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Supplementary Table S4. Synthetic lethal siRNAs that are associated with the drug classes identified as being CDH1 synthetic lethal in the known drug screen.

Supplementary Figure S3. Alternative real time assays.

Supplementary Data from Development of a Multiplex RNA Urine Test for the Detection and Stratification of Transitional Cell Carcinoma of the Bladder

Supplementary Table S1 from Secreted CXCL1 Is a Potential Mediator and Marker of the Tumor Invasion of Bladder Cancer

Purpose: New markers that enable the percentage of transitional cell carcinomas (TCC) of the bladder that are diagnosed before invasion of the bladder muscle layers to be increased would reduce the morbidity and mortality associated with this disease. The purpose of this study was to develop a simple, accurate urine test based on mRNA markers and simple gene signatures that (a) could detect TCC before muscle invasion while maintaining high specificity in patients with hematuria or urinary tract infections and (b) identify patients most likely to have grade 3 or stage ≥T1 disease.Experimental Design: RNA markers with high overexpression in stage Ta tumors and/or T1 to T4 tumors but low expression in blood or inflammatory cells were characterized by quantitative reverse transcription-PCR using 2 mL of voided urine from 75 TCC patients and 77 control patients with other urological diseases.Results: A combination of the RNAs CDC2, MDK, IGFBP5, and HOXA13 detected 48%, 90%, and 100% of stage Ta, T1, and >T1 TCCs, respectively, at a specificity of 85%. Detection of Ta tumors increased to 60% for primary (non-recurrent) Ta tumors and 76% for Ta tumors ≥1 cm in diameter. Test specificity was 80% for the 20 control patients with urinary tract infections. The combination of CDC2 and HOXA13 distinguished between grade 1 to 2 TCCs and grade 3 or stage ≥T1 TCCs with ∼80% specificity and sensitivity.Conclusions: Simple gene expression signatures can be used as urine markers for the accurate detection and characterization of bladder cancer.

Purpose: The purpose of this study was to identify proteins that are potentially involved in the tumor invasion of bladder cancer.Experimental Design: We searched for the candidate proteins by comparing the profiles of secreted proteins among the poorly invasive human bladder carcinoma cell line RT112 and the highly invasive cell line T24. The proteins isolated from cell culture supernatants were identified by shotgun proteomics. We found that CXCL1 is related to the tumor invasion of bladder cancer cells. We also evaluated whether the amount of the chemokine CXCL1 in the urine would be a potential marker for predicting the existence of invasive bladder tumors.Results: Higher amount of CXCL1 was secreted from highly invasive bladder carcinoma cell lines and this chemokine modulated the invasive ability of those cells in vitro. It was revealed that CXCL1 regulated the expression of matrix metalloproteinase-13 in vitro and higher expression of CXCL1 was associated with higher pathologic stages in bladder cancer in vivo. We also showed that urinary CXCL1 levels were significantly higher in patients with invasive bladder cancer (pT1-4) than those with noninvasive pTa tumors (P = 0.0028) and normal control (P < 0.0001). Finally, it was shown that CXCL1 was an independent factor for predicting the bladder cancer with invasive phenotype.Conclusions: Our results suggest that CXCL1 modulates the invasive abilities of bladder cancer cells and this chemokine may be a potential candidate of urinary biomarker for invasive bladder cancer and a possible therapeutic target for preventing tumor invasion.

The importance of loss of the cell-cell adhesion molecule E-cadherin (encoded by CDH1) to tumor progression is well established. However, CDH1 germ-line mutations predispose to the cancer susceptibility syndrome hereditary diffuse gastric cancer (HDGC), suggesting a role for E-cadherin in tumor initiation. The earliest indications of cancer in the stomachs of CDH1 mutation carriers are microscopic foci of intramucosal signet-ring cell carcinoma (SRCC; designated “eHDGC”). Here, we used N-methyl-N-nitrosourea (MNU) to promote gastric carcinogenesis in wild-type (wt) and cdh1+/− mice. MNU induced a variety of gastric tumors; however, intramucosal SRCC developed with an 11 times higher incidence in cdh1+/− mice compared with wt mice. The murine SRCC resembled the human eHDGCs in that they were hypoproliferative, lacked nuclear β-catenin accumulation, and had reduced membrane localization of E-cadherin and its interacting junctional proteins. The down-regulation of E-cadherin in the murine SRCCs confirmed the importance of the second CDH1 hit to the initiation of diffuse gastric cancer. CDH1 promoter hypermethylation has been proposed to be a major second hit in advanced HDGC; however, its contribution to eHDGC was unknown. We thus examined a series of human eHDGC and detected CDH1 promoter methylation in 50% of foci. Promoter methylation was accompanied by reduced wt CDH1 mRNA levels in the foci and had a monoclonal pattern, consistent with an epigenetic initiation of disease. Together, these findings provide compelling evidence for a deficiency in cell-to-cell adhesion being sufficient to initiate diffuse gastric cancer in the absence of hyperproliferation and β-catenin activation. [Cancer Res 2009;69(5):2050–6]

