A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance.

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

High levels of childhood trauma associated with changes in hippocampal functional activity and connectivity in young adults during novelty salience.

Childhood trauma (CT) has been linked to increased risk for psychosis. Moreover, CT has been linked to psychosis phenotypes such as impaired cognitive and sensory functions involved in the detection of novel sensory stimuli. Our objective was to investigate if CT was associated with changes in hippocampal and superior temporal gyrus functional activation and connectivity during a novelty detection task. Fifty-eight young adults were assigned to High-CT (n = 28) and Low-CT (n = 24) groups based on their scores on the childhood trauma questionnaire (CTQ) and underwent functional Magnetic Resonance Imaging during an auditory oddball task (AOT). Relative to the Low CT group, High CT participants showed reduced functional activation in the left hippocampus during the unpredictable tone condition of the AOT. Furthermore, in the High CT group, psychophysiological interaction analysis revealed hypoconnectivity between the hippocampus and temporal and medial regions. The present study indicates both altered hippocampal activation and hippocampal-temporal-prefrontal connectivity during novelty detection in individuals that experienced CT, similarly to that reported in psychosis risk populations. Early stressful experiences and environments may alter hippocampal function during salient events, mediating the relationship between childhood trauma and psychosis risk.

Genomics of gynaecological carcinosarcomas and future treatment options.

Gynaecological carcinosarcomas are the most lethal gynaecological malignancies that are often highly resistant to standard chemotherapy. They are composed of both carcinomatous and sarcomatous components and are associated with high rates of metastatic disease. Due to their rarity, molecular studies have been carried out on relatively few tumours, revealing a broad spectrum of heterogeneity. In this review, we have collated the gene mutations, gene expression, epigenetic regulation and protein expression reported by a number of studies on gynaecological carcinosarcomas. Based on these results, we describe potential therapeutics that may demonstrate efficacy and present any pre-clinical studies that have been carried out. We also describe the pre-clinical models currently available for future research to assess the potential of molecularly matched therapies. Interestingly, over-expression of many biomarkers in carcinosarcoma tumours often doesn't correlate with a worse prognosis. Therefore, we propose that profiling the mutational landscape, gene expression, and gene amplification/deletion may better indicate potential treatment strategies and predict response, thus improving outcomes for women with this rare, aggressive disease.

Daily and intermittent smoking are associated with low prefrontal volume and low concentrations of prefrontal glutamate, creatine, myo-inositol, and N-acetylaspartate.

Cigarette smoking is still the largest contributor to disease and death worldwide. Successful cessation is hindered by decreases in prefrontal glutamate concentrations and gray matter volume due to daily smoking. Because nondaily, intermittent smoking also contributes greatly to disease and death, understanding whether infrequent tobacco use is associated with reductions in prefrontal glutamate concentrations and gray matter volume may aid public health. Eighty-five young participants (41 nonsmokers, 24 intermittent smokers, 20 daily smokers, mean age ~23 years old), underwent 1 H-magnetic resonance spectroscopy of the medial prefrontal cortex, as well as structural magnetic resonance imaging (MRI) to determine whole-brain gray matter volume. Compared with nonsmokers, both daily and intermittent smokers exhibited lower concentrations of glutamate, creatine, N-acetylaspartate, and myo-inositol in the medial prefrontal cortex, and lower gray matter volume in the right inferior frontal gyrus; these measures of prefrontal metabolites and structure did not differ between daily and intermittent smokers. Finally, medial prefrontal metabolite concentrations and right inferior frontal gray matter volume were positively correlated, but these relationships were not influenced by smoking status. This study provides the first evidence that both daily and intermittent smoking are associated with low concentrations of glutamate, creatine, N-acetylaspartate, and myo-inositol and low gray matter volume in the prefrontal cortex. Future tobacco cessation efforts should not ignore potential deleterious effects of intermittent smoking by considering only daily smokers. Finally, because low glutamate concentrations hinder cessation, treatments that can normalize tonic levels of prefrontal glutamate, such as N-acetylcysteine, may help intermittent and daily smokers to quit.

