Clonal dynamics limits detection of selection in tumour xenograft CRISPR/Cas9 screens.

Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are edited in vitro and inoculated into mice to form tumours, allow evaluation of gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. To improve our understanding of the key parameters for success with this method, we investigated the choice of cell line, mouse host, tumour harvesting timepoint and guide RNA (gRNA) library size. We found that high gRNA (80-95%) representation was maintained in a HCT116 subline transduced with the GeCKOv2 whole-genome gRNA library and transplanted into NSG mice when tumours were harvested at early (14 d) but not late time points (38-43 d). The decreased representation in older tumours was accompanied by large increases in variance in gRNA read counts, with notable expansion of a small number of random clones in each sample. The variable clonal dynamics resulted in a high level of 'noise' that limited the detection of gRNA-based selection. Using simulated datasets derived from our experimental data, we show that considerable reductions in count variance would be achieved with smaller library sizes. Based on our findings, we suggest a pathway to rationally design adequately powered in vivo CRISPR screens for successful evaluation of gene function.

Dynamic ctDNA Mutational Complexity in Patients with Melanoma Receiving Immunotherapy.

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

Complex Patterns of Genomic Heterogeneity Identified in 42 Tumor Samples and ctDNA of a Pulmonary Atypical Carcinoid Patient.

Tumor evolution underlies many challenges facing precision oncology, and improving our understanding has the potential to improve clinical care. This study represents a rare opportunity to study tumor heterogeneity and evolution in a patient with an understudied cancer type. A patient with pulmonary atypical carcinoid, a neuroendocrine tumor, metastatic to 90 sites, requested and consented to donate tissues for research. 42 tumor samples collected at rapid autopsy from 14 anatomically distinct sites were analyzed through DNA whole-exome sequencing and RNA sequencing, and five analyzed through linked-read sequencing. Targeted DNA sequencing was completed on two clinical tissue biopsies and one blood plasma sample. Chromosomal alterations and gene variants accumulated over time, and specific chromosomal alterations preceded the single predicted gene driver variant (ARID1A). At the time of autopsy, all sites shared the gain of one copy of Chr 5, loss of one copy of Chr 6 and 21, chromothripsis of one copy of Chr 11, and 39 small variants. Two tumor clones (carrying additional variants) were detected at metastatic sites, and occasionally in different regions of the same organ (e.g., within the pancreas). Circulating tumor DNA (ctDNA) sequencing detected shared tumor variants in the blood plasma and captured marked genomic heterogeneity, including all metastatic clones but few private tumor variants. This study describes genomic tumor evolution and dissemination of a pulmonary atypical carcinoid donated by a single generous patient. It highlights the critical role of chromosomal alterations in tumor initiation and explores the potential of ctDNA analysis to represent genomically heterogeneous disease. Significance: DNA sequencing data from tumor samples and blood plasma from a single patient highlighted the critical early role of chromosomal alterations in atypical carcinoid tumor development. Common tumor variants were readily detected in the blood plasma, unlike emerging tumor variants, which has implications for using ctDNA to capture cancer evolution.

Chromosomal Aberrations Accumulate during Metastasis of Virus-Negative Merkel Cell Carcinoma.

Merkel cell carcinoma is a rare, aggressive skin tumor initiated by polyomavirus integration or UV light DNA damage. In New Zealand, there is a propensity toward the UV-driven form (31 of 107, 29% virus positive). Using archival formalin-fixed, paraffin-embedded tissues, we report targeted DNA sequencing covering 246 cancer genes on 71 tumor tissues and 38 nonmalignant tissues from 37 individuals, with 33 of 37 being negative for the virus. Somatic variants of New Zealand virus-negative Merkel cell carcinomas partially overlapped with those reported overseas, including TP53 variants in all tumors and RB1, LRP1B, NOTCH1, and EPHA3/7 variants each found in over half of the cohort. Variants in genes not analyzed or reported in previous studies were also found. Cataloging variants in TP53 and RB1 from published datasets revealed a broad distribution across these genes. Chr 1p gain and Chr 3p loss were identified in around 50% of New Zealand virus-negative Merkel cell carcinomas, and RB1 loss of heterozygosity was found in 90% of cases. Copy number variants accumulate in most metastases. Virus-negative Merkel cell carcinomas have complex combinations of somatic DNA-sequence variants and copy number variants. They likely carry the small genomic changes permissive for metastasis from early tumor development; however, chromosomal alterations may contribute to driving metastatic progression.

