide sterilization . Models The collection of articles displayed a diverse set of cutting-edge applications that spanned medical training, interventional planning, medical devices, augmented reality, and quality assurance. This is only a subset of the potential pool of medical 3D printing applications, but still reflects the potential spectrum of the technology.


Literature review demonstrated a surprising lack of publications on digital e-learning pathology resources for senior medical undergraduates and interns. An interactive Digital Pathology Repository (iDPR) integrating two- and three-dimensional  high-resolution anatomical pathology images with correlated digital histopathology was developed. The novel iDPR was rigorously evaluated using mixed methods to assess pathology knowledge gains (pre- and post-tests), quality impact analysis (questionnaire), user feedback (focus group discussions) and user visual behaviour .n = 69, p < 0.001). In addition, most users were highly satisfied with the key design elements of the iDPR tool. Focus group discussion revealed the iDPR was regarded as a relevant online learning resource, although some minor technical issues were also noted. Interestingly, visual behaviour trends indicated that specific diagnostic pathological lesions could be correctly identified faster in 3D images, when compared to 2D images.Exposure to iDPR appeared to improve user pathology knowledge, as observed by significantly increased test scores on topic-related quizzes (The iDPR offers promise and potential in pathology education for senior clinical students and interns, gauging from both qualitative and quantitative positive user feedback. With incorporation of image annotations and interactive functionality, and with further technology development, this would prove a useful tool for diagnostic pathology and telepathology. As images with added visual-spatial dimension can provide enhanced detail and aid more rapid diagnosis, future applications of the iDPR could include virtual reality or holographic images of anatomical pathology specimens.The online version contains supplementary material available at 10.1186/s12909-022-03545-x. Irrespective of medical specialty or general practice, competency as a practising doctor is undeniably dependent on a firm foundation in pathology. Whilst recognized as a core subject encompassing the aetiology, behaviour and effects of human disease, pathology is taught primarily in the early pre-clinical years of most medical schools , 2, whetUsage of digital learning resources in medical education, especially e-learning resources, has been reported to be widely accepted amongst pre-clinical medical students studying conceptually difficult topics like leukaemia . In a raThe incorporation of 3D technologies to enhance learner experience and performance has offered many advantages of traditional gross teaching such as ability to rotate and orient the portable specimen without the restrictions imposed by biohazardous specimens , 16. OwiIn this paper, the research paradigms of cognitive load theory and the cognitive theory of multimedia learning are used to allow more effective instructional visualisation . Using tDespite increased implementation of e-learning in medical schools, systematic literature review , 23 ideni)Can an iDPR tool improve clinical pathology learning to effectively address the reported ‘pathology gap’ in clinical education and internship?ii)Is gaze behaviour and recognition of key identifying features enhanced when viewing 3D vs 2D pathology images?The research questions for this study are as follows:i)A digital pathology repository integrating annotated 2D and 3D images of gross anatomical specimens and histopathology slides may provide a useful online tool to bridge the ‘pathology gap’ in clinical education.ii)Visualisation of dynamic 3D images provides a superior/enhanced learning experience compared to static 2D images of pathological specimens.The hypotheses of this study are as follows:i)designed.Upon identification of a ‘pathology gap’, the framework of the iDPR website was ii)developed to address the aforementioned gap.Subsequently, the iDPR website was iii)analysis and evaluation. The findings from this study can be used to redefine the problem and forms an iterative process typical of the DBR model, aimed to ultimately develop solutions to a problem.In order to effectively implement the iDPR website in the curriculum, the iDPR website underwent vigorous The study was based on the Design-Based Research (DBR) model define problem(s), plan action, implement, evaluate and specifying findings) , and wasJumbo Mode360, Poland) prior to following a systematic process flow for the iDPR website development  website was designed as an e-learning tool with a focus on key female reproductive tract pathologies, topics previously identified by clinical year medical students as somewhat neglected and needing improved correlation with gynaecology postings. Specimens from the female genital tract were selected, once the reports had been finalised, and after permission for the release of tissue had been obtained from the Pathology Laboratory, Subang Jaya Medical Centre. As a learning resource, the iDPR module integrated both two- and