HSP90 inhibition sensitizes head and neck cancer to platin-based chemoradiotherapy by modulation of the DNA damage response resulting in chromosomal fragmentation.

BACKGROUND: Concurrent cisplatin radiotherapy (CCRT) is a current standard-of-care for locally advanced head and neck squamous cell carcinoma (HNSCC). However, CCRT is frequently ineffective in patients with advanced disease. It has previously been shown that HSP90 inhibitors act as radiosensitizers, but these studies have not focused on CCRT in HNSCC. Here, we evaluated the HSP90 inhibitor, AUY922, combined with CCRT. METHODS: The ability of AUY922 to sensitize to CCRT was assessed in p53 mutant head and neck cell lines by clonogenic assay. Modulation of the CCRT induced DNA damage response (DDR) by AUY922 was characterized by confocal image analysis of RAD51, BRCA1, 53BP1, ATM and mutant p53 signaling. The role of FANCA depletion by AUY922 was examined using shRNA. Cell cycle checkpoint abrogation and chromosomal fragmentation was assessed by western blot, FACS and confocal. The role of ATM was also assessed by shRNA. AUY922 in combination with CCRT was assessed in vivo. RESULTS: The combination of AUY922 with cisplatin, radiation and CCRT was found to be synergistic in p53 mutant HNSCC. AUY922 leads to significant alterations to the DDR induced by CCRT. This comprises inhibition of homologous recombination through decreased RAD51 and pS1524 BRCA1 with a corresponding increase in 53BP1 foci, activation of ATM and signaling into mutant p53. A shift to more error prone repair combined with a loss of checkpoint function leads to fragmentation of chromosomal material. The degree of disruption to DDR signalling correlated to chromosomal fragmentation and loss of clonogenicity. ATM shRNA indicated a possible rationale for the combination of AUY922 and CCRT in cells lacking ATM function. CONCLUSIONS: This study supports future clinical studies combining AUY922 and CCRT in p53 mutant HNSCC. Modulation of the DDR and chromosomal fragmentation are likely to be analytical points of interest in such trials.

Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model.

The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three-dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non-malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1-HRAS and UBA2-PDCD2L expressed in-frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer-relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Identification of hypoxia-regulated proteins using MALDI-mass spectrometry imaging combined with quantitative proteomics.

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed.

Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation.

The transcription factor Gata-3 is a defining marker of the 'luminal' subtypes of breast cancer. To gain insight into the role of Gata-3 in breast epithelial development and oncogenesis, we have explored its normal function within the mammary gland by conditionally deleting Gata-3 at different stages of development. We report that Gata-3 has essential roles in the morphogenesis of the mammary gland in both the embryo and adult. Through the discovery of a novel marker (beta3-integrin) of luminal progenitor cells and their purification, we demonstrate that Gata-3 deficiency leads to an expansion of luminal progenitors and a concomitant block in differentiation. Remarkably, introduction of Gata-3 into a stem cell-enriched population induced maturation along the alveolar luminal lineage. These studies provide evidence for the existence of an epithelial hierarchy within the mammary gland and establish Gata-3 as a critical regulator of luminal differentiation.

CHK1 inhibitor SRA737 is active in PARP inhibitor resistant and CCNE1 amplified ovarian cancer.

High-grade serous ovarian cancers (HGSOCs) with homologous recombination deficiency (HRD) are initially responsive to poly (ADP-ribose) polymerase inhibitors (PARPi), but resistance ultimately emerges. HGSOC with CCNE1 amplification (CCNE1 amp) are associated with resistance to PARPi and platinum treatments. High replication stress in HRD and CCNE1 amp HGSOC leads to increased reliance on checkpoint kinase 1 (CHK1), a key regulator of cell cycle progression and the replication stress response. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable CHK1 inhibitor (CHK1i), SRA737, in both acquired PARPi-resistant BRCA1/2 mutant and CCNE1 amp HGSOC models. We demonstrated that SRA737 increased replication stress and induced subsequent cell death in vitro. SRA737 monotherapy in vivo prolonged survival in CCNE1 amp models, suggesting a potential biomarker for CHK1i therapy. Combination SRA737 and PARPi therapy increased tumor regression in both PARPi-resistant and CCNE1 amp patient-derived xenograft models, warranting further study in these HGSOC subgroups.