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body's immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.

Tuberous sclerosis complex: a complex case.

Tuberous sclerosis complex (TSC) is an inheritable disorder characterized by the formation of benign yet disorganized tumors in multiple organ systems. Germline mutations in the TSC1 (hamartin) or more frequently TSC2 (tuberin) genes are causative for TSC. The malignant manifestations of TSC, pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipoma (AML), may also occur as independent sporadic perivascular epithelial cell tumor (PEComa) characterized by somatic TSC2 mutations. Thus, discerning TSC from the copresentation of sporadic LAM and sporadic AML may be obscured in TSC patients lacking additional features. In this report, we present a case study on a single patient initially reported to have sporadic LAM and a mucinous duodenal adenocarcinoma deficient in DNA mismatch repair proteins. Moreover, the patient had a history of Wilms' tumor, which was reclassified as AML following the LAM diagnosis. Therefore, we investigated the origins and relatedness of these tumors. Using germline whole-genome sequencing, we identified a premature truncation in one of the patient's TSC2 alleles. Using immunohistochemistry, loss of tuberin expression was observed in AML and LAM tissue. However, no evidence of a somatic loss of heterozygosity or DNA methylation epimutations was observed at the TSC2 locus, suggesting alternate mechanisms may contribute to loss of the tumor suppressor protein. In the mucinous duodenal adenocarcinoma, no causative mutations were found in the DNA mismatch repair genes MLH1, MSH2, MSH6, or PMS2 Rather, clonal deconvolution analyses were used to identify mutations contributing to pathogenesis. This report highlights both the utility of using multiple sequencing techniques and the complexity of interpreting the data in a clinical context.

Clinically Relevant Biology of Hyaluronic Acid in the Desmoplastic Stroma of Pancreatic Ductal Adenocarcinoma.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor outcome. The presence of a dense desmoplastic stroma is a hallmark of this malignancy, and abundant hyaluronic acid (HA) within this stroma is a common feature of PDAC. At the end of 2019, an HA-targeting drug, after initial promise, failed phase 3 clinical trials in PDAC. This failure in the face of such strong evidence for biological importance forces us to turn back to the research and seek a better understanding of HA biology in PDAC. Therefore, in this review, we reexamine what is known about HA biology, the methods used to detect and quantify HA, and the ability of the biological models in which HA has been investigated to recapitulate an HA-rich desmoplastic tumor stroma. The role of HA in PDAC relies on its complex interplay with a range of HA-associated molecules, which have not been as extensively investigated as HA itself. Therefore, using large genomic data sets, we cataloged the abundance and activity in PDAC of molecules that modulate HA synthesis, degradation, protein interactions, and receptor binding. Based on their association with clinical characteristics and individual patient outcomes, we suggest a small number of HA-associated molecules that warrant further investigation as biomarkers and drug targets.

Breast Cancer Patient Prognosis Is Determined by the Interplay between TP53 Mutation and Alternative Transcript Expression: Insights from TP53 Long Amplicon Digital PCR Assays.

