Misspellings or "miscellings"-Non-verifiable and unknown cell lines in cancer research publications.

Reproducible laboratory research relies on correctly identified reagents. We have previously described gene research papers with wrongly identified nucleotide sequence(s), including papers studying miR-145. Manually verifying reagent identities in 36 recent miR-145 papers found that 56% and 17% of papers described misidentified nucleotide sequences and cell lines, respectively. We also found 5 cell line identifiers in miR-145 papers with misidentified nucleotide sequences and cell lines, and 18 cell line identifiers published elsewhere, that did not represent indexed human cell lines. These 23 identifiers were described as non-verifiable (NV), as their identities were unclear. Studying 420 papers that mentioned 8 NV identifier(s) found 235 papers (56%) that referred to 7 identifiers (BGC-803, BSG-803, BSG-823, GSE-1, HGC-7901, HGC-803, and MGC-823) as independent cell lines. We could not find any publications describing how these cell lines were established. Six cell lines were sourced from cell line repositories with externally accessible online catalogs, but these cell lines were not indexed as claimed. Some papers also stated that short tandem repeat (STR) profiles had been generated for three cell lines, yet no STR profiles could be identified. In summary, as NV cell lines represent new challenges to research integrity and reproducibility, further investigations are required to clarify their status and identities.

An Approach to Evaluate the Costs and Outputs of Academic Biobanks.

Academic biobanks commonly report sustainability challenges, which may be exacerbated by a lack of information on biobank value. To better understand the costs and supported outputs that contribute to biobank value, we developed a systematic, generalizable methodology to determine biobank inputs and publications arising from biobank-supported research. We then tested this in a small cohort (n = 12) of academic cancer biobanks in New South Wales, Australia. A proforma was developed to capture monetary and in-kind biobank costing data from biobank managers and publicly available sources. Participating biobanks were grouped and compared according to the following two classifications: open- versus restricted-access and high versus low total annual costs. Our methodology provides a feasible approach for capturing comprehensive costing data for a defined period. Characterization of biobanks using this approach showed that median total costs, as well as median staffing and in-kind costs, were comparable for open- and restricted-access biobanks, as were the quantity and journal impact metrics of supported publications. High- and low-cost biobanks supported similar median numbers of publications; however, high-cost biobanks supported publications with higher median journal impact factor and Altmetric scores. Overall, 9 of 10 biobanks had higher Field-Weighted Citation Impact scores than the global average for similar publications. This is the first tested, generalizable approach to analyze the costs and publications arising from biobank-supported research. By determining explicit cost and output data, academic biobanks, funders, and policymakers can engage in or support informed redirection of resourcing and/or benchmark setting with the aim of improving biobank support of research.

Verification of nucleotide sequence reagent identities in original publications in high impact factor cancer research journals.

Human gene research studies that describe wrongly identified nucleotide sequence reagents have been mostly identified in journals of low to moderate impact factor, where unreliable findings could be considered to have limited influence on future research. This study examined whether papers describing wrongly identified nucleotide sequences are also published in high-impact-factor cancer research journals. We manually verified nucleotide sequence identities in original Molecular Cancer articles published in 2014, 2016, 2018, and 2020, including nucleotide sequence reagents that were claimed to target circRNAs. Using keywords identified in some 2018 and 2020 Molecular Cancer papers, we also verified nucleotide sequence identities in 2020 Oncogene papers that studied miRNA(s) and/or circRNA(s). Overall, 3.8% (251/6647) and 4.0% (47/1165) nucleotide sequences that were verified in Molecular Cancer and Oncogene papers, respectively, were found to be wrongly identified. Wrongly identified nucleotide sequences were distributed across 18% (91/500) original Molecular Cancer papers, including 38% (31/82) Molecular Cancer papers from 2020, and 40% (21/52) selected Oncogene papers from 2020. Original papers with wrongly identified nucleotide sequences were therefore unexpectedly frequent in two high-impact-factor cancer research journals, highlighting the risks of employing journal impact factors or citations as proxies for research quality.