Supplementary Figure 4 from E-Cadherin Deficiency Initiates Gastric Signet-Ring Cell Carcinoma in Mice and Man

Supplementary Materials, Tables 1-9, Figures 1-8 from The Bioreductive Prodrug PR-104A Is Activated under Aerobic Conditions by Human Aldo-Keto Reductase 1C3

Supplementary Legends for Figures 1-5 from E-Cadherin Deficiency Initiates Gastric Signet-Ring Cell Carcinoma in Mice and Man

Circulating tumour DNA (ctDNA) analysis promises to improve the care of people with cancer, address health inequities and guide translational research. This observational cohort study used ctDNA to follow 29 New Zealand (NZ) unresectable advanced-stage cutaneous melanoma patients through multiple cycles of immunotherapy, to identify the breadth and complexity of tumour genomic information that ctDNA analysis can reliably report. During the course of treatment, a high level of dynamic mutational complexity was identified in blood plasma of these patients, including: multiple BRAF mutations in the same patient, clinically-relevant BRAF mutations emerging through therapy, and co-occurring sub-clonal BRAF and NRAS mutations. The technical validity of this ctDNA analysis was supported by high sample analysis-reanalysis concordance as well as by concordance between three ctDNA measurement technologies: droplet digital polymerase chain reaction (ddPCR), a custom melanoma-specific amplicon next-generation sequencing (NGS) panel and mass spectrometry. In addition, we observed >90% concordance in the detection of ctDNA when using cell-stabilising collection tubes followed by 7-day delayed processing, compared to standard EDTA blood collection protocols with rapid processing. We also found that undetectability of ctDNA at a proportion of treatment cycles was associated with both clinical benefit (best RECIST response) and prognosis (disease-specific survival). In summary, we found that multiple ctDNA processing and analysis methods consistently identified complex longitudinal patterns of clinically-relevant mutations, adding support for expanded implementation of this technology to guide in-treatment tailored cancer therapy.

Accurate assessment of chemotherapy response provides the means to terminate ineffective treatment, trial alternative drug regimens or schedules and reduce dose to minimize toxicity. Here, we have compared circulating tumor DNA (ctDNA) with carcinoembryonic antigen (CEA) for the cycle by cycle assessment of chemotherapy response in 30 patients with metastatic colorectal cancer. CtDNA (quantified using individualized digital droplet PCR (ddPCR) assays) and CEA levels were determined immediately prior to each chemotherapy cycle over time periods ranging from 42–548 days (average of 10 time points/patient). Twenty-nine/thirty (97%) patients had detectable ctDNA compared with 83% whose tumors were CEA-positive (>5 ng/ml) during the monitoring course. Over the course of treatment, 20 disease progression events were detected by computed tomography; ctDNA predicted significantly more of these events than CEA (16 (80%) versus 6 (30%), respectively; P-value = 0.004). When progression was detected by both ctDNA and CEA, the rise in ctDNA occurred significantly earlier than CEA (P-value = 0.046). Partial responses to chemotherapy were also detected more frequently by ctDNA, although this was not significant (P-value = 0.07). In addition, another 28 colorectal cancer patients who underwent potentially curative surgery and showed no evidence of residual disease were monitored with ctDNA for up to 2 years. Clinical relapse was observed in 6/28 (21%) patients. Four out of 6 of these patients showed a significant increase in ctDNA at or prior to relapse. Overall, ctDNA analyses were able to be performed in a clinically relevant timeline and were a more sensitive and responsive measure of tumor burden than CEA.

