Looking beyond workforce parity: addressing gender inequity in pathology.

While women pathologists have made up over one-third of pathologists in the Australian workforce for over 15 years and at least 50% since 2019, they are under-represented in senior leadership roles, scientific publications, grant recipients, editorial boards, key presentations, and professional awards. This is not unique to pathology and is seen in the broader medical and academic community. Barriers to gender equity and equality in pathology, medicine and academia include gender stereotypes, gender-based discrimination, structural and organisational barriers as well as broader social and cultural barriers. A diverse leadership reflective of the whole professional body and the broader community is important for optimal health outcomes. It is the responsibility and moral duty of individuals and organisations to address any gender disparities, inequities, and inequalities by monitoring, identifying, and acting on gender biases and systemic barriers that hinder appropriate levels of representation by women.

Heatwaves and wildfires suffocate our healthy start to life: time to assess impact and take action.

Adverse environmental exposures in utero and early childhood are known to programme long-term health. Climate change, by contributing to severe heatwaves, wildfires, and other natural disasters, is plausibly associated with adverse pregnancy outcomes and an increase in the future burden of chronic diseases in both mothers and their babies. In this Personal View, we highlight the limitations of existing evidence, specifically on the effects of severe heatwave and wildfire events, and compounding syndemic events such as the COVID-19 pandemic, on the short-term and long-term physical and mental health of pregnant women and their babies, taking into account the interactions with individual and community vulnerabilities. We highlight a need for an international, interdisciplinary collaborative effort to systematically study the effects of severe climate-related environmental crises on maternal and child health. This will enable informed changes to public health policy and clinical practice necessary to safeguard the health and wellbeing of current and future generations.

Validation of a tool for determining the clinical utility of stillbirth investigations.

BACKGROUND: Up to 20% of all stillbirths and 45% of term stillbirths are currently classified as unexplained. Many of these stillbirths do not undergo currently recommended investigations. This may leave questions unanswered and not identify stillbirths with a recurrence risk in subsequent pregnancies. AIMS: To validate a new tool (Stillbirth Investigation Utility Tool) to identify the clinical utility of investigations in stillbirth and the inter-rater agreement on cause of stillbirth using the Perinatal Society of Australia and New Zealand-Perinatal Death Classification (PSANZ-PDC). MATERIALS AND METHODS: Thirty-four stillbirths were randomly selected for inclusion, each assessed independently by five blinded assessors. The investigations were grouped into three categories: clinical and laboratory; placental pathology; and autopsy examination. The cause of death was assigned at the end of each group. Outcome measures were clinical utility of investigations measured by assessor rated usefulness and inter-rater agreement on the assigned cause of death. RESULTS: Comprehensive maternal history, maternal full blood count, maternal blood group and screen and placenta histopathology were useful in all cases. Clinical photographs were not performed and should have been performed in 50% of cases. The inter-rater agreement on cause of death assigned after all investigation results was 0.93 (95% CI 0.87-1.0). CONCLUSIONS: The new Stillbirth Investigation Utility Tool showed very good agreement in assigning the cause of death using PSANZ-PDC. Four investigations were useful in all cases. Minor refinements will be made based on feedback to enhance usability for wider implementation in research studies to assess the yield of investigations in stillbirths.

High Dietary Iron in Western Diet-Fed Male Rats Causes Pancreatic Islet Injury and Acute Pancreatitis.

BACKGROUND: High dietary iron has been linked to an increased type 2 diabetes risk. We have previously shown that intrauterine growth restriction (IUGR) and feeding a Western diet (WD) to male Sprague-Dawley rats independently, as well as together, cause pancreatic islet inflammation, fibrosis, and hemosiderosis. OBJECTIVES: To investigate whether iron has a role in the pathogenesis of this inflammatory islet injury caused by IUGR and WD intake. METHODS: Male Sprague-Dawley offspring of bilateral uterine artery ligated (IUGR) and sham-operated (Sham) dams, fostered to nonoperated dams, were fed a WD [45% sucrose, 19.4% protein and 23% fat (w/w)] containing low iron (LI, 20 mg/kg) or high iron (HI, 500 mg/kg) from weaning. Four groups were studied: Sham-LI, Sham-HI, IUGR-LI, and IUGR-HI. Serial measurements of rat body weight, blood glucose, lipids and insulin, an intraperitoneal glucose tolerance test (age 13 wk), and histological analysis of pancreas and liver (age 14 wk) were recorded. The effects of iron, IUGR, and their interaction, on these measurements have been analyzed. RESULTS: WD with HI compared with LI caused an 11% greater weight gain by age 14 wk (P < 0.001), impaired glucose tolerance [AUC for glucose (G-AUC) 17% higher; P < 0.001), acute pancreatitis (17/18, HI; 6/17, LI; P < 0.001), pancreas-associated fat necrosis and saponification (7/18, HI; 0/17 LI; P < 0.01), and a trend to islet fibrotic injury (7/18, HI; 1/17 LI; P = 0.051). Although pancreatic and hepatic steatosis was evident in almost all WD-fed rats, pancreatic and hepatic iron accumulation was prevalent only in HI-fed rats (P < 0.0001 for both), being only mild in the livers. IUGR, independent of dietary iron, also caused impairment in glucose tolerance (G-AUC: 17% higher; P < 0.05). CONCLUSIONS: A postweaning WD containing HI, independent of IUGR, causes acute pancreatitis and islet injury in Sprague-Dawley rats suggesting a role of dietary iron in the development of steatopancreatitis.