The TP53 gene locus is capable of producing multiple RNA transcripts encoding the different p53 protein isoforms. We recently described multiplex long amplicon droplet digital PCR (ddPCR) assays to quantify seven of eight TP53 reference transcripts in human tumors. Here, we describe a new long amplicon ddPCR assay to quantify expression of the eighth TP53 reference transcript encoding ∆40p53α. We then applied these assays, alongside DNA sequencing of the TP53 gene locus, to tumors from a cohort of New Zealand (NZ) breast cancer patients. We found a high prevalence of mutations at TP53 splice sites in the NZ breast cancer cohort. Mutations at TP53 intron 4 splice sites were associated with overexpression of ∆133TP53 transcripts. Cox proportional hazards survival analysis showed that interplay between TP53 mutation status and expression of TP53 transcript variants was significantly associated with patient outcome, over and above standard clinical and pathological information. In particular, patients with no TP53 mutation and a low ratio of TP53 transcripts t2 to t1, which derive from alternative intron 1 acceptor splice sites, had a remarkably good outcome. We suggest that this type of analysis, integrating mutation and transcript expression, provides a step-change in our understanding of TP53 in cancer.

Genomic and signalling pathway characterization of the NZM panel of melanoma cell lines: A valuable model for studying the impact of genetic diversity in melanoma.

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole-exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer-testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell-based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.

Specialized Cell-Free DNA Blood Collection Tubes Can Be Repurposed for Extracellular Vesicle Isolation: A Pilot Study.

Background: Liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. However, these are highly affected by preprocessing variables with many protocols designed for downstream analysis of a single molecular biomarker. Here we investigate whether specialized blood tubes could be repurposed for the analysis of an increasingly valuable biomarker, extracellular vesicles (EVs). Methods: Blood was collected from three donors into K3-EDTA, Roche, or Streck cell-free DNA (cfDNA) collection tubes and processed using sequential centrifugation either immediately or after storage for 3 days. MicroEV were collected from platelet-poor plasma by 10,000 g centrifugation and NanoEVs isolated using size exclusion chromatography. Particle size and counts were assessed by Nanoparticle Tracking Analysis, protein quantitation by bicinchoninic acid assay (BCA) assay, and dot blotting for blood cell surface proteins. Results: MicroEVs and NanoEVs could be isolated from plasma collected using all three tube types. Major variations were seen with delayed time to processing. Both MicroEV particle number and protein content increased with the processing delay. The NanoEV number did not change with the time-delay but their protein quantity increased. EV-associated proteins predominantly arose from platelets (CD61) and erythrocytes (CD235a). However, leukocyte marker CD45 was only increased in NanoEVs from ethylenediaminetetraacetic acid (EDTA) tubes, suggestive of stabilization of nucleated cells by the specialized blood tubes. Epithelial cell surface marker EpCAM, often used as a marker of cancer, remained the same across conditions in both MicroEV and NanoEV preparations indicating that these EVs were stable with time. Conclusions: Specialized cfDNA collection tubes can be repurposed for MicroEV and NanoEV analysis; however, simple counting or using protein quantity as a surrogate of EV number may be confounded by preanalytical processing. The EVs would be suitable for disease selective EV subtype analysis if the molecular target of interest is not present in blood cells.

Accessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual TP53 Transcripts.

TP53, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53's alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5' and 3' ends, but because they have a common central region of 618 bp, the individual TP53 transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major TP53 transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms.

Transcriptomic Features of T Cell-Barren Tumors Are Conserved Across Diverse Tumor Types.