LOXL2 induces aberrant acinar morphogenesis via ErbB2 signaling.

INTRODUCTION: Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has been shown to play a key role in invasion and metastasis of breast carcinoma cells. However, very little is known about its role in normal tissue homeostasis. Here, we investigated the effects of LOXL2 expression in normal mammary epithelial cells to gain insight into how LOXL2 mediates cancer progression. METHODS: LOXL2 was expressed in MCF10A normal human mammary epithelial cells. The 3D acinar morphogenesis of these cells was assessed, as well as the ability of the cells to form branching structures on extracellular matrix (ECM)-coated surfaces. Transwell-invasion assays were used to assess the invasive properties of the cells. Clinically relevant inhibitors of ErbB2, lapatinib and Herceptin (traztuzumab), were used to investigate the role of ErbB2 signaling in this model. A retrospective study on a previously published breast cancer patient dataset was carried out by using Disease Specific Genomic Analysis (DSGA) to investigate the correlation of LOXL2 mRNA expression level with metastasis and survival of ErbB2-positive breast cancer patients. RESULTS: Fluorescence staining of the acini revealed increased proliferation, decreased apoptosis, and disrupted polarity, leading to abnormal lumen formation in response to LOXL2 expression in MCF10A cells. When plated onto ECM, the LOXL2-expressing cells formed branching structures and displayed increased invasion. We noted that LOXL2 induced ErbB2 activation through reactive oxygen species (ROS) production, and ErbB2 inhibition by using Herceptin or lapatinib abrogated the effects of LOXL2 on MCF10A cells. Finally, we found LOXL2 expression to be correlated with decreased overall survival and metastasis-free survival in breast cancer patients with ErbB2-positive tumors. CONCLUSIONS: These findings suggest that LOXL2 expression in normal epithelial cells can induce abnormal changes that resemble oncogenic transformation and cancer progression, and that these effects are driven by LOXL2-mediated activation of ErbB2. LOXL2 may also be a beneficial marker for breast cancer patients that could benefit most from anti-ErbB2 therapy.

Signatures of exposure to childhood trauma in young adults in the structure and neurochemistry of the superior temporal gyrus.

BACKGROUND: Childhood trauma (CT) has been linked to increased risk for mental illness in adulthood. Although work in experimental animals has shown that early life stressors can affect inhibitory and excitatory neurotransmission in adult rodents, with possible excitotoxic effects on local grey matter volumes (GMV), the neurobiological mechanisms that mediate this relationship in humans remain poorly understood. AIM: To examine glutamate and gamma-aminobutyric acid (GABA) metabolite concentrations and potential excitotoxic effects on GMV, in adults who experienced CT. METHODS: Fifty-six young adults (Mage = 20.41) were assigned to High CT (n = 29) and Low CT (n = 27) groups (by using the CT questionnaire) and underwent magnetic resonance spectroscopy (1H-MRS) to measure temporal lobe metabolite concentrations and volumetric imaging to measure GMV. RESULTS: Glutamate concentrations did not differ between groups; however, relative to the Low CT group, participants in the High CT group had reduced GABA concentrations in the left superior temporal gyrus (STG) voxel. Furthermore, logistic regression showed that participants with low left STG GABA concentrations and low left STG volumes were significantly more likely to be in the high CT group. CONCLUSIONS: This study provides the first evidence that both low GABA concentrations and its interaction with GMV in the left STG are associated with high levels of CT and suggest that altered inhibitory neurotransmission/metabolism may be linked to a lower GMV in the left STG in adults who experienced CT. Future studies are warranted to establish if utilizing these measures can stratify clinical high-risk and predict future clinical outcomes in high CT individuals.

A Matched Molecular and Clinical Analysis of the Epithelioid Haemangioendothelioma Cohort in the Stafford Fox Rare Cancer Program and Contextual Literature Review.