Normative values of renin and aldosterone in clinically stable preterm neonates.

BACKGROUND: There is a paucity of literature on the normative levels of plasma renin concentration (PRC) and serum aldosterone (SA) in premature neonates. This study aims to provide normative data on PRC and SA levels in preterm neonates in the first 2 weeks after birth and explore associations with maternal, perinatal, or postnatal factors. METHODS: Neonates born at 26- to 34-week gestation were recruited from two neonatal intensive care units in Canada and Australia. The direct renin assay PRC and SA were analyzed on day 1 and days 14-21 after birth to compare across categorical variables and to produce normative values. RESULTS: A total of 262 subjects were enrolled from the Canadian (29%) and Australian (71%) sites. The mean gestational age was 30 weeks, with a mean birth weight of 1457 g. The normative values of PRC and SA for neonates born between 26 + 0 and 29 + 6 weeks and 30 + 0 and 34 + 0 weeks of gestation were produced for day 1 and day 14-21 after birth. Both PRC and SA increased from day 1 to day 14-21. The more premature neonates reached a higher PRC on days 14-21 after birth but exhibited lower SA levels on day 1 after birth. When comparing gender, birth weight, and maternal risk factor categories, no statistical differences in PRC or SA were found. A small but significant decrease in PRC, but not SA, was noted for neonates with placental pathology. CONCLUSIONS: This study produced normative values of PRA and SA in clinically stable preterm neonates that can be referenced for use in clinical practice. A higher resolution version of the Graphical abstract is available as Supplementary information.

Bushfires and Mothers' Mental Health in Pregnancy and Recent Post-Partum.

BACKGROUND: The compounding effects of climate change catastrophes such as bushfires and pandemics impose significant burden on individuals, societies, and their economies. The enduring effects of such syndemics on mental health remain poorly understood, particularly for at-risk populations (e.g., pregnant women and newborns). The aim of this study was to investigate the impact of direct and indirect exposure to the 2019/20 Australian Capital Territory and South-Eastern New South Wales bushfires followed by COVID-19 on the mental health and wellbeing of pregnant women and mothers with newborn babies. METHODS: All women who were pregnant, had given birth, or were within three months of conceiving during the 2019/2020 bushfires, lived within the catchment area, and provided consent were invited to participate. Those who consented were asked to complete three online surveys. Mental health was assessed with the DASS-21 and the WHO-5. Bushfire, smoke, and COVID-19 exposures were assessed by self-report. Cross-sectional associations between exposures and mental health measures were tested with hierarchical regression models. RESULTS: Of the women who participated, and had minimum data (n = 919), most (>75%) reported at least one acute bushfire exposure and 63% reported severe smoke exposure. Compared to Australian norms, participants had higher depression (+12%), anxiety (+35%), and stress (+43%) scores. Women with greater exposure to bushfires/smoke but not COVID-19 had poorer scores on all mental health measures. CONCLUSIONS: These findings provide novel evidence that the mental health of pregnant women and mothers of newborn babies is vulnerable to major climate catastrophes such as bushfires.

Neonatal hypoxic ischemic encephalopathy increases acute kidney injury urinary biomarkers in a rat model.