Background: Understanding how tumors subvert immune destruction is essential to the development of cancer immunotherapies. New evidence suggests that tumors limit anti-tumor immunity by exploiting transcriptional programs that regulate intratumoral trafficking and accumulation of effector cells. Here, we investigated the gene expression profiles that distinguish immunologically "cold" and "hot" tumors across diverse tumor types. Methods: RNAseq profiles of tumors (n = 8,920) representing 23 solid tumor types were analyzed using immune gene signatures that quantify CD8+ T cell abundance. Genes and pathways associated with a low CD8+ T cell infiltration profile (CD8-Low) were identified by correlation, differential expression, and statistical ranking methods. Gene subsets were evaluated in immunotherapy treatment cohorts and functionally characterized in cell lines and mouse tumor models. Results: Among different cancer types, we observed highly significant overlap of genes enriched in CD8-Low tumors, which included known immunomodulatory genes (e.g., BMP7, CMTM4, KDM5B, RCOR2) and exhibited significant associations with Wnt signaling, neurogenesis, cell-cell junctions, lipid biosynthesis, epidermal development, and cancer-testis antigens. Analysis of mutually exclusive gene clusters demonstrated that different transcriptional programs may converge on the T cell-cold phenotype as well as predict for response and survival of patients to Nivo treatment. Furthermore, we confirmed that a top-ranking candidate belonging to the TGF-ß superfamily, BMP7, negatively regulates CD8+ T cell abundance in immunocompetent murine tumor models, with and without anti-PD-L1 treatment. Conclusions: This study presents the first evidence that solid tumors of diverse anatomical origin acquire conserved transcriptional alterations that may be operative in the T cell-cold state. Our findings demonstrate the potential clinical utility of CD8-Low tumor-associated genes for predicting patient immunotherapy outcomes and point to novel mechanisms with potential for broad therapeutic exploitation.

A Predictor of Early Disease Recurrence in Patients With Breast Cancer Using a Cell-free RNA and Protein Liquid Biopsy.

INTRODUCTION: Circulating biomarkers have been increasingly used in the clinical management of breast cancer. The present study evaluated whether RNAs and a protein present in the plasma of patients with breast cancer might have utility as prognostic biomarkers complementary to existing clinical tests. PATIENTS AND METHODS: We performed microarray profiling of small noncoding RNAs in plasma samples from 30 patients with breast cancer and 10 control individuals. Two small noncoding RNAs, including microRNA (miR)-923, were selected and quantified in plasma samples from an evaluation cohort of 253 patients with breast cancer, using droplet digital polymerase chain reaction. We also measured cancer antigen (CA) 15-3 protein levels in these samples. Cox regression survival analysis was used to determine which markers were associated with patient prognosis. RESULTS: As independent markers of prognosis, the plasma levels of miR-923 and CA 15-3 at the time of surgery for breast cancer were significantly associated with prognosis, irrespective of treatment (Cox proportional hazards, P = 3.9 × 10-3 and 1.9 × 10-9, respectively). After building a multivariable model with standard clinical and pathological features, the addition of miR-923 and CA 15-3 information into the model resulted in a significantly better predictor of disease recurrence in patients, irrespective of treatment, compared with the use of clinicopathological data alone (area under the curve at 3 years, 0.858 vs. 0.770 with clinicopathological markers only; P = .017). CONCLUSION: We propose that the plasma levels of miR-923 and CA 15-3, combined with standard clinicopathological predictors, could be used as a preoperative, noninvasive estimate of patient prognosis to identify which women might need more aggressive treatment or closer surveillance after surgery for breast cancer.

Systems immunology reveals a linked IgG3-C4 response in patients with acute rheumatic fever.

Acute rheumatic fever (ARF) and chronic rheumatic heart disease (RHD) are autoimmune sequelae of a Group A streptococcal infection with significant global mortality and poorly understood pathogenesis. Immunoglobulin and complement deposition were observed in ARF/RHD valve tissue over 50 years ago, yet contemporary investigations have been lacking. This study applied systems immunology to investigate the relationships between the complement system and immunoglobulin in ARF. Patients were stratified by C-reactive protein (CRP) concentration into high (≥10 µg mL-1 ) and low (<10 µg mL-1 ) groups to distinguish those with clinically significant inflammatory processes from those with abating inflammation. The circulating concentrations of 17 complement factors and six immunoglobulin isotypes and subclasses were measured in ARF patients and highly matched healthy controls using multiplex bead-based immunoassays. An integrative statistical approach combining feature selection and principal component analysis revealed a linked IgG3-C4 response in ARF patients with high CRP that was absent in controls. Strikingly, both IgG3 and C4 were elevated above clinical reference ranges, suggesting these features are a marker of ARF-associated inflammation. Humoral immunity in response to M protein, an antigen implicated in ARF pathogenesis, was completely polarized to IgG3 in the patient group. Furthermore, the anti-M-protein IgG3 response was correlated with circulating IgG3 concentration, highlighting a potential role for this potent immunoglobulin subclass in disease. In conclusion, a linked IgG3-C4 response appears important in the initial, inflammatory stage of ARF and may have immediate utility as a clinical biomarker given the lack of specific diagnostic tests currently available.