BACKGROUND: Epithelioid haemangioendothelioma (EHE) is an ultra-rare malignant vascular tumour with a prevalence of 1 per 1,000,000. It is typically molecularly characterised by a WWTR1::CAMTA1 gene fusion in approximately 90% of cases, or a YAP1::TFE3 gene fusion in approximately 10% of cases. EHE cases are typically refractory to therapies, and no anticancer agents are reimbursed for EHE in Australia. METHODS: We report a cohort of nine EHE cases with comprehensive histologic and molecular profiling from the Walter and Eliza Hall Institute of Medical Research Stafford Fox Rare Cancer Program (WEHI-SFRCP) collated via nation-wide referral to the Australian Rare Cancer (ARC) Portal. The diagnoses of EHE were confirmed by histopathological and immunohistochemical (IHC) examination. Molecular profiling was performed using the TruSight Oncology 500 assay, the TruSight RNA fusion panel, whole genome sequencing (WGS), or whole exome sequencing (WES). RESULTS: Molecular analysis of RNA, DNA or both was possible in seven of nine cases. The WWTR1::CAMTA1 fusion was identified in five cases. The YAP1::TFE3 fusion was identified in one case, demonstrating unique morphology compared to cases with the more common WWTR1::CAMTA1 fusion. All tumours expressed typical endothelial markers CD31, ERG, and CD34 and were negative for pan-cytokeratin. Cases with a WWTR1::CAMTA1 fusion displayed high expression of CAMTA1 and the single case with a YAP1::TFE3 fusion displayed high expression of TFE3. Survival was highly variable and unrelated to molecular profile. CONCLUSIONS: This cohort of EHE cases provides molecular and histopathological characterisation and matching clinical information that emphasises the molecular patterns and variable clinical outcomes and adds to our knowledge of this ultra-rare cancer. Such information from multiple studies will advance our understanding, potentially improving treatment options.

The microtubule inhibitor eribulin demonstrates efficacy in platinum-resistant and refractory high-grade serous ovarian cancer patient-derived xenograft models.

Background: Despite initial response to platinum-based chemotherapy and PARP inhibitor therapy (PARPi), nearly all recurrent high-grade serous ovarian cancer (HGSC) will acquire lethal drug resistance; indeed, ~15% of individuals have de novo platinum-refractory disease. Objectives: To determine the potential of anti-microtubule agent (AMA) therapy (paclitaxel, vinorelbine and eribulin) in platinum-resistant or refractory (PRR) HGSC by assessing response in patient-derived xenograft (PDX) models of HGSC. Design and methods: Of 13 PRR HGSC PDX, six were primary PRR, derived from chemotherapy-naïve samples (one was BRCA2 mutant) and seven were from samples obtained following chemotherapy treatment in the clinic (five were mutant for either BRCA1 or BRCA2 (BRCA1/2), four with prior PARPi exposure), recapitulating the population of individuals with aggressive treatment-resistant HGSC in the clinic. Molecular analyses and in vivo treatment studies were undertaken. Results: Seven out of thirteen PRR PDX (54%) were sensitive to treatment with the AMA, eribulin (time to progressive disease (PD) ⩾100 days from the start of treatment) and 11 out of 13 PDX (85%) derived significant benefit from eribulin [time to harvest (TTH) for each PDX with p < 0.002]. In 5 out of 10 platinum-refractory HGSC PDX (50%) and one out of three platinum-resistant PDX (33%), eribulin was more efficacious than was cisplatin, with longer time to PD and significantly extended TTH (each PDX p < 0.02). Furthermore, four of these models were extremely sensitive to all three AMA tested, maintaining response until the end of the experiment (120d post-treatment start). Despite harbouring secondary BRCA2 mutations, two BRCA2-mutant PDX models derived from heavily pre-treated individuals were sensitive to AMA. PRR HGSC PDX models showing greater sensitivity to AMA had high proliferative indices and oncogene expression. Two PDX models, both with prior chemotherapy and/or PARPi exposure, were refractory to all AMA, one of which harboured the SLC25A40-ABCB1 fusion, known to upregulate drug efflux via MDR1. Conclusion: The efficacy observed for eribulin in PRR HGSC PDX was similar to that observed for paclitaxel, which transformed ovarian cancer clinical practice. Eribulin is therefore worthy of further consideration in clinical trials, particularly in ovarian carcinoma with early failure of carboplatin/paclitaxel chemotherapy.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance.