Hypoxic ischemic encephalopathy (HIE) is associated with acute kidney injury (AKI) in neonates with birth asphyxia. This study aimed to utilize urinary biomarkers to characterize AKI in an established neonatal rat model of HIE. Day 7 Sprague-Dawley rat pups underwent HIE using the Rice-Vannucci model (unilateral carotid ligation followed by 120 mins of 8% oxygen). Controls included no surgery and sham surgery. Weights and urine for biomarkers (NGAL, osteopontin, KIM-1, albumin) were collected the day prior, daily for 3 days post-intervention, and at sacrifice day 14. Kidneys and brains were processed for histology. HIE pups displayed histological evidence of kidney injury including damage to the proximal tubules, consistent with resolving acute tubular necrosis, and had significantly elevated urinary levels of NGAL and albumin compared to sham or controls 1-day post-insult that elevated for 3 days. KIM-1 significantly increased for 2 days post-HIE. HIE did not significantly alter osteopontin levels. Seven days post-start of experiment, controls were 81.2% above starting weight compared to 52.1% in HIE pups. NGAL and albumin levels inversely correlated with body weight following HIE injury. The AKI produced by the Rice-Vannucci HIE model is detectable by urinary biomarkers, which can be used for future studies of treatments to reduce kidney injury.

Margin status and survival outcomes after breast cancer conservation surgery: prospectively registered systematic review and meta-analysis.

OBJECTIVE: To determine if margin involvement is associated with distant recurrence and to determine the required margin to minimise both local recurrence and distant recurrence in early stage invasive breast cancer. DESIGN: Prospectively registered systematic review and meta-analysis of literature. DATA SOURCES: Medline (PubMed), Embase, and Proquest online databases. Unpublished data were sought from study authors. ELIGIBILITY CRITERIA: Eligible studies reported on patients undergoing breast conserving surgery (for stages I-III breast cancer), allowed an estimation of outcomes in relation to margin status, and followed up patients for a minimum of 60 months. Patients with ductal carcinoma in situ only or treated with neoadjuvant chemotherapy or by mastectomy were excluded. Where applicable, margins were categorised as tumour on ink (involved), close margins (no tumour on ink but <2 mm), and negative margins (≥2 mm). RESULTS: 68 studies from 1 January 1980 to 31 December 2021, comprising 112 140 patients with breast cancer, were included. Across all studies, 9.4% (95% confidence interval 6.8% to 12.8%) of patients had involved (tumour on ink) margins and 17.8% (13.0% to 23.9%) had tumour on ink or a close margin. The rate of distant recurrence was 25.4% (14.5% to 40.6%) in patients with tumour on ink, 8.4% (4.4% to 15.5%) in patients with tumour on ink or close, and 7.4% (3.9% to 13.6%) in patients with negative margins. Compared with negative margins, tumour on ink margins were associated with increased distant recurrence (hazard ratio 2.10, 95% confidence interval 1.65 to 2.69, P<0.001) and local recurrence (1.98, 1.66 to 2.36, P<0.001). Close margins were associated with increased distant recurrence (1.38, 1.13 to 1.69, P<0.001) and local recurrence (2.09, 1.39 to 3.13, P<0.001) compared with negative margins, after adjusting for receipt of adjuvant chemotherapy and radiotherapy. In five studies published since 2010, tumour on ink margins were associated with increased distant recurrence (2.41, 1.81 to 3.21, P<0.001) as were tumour on ink and close margins (1.44, 1.22 to 1.71, P<0.001) compared with negative margins. CONCLUSIONS: Involved or close pathological margins after breast conserving surgery for early stage, invasive breast cancer are associated with increased distant recurrence and local recurrence. Surgeons should aim to achieve a minimum clear margin of at least 1 mm. On the basis of current evidence, international guidelines should be revised. SYSTEMATIC REVIEW REGISTRATION: CRD42021232115.

Superior Hemostatic and Wound-Healing Properties of Gel and Sponge Forms of Nonoxidized Cellulose Nanofibers: In Vitro and In Vivo Studies.

Many materials have been engineered and commercialized as hemostatic agents. However, there is still a gap in the availability of hemostats that offer biocompatibility and biodegradability in combination with effective hemostatic properties. Cellulose nanofibers are investigated as hemostatic materials with most studies focusing on oxidized cellulose-derived hemostats. The recent studies demonstrate that by optimizing the morphological properties of nonoxidized cellulose nanofibers (CNFs) enhanced hemostasis is achieved. Herein, the hemostatic and wound-healing properties of CNFs with optimized morphology using two forms, gel, and sponge is investigated. In vitro thromboelastometry studies demonstrate that CNFs reduce clotting time by 68% (±SE 2%) and 88% (±SE 5%) in gel and sponge forms, respectively. In an in vivo murine liver injury model, CNFs significantly reduce blood loss by 38% (±SE 10%). The pH-neutral CNFs do not damage red blood cells, nor do they impede the proliferation of fibroblast or endothelial cells. Subcutaneously-implanted CNFs show a foreign body reaction resolving with the degradation of CNFs on histological examination and there is no scarring in the skin after 8 weeks. Demonstrating superior hemostatic performance in a variety of forms, as well as biocompatibility and biodegradability, CNFs hold significant potential for use in surgical and first-aid environments.