three-dimensional images of selected gross pathology specimens ; related histopathology slide sections; histopathology examination reports; relevant high-yield facts, fun facts and online links to an evidence-based clinical decision support system. The website design was initially beta-tested and later, implemented as an e-learning tool and evaluated for its pathology content and pedagogical value and technology enhancements. High-resolution 2D and 3D images of selected gross and microscopic pathology specimens of the female reproductive tract  while 3D images of gross specimens were annotated using a custom software . Both 2D and 3D images along with histopathology reports, explanatory notes including fun facts and related clinical links were subsequently uploaded onto a secure server and web domain (www.i-dpr.com) prior to beta-testing. In order to measure any gains in pathology knowledge, students were subjected to pre-/ post-tests based solely on website content. The impact quality of the iDPR website on users was evaluated qualitatively through both a questionnaire and focus group discussion. In addition, comparative eye tracking of 2D vs 3D images was measured quantitatively through visual behaviour analysis of participants using video-based eye tracking equipment , while the partial qualitative data, which can be observed in real-time on the researcher’s screen . All methods were performed in accordance with the relevant ethics and institution guidelines and regulations.The study was approved by the Monash University Human Research Ethics Committee (MUHREC) and the MBBS Ethics Committee on 10n = 3) not involved in the study. The “knowledge” domains assessed in pre-tests  served to determine basic identification and comprehension of core pathologies related to the female reproductive tract  using n = 69) who reviewed the iDPR website were subjected to a series of pre- and post-tests to measure the suitability of the e-learning tool for pathology learning needs and learning outcomes.Consenting study participants  and if the data was skewed, comparison of the total pre- and total post-test scores (matched data) was analysed using Wilcoxon Signed Ranks test. Conversely, if the matched data were not skewed, paired t-test would be used.The total pre- and post-test scores were checked for skewness using One-Sample Kolmogorov–Smirnov test (n = 69) remained anonymous, socio-demographic data were included in this questionnaire. Respondents were also given the opportunity to include open-ended written feedback in an additional comments section. Thirty positive statements contained in the survey instrument  was assessed for its educational content, multimedia and user interface, navigation, accessibility/technical issues, interactivity and user motivation. Although participants (n = 5) and pre-interns/ fresh medical graduates awaiting housemanship (n = 5), to discuss their recent experiences with the iDPR website. The traditional size of a focus group of 10–12 people can be considered too large for education-related topics of discussion and hence, 5–8 participants are usually recommended, especially if the discussion involves expertise or strong emotions [QSR NVivo 10). The focus group discussions were based on a standardised set of questions:1. Do you feel that there is a pathology gap, i.e. certain parts of pathology are not covered in the curriculum?2. Have you used any e-learning tools for pathology? How effective would an interactive e-learning tool be in aiding clinical pathology learning?3. Which image(s)  vs 3-Dimensional (3D)) was better in terms of understanding and recognition of the lesions on the specimens?4. In your opinion, how did you find the interactive Digital Pathology Repository (iDPR) website?5. What were the strengths of the iDPR website?6. What were the limitations of the iDPR website?7. How could the iDPR website be improved?8. How was your learning experience for pathology after visiting the iDPR website?9. Overall, did you benefit from the iDPR website and are there any distinct advantages compared to other sources?10. Any comments or suggestions?Ten voluntary participants were recruited to form two focus groups, comprising fourth year medical students , was utilised to collect accurate gaze data with an in-built camera recording for visual behaviour analysis including fixation counts. For this part of the study, 2D and 3D images were taken of a separate gross pathology specimen (not included in iDPR pathology modules); i.e. uterus with multiple endometriotic cysts. Participants were required to ascertain the pathological diagnosis and count the total number of pathologic lesions. Calibration and refinement of the eye tracker system were carried out previously in a preliminary study (n = 4).High precision video-based eye-tracking equipment (n = 10) was voluntarily recruited comprising fresh medical graduates/ pre-house officers. Participants all read a standardised information sheet containing a summary of common pathologies of the female reproductive tract, in order to refresh and align their preliminary knowledge levels prior to undergoing eye tracking analysis. The setup, calibration and positioning of equipment, participant and researcher are illustrated in Fig. Subsequently, a