Chromosomal instability and its effect on cell lines.

BACKGROUND: Cancer cell lines are invaluable model systems for biomedical research because they provide an almost unlimited supply of biological materials. However, there is considerable skepticism regarding the reproducibility of data derived from these in vitro models. RECENT FINDINGS: Chromosomal instability (CIN) is one of the primary issues associated with cell lines, which can cause genetic heterogeneity and unstable cell properties within a cell population. Many of these problems can be avoided with some precautions. Here we review the underlying causes of CIN, including merotelic attachment, telomere dysfunction, DNA damage response defects, mitotic checkpoint defects and cell cycle disturbances. CONCLUSION: In this review we summarize studies highlighting the consequences of CIN in various cell lines and provide suggestions on monitoring and controlling CIN during cell culture.

LOT and HOT or not. The proliferation of clinically insignificant and poorly characterised types of renal neoplasia.

The classification of malignant tumours is influenced by both immunohistochemical and molecular genetic findings. This is highlighted in the latest World Health Organization classification of renal neoplasia, which has a tumour category of 'tumours that are molecularly defined'. This implies that the defining molecular features are integral to tumourigenesis, which may not necessarily be the case. Renal oncocytoma is recognised as a benign tumour with variable morphology and immunoexpression. A variant of these tumours is hybrid oncocytic chromophobe tumour, which has features of both oncocytoma and chromophobe renal cell carcinoma and may, on rare occasions, show malignant behaviour. Recent reports have proposed two further entities with eosinophilic cytoplasm and varying nuclear pleomorphism, designated low grade oncocytic tumour (LOT) and eosinophilic vacuolated tumour (EVT), formally known as high grade oncocytic tumour (HOT). The diagnosis of these apparently benign tumours was made on the basis of morphological and immunohistochemical features. More recently it has been claimed that the mutations in the mTOR pathway are also a diagnostic feature and it is further suggested that these mutations are key to the pathogenesis of these tumours. As is seen in oncocytoma, immunohistochemical expression of tumours included in series of LOT and EVT is variable. The mutations in the mTOR pathway, where detected, were not constant, with any combination of mTOR, TSC1 and/or TSC2 being involved. A major issue is that in many of the studies full comparative genomic hybridisation results are not presented. In addition it is well recognised that mTOR mutations are seen in a variety of renal tumours. In view of these conflicting results, the rarity of these tumours and their apparent benign nature, raises questions as to why these tumours should be considered specific entities.

Prognostic significance of percentage Gleason grade 5 prostatic adenocarcinoma in needle biopsies from patients treated by radical prostatectomy.

Previous studies have shown that the percentage of high grade prostatic adenocarcinoma (Gleason patterns 4 and 5) in a biopsy correlates with outcome parameters. It has also been shown that the percentage Gleason pattern 4/5 tumour correlates with biochemical failure and overall survival. There are little data relating to the prognostic significance of quantifying the percentage of Gleason pattern 5 in isolation. We investigated the prognostic predictive value of quantifying the percentage of Gleason pattern 5 tumour in needle biopsies from a series of 196 cases of Gleason score 4+5=9 prostate adenocarcinoma from patients who had also undergone radical prostatectomy. Division of cases according to the percentage of Gleason pattern 5 present (based upon the core with the highest grade) and analysing these with tumour grouped as Gleason score 4+5 with <5% pattern 5 (GS 4+5 <5%), Gleason score 4+5 with 5-20% pattern 5 (GS 4+5 5-20%) and Gleason score 4+5 with 21-49% pattern 5 (GS 4+5 21-49%) showed no difference in outcome determined as time interval to prostate specific antigen biochemical failure. The results showed that each of the subgroups of GS 4+5 tumours had a significantly shorter biochemical recurrence-free survival than for a control group of 179 patients with Gleason score 4+3=7 (GS 4+3) cancer. Similar results were obtained when grading was based upon percentage of Gleason pattern 5 present in all the cores taken from the same patient (case-based grade). Adverse findings at radical prostatectomy showed each of the subgroups of GS 4+5 tumours to have a higher incidence of extraprostatic extension and seminal vesicle invasion than the GS 4+3 group of controls. Further, the differences in incidence between each of the subgroups were not significant for either extraprostatic extension or seminal vesicle invasion. These observations applied to both the highest core-based grade and the case-based grade. Our study has shown that any proportion of Gleason pattern 5 tumour in a needle biopsy is associated with a worse prognosis when compared to GS4+3 tumours and that these results are similar for grading that is core- or case-based.