Introduction: Cell invasion is a critical step for tumor progression and dissemination into neighboring and distant tissues, which constitute the foremost cause of cancer mortality. Loss of the cell-cell adhesion molecule E-cadherin occurs in 70% of carcinomas and is widely described as an initiating event in the invasion process. Recently, data has emerged demonstrating that this milestone phenomenon is accompanied by an abnormal affair between cancer cells and the extracellular matrix (ECM). In fact, cancer cells display increased plasticity and a remarkable ability to overcome the fibrous structure of the ECM. Since the nucleus is the largest and stiffest organelle in the cell, we hypothesize that it represents a major determinant of cell reshaping and spreading capacity. Our main aim is thus to dissect the mechanisms through which the nucleus supports invasion of cancer cells. Material and Methods: To determine alterations in nuclear composition and architecture that are distinctive of cells with E-cadherin impairment, we have used cell lines and Drosophila strains expressing either wild-type E-cadherin or the Y755C variant identified in Hereditary Diffuse Gastric Cancer (HDGC) patients. Nuclear morphological features and migratory rates were assessed by fluorescent markers coupled with confocal microscopy and advanced bioimaging techniques. The molecular profile of nuclei envelope from E-cadherin mutant and wild-type cells was subsequently investigated by implementation of a proteomic workflow, encompassing cellular fractionation and processing through high-resolution Mass Spectrometry (LC-MS). Results: We have shown that the Y755C E-cadherin mutation induces evident changes in nuclear morphology, and an increased migration performance of border cells in Drosophila. In particular, detailed quantitative measurements revealed differences in nuclear area, mean intensity, perimeter, solidity and relative position to basal surface of E-cadherin mutant cells, when compared with those expressing the wild-type protein. Further, proteomic analysis of enriched nuclear fractions disclosed that the composition of the nuclear envelope from E-cadherin mutant cells is significantly different from that of the wild-type counterparts. Differentially abundant molecules include proteins relevant for spatial and structural integrity of the nucleus. Conclusion: Overall, this work provides evidence that cells with E-cadherin loss of function activate a specific mechanotransduction program that imposes nuclear remodeling at physical and biochemical levels, endowing cells with a pro-invasive signature. Citation Format: Joana Figueiredo, Joana Pereira, Rui M. Ferreira, Luísa Carvalho, Margarida Gonçalves, Soraia Melo, Patrícia Carneiro, Parry Guilford, Eurico Morais-de-Sá, Raquel Seruca. Nucleus positioning and composition dictate the invasion performance of E-cadherin mutant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5815.

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for the cancer syndrome hereditary diffuse gastric cancer (HDGC). CDH1-deficient gastric cancers exhibit high AKT serine/threonine kinase 3 (AKT3) expression, but specific drugs against this AKT isoform are not available. We therefore used two publicly available datasets to identify AKT3-associated genes which could be used to indirectly target AKT3. Reactome analysis identified an enrichment of extracellular matrix remodelling genes in AKT3-high gastric cancers. Of the 51 genes that were significantly correlated with AKT3 (but not AKT1), discoidin domain receptor tyrosine kinase 2 (DDR2) showed the strongest positive association. Treatment of isogenic human cells and mouse gastric and mammary organoids with dasatinib, a small molecule inhibitor of multiple kinases including SRC, BCR-ABL and DDR2, preferentially slowed the growth and induced apoptosis of E-cadherin-deficient cells. Dasatinib treatment also preferentially slowed the growth of gastric and mammary organoids harbouring both Cdh1 and Tp53 mutations. In organoid models, dasatinib treatment was associated with decreased phosphorylation of total AKT, with a stronger effect seen in Cdh1-deficient organoids. Treatment with combinations of dasatinib and an inhibitor of AKT, MK2206, enhanced the effect of dasatinib in breast MCF10A cells. In conclusion, targeting the DDR2-SRC-AKT3 axis with dasatinib represents a promising approach for the chemoprevention and chemotherapy of gastric and breast cancers lacking E-cadherin.

Simple Summary Inactivating mutations in the CDH1 gene cause the cancer syndrome hereditary diffuse gastric cancer and are also frequent in sporadic diffuse gastric and lobular breast cancers. This research aimed to test whether cancers with CDH1 mutations have heightened sensitivity to histone deacetylase (HDAC) inhibitors. The impact of these drugs was tested on several gastric and breast preclinical models with and without CDH1 mutations. One HDAC inhibitor, entinostat, showed a strong inhibitory effect on each of the models with CDH1 mutations. This study highlighted the potential beneficial impact of entinostat for the chemoprevention and/or treatment of gastric and breast cancers carrying CDH1 mutations. Abstract Inactivating germline mutations in the CDH1 gene (encoding the E-cadherin protein) are the genetic hallmark of hereditary diffuse gastric cancer (HDGC), and somatic CDH1 mutations are an early event in the development of sporadic diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In this study, histone deacetylase (HDAC) inhibitors were tested for their ability to preferentially inhibit the growth of human cell lines (MCF10A and NCI-N87) and murine organoids lacking CDH1 expression. CDH1−/− breast and gastric cells were more sensitive to the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat than wild-type cells, with an elevated growth inhibition that was, in part, attributable to increased apoptosis. CDH1-null cells were also sensitive to more class-specific HDAC inhibitors, but compared to the pan-inhibitors, these effects were less robust to genetic background. Increased sensitivity to entinostat was also observed in gastric organoids with both Cdh1 and Tp53 deletions. However, the deletion of Tp53 largely abrogated the sensitivity of the Cdh1-null organoids to pracinostat and mocetinostat. Finally, entinostat enhanced Cdh1 expression in heterozygous Cdh1+/− murine organoids. In conclusion, entinostat is a promising drug for the chemoprevention and/or treatment of HDGC and may also be beneficial for the treatment of sporadic CDH1-deficient cancers.