Identification of human gene research articles with wrongly identified nucleotide sequences.

Nucleotide sequence reagents underpin molecular techniques that have been applied across hundreds of thousands of publications. We have previously reported wrongly identified nucleotide sequence reagents in human research publications and described a semi-automated screening tool Seek & Blastn to fact-check their claimed status. We applied Seek & Blastn to screen >11,700 publications across five literature corpora, including all original publications in Gene from 2007 to 2018 and all original open-access publications in Oncology Reports from 2014 to 2018. After manually checking Seek & Blastn outputs for >3,400 human research articles, we identified 712 articles across 78 journals that described at least one wrongly identified nucleotide sequence. Verifying the claimed identities of >13,700 sequences highlighted 1,535 wrongly identified sequences, most of which were claimed targeting reagents for the analysis of 365 human protein-coding genes and 120 non-coding RNAs. The 712 problematic articles have received >17,000 citations, including citations by human clinical trials. Given our estimate that approximately one-quarter of problematic articles may misinform the future development of human therapies, urgent measures are required to address unreliable gene research articles.

Rare germline variants in childhood cancer patients suspected of genetic predisposition to cancer.

Identification of cancer-predisposing germline variants in childhood cancer patients is important for therapeutic decisions, disease surveillance and risk assessment for patients, and potentially, also for family members. We investigated the spectrum and prevalence of pathogenic germline variants in selected childhood cancer patients with features suggestive of genetic predisposition to cancer. Germline DNA was subjected to exome sequencing to filter variants in 1048 genes of interest including 176 known cancer predisposition genes (CPGs). An enrichment burden analysis compared rare deleterious germline CPG variants in the patient cohort with those in a healthy aged control population. A subset of predicted deleterious variants in novel candidate CPGs was investigated further by examining matched tumor samples, and the functional impact of AXIN1 variants was analyzed in cultured cells. Twenty-two pathogenic/likely pathogenic (P/LP) germline variants detected in 13 CPGs were identified in 19 of 76 patients (25.0%). Unclear association with the diagnosed cancer types was observed in 11 of 19 patients carrying P/LP CPG variants. The burden of rare deleterious germline variants in autosomal dominant CPGs was significantly higher in study patients versus healthy aged controls. A novel AXIN1 frameshift variant (Ser321fs) may impact the regulation of ß-catenin levels. Selection of childhood cancer patients for germline testing based on features suggestive of an underlying genetic predisposition could help to identify carriers of clinically relevant germline CPG variants, and streamline the integration of germline genomic testing in the pediatric oncology clinic.

Vignettes to Illustrate the Value of Tumor Biobanks in Cancer Research in Canada.

Background: Tumor biobanks are a common research infrastructure. As a collection of biospecimens and annotated data collected to support a multitude of research projects, biobanks facilitate access to materials that are the critical fuel for the generation of data in up to 40% of cancer research publications. However, quantifying how to measure biobanks' impact and their value on the field of cancer research discoveries and findings, has not been well elucidated. Methods: We have used a qualitative case study approach to illustrate the impact of tumor biobanks. We assessed the impact of three research studies published between 2010 and 2012 that required easily accessible "classic" biobanks. Each study utilized preassembled collections of tumor biospecimens with associated patient outcomes data at the outset of the research project. We compared the resulting journal impact factor, altmetric and field-weighted citation impact factor scores for each article to a set of six "benchmark" articles that represent cancer research and treatment discoveries from the same time period and two sentinel scientific discovery articles. Results: We developed a value model using a literature search and design-thinking methodologies to illustrate the contributions of these "classic" model biobanks to these research studies. Assessment of the three example articles supported by biobanks demonstrates that the output can have impact that is comparable to the impact of a set of benchmark articles describing milestones in the field of cancer research and cancer care. Conclusions: These case studies illustrate the value of the sustained investment of funds, planning, time, and effort on the part of the biobanks before the conduct of the research study to be able to ultimately support high-value research. The "value" model will enable further discussion around impact and may be useful in better delineating qualitative metrics of biobank value in the future.