separate sample cohort (n = 10) comparing number of pathologic lesions mentally counted (e.g. endometriotic cysts) when viewing 2D vs 3D images were analysed for inter-group association (Fisher’s Exact Test), while mean differences in fixation count also when viewing 2D vs 3D images were analysed via continuous variables, i.e. Independent t-test, if the differences are not skewed using One-Sample Kolmogorov–Smirnov test (n = 10).Quantitative data  and quality impact analysis of the iDPR website with the following study cohort demographics: ages ranged from 22–27 years , 60% being female and more than two-thirds of Chinese ethnic origin. Out of the 69 participants, 19 (27.5%) fourth year medical students, 12 (17.4%) final year medical students and 38 (55.1%) fresh medical graduates/ pre-house officers were involved.p < 0.001, n = 69), analysed using Wilcoxon Signed Ranks test, as the participant scores were skewed, Kolmogorov–Smirnov test .After viewing the designated pathology modules on female reproductive tract pathology via the iDPR website, participant scores increased significantly in relation to content knowledge of these topics, i.e. overall median scores: pre-test 2.0 (IQR =  ± 1) vs post-test 3.0 (IQR =  ± 2) (n = 69) responded positively (strongly agreed/agreed) to the educational content, multimedia and user interface, navigation, accessibility, interactivity and motivation to learn that the iDPR website encouraged  using Kolmogorov–Smirnov test and subsequently, analysed using Independent t-test , this finding is not statistically significant  is defined as the number of times the participants fixate (direct one's eyes towards) an area of interest. It is related to a cluster of gaze points which are temporally and spatially close together and are useful measures of visual attention. Out of the 10 participants involved in eye tracking analysis, 7 participants had higher fixation counts when viewing, identifying and counting endometriotic cysts from a 2D image compared to a 3D image of a pathological uterus specimen. Of the remaining 3 participants, 2 participants had less fixation counts for 3D image viewing while one participant had the same frequency of gaze points when exposed to both 2D and 3D images. Overall, although most participants demonstrated higher fixation counts for viewing 2D compared to 3D pathology images . The mean difference was not skewed,  Specimens are immortalised via 3D photography before sectioning and placed on cassettes and through wireless connection or holographic projection, the 3D photograph of the specimen with the margins can be broadcast into the Operating Theatre for the surgeon to view and decide whether or not further resection needs to be done. This technology is already used in telepathology. The presence of 3D photographic images in the histopathology report can be given to the patient in the form of a compact disk (CD). It will enable easy referral, good clinical records when the need to trace the results arises, or for any second opinions. Horn et al. have shown improvement in diagnostic accuracy with 3D imagery . The digThe novelty of the iDPR website is to visualize specimens in a manner in which they could be manipulated in their hands via 3D rendering. Even with 2D imagery, identification of subtle pathological changes compared with areas of normal tissues in gross anatomical pathology specimens remains difficult to be visualised in a non-transparent/solid organ, especially for junior medical doctors . TechnolIn Iowa, Peterson and Mlynarczyk reported that 3D augmented curriculum in anatomy teaching has improved long-term retention of gross anatomy material and proven beneficial in supplementing anatomy in veterinary medicine . This isOther visual behaviour analyses have included the use of complex radiological imaging such as Positron Emission Tomography (PET) scans and functional Magnetic Resonance Imaging (fMRI) to study the brain to delineate which cortical areas are involved in 2D vs 3D perception of gross pathology images, in order to improve pathology diagnostic accuracy. Cottereau et al. have utilised MRI and high-density electroencephalogram (EEG) imaging techniques to successfully map brain areas involved in 2D motion vs 3D motion in particular V1, V4, lateral occipital complex, V3, hMT + and inferior and superior parietal complex . Lee et In addressing the main research question of whether the iDPR e-learning tool can effectively address the reported ‘pathology gap’ in clinical education and internship, the voluntary participants have been from a private medical school university. As a result, the data may not be extrapolated to other interns from other medical schools with different clinical curricula. Based on the experience of the two co-authors, who are clinical pathologists, with vast experience with curricula contributions of many medical schools, the ‘pathology gap’ is evident. Future studies to roll out the iDPR website to other medical schools and interns and subsequently, confirming the true extent of the ‘pathology gap’.Quality impact analysis and focus group discussion formed a major part of gathering data for quantitative and qualitative analysis of the iDPR website. The typical thematic analysis approach could also