Simple Summary Germline loss of the CDH1 gene is the primary genetic basis for hereditary diffuse gastric cancer, a disease resulting in elevated risk of both diffuse gastric cancer and lobular breast cancer. Current preventative treatment consists of prophylactic total gastrectomy, a therapy with several associated long-term morbidities. To address the lack of targeted molecular therapies for hereditary diffuse gastric cancer, we have utilized a synthetic lethal approach to identify candidate compounds that can specifically kill CDH1-null cells. Inhibitors of sphingolipid metabolism and vesicle trafficking pathways were identified as promising candidate compounds in a cell line model of CDH1 loss, then further validated in murine-derived organoid models of hereditary diffuse gastric cancer. With further research, these findings may lead to the development of novel chemoprevention strategies for the treatment of hereditary diffuse gastric cancer. Abstract Germline inactivating variants of CDH1 are causative of hereditary diffuse gastric cancer (HDGC), a cancer syndrome characterized by an increased risk of both diffuse gastric cancer and lobular breast cancer. Because loss of function mutations are difficult to target therapeutically, we have taken a synthetic lethal approach to identify targetable vulnerabilities in CDH1-null cells. We have previously observed that CDH1-null MCF10A cells exhibit a reduced rate of endocytosis relative to wildtype MCF10A cells. To determine whether this deficiency is associated with wider vulnerabilities in vesicle trafficking, we screened isogenic MCF10A cell lines with known inhibitors of autophagy, endocytosis, and sphingolipid metabolism. Relative to wildtype MCF10A cells, CDH1−/− MCF10A cells showed significantly greater sensitivity to several drugs targeting these processes, including the autophagy inhibitor chloroquine, the endocytosis inhibitors chlorpromazine and PP1, and the sphingosine kinase 1 inhibitor PF-543. Synthetic lethality was confirmed in both gastric and mammary organoid models of CDH1 loss, derived from CD44-Cre/Cdh1fl/fl/tdTomato mice. Collectively, these results suggest that both sphingolipid metabolism and vesicle trafficking represent previously unrecognised druggable vulnerabilities in CDH1-null cells and may lead to the development of new therapies for HDGC.

Contains fulltext : 225261.pdf (Publisher’s version ) (Closed access) Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant hereditary syndrome associated with an increased risk of diffuse gastric cancer and lobular breast cancer. In this chapter, a multidisciplinary and international team of experts on HDGC, including pathologists, a surgeon, a gastroenterologist, molecular biologists, and dedicated researchers gathered to assess the available knowledge on HDGC and areas of emerging research that may be relevant for the translation into clinical practice. Particularly, in this chapter the authors will cover: first, the changes introduced in the updated clinical practice Guidelines on HDGC; second, the HDGC clinical spectrum; third, the models of development and the morphological heterogeneity of HDGC; fourth, the main causal germline defects of HDGC, including CDH1 and CTNNA1 germline variants; fifth, the state-of-art of HDGC-like syndromes; sixth, endoscopic and surgical approaches; seventh, emerging strategies of chemoprevention.

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.

Increases in cancer survival may increase cancer prevalence and demand for healthcare. We aimed to estimate cancer prevalence in the New Zealand population.We used national linked health, social and census datasets from the Stats NZ Integrated Data Infrastructure to identify the number of New Zealand residents who had at least one cancer diagnosis in New Zealand. We included all primary cancers recorded on the New Zealand Cancer Registry from January 1995 to June 2013, and used the 2013 census for demographic and socioeconomic data.On 30 June 2013, 140,600 of 4,438,900 (3.2%) New Zealand residents had been diagnosed with cancer in the last 18.5 years. In ≥15 year olds, the age-standardised prevalence of cancer diagnosed 0 to ≤1 year, and1 to ≤5 years, prior to 30 June 2013 was 0.4% and 1.1% in men and 0.3% and 0.9% in women, respectively. Over the 18.5-year period prevalence was greatest in the oldest ages, European/Other, highest qualified, highest income, least deprived, ex-smokers, and Canterbury, Bay of Plenty and Nelson/Marlborough District Health Boards (age-standardised).Groups with the highest survival and the greatest access to healthcare had the highest cancer prevalences.

CDH1encodes E-cadherin, a key protein in adherens junctions. Given that E-cadherin is involved in major cellular processes such as embryogenesis and maintenance of tissue architecture, it is no surprise that deleterious effects arise from its loss of function. E-cadherin is recognised as a tumour suppressor gene, and it is well established thatCDH1genetic alterations cause diffuse gastric cancer and lobular breast cancer—the foremost manifestations of the hereditary diffuse gastric cancer syndrome. However, in the last decade, evidence has emerged demonstrating thatCDH1mutations can be associated with lobular breast cancer and/or several congenital abnormalities, without any personal or family history of diffuse gastric cancer. To date, no genotype–phenotype correlations have been observed. Remarkably, there are reports of mutations affecting the same nucleotide but inducing distinct clinical outcomes. In this review, we bring together a comprehensive analysis ofCDH1-associated disorders and germline alterations found in each trait, providing important insights into the biological mechanisms underlying E-cadherin’s pleiotropic effects. Ultimately, this knowledge will impact genetic counselling and will be relevant to the assessment of risk of cancer development or congenital malformations inCDH1mutation carriers.