In vitro and in vivo drug screens of tumor cells identify novel therapies for high-risk child cancer.

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid high-throughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

Analysis of the CD8+ IL-10+ T cell response elicited by vaccination with the oncogenic tumor-self protein D52.

Development of cancer vaccines targeting tumor self-antigens is complex and challenging due to the difficulty of overcoming immune tolerance to self-proteins. Vaccination against tumor self-protein D52 (D52) has been successful, although complete protection appears impaired by immune regulation. Our previous studies suggest that vaccine elicited CD8 + T cells producing interleukin 10 (IL-10) may have a negative impact on tumor protection. Understanding the role CD8+ IL-10 + T cells play in the immune response following vaccination with D52 could result in a more potent vaccine. To address this, we vaccinated IL-10 deficient mice with the murine orthologue of D52; vaccination of wild type (wt) C57BL/6J served as a control for comparison. In separate experiments, D52 vaccinated wt mice were administered IL-10R-specific mAb to neutralize IL-10 function. Interestingly, we observed similar protection against primary tumor challenge in the experimental groups compared to the controls. However, individual IL-10 deficient mice that rejected the primary tumor challenge were re-challenged 140 days post-primary challenge to access vaccine durability and immunologic memory against tumor recurrence. Mice deficient in IL-10 demonstrated a memory response in which 100% of the mice were protected from secondary tumor challenge, while wt mice had diminished recall response (25%) against tumor recurrence. These results with analysis of vaccine-elicited CD8 + T cells for tumor-specific killing and regulatory cell marker expression, add further support to our premise that CD8+ IL-10 + T cells elicited by D52 tumor-self protein vaccine contribute to the suppression of a memory CTL responses and durable tumor immunity.

Delayed recruiting of TPD52 to lipid droplets - evidence for a "second wave" of lipid droplet-associated proteins that respond to altered lipid storage induced by Brefeldin A treatment.

Tumor protein D52 (TPD52) is amplified and overexpressed in breast and prostate cancers which are frequently characterised by dysregulated lipid storage and metabolism. TPD52 expression increases lipid storage in mouse 3T3 fibroblasts, and co-distributes with the Golgi marker GM130 and lipid droplets (LDs). We examined the effects of Brefeldin A (BFA), a fungal metabolite known to disrupt the Golgi structure, in TPD52-expressing 3T3 cells, and in human AU565 and HMC-1-8 breast cancer cells that endogenously express TPD52. Five-hour BFA treatment reduced median LD numbers, but increased LD sizes. TPD52 knockdown decreased both LD sizes and numbers, and blunted BFA's effects on LD numbers. Following BFA treatment for 1-3 hours, TPD52 co-localised with the trans-Golgi network protein syntaxin 6, but after 5 hours BFA treatment, TPD52 showed increased co-localisation with LDs, which was disrupted by microtubule depolymerising agent nocodazole. BFA treatment also increased perilipin (PLIN) family protein PLIN3 but reduced PLIN2 detection at LDs in TPD52-expressing 3T3 cells, with PLIN3 recruitment to LDs preceding that of TPD52. An N-terminally deleted HA-TPD52 mutant (residues 40-184) almost exclusively targeted to LDs in both vehicle and BFA treated cells. In summary, delayed recruitment of TPD52 to LDs suggests that TPD52 participates in a temporal hierarchy of LD-associated proteins that responds to altered LD packaging requirements induced by BFA treatment.

"I remember how I felt, but I don't remember the gene": Families' experiences of cancer-related genetic testing in childhood.