Orthogonal assays for the identification of inhibitors of the single-stranded nucleic acid binding protein YB-1.

We have previously shown that high expression of the nucleic acid binding factor YB-1 is strongly associated with poor prognosis in a variety of cancer types. The 3-dimensional protein structure of YB-1 has yet to be determined and its role in transcriptional regulation remains elusive. Drug targeting of transcription factors is often thought to be difficult and there are very few published high-throughput screening approaches. YB-1 predominantly binds to single-stranded nucleic acids, adding further difficulty to drug discovery. Therefore, we have developed two novel screening assays to detect compounds that interfere with the transcriptional activation properties of YB-1, both of which may be generalizable to screen for inhibitors of other nucleic acid binding molecules. The first approach is a cell-based luciferase reporter gene assay that measures the level of activation of a fragment of the E2F1 promoter by YB-1. The second approach is a novel application of the AlphaScreen system, to detect interference of YB-1 interaction with a single-stranded DNA binding site. These complementary assays examine YB-1 binding to two discrete nucleic acid sequences using two different luminescent signal outputs and were employed sequentially to screen 7360 small molecule compounds leading to the identification of three putative YB-1 inhibitors.

Mapping a route to Indigenous engagement in cancer genomic research.

Precision oncology guided by genomic research has an increasingly important role in the care of people with cancer. However, substantial inequities remain in cancer outcomes of Indigenous peoples, including Indigenous Maori in Aotearoa New Zealand (New Zealand). These inequities will be perpetuated unless deliberate steps are taken to include Indigenous peoples in all parts of cancer research-as research participants, in research leadership, and in research governance. This approach is especially important when there have been historical breaches of trust that have discouraged their participation in health research. This Personal View describes a precision oncology research roadmap for neuroendocrine tumour research, which seeks to reflect the values of New Zealand's Indigenous Maori people. This roadmap includes facilitating ongoing dialogue, Maori leadership, reciprocity, agreed kawa (guiding principles), tikanga (cultural protocols), and honest monitoring of what is and what is not being achieved. We challenge cancer researchers worldwide to generate locally appropriate roadmaps that honestly assess their practices to benefit Indigenous people internationally.

Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.

Plasma miRNAs Display Limited Potential as Diagnostic Tools for Endometriosis.

CONTEXT: Despite extensive searches for novel noninvasive diagnostics, laparoscopy remains the reference test for endometriosis. Circulating miRNAs are purported endometriosis biomarkers; however, the miRNA species and their diagnostic accuracy differ between studies and have not been validated in independent cohorts. OBJECTIVE: Identify endometriosis-specific plasma miRNAs and determine their diagnostic test accuracy. SETTING: Two university-based, public hospitals and a private gynecology practice in Australia. DESIGN AND PARTICIPANTS: Four phases: (i) Explorative phase. Plasma miRNA menstrual cycle fluctuations were evaluated in women with endometriosis and asymptomatic controls (n = 16). (ii) Biomarker discovery. Endometriosis-specific plasma miRNAs were identified in (a) women with endometriosis and asymptomatic controls (n = 16) and (b) women with and without surgically defined endometriosis (n = 20). (iii) Biomarker selection. Plasma miRNAs with the best diagnostic potential for endometriosis were selected in a surgically defined selection cohort (n = 78). (iv) Biomarker validation. The diagnostic test accuracy of these miRNAs was calculated in an independent, surgically defined validation cohort (n = 119). RESULTS: Forty-nine miRNAs were differentially expressed in women with endometriosis. Nine maintained dysregulation in the selection cohort, but only three (miR-155, miR574-3p and miR139-3p) did so in the validation cohort. Combined, these