Molecular Profiling of Endometrial Cancer: An Exploratory Study in Aotearoa, New Zealand.

BACKGROUND: Aotearoa, New Zealand, has one of the fastest-rising rates of endometrial cancer (EC) worldwide, increasing particularly in younger Maori and Pasifika women. There is a move towards using molecular profiling to direct treatment for each EC subtype. AIM: This study aimed to explore the molecular profiling of primary EC tissue in Aotearoa. METHODS: We used the PORTEC guidelines for the molecular subtyping of 90 patients' samples into four categories: POLE-mutated, p53 abnormal, mismatch repair deficient (MMRd) and no specific molecular profile (NSMP). The CTNNB1 mutation and L1CAM expression were also included in the analysis. POLE and CTNNB1 mutations were analysed using targeted next-generation sequencing (NGS). Novel mutations were assessed using VarSome. MMRd, L1CAM and p53 abnormalities were analysed using immunohistochemistry. RESULTS: In total, 15 samples were MMRd, 9 were p53 abnormal, 8 were POLE-mutated and the rest (56) were NSMP. Eleven samples had exon 3 CTNNB1 mutations and eleven novel POLE mutations were described. CONCLUSION: Surrogate markers for POLE mutations should be investigated. The validation of POLE variants and CTNNB1 mutations as part of an Aotearoa-based molecular panel is warranted.

Prognostic significance of morphological patterns of Gleason grade 5 prostatic adenocarcinoma diagnosed on needle biopsy.

Grading is one of the best prognostic indicators of prostate cancer with Gleason grade 5 having the worst outcome. The prognostic influence of grade 5 patterns remains uncertain. A total of 646 prostate needle biopsy sets with Gleason score (GS) 9-10 prostatic adenocarcinoma were prospectively analysed. Patterns of grade 5 were correlated with radical prostatectomy (RP) adverse findings of high tumour volume (TV), extra-prostatic extension (EPE), seminal vesicle invasion (SVI) and lymph node involvement (LNI) in 472 and biochemical recurrence (BCR) in 338 patients after RP. Mean age and serum PSA were 69 years (range 37-91) and 26.1 ng/mL (range 1.4-1800), respectively. Gleason scores were 4+5=9 in 539 (83%), 5+4=9 in 94 (15%) and 5+5=10 in 13 cases (2%). Clusters/cords, single cells, sheets and comedocarcinoma were found in 86%, 69%, 26% and 18% with a pure pattern in 25% of cases. Comparing cases with and without sheets, there were no significant differences with RP high TV (p=0.8577), EPE (p=0.5372), SVI (p=0.5183) and LNI (p=0.4323). However, the presence of sheets predicted a significantly higher BCR rate (p=0.0033), while for tumours with single cells, the interval to BCR was significantly shorter (p<0.0001). Comparing cases with and without the other patterns, two other significant differences were found. Comedocarcinoma predicted high TV (p=0.0230) and single cells predicted EPE (p=0.0101). This study shows that all patterns currently used to assign a Gleason grade 5, including sheets, comedocarcinoma, single cells and clusters/cords, are associated with aggressive outcomes validating their inclusion in grade 5.

Gene of the month: DICER1: ruler and controller.

DICER1 is a highly conserved RNaseIII endoribonuclease that has a critical role in the biogenesis of microRNAs (miRNAs). miRNAs are small regulatory RNAs responsible for post-transcriptional gene silencing, controlling more than half of human protein-coding genes. This is achieved through the targeting and regulation of complementary RNA transcripts and has a well-documented role in post-transcriptional gene regulation and transposon repression. DICER1 deficiency results in dysregulation of miRNAs, changing the expression of many genes. DICER1 syndrome represents a collection of benign and malignant tumours arising from an autosomally inherited germline mutation leading to an inherited predisposition to cancer. The syndrome represents an unusual form of Knudson's two-hit hypothesis, where individuals with a pathogenic germline DICER1 variant acquire a second trans-somatic missense DICER1 mutation. This somatic mutation appears to have to occur in one of five hotspots codons and may contribute towards the incomplete penetrance observed within DICER1 syndrome families. In this case, DICER1 is haploinsuffcient with only one deletion required and partial loss of function being advantageous to tumours over complete loss of function. As increasing data emerge reaffirming the pivotal role of DICER1 in the maintenance of human physiology, DICER1 is likely to become an increasingly attractive target for novel therapeutic strategies.

Staging of renal cell carcinoma: current progress and potential advances.