BACKGROUND: Genetic testing in children for hereditary cancer predisposition syndromes (CPSs) involves unique psychosocial and family-systems considerations. This retrospective study explored the perspectives and emotional reactions of parents and young adults about cancer-related genetic counseling and testing offered to children in the family. METHODS: Families were eligible if they had considered genetic testing for a child (≤18 years) within the family. Parents and young adults ≥16 years participated in semistructured interviews that we coded and identified key themes. We also quantitively assessed emotional distress, quality of life, impact of receiving genetic cancer risk information, and service-related satisfaction. RESULTS: From 35 interviews (26 parents, nine young adults), we identified themes spanning families' experiences from referral to genetic services to the longer term impact of receiving information about family cancer risk from testing of children. Supported by quantitative data, families generally described positive experiences of genetic services and reported benefits to genetic testing. Nevertheless, families faced unique emotional and relational challenges that changed over the family lifecycle. Those challenges differed according to whether the child was asymptomatic or had a cancer diagnosis at testing. Parents of children with cancer described genetic consultations as a secondary concern to the immediate stressors of their child's treatment. CONCLUSIONS: We conclude that the successful integration of cancer genetics into pediatric cancer care requires specialist pediatric genetic counseling and psychosocial support services that are able to respond to families' changing needs.

The Possibility of Systematic Research Fraud Targeting Under-Studied Human Genes: Causes, Consequences, and Potential Solutions.

A major reason for biomarker failure is the selection of candidate biomarkers based on inaccurate or incorrect published results. Incorrect research results leading to the selection of unproductive biomarker candidates are largely considered to stem from unintentional research errors. The additional possibility that biomarker research may be actively misdirected by research fraud has been given comparatively little consideration. This review discusses what we believe to be a new threat to biomarker research, namely, the possible systematic production of fraudulent gene knockdown studies that target under-studied human genes. We describe how fraudulent papers may be produced in series by paper mills using what we have described as a 'theme and variations' model, which could also be considered a form of salami slicing. We describe features of these single-gene knockdown publications that may allow them to evade detection by journal editors, peer reviewers, and readers. We then propose a number of approaches to facilitate their detection, including improved awareness of the features of publications constructed in series, broader requirements to post submitted manuscripts to preprint servers, and the use of semi-automated literature screening tools. These approaches may collectively improve the detection of fraudulent studies that might otherwise impede future biomarker research.

Research Perspective on Utilizing and Valuing Tumor Biobanks.

Tumor biobanks have become critical components of the cancer research infrastructure. Consideration of how to place appropriate values on tumor biobanks is important for all stakeholders. At the level of individual biobanks, value is important in determining how to contribute to, utilize, and fund biobanks. At the level of the research system, value is important in determining how to evaluate, rationalize, and sustain or modify the investments in this infrastructure. This review considers approaches and indicators for evaluation of a biobank with a particular focus on utilization, as one important indicator of value, from the perspective of the researcher and funder. The patterns of utilization and the influence of different phases and approaches of research, and types of biobank are described, as well as strategies for biobanks to increase utilization and therefore their value to research.

Investigation of clinically relevant germline variants detected by next-generation sequencing in patients with childhood cancer: a review of the literature.

Genetic predisposition is an important underlying cause of childhood cancer, although the proportion of patients with childhood cancer carrying predisposing pathogenic germline variants is uncertain. This review considers the pathogenic or likely pathogenic germline variants reported by six studies that used next-generation sequencing to investigate genetic predisposition in selected cohorts of patients with childhood cancer and used incompletely overlapping gene sets for analysis and interpretation. These six studies reported that 8.5%-35.5% of patients with childhood cancer carried clinically relevant germline variants. Analysis of 52 autosomal dominant cancer predisposition genes assumed common to all six studies showed that 5.5%-25.8% of patients with childhood cancer carried pathogenic or likely pathogenic germline variants in at least one of these genes. When only non-central nervous system solid tumours (excluding adrenocortical carcinomas) were considered, 8.5%-10.3% of the patients carried pathogenic or likely pathogenic germline variants in at least one of 52 autosomal dominant cancer predisposition genes. There was a lack of concordance between the genotype and phenotype in 33.3%-57.1% of the patients reported with pathogenic or likely pathogenic germline variants, most of which represented variants in autosomal dominant cancer predisposition genes associated with adult onset cancers. In summary, germline genetic testing in patients with childhood cancer requires clear definition of phenotypes and genes considered for interpretation, with potential to inform and broaden childhood cancer predisposition syndromes.