Background The E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers. Methods Density distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1−/− cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression. Results MCF10A-CDH1−/− cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1−/− and NCI-N87-CDH1−/− cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1−/− cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes. Conclusions E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities. Electronic supplementary material The online version of this article (10.1007/s10120-018-0859-1) contains supplementary material, which is available to authorised users.

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for hereditary diffuse gastric cancer syndrome (HDGC). Using cell viability assays, we identified that breast (MCF10A) and gastric (NCI-N87) cells lacking CDH1 expression are more sensitive to allosteric AKT inhibitors than their CDH1-expressing isogenic counterparts. Apoptosis priming and total apoptosis assays in the isogenic MCF10A cells confirmed the enhanced sensitivity of E-cadherin-null cells to the AKT inhibitors. In addition, two of these inhibitors, ARQ-092 and MK2206, preferentially targeted mouse-derived gastric Cdh1&minus, /&minus, organoids for growth arrest. AKT protein expression and activation (as measured by phosphorylation of serine 473) were differentially regulated in E-cadherin-null MCF10A and NCI-N87 cells, with downregulation in the normal breast cells, but upregulation in the gastric cancer cells. Bioinformatic analysis of the TCGA STAD dataset revealed that AKT3, but not AKT1 or AKT2, is upregulated in the majority of E-cadherin-deficient gastric cancers. In conclusion, allosteric AKT inhibitors represent a promising class of drugs for chemoprevention and chemotherapy of cancers with E-cadherin loss.

For Pacific Peoples living in Aotearoa/New Zealand, the burden of cancer remains a serious health concern. A promising early cancer detection approach involves the use of circulating tumour DNA (ctDNA) found in the blood circulation. Current ctDNA approaches focus on the analysis of ctDNA derived from established tumours in patients, to help provide information relating to the cancer’s size and progression using specific laboratory isolation and quantification techniques. Adaptation of these current ctDNA technologies used in the cancer monitoring setting, for early diagnosis, would generate a cancer detection approach easily initiated by obtaining ctDNA from DNA fragments found within a blood sample. To understand the potential utility of ctDNA technology for early cancer detection within the Pacific setting in Aotearoa/New Zealand, engagement was sought with Pacific leaders using Talanoa, focused on cancer and early cancer detection. Interim analyses highlighted factors considered directly relevant to Pacific Communities and the cancer setting including the need for health technologies and systems that align with cultural contexts for Pacific Peoples and their families. Support was also expressed for the development of an early cancer detection tool utilising ctDNA as a simple methodological approach, with acknowledgement of the potential for this method to address current barriers to cancer care and services. This work highlights the importance of ensuring cultural context is understood and respected when engaging with Pacific Communities, and also within the cancer and health care setting.

Circulating tumour DNA (ctDNA) refers to small fragments of tumour-derived DNA that escape into the bloodstream. Analysis of these ctDNA fragments can be utilized to monitor responses to treatment by providing information relating to tumour size and progression and can support clinical decision making. A potential adaptation of the ctDNA monitoring technology is proposed to support utility of ctDNA for early cancer diagnostic efforts. This would comprise a simple method for early cancer detection that would be helpful across multiple settings, including that for Pacific Peoples. Pacific Peoples living in Aotearoa/New Zealand and in the Pacific Region are disproportionately affected by specific cancer types. The potential utility of ctDNA for early cancer detection is an approach that could help reduce adverse health outcomes if successful particularly for Pacific communities. To understand more about the diagnostic approaches applicable for early cancer diagnostics for Pacific young people in living in Aotearoa/New Zealand, feedback was sought on the aspects requiring attention in the Youth setting and also in relation to the possible utility of ctDNA technology as an example. Preliminary aspects of the cancer setting identified as important for early cancer diagnosis for Pacific young people, included the need for improved accessibility, the importance of cultural context and awareness, and the need for simplicity with any technological approach used for early cancer diagnosis, such as that being proposed using ctDNA.