BRCA1 splice isoforms Δ11 and Δ11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out the mutation-containing exon, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs), predicted in silico to drive exon skipping. Predictions were confirmed using qRT-PCR, RNA sequencing, western blots and BRCA1 minigene modelling. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. We demonstrate that SSMs drive BRCA1 exon 11 skipping and PARPi resistance, and should be clinically monitored, along with frame-restoring secondary mutations.

Targeting homologous recombination deficiency in uterine leiomyosarcoma.

BACKGROUND: Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of < 10%. Mutations in the homologous recombination (HR) DNA repair pathway have been observed in ~ 10% of uLMS cases, with reports of some individuals benefiting from poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy, which targets this DNA repair defect. In this report, we screened individuals with uLMS, accrued nationally, for mutations in the HR repair pathway and explored new approaches to therapeutic targeting. METHODS: A cohort of 58 individuals with uLMS were screened for HR Deficiency (HRD) using whole genome sequencing (WGS), whole exome sequencing (WES) or NGS panel testing. Individuals identified to have HRD uLMS were offered PARPi therapy and clinical outcome details collected. Patient-derived xenografts (PDX) were generated for therapeutic targeting. RESULTS: All 13 uLMS samples analysed by WGS had a dominant COSMIC mutational signature 3; 11 of these had high genome-wide loss of heterozygosity (LOH) (> 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs. CONCLUSIONS: Our work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.

Interrogating the Genomic Landscape of Uterine Leiomyosarcoma: A Potential for Patient Benefit.

Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy. Surgical removal and chemotherapy are commonly used to treat uLMS, but recurrence rates are high. Over the last few decades, clarification of the genomic landscape of uLMS has revealed a number of recurring mutations, including TP53, RB1, ATRX, PTEN, and MED12. Such genomic aberrations are difficult to target therapeutically or are actively targeted in other malignancies, and their potential as targets for the treatment of uLMS remains largely unexplored. Recent identification of deficiencies in homologous recombination in a minority of these tumours, however, has provided a rationale for investigation of PARP inhibitors in this sub-set. Here, we review these mutations and the evidence for therapeutic avenues that may be applied in uLMS. We also provide a comprehensive background on diagnosis and current therapeutic strategies as well as reviewing preclinical models of uLMS, which may be employed not only in testing emerging therapies but also in understanding this challenging and deadly disease.

Epithelial-to-Mesenchymal Transition Supports Ovarian Carcinosarcoma Tumorigenesis and Confers Sensitivity to Microtubule Targeting with Eribulin.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. SIGNIFICANCE: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Relationship between depression, prefrontal creatine and grey matter volume.

BACKGROUND: Depression and low mood are leading contributors to disability worldwide. Research indicates that clinical depression may be associated with low creatine concentrations in the brain and low prefrontal grey matter volume. Because subclinical depression also contributes to difficulties in day-to-day life, understanding the neural mechanisms of depressive symptoms in all individuals, even at a subclinical level, may aid public health. METHODS: Eighty-four young adult participants completed the Depression, Anxiety and Stress Scale (DASS) to quantify severity of depression, anxiety and stress, and underwent 1H-Magnetic Resonance Spectroscopy of the medial prefrontal cortex and structural magnetic resonance imaging (MRI) to determine whole-brain grey matter volume. RESULTS/OUTCOMES: DASS depression scores were negatively associated (a) with concentrations of creatine (but not other metabolites) in the prefrontal cortex and (b) with grey matter volume in the right superior medial frontal gyrus. Medial prefrontal creatine concentrations and right superior medial frontal grey matter volume were positively correlated. DASS anxiety and DASS stress scores were not related to prefrontal metabolite concentrations or whole-brain grey matter volume. CONCLUSIONS/INTERPRETATIONS: This study provides preliminary evidence from a representative group of individuals who exhibit a range of depression levels that prefrontal creatine and grey matter volume are negatively associated with depression. While future research is needed to fully understand this relationship, these results provide support for previous findings, which indicate that increasing creatine concentrations in the prefrontal cortex may improve mood and well-being.