MCC Gene Silencing Is a CpG Island Methylator Phenotype-Associated Factor That Predisposes Colon Cancer Cells to Irinotecan and Olaparib.

Chemotherapy is a mainstay of colorectal cancer treatment, and often involves a combination drug regime. CpG island methylator phenotype (CIMP)-positive tumors are potentially more responsive to the topoisomerase-inhibitor irinotecan. The mechanistic basis of the increased sensitivity of CIMP cancers to irinotecan is poorly understood. Mutated in Colorectal Cancer (MCC) is emerging as a multifunctional tumor suppressor gene in colorectal and liver cancers, and has been implicated in drug responsiveness. Here, we found that CIMP tumors undergo MCC loss almost exclusively via promoter hypermethylation rather than copy number variation or mutations. A subset of cancers display hypomethylation which is also associated with low MCC expression, particularly in rectal cancer, where CIMP is rare. MCC knockdown or deletion was found to sensitize cells to SN38 (the active metabolite of irinotecan) or the PARP-inhibitor Olaparib. A synergistic effect on cell death was evident when these drugs were used concurrently. The improved SN38/irinotecan efficacy was accompanied by the down-regulation of DNA repair genes. Thus, differential methylation of MCC is potentially a valuable biomarker to identify colorectal cancers suitable for irinotecan therapy, possibly in combination with PARP inhibitors.

A Phase 1 Proof of Concept Study Evaluating the Addition of an LSD1 Inhibitor to Nab-Paclitaxel in Advanced or Metastatic Breast Cancer (EPI-PRIMED).

Objective: Lysine-Specific Demethylase-1 (LSD1) is overexpressed in breast cancer cells and facilitate mesenchymal properties which may contribute to therapeutic resistance and cancer progression. The purpose of this study was to investigate the safety of combination, nab-paclitaxel and phenelzine, an irreversible LSD1 inhibitor in patients with metastatic breast cancer (mBC). Methods: Eligible patients with mBC were treated with nab-paclitaxel (100mg/m2) weekly for 3 weeks with one week break in a 28-day cycle. Dose escalation of phenelzine followed the Cumulative Cohort Design and phenelzine treatment commenced from day 2 of first cycle. Eleven patients were screened, and eligible patients were enrolled in cohorts with the dose of phenelzine ranging from 45mg to 90mg. Results: The Optimum Biological Dose was established at 60mg of phenelzine daily in combination with nab-paclitaxel and considered as the recommended phase 2 dose. Most (95%) of adverse events were grade 1 or 2 with two grade 3 events being diarrhea and neutropenia at 45mg and 60mg phenelzine respectively, with no unexpected toxicity/deaths. Commonly reported toxicities were fatigue (n=4,50%), dizziness (n=6,75%), neutropenia (n=3,37.5%), peripheral neuropathy (n=3,37.5%), diarrhea (n=2,25%), and hallucination (n=2,25%). After a median follow up of 113 weeks, all patients showed disease progression on trial with 4 patients being alive at the time of data cut off, including one patient with triple negative breast cancer. Median progression-free survival was 34 weeks. Significant inhibition of LSD1 and suppression of mesenchymal markers in circulating tumor cells were noted. Conclusion: Phenelzine in combination with nab-paclitaxel was well tolerated, without any unexpected toxicities in patients with mBC and demonstrated evidence of antitumor activity. For the first time, this proof-of-concept study showed in-vivo inhibition of LSD1 suppressed mesenchymal markers, which are known to facilitate generation of cancer stem cells with metastatic potential. Clinical Trial Registration: ClinicalTrials.Gov NCT03505528, UTN of U1111-1197-5518.

Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8+ T Cells in Immunotherapy-Resistant and Metastatic Cancers.

Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.

The Antiviral Drug Efavirenz in Breast Cancer Stem Cell Therapy.