Pacific Peoples living in Aotearoa/New Zealand and in the Pacific Region, remain disproportionately affected by some cancers. In addition to ensuring effective treatments and therapies are available and continue to improve, continued early cancer diagnostic efforts are also critical for supporting better health outcomes for Pacific Peoples within the cancer setting. A novel and promising early cancer detection method known as circulating tumour DNA (ctDNA) analyses small fragments of tumour DNA found in the blood circulation to provide information about a tumour. This approach is more commonly associated with and utilised for monitoring patients being treated for their cancers and is being adapted to support early cancer detection efforts. To support the possible acceptability of this approach for early cancer diagnosis for Pacific Peoples, it is imperative to better understand the clinical considerations relevant to the Pacific setting. To identify the clinical considerations needed for early cancer detection with ctDNA approaches within the Pacific setting for Pacific Peoples living in Aotearoa/New Zealand, researchers and Pacific Health Professionals were consulted. Relevant considerations included the immediate need to support early cancer diagnostic efforts to avoid late presentations, better understanding of the cultural context for Pacific Peoples within the healthcare setting more broadly, with strong support expressed for a simple approach for early cancer detection such as that being proposed with ctDNA technology. These findings highlight the critical need to consider the clinical context within the cancer setting for Pacific Peoples. Health Research Council of New Zealand

Colorectal cancer (CRC) is one of the biggest cause of death world-wide and the second-highest cause of death by cancer in New Zealand with a mortality rate of approximately four deaths/day and 90% of CRC-associated mortality is caused by metastasis. Development of metastasis and the modifications that occur during various stages in the CRC metastatic cascade remains obscure. Emerging evidence indicates that epigenetic alterations have a greater influence on the development of metastasis than genetic mutations, but the current understanding of epigenetic drivers that cause CRC metastasis is limited. In the current project, as a first step in identifying epigenetic drivers for metastasis, whole-genome methylation and transcriptome profiles from paired primary and metastatic samples from the same NZ patients will be compared. Subsequently, the epigenetic drivers will be validated, and it can potentially be used as CRC metastatic markers. Circulating tumour cells (CTCs) represent the conduit between primary and metastatic tumours. They could be used to identify driver epigenetic changes associated with epithelial-to-mesenchymal transition (EMT), migration and invasion which occur during the metastatic cascade. CTCs are obtained from the blood and would provide a clinically less invasive test rather than requiring tissue samples. The recent development of single-cell sequencing technologies has now made it feasible to identify epigenetic changes in individual CTCs. In the second part of this project, protocols for single-cell DNA methylation and expression analysis will be developed using human colon cancer cell lines as a model. These optimised protocols will then be applied to CTCs from patient blood samples to identify epigenetic changes that predispose patients to metastasis. Identification of epigenetic drivers will help to detect individuals at high risk for metastasis prior to metastasis being clinically apparent, allowing for more personalised treatment and contribute to improving survival. Citation Format: Aniruddha Chatterjee, John McCall, Parry Guilford, Sharon Pattison, Euan Rodger, Priyadarshana Ajithkumar. Epigenomic profiling of colorectal cancer to predict patients at high risk of metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2434.

Circulating tumour DNA (ctDNA) refers to small fragments of tumour DNA found in the blood circulation, recognised by the presence of mutations in cancer genes. These DNA fragments can be isolated and quantified to obtain data about the cancer’s size and progression. Since gaining access to and obtaining biopsy samples from solid cancers in people is not always possible, the possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth is highly desirable. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer diagnostics and surveillance and also lead to improved cancer health outcomes for Pacific Peoples. The purpose of this work was to support engagement with Pacific communities and share, and discuss and explore current research efforts, that included the potential utilization of ctDNA to improve the diagnosis, care and health outcomes of Pacific Peoples. A Pacific Fono (gathering) was held in the Hutt Valley region to promote appropriate knowledge translation activity, engagement and meaningful dialogue between researchers and the Pacific communities regarding current research works and findings of direct relevance and significance to Pacific communities. The Pacific community response to this research outreach effort was overwhelmingly positive and encouraging. Discussions were respectful and informative with shared information being exchanged between researchers and those in attendance. Feedback included expressed support for regarding the ctDNA research and for other areas of research focus that would be of benefit to Pacific communities. Requests from Pacific community members were also made for future similar events to be held.

A novel cancer detection method known as circulating tumour DNA (ctDNA) analyses the small fragments of tumour DNA found in the blood circulation. Information relating to a cancer’s size and progression can be easily obtained from the DNA fragments found in a blood sample using specific isolation and quantification techniques. This is a relatively simple method for monitoring cancer and is being adapted to support early cancer detection efforts. Pacific Peoples living in New Zealand (NZ) and in the Pacific Region, remain disproportionately affected by some cancers. Given the increasing cancer rates worldwide and the medical situation for cancer patients in NZ and in the Pacific, the burden of cancer remains concerning as significant health inequalities exist for Pacific Peoples in particular. To support meaningful and respectful scientific knowledge translation activity with appropriate engagement promoting dialogue between researchers and Pacific communities, a Pacific Fono (gathering) event was held in the Wellington Region that included discussion of the ctDNA early cancer detection technology. Feedback from members of the various Pacific communities who attended indicated that researchers had engaged respectfully and facilitated discussion appropriately that was helpful, and also supported understanding of the ctDNA research work. Strong support was also expressed for the development of an early detection tool that involved a simple blood test methodology. The importance of taking into account the cultural context, combined with the appropriate scientific communication was shown to build meaningful connections and support respectful dialogue with Pacific communities.