An Australian Biobank Certification Scheme: A Study of Economic Costs to Participating Biobanks.

Biobanks face increasing demands for research materials of consistent quality, which can be used in collaborative studies. Several countries and some international agencies have made formal efforts to standardize biobank operations and outputs. These include the establishment of best practice guidelines for collection management, and certification programs. Such guidelines and programs increase biobanks' opportunities for participation in high impact research and funding. However, they also impose economic and time costs, which may burden biobanks. This study aimed to estimate the costs of gaining certification and maintaining certification (i.e., committing extra resources to continue standards) for three cancer biobanks participating in a biobank certification program in New South Wales, Australia. To gather cost data for a range of cancer biobanks, we recruited three with different full time equivalent (FTE) staff levels (1.0-3.0), recognizing FTE staff level as an indicator of resources and operating scale. In extended interviews with staff, we gathered biobanks' expected costs in obtaining and annually maintaining certification. The biobank with the highest staff level reported the lowest expected costs in gaining certification, due to the strong prealignment of its present operations with certification requirements. The other biobanks expected higher costs as their operations required greater adjustments. Overall, relative costs of gaining certification were between 2% and 6% of current total annual wage costs. To the authors' knowledge, this is the first such costing study of a biobank certification program. Supplementary Data include the interview schedule that other biobanks may use to estimate their own economic certification costs.

Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo.

The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates.

Acute acinar pancreatitis blocks vesicle-associated membrane protein 8 (VAMP8)-dependent secretion, resulting in intracellular trypsin accumulation.

Zymogen secretory granules in pancreatic acinar cells express two vesicle-associated membrane proteins (VAMP), VAMP2 and -8, each controlling 50% of stimulated secretion. Analysis of secretion kinetics identified a first phase (0-2 min) mediated by VAMP2 and second (2-10 min) and third phases (10-30 min) mediated by VAMP8. Induction of acinar pancreatitis by supramaximal cholecystokinin (CCK-8) stimulation inhibits VAMP8-mediated mid- and late-phase but not VAMP2-mediated early-phase secretion. Elevation of cAMP during supramaximal CCK-8 mitigates third-phase secretory inhibition and acinar damage caused by the accumulation of prematurely activated trypsin. VAMP8-/- acini are resistant to secretory inhibition by supramaximal CCK-8, and despite a 4.5-fold increase in total cellular trypsinogen levels, are fully protected from intracellular trypsin accumulation and acinar damage. VAMP8-mediated secretion is dependent on expression of the early endosomal proteins Rab5, D52, and EEA1. Supramaximal CCK-8 (60 min) caused a 60% reduction in the expression of D52 followed by Rab5 and EEA1 in isolated acini and in in vivo The loss of D52 occurred as a consequence of its entry into autophagic vacuoles and was blocked by lysosomal cathepsin B and L inhibition. Accordingly, adenoviral overexpression of Rab5 or D52 enhanced secretion in response to supramaximal CCK-8 and prevented accumulation of activated trypsin. These data support that acute inhibition of VAMP8-mediated secretion during pancreatitis triggers intracellular trypsin accumulation and loss of the early endosomal compartment. Maintaining anterograde endosomal trafficking during pancreatitis maintains VAMP8-dependent secretion, thereby preventing accumulation of activated trypsin.

Programmed Death-Ligand 1 Expression in a Large Cohort of Pediatric Patients With Solid Tumor and Association With Clinicopathologic Features in Neuroblastoma.