Formal staging classifications for renal cell carcinoma (RCC) were first proposed in 1978 and were incorporated into the Tumour, Nodes, Metastases (TNM) system initially published by the Union Internationale Contre le Cancer (UICC) in 1978. There has been a gradual evolution of grading criteria through six separate editions of the UICC TNM Classification, with the latest edition being published in 2016. Somewhat surprisingly there were no changes to the T category criteria from the 2009 to the 2016 editions of the classification, although an erratum has subsequently been published that incorporated the minor changes included in the eighth edition of the TNM Classification published by the American Joint Committee on Cancer. Localised tumours are staged according to the size of the primary tumour, with the TNM classification recognising that these tumours may exceed 10 cm in diameter. This is unfortunate as there is good evidence to demonstrate that, for clear cell RCC, virtually all tumours >7 cm in diameter and a substantial proportion of tumours <7 cm in diameter, show extra-renal spread. Infiltration of tumour beyond the renal capsule into the peri-renal fat is also categorised as T3a, however the clinical importance of this remains unclear. The classification of microvascular invasion within the renal sinus requires clarification, as does the prognostic significance of tumour in small vessels within the kidney.

Gene of the month: TMPRSS2 (transmembrane serine protease 2).

Transmembrane serine protease 2 is encoded by the TMPRSS2 gene. The gene is widely conserved and has two isoforms, both being autocatalytically activated from the inactive zymogen form. A fusion gene between the TMPRSS2 gene and ERG (erythroblast-specific-related gene), an oncogenic transcription factor, is the most common chromosomal aberration detected in prostate cancer, responsible for driving carcinogenesis. The other key role of TMPRSS2 is in priming the viral spike protein which facilitates viral entry essential for viral infectivity. The protease activates a diverse range of viruses. Both SARS-CoV and SARS-CoV-2 (COVID-19) use angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 to facilitate entry to cells, but with SARS-CoV-2 human-to-human transmission is much higher than SARS-CoV. As TMPRSS2 is expressed outside of the lung, and can therefore contribute to extrapulmonary spread of viruses, it warrants further exploration as a potential target for limiting viral spread and infectivity.

Granular necrosis: a distinctive form of cell death in malignant tumours.

Foci of necrosis are frequently seen in malignant tumours and may be due to a variety of causes. Different types of necrosis are given various names based upon their morphological features and presumed pathogenesis, such as coagulative, liquefactive and fibrinoid necrosis. Here, we propose the term 'granular necrosis' (GN) for a specific form of tumour necrosis characterised by the presence of well-defined necrotic foci being sharply demarcated from adjacent viable tumour. A constant feature is loss of architecture resulting in an amorphous necrotic mass containing granular nuclear and cytoplasmic debris, without an associated neutrophilic infiltrate. There is usually extensive karyorrhexis, which in larger tumours is more prominent at the periphery. These foci are often microscopic but may range up to several millimetres or larger in size. This distinctive form of necrosis has been erroneously given a variety of names in the literature including coagulative necrosis and microscopic necrosis, which on the basis of the aforementioned gross and microscopic findings is inappropriate. It is apparent that this is a specific form of necrosis, hence the descriptive term 'granular necrosis' that differentiates this form of necrosis from other types. The presence of GN is recognised as occurring in a variety of tumour types, being commonly seen in renal cell carcinoma, where it has been shown to have independent prognostic significance. In some epithelial and stromal tumours of the uterus, the presence of GN also has prognostic significance and is a defining feature for the differentiation of uterine leiomyoma and leiomyosarcoma. The pathogenesis of GN is unresolved. It does not show the features of apoptosis and in recent studies has been shown to have some of the molecular changes associated with necroptosis.

Intraductal carcinoma of the prostate is an aggressive form of invasive carcinoma and should be graded.

Infiltration of the prostatic ducts by prostatic adenocarcinoma occurs relatively frequently, being most commonly associated with high grade disease. It is now recognised that intraductal carcinoma of the prostate (IDCP) has an associated poor prognosis and this is reflected in its histological, molecular and immunohistochemical features. The current recommendation of the World Health Organization is that IDCP not be taken into consideration when grading prostate adenocarcinoma. It is apparent that Gleason did not differentiate between IDCP and stromal invasive carcinoma when developing and validating his grading system, and recent studies suggest that the incorporation of IDCP grading into the overall grading of the specimen provides additional prognostic information.

Reflection on the current status of endometrial cancer in New Zealand.

Incidence of endometrial cancer is increasing rapidly in the developed world and is the most common gynaecological cancer in Australia and New Zealand. In line with obesity rates, the landscape and average age of women diagnosed with endometrial cancer are changing. There is still unmet need in early diagnosis, directed treatment, management of comorbidities and prevention strategies. This opinion piece aims to reflect on the current status of endometrial cancer in New Zealand in parallel to Australia, drawing out areas for future research and discussion.