Item does not contain fulltext Around 10-20% of gastric cancer patients have relatives with a diagnosis of GC and in 1-3% of patients a genetic cause can be confirmed. Histopathologically, GC is classified into intestinal-type, with glandular growth, and diffuse-type with poorly cohesive growth pattern often with signet ring cells. Familial or hereditary GC is classified into hereditary diffuse GC (HDGC), familial intestinal GC (FIGC) and polyposis forms. This review focuses on recent research findings and new concepts of hereditary GC.

Several methods exist for constructing sequencing libraries from single-cell amounts of RNA. However, different library preparation methods are required to enable full length or strand specific end profiling. The Integrated Cell Expression Toolkit (ICE-T) maximizes functionality by integrating these approaches with novel strategies that provide a range of profiling options.

Objectives To compare the Roche Cell-Free DNA Collection Tubes® against the Streck Cell-Free DNA BCT®s for sample stability using Cell Free DNA (cfDNA) from healthy volunteers (n = 20). Design & methods Whole blood was drawn into five Roche and five Streck tubes per volunteer, stored at room temperature and processed at five different time points (Days 0, 4, 7, 10 and 14). One volunteer had blood drawn into ×10 K3EDTA tubes to observe the effect of no preservation buffer on White Blood Cell (WBC) lysis. DNA was extracted from the plasma and the concentration (ng/μL) measured using the Qubit Fluorometer® at each time point. The eluted DNA was further analysed by capillary electrophoresis to determine the proportion of cfDNA and gDNA contamination in the samples over the 14 days. Results There was no difference in individual (p = 0.097) and median paired (p = 0.26) DNA concentration across the five time points between the two tubes. However, a difference was observed for samples in the Roche tubes for pair days 0–7 (p = 0.01), 0 to 10 (p = 0.046) and 0 to 14 (p = 0.0016) in contrast to the Streck tubes after adjustment for multiple testing. Conclusion The findings of this study indicate that the Roche Cell-Free DNA Collection Tubes® are a suitable alternative for sample collection and storage at room temperature, albeit for a duration of less than 7 days., Highlights • We compared two commercial cfDNA Blood Collection Tubes; Streck Cell Free BCT®s and Roche Cell Free DNA Collection Tubes®. • Samples were drawn from healthy volunteers and stored at room temperature for 14 days. • The concentration of plasma DNA was measured at 5 different time points (Day 0, 4, 7, 10 and 14). • gDNA contamination was measured using capillary electrophoresis. • The Roche tubes are suitable for sample collection and storage at room temperature for a period within 7 days.

The aim of this study was to uncover the pathogenic relevance and the underlying molecular mechanism of a novel CDH1 variant found in a Hereditary Diffuse Gastric Cancer family (p.L13_L15del), which affects the signal peptide of E-cadherin without changing the remaining predicted sequence. We verified that p.L13_L15del cells yield low levels of E-cadherin, decreased cell adhesion and enhanced cell invasion. Further, we demonstrated that the disruption of the highly conserved hydrophobic core of the signal peptide hampers the binding of cellular components crucial for E-cadherin translation and translocation into the endoplasmic reticulum, constituting a new molecular basis for the loss of a tumour suppressor gene causative of hereditary cancer. This work was financed by FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE), Programa Operacional Regional do Norte (Norte 2020) and by National Funds through the Portuguese Foundation for Science and Technology (FCT), under the projects PTDC/BIM-ONC/0171/2012, PTDC/BIM-ONC/0281/2014, PTDC/BBB-IMG/0283/2014, NORTE-01-0145-FEDER-000029; Post-Doctoral grant SFRH/BPD/87705/2012-JF and Doctoral grant SFRH/BD/108009/2015-SM. We acknowledge the American Association of Patients with Hereditary Gastric Cancer “No Stomach for Cancer” for funding Seruca and Figueiredo research.

New Zealand Māori have a considerably higher incidence of gastric cancer compared to non-Māori, and are one of the few populations worldwide with a higher prevalence of diffuse-type disease. Pathogenic germline CDH1 mutations are causative of hereditary diffuse gastric cancer, a cancer predisposition syndrome primarily characterised by an extreme lifetime risk of developing diffuse gastric cancer. Pathogenic CDH1 mutations are well described in Māori families in New Zealand. However, the contribution of these mutations to the high incidence of gastric cancer is unknown. We have used next-generation sequencing, Sanger sequencing, and Multiplex Ligation-dependent Probe Amplification to examine germline CDH1 in an unselected series of 94 Māori gastric cancer patients and 200 healthy matched controls. Overall, 18% of all cases, 34% of cases diagnosed with diffuse-type gastric cancer, and 67% of cases diagnosed aged less than 45 years carried pathogenic CDH1 mutations. After adjusting for the effect of screening known HDGC families, we estimate that 6% of all advanced gastric cancers and 13% of all advanced diffuse-type gastric cancers would carry germline CDH1 mutations. Our results demonstrate that germline CDH1 mutations are a significant contributor to the high frequency of diffuse gastric cancer in New Zealand Māori. Electronic supplementary material The online version of this article (10.1007/s10689-018-0080-8) contains supplementary material, which is available to authorized users.