PURPOSE: Programmed death-ligand 1 (PD-L1) expression represents a potential predictive biomarker of immune checkpoint blockade response. However, literature about the prevalence of PD-L1 expression in the pediatric cancer setting is discordant. METHODS: PD-L1 expression was analyzed using immunohistochemistry in 500 pediatric tumors (including neuroblastoma, sarcomas, and brain cancers). Tumors with ≥ 1% cells showing PD-L1 membrane staining of any intensity were scored as positive. Positive cases were further characterized, with cases with weak intensity PD-L1 staining reported as having low PD-L1 expression and cases with a moderate or strong intensity of staining considered to have high PD-L1 expression. RESULTS: PD-L1-positive staining was identified in 13% of cases, whereas high PD-L1 expression was found in 3% of cases. Neuroblastoma (n = 254) showed PD-L1 expression of any intensity in 18.9% of cases and was associated with longer overall survival (P = .045). However, high PD-L1 expression in neuroblastoma (3.1%) was significantly associated with an increased risk of relapse (P = .002). Positive PD-L1 staining was observed more frequently in low- and intermediate-risk patients (P = .037) and in cases lacking MYCN amplification (P = .002). CONCLUSION: In summary, high PD-L1 expression in patients with neuroblastoma may represent an unfavorable prognostic factor associated with a higher risk of cancer relapse. This work proposes PD-L1 immunohistochemical assessment as a novel parameter for identifying patients with an increased likelihood of cancer recurrence.

The BET bromodomain inhibitor exerts the most potent synergistic anticancer effects with quinone-containing compounds and anti-microtubule drugs.

BET bromodomain inhibitors are very promising novel anticancer agents, however, single therapy does not cause tumor regression in mice, suggesting the need for combination therapy. After screening a library of 2697 small molecule compounds, we found that two classes of compounds, the quinone-containing compounds such as nanaomycin and anti-microtubule drugs such as vincristine, exerted the best synergistic anticancer effects with the BET bromodomain inhibitor JQ1 in neuroblastoma cells. Mechanistically, the quinone-containing compound nanaomycin induced neuroblastoma cell death but also activated the Nrf2-antioxidant signaling pathway, and the BET bromodomain proteins BRD3 and BRD4 formed a protein complex with Nrf2. Treatment with JQ1 blocked the recruitment of Nrf2 to the antioxidant responsive elements at Nrf2 target gene promoters, and JQ1 exerted synergistic anticancer effects with nanaomycin by blocking the Nrf2-antioxidant signaling pathway. JQ1 and vincristine synergistically induced neuroblastoma cell cycle arrest at the G2/M phase, aberrant mitotic spindle assembly formation and apoptosis, but showed no effect on cell survival in normal non-malignant cells. Importantly, co-treatment with JQ1 and vincristine synergistically suppressed tumor progression in neuroblastoma-bearing mice. These results strongly suggest that patients treated with BET bromodomain inhibitors in clinical trials should be co-treated with vincristine.

Dropping in on the lipid droplet- tumor protein D52 (TPD52) as a new regulator and resident protein.

Lipid droplets are essential for both the storage and retrieval of excess cellular nutrients, and their biology is regulated by a diverse range of cellular proteins, some of which function at the lipid droplet. Numerous studies have characterized lipid droplet proteomes in different organisms and cell types, and RNAi whole genome screening studies have examined the genetic regulation of lipid storage in C. elegans and D. melanogaster. While tumor protein D52 (TPD52) did not emerge from earlier studies as a strong candidate, exogenous expression of human TPD52 in cultured cells resulted in significantly increased numbers of lipid droplets, and oleic acid supplementation increased TPD52 detection at both lipid droplets and the Golgi apparatus. These results suggest that direct testing of proteins that are infrequently but recurrently identified in proteomic and RNAi screening studies may identify novel lipid droplet regulators. While the analysis of these possibly lower-abundance or itinerant lipid droplet proteins may be more technically challenging, such proteins could facilitate a more detailed interrogation of emerging aspects of lipid droplet biology.