Although many breast cancer therapies show initial success in the treatment of the primary tumour, they often fail to eliminate a sub-population of cells known as cancer stem cells (CSCs). These cells are recognised for their self-renewal properties and for their capacity for differentiation often leading to chemo/radio-resistance. The antiviral drug Efavirenz has been shown to be effective in eliminating triple-negative breast cancer cells, and here we examine its effect on breast CSCs. The effects of Efavirenz on CSCs for several breast cancer cell lines were investigated by examining cellular changes upon drug treatment, including CSC numbers, morphology, RNA/microRNA expression and levels of epithelial/mesenchymal CSC subtypes. Efavirenz treatment resulted in a decrease in the size and number of tumorspheres and a reduction in epithelial-type CSC levels, but an increase in mesenchymal-type CSCs. Efavirenz caused upregulation of several CSC-related genes as well as miR-21, a CSC marker and miR-182, a CSC suppressor gene. We conclude that Efavirenz alters the phenotype and expression of key genes in breast CSCs, which has important potential therapeutic implications.

Knockout of the Amino Acid Transporter SLC6A19 and Autoimmune Diabetes Incidence in Female Non-Obese Diabetic (NOD) Mice.

High protein feeding has been shown to accelerate the development of type 1 diabetes in female non-obese diabetic (NOD) mice. Here, we investigated whether reducing systemic amino acid availability via knockout of the Slc6a19 gene encoding the system B(0) neutral amino acid transporter AT1 would reduce the incidence or delay the onset of type 1 diabetes in female NOD mice. Slc6a19 gene deficient NOD mice were generated using the CRISPR-Cas9 system which resulted in marked aminoaciduria. The incidence of diabetes by week 30 was 59.5% (22/37) and 69.0% (20/29) in NOD.Slc6a19+/+ and NOD.Slc6a19-/- mice, respectively (hazard ratio 0.77, 95% confidence interval 0.41-1.42; Mantel-Cox log rank test: p = 0.37). The median survival time without diabetes was 28 and 25 weeks for NOD.Slc6a19+/+ and NOD.Slc6a19-/- mice, respectively (ratio 1.1, 95% confidence interval 0.6-2.0). Histological analysis did not show differences in islet number or the degree of insulitis between wild type and Slc6a19 deficient NOD mice. We conclude that Slc6a19 deficiency does not prevent or delay the development of type 1 diabetes in female NOD mice.

Simple and effective bacterial-based intratumoral cancer immunotherapy.

BACKGROUND: We describe intratumoral injection of a slow-release emulsion of killed mycobacteria (complete Freund's adjuvant (CFA)) in three preclinical species and in human cancer patients. METHODS: Efficacy and safety were tested in mammary tumors in mice, in mastocytomas in mice and dogs, and in equine melanomas. In mice, survival, tumor growth, and tumor infiltration by six immune cell subsets (by flow cytometry) were investigated and analyzed using Cox proportional hazards, a random slopes model, and a full factorial model, respectively. Tumor growth and histology were investigated in dogs and horses, as well as survival and tumor immunohistochemistry in dogs. Tumor biopsies were taken from human cancer patients on day 5 (all patients) and day 28 (some patients) of treatment and analyzed by histology. CT scans are provided from one patient. RESULTS: Significantly extended survival was observed in mouse P815 and 4T1 tumor models. Complete tumor regressions were observed in all three non-human species (6/186 (3%) of mouse mastocytomas; 3/14 (21%) of canine mastocytomas and 2/11 (18%) of equine melanomas). Evidence of systemic immune responses (regression of non-injected metastases) was also observed. Analysis of immune cells infiltrating mastocytoma tumors in mice showed that early neutrophil infiltration was predictive of treatment benefit. Analysis of the site of mastocytoma regression in dogs weeks or months after treatment demonstrated increased B and T cell infiltrates. Thus, activation of the innate immune system alone may be sufficient for regression of some injected tumors, followed by activation of the acquired immune system which can mediate regression of non-injected metastases. Finally, we report on the use of CFA in 12 human cancer patients. Treatment was well tolerated. CT scans showing tumor regression in a patient with late-stage renal cancer are provided. CONCLUSION: Our data demonstrate that intratumoral injection of CFA has major antitumor effects in a proportion of treated animals and is safe for use in human cancer patients. Further trials in human cancer patients are therefore warranted. Our novel treatment provides a simple and inexpensive cancer immunotherapy, immediately applicable to a wide range of solid tumors, and is suitable to patients in developing countries and advanced care settings.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Tetranucleotide and Low Microsatellite Instability Are Inversely Associated with the CpG Island Methylator Phenotype in Colorectal Cancer.