Maternal cadmium exposure and impact on foetal gene expression through methylation changes.

Cadmium (Cd) exposure is not easily avoidable; it is a common contaminant found in many food sources, accumulates throughout life and, in high doses, is a significant health hazard for humans. Women are highly vulnerable to Cd because of their relatively higher absorption rate than men. High levels of Cd accumulated in the mother could potentially cause harm to both the mother and new-born child. The foetal genome is vulnerable to external signals; Cd partially crosses the placental barrier and can impact on foetal development, potentially, through epigenetic mechanisms causing changes to foetal gene expression. This review explores current research on Cd induced methylation changes to maternal and foetal genomes. Cd is significantly associated with differential methylation of both maternal and foetal genomes. Some studies have described infant sex-specific changes in DNA methylation in association with maternal Cd burden. However, research on methylation changes to the foetal genome due to prenatal Cd exposure is scarce. More research is required to explore the impact of maternal Cd accumulation on differential methylation of the foetal genome.

De novo transcriptome assembly, functional annotation and differential gene expression analysis of juvenile and adult E. fetida, a model oligochaete used in ecotoxicological studies

BACKGROUND: Earthworms are sensitive to toxic chemicals present in the soil and so are useful indicator organisms for soil health. Eisenia fetida are commonly used in ecotoxicological studies; therefore the assembly of a baseline transcriptome is important for subsequent analyses exploring the impact of toxin exposure on genome wide gene expression. RESULTS: This paper reports on the de novo transcriptome assembly of E. fetida using Trinity, a freely available software tool. Trinotate was used to carry out functional annotation of the Trinity generated transcriptome file and the transdecoder generated peptide sequence file along with BLASTX, BLASTP and HMMER searches and were loaded into a Sqlite3 database. To identify differentially expressed transcripts; each of the original sequence files were aligned to the de novo assembled transcriptome using Bowtie and then RSEM was used to estimate expression values based on the alignment. EdgeR was used to calculate differential expression between the two conditions, with an FDR corrected P value cut off of 0.001, this returned six significantly differentially expressed genes. Initial BLASTX hits of these putative genes included hits with annelid ferritin and lysozyme proteins, as well as fungal NADH cytochrome b5 reductase and senescence associated proteins. At a cut off of P = 0.01 there were a further 26 differentially expressed genes. CONCLUSION: These data have been made publicly available, and to our knowledge represent the most comprehensive available transcriptome for E. fetida assembled from RNA sequencing data. This provides important groundwork for subsequent ecotoxicogenomic studies exploring the impact of the environment on global gene expression in E. fetida and other earthworm species.

Pre-Adolescent Cardio-Metabolic Associations and Correlates: PACMAC methodology and study protocol.

INTRODUCTION: Although cardiovascular disease is typically associated with middle or old age, the atherosclerotic process often initiates early in childhood. The process of atherosclerosis appears to be occurring at an increasing rate, even in pre-adolescents, and has been linked to the childhood obesity epidemic. This study will investigate the relationships between obesity, lifestyle behaviours and cardiometabolic health in pre-pubescent children aged 8-10 years, and investigates whether there are differences in the correlates of cardiometabolic health between Maori and Caucasian children. Details of the methodological aspects of recruitment, inclusion/exclusion criteria, assessments, statistical analyses, dissemination of findings and anticipated impact are described. METHODS AND ANALYSIS: Phase 1: a cross-sectional study design will be used to investigate relationships between obesity, lifestyle behaviours (nutrition, physical activity/fitness, sleep behaviour, psychosocial influences) and cardiometabolic health in a sample of 400 pre-pubescent (8-10 years old) children. Phase 2: in a subgroup (50 Caucasian, 50 Maori children), additional measurements of cardiometabolic health and lifestyle behaviours will be obtained to provide objective and detailed data. General linear models and logistic regression will be used to investigate the strongest correlate of (1) fatness; (2) physical activity; (3) nutritional behaviours and (4) cardiometabolic health. ETHICS AND DISSEMINATION: Ethical approval will be obtained from the New Zealand Health and Disabilities Ethics Committee. The findings from this study will elucidate targets for decreasing obesity and improving cardiometabolic health among preadolescent children in New Zealand. The aim is to ensure an immediate impact by disseminating these findings in an applicable manner via popular media and traditional academic forums. Most importantly, results from the study will be disseminated to participating schools and relevant Maori health entities.