A family history of disease is a strong risk factor for testicular germ cell tumour (TGCT). In order to identify the location of putative TGCT susceptibility gene(s) we conducted a linkage search in 237 pedigrees with two or more cases of TGCT. One hundred and seventy-nine pedigrees were evaluated genome-wide with an average inter-marker distance of 10 cM. An additional 58 pedigrees were used to more intensively investigate several genomic regions of interest. Genetic linkage analysis was performed with the ALLEGRO software using two model-based parametric analyses and a non-parametric analysis. Six genomic regions on chromosomes 2p23, 3p12, 3q26, 12p13-q21, 18q21-q23 and Xq27 showed heterogeneity LOD (HLOD) scores of greater than 1, with a maximum HLOD of 1.94 at 3q26. Genome-wide simulation studies indicate that the observed number of HLOD peaks greater than one does not differ significantly from that expected by chance. A TGCT locus at Xq27 has been previously reported. Of the 237 pedigrees examined in this study, 66 were previously unstudied at Xq27, no evidence for linkage to this region was observed in this new pedigree set. Overall, the results indicate that no single major locus can account for the majority of the familial aggregation of TGCT, and suggests that multiple susceptibility loci with weak effects contribute to the disease.

Māori, the indigenous people of New Zealand, experience disproportionate rates of stomach cancer, compared to non-Māori. The overall aim of the study was to better understand the reasons for the considerable excess of stomach cancer in Māori and to identify priorities for prevention. Māori stomach cancer cases from the New Zealand Cancer Registry between 1 February 2009 and 31 October 2013 and Māori controls, randomly selected from the New Zealand electoral roll were matched by 5-year age bands to cases. Logistic regression was used to estimate odd ratios (OR) and 95% confidence intervals (CI) between exposures and stomach cancer risk. Post-stratification weighting of controls was used to account for differential non-response by deprivation category. The study comprised 165 cases and 480 controls. Nearly half (47.9%) of cases were of the diffuse subtype. There were differences in the distribution of risk factors between cases and controls. Of interest were the strong relationships seen with increased stomach risk and having >2 people sharing a bedroom in childhood (OR 3.30, 95%CI 1.95-5.59), testing for H pylori (OR 12.17, 95%CI 6.15-24.08), being an ex-smoker (OR 2.26, 95%CI 1.44-3.54) and exposure to environmental tobacco smoke in adulthood (OR 3.29, 95%CI 1.94-5.59). Some results were attenuated following post-stratification weighting. This is the first national study of stomach cancer in any indigenous population and the first Māori-only population-based study of stomach cancer undertaken in New Zealand. We emphasize caution in interpreting the findings given the possibility of selection bias. Population-level strategies to reduce the incidence of stomach cancer in Māori include expanding measures to screen and treat those infected with H pylori and a continued policy focus on reducing tobacco consumption and uptake.

Ongoing improvements in digital data acquisition and storage has led to the phenomenon of e(lectronic)-mortality, where digital data can now exist for a potentially infinite period. Globally, many countries are facilitating the acquisition and researcher-led access to large-scale, population-based digitised healthcare data sets. Their utilisation has led to numerous positive advances in healthcare. New Zealand's medical record system is becoming increasingly digitised, and as a consequence there will be an ever-increasing resource of posthumous healthcare data stored digitally, including genomic information. Such data could be utilised for research purposes, and developing such a consolidated resource could improve healthcare outcomes in our own nation and allow us to parallel global progress in healthcare research trends. This viewpoint article explores the issues surrounding, and potential for utilisation of, a national resource of posthumous digital healthcare data. Currently, there appear to be no legal barriers to the large-scale acquisition and utilisation of posthumous healthcare data in New Zealand, however, previous legislation may not have been developed with developments in technology or e-mortality in mind. Ethically, culturally and socially there are many challenges to address, including issues surrounding obtaining consent, respecting privacy, management of incidental findings, maintaining anonymity and ensuring community support for such a resource. Despite the potential for widespread health benefits that utilisation of posthumous healthcare data in this country may facilitate, wide and ongoing consultation is required to examine how such a precious resource can be enabled for the downstream benefit of all New Zealanders.

Background Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. Methods DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. Results In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). Conclusions Our genome-wide characterization of DNA methylation in colorectal cancer has identified 132 genes hypermethylated in 100% of CIMP-H samples. Three genes, EYA4, TLX1 and TFPI2 are hypermethylated in >90% of all tumour samples, regardless of CIMP subtype. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3226-4) contains supplementary material, which is available to authorized users.