MSH3 gene or protein deficiency or loss-of-function in colorectal cancer can cause a DNA mismatch repair defect known as "elevated microsatellite alterations at selected tetranucleotide repeats" (EMAST). A high percentage of MSI-H tumors exhibit EMAST, while MSI-L is also linked with EMAST. However, the distribution of CpG island methylator phenotype (CIMP) within the EMAST spectrum is not known. Five tetranucleotide repeat and five MSI markers were used to classify 100 sporadic colorectal tumours for EMAST, MSI-H and MSI-L according to the number of unstable markers detected. Promoter methylation was determined using methylation-specific PCR for MSH3, MCC, CDKN2A (p16) and five CIMP marker genes. EMAST was found in 55% of sporadic colorectal carcinomas. Carcinomas with only one positive marker (EMAST-1/5, 26%) were associated with advanced tumour stage, increased lymph node metastasis, MSI-L and lack of CIMP-H. EMAST-2/5 (16%) carcinomas displayed some methylation but MSI was rare. Carcinomas with ≥3 positive EMAST markers (13%) were more likely to have a proximal colon location and be MSI-H and CIMP-H. Our study suggests that EMAST/MSI-L is a valuable prognostic and predictive marker for colorectal carcinomas that do not display the high methylation phenotype CIMP-H.

Glutathione transferase Omega 1 confers protection against azoxymethane-induced colorectal tumour formation.

Inflammatory bowel disease (IBD) is characterized by multiple alterations in cytokine expression and is a risk factor for colon cancer. The Omega class glutathione transferase GSTO1-1 regulates the release of the pro-inflammatory cytokines interleukin 1ß (IL-1ß) and interleukin 18 (IL-18) by deglutathionylating NEK7 in the NLRP3 inflammasome. When treated with azoxymethane and dextran sodium sulphate (AOM/DSS) as a model of IBD, Gsto1-/- mice were highly sensitive to colitis and showed a significant increase in the size and number of colon tumours compared with wild-type (WT) mice. Gsto1-/- mice treated with AOM/DSS had significantly lower serum IL-1ß and IL-18 levels as well as significantly decreased interferon (IFN)-γ, decreased pSTAT1 and increased pSTAT3 levels in the distal colon compared with similarly treated WT mice. Histologically, AOM/DSS treated Gsto1-/- mice showed increased active chronic inflammation with macrophage infiltration, epithelial dysplasia and invasive adenocarcinoma compared with AOM/DSS treated WT mice. Thus, this study shows that GSTO1-1 regulates IL-1ß and IL-18 activation and protects against colorectal cancer formation in the AOM/DSS model of IBD. The data suggest that while GSTO1-1 is a new target for the regulation of the NLRP3 inflammasome-associated cytokines IL-1ß and IL-18 by small molecule inhibitors, there is a possibility that anti-inflammatory drugs targeting these cytokines may potentiate colon cancer in some situations.

Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch.

Lysine specific demethylase 1 (LSD1) is a key epigenetic eraser enzyme implicated in cancer metastases and recurrence. Nuclear LSD1 phosphorylated at serine 111 (nLSD1p) has been shown to be critical for the development of breast cancer stem cells. Here we show that circulating tumor cells isolated from immunotherapy-resistant metastatic melanoma patients express higher levels of nLSD1p compared to responders, which is associated with co-expression of stem-like, mesenchymal genes. Targeting nLSD1p with selective nLSD1 inhibitors better inhibits the stem-like mesenchymal signature than traditional FAD-specific LSD1 catalytic inhibitors such as GSK2879552. We also demonstrate that nLSD1p is enriched in PD-1+CD8+ T cells from resistant melanoma patients and 4T1 immunotherapy-resistant mice. Targeting the LSD1p nuclear axis induces IFN-γ/TNF-α-expressing CD8+ T cell infiltration into the tumors of 4T1 immunotherapy-resistant mice, which is further augmented by combined immunotherapy. Underpinning these observations, nLSD1p is regulated by the key T cell exhaustion transcription factor EOMES in dysfunctional CD8+ T cells. EOMES co-exists with nLSD1p in PD-1+CD8+ T cells in resistant patients, and nLSD1p regulates EOMES nuclear dynamics via demethylation/acetylation switching of critical EOMES residues. Using novel antibodies to target these post-translational modifications, we show that EOMES demethylation/acetylation is reciprocally expressed in resistant and responder patients. Overall, we show for the first time that dual inhibition of metastatic cancer cells and re-invigoration of the immune system requires LSD1 inhibitors that target the nLSD1p axis.