be used as a potential alternative to analyse focus group transcripts. There is an inherent limitation of self-reported or self-recalled data as it is limited by the fact that it rarely can be verified independently but accept the voluntary participants to say or write in focus groups or on questionnaires, at face value. These self-reported data contain potential sources of selective memory bias—voluntary participants have to recall their experience on the iDPR website after some time lapse.Visual based analysis was used to study the impact of visualisation of 3D objects when compared to the traditional 2D approach. As evident from the literature review and discussion, the 2D and 3D comparisons of gross pathology were either by complex radiological imaging or EEG, or VR headset. Specifically, there was a scarcity of comparative research studies on the use of eye-tracking tools to study 3D anatomical pathology specimens. Hence, usage of eye tracking analyses in this study is an innovative approach to assess visualisation in pathology education. However, this limitation can serve as an important opportunity to explore such methods in larger research on visual impact of such 3D e-learning tools in the near future.Tobii tool which was used for the collection of data for the visual based analysis included:i)Incompatibility with all types of files and media.ii)Inability to support the actual annotated 3D pathology image, the researcher compromised by using a screen recorder to record the 3D rotation of the pathology image, hence, the annotations were lost and had to be supplemented with an extra 5-s slide of 2D pathology image with labels (A-D) for answering the questions.iii)Inability to track gaze plots (the first object the participant focuses on and subsequently when the participant stops focusing) for moving videos (3D pathology image) hence, the comparison with the 2D pathology image could not be accomplished.iv)Inability to apply other methods of variables to the analyses, i.e. ‘time to first fixation’, to be recorded and computed as the participant will need to close their eyes before looking at the images; and ‘time for mouse clicks’. This was beyond the scope of this study according to the research questions relevant to the researcher but something to consider in different studies in the near future.The limitations of the n = 10) were recruited. Even though, the results demonstrated a rather large mean difference of 5.70 when comparing the standardised 2D and 3D images of pathological uterus with endometriotic cysts, participants were mostly faster when viewing and detecting pathological lesions for 3D compared to 2D images, however, this data is not statistically significant, given the constraint of small sample size. Thus, 3D imaging may be more suitable when making a diagnosis when compared to 2D gross pathology images. Although, the eye tracking device is powerful enough to measure the fixation count for both ‘still pictures’ (2D pathology image) and ‘motion pictures’ (3D pathology image), however, the ability to track gaze plots (the first object the participant focuses on and subsequently when the participant stops focusing) can only be utilised in the former with the current paraphernalia. Thus, hindering the comparison of gaze plots between 2D image and 3D image . Hence, it can be concluded that the iDPR was indeed an effective learning tool for Pathology. Quality impact analysis of the iDPR website quantitatively was largely positive with strengths mostly outweighing the reported limitations. Focus group discussions on the iDPR website were exhaustive and various strengths such as high-resolution 2D and 3D gross pathology images and histopathology slides and the presence of histopathology reports together with feedback on limitations and scope for further improvements. Although not statistically significant, the time taken to view and detect pathological lesions was mostly less for 3D compared to 2D images. Hence, 3D may be better at making a diagnosis when compared to 2D gross pathology images. To make definitive conclusions on the use of 3D imagery as in the present project, future work on EEG combined with a more versatile eye tracking analysis or fMRI may be deployed for a larger group of participants to investigate the effectiveness of anatomic perception of the brain and visual gaze evaluation of 3D imagery.The literature review findings identified a scarcity of peer-reviewed publications on digital learning resources for pathology education geared towards clinical medical undergraduates/ interns. Addressing such a gap in the literature, an e-learning tool for pathology, iDPR website has been designed and developed in the present project. Pathology knowledge of users after viewing the iDPR website showed significant improvement, i.e. increased knowledge scores  tools, VR environment or even holographic images of gross pathology specimens to view such 3D morphology of the gross pathology along with histopathology slides.Additional file 1. Pre- and Post-Knowledge Gain Test and Quality Impact Analysis for the iDPR Website.Additional file 2. Participant responses to Quality Impact Analysis Survey Questionnaire for iDPR Website According to Themes & Sub-Themes (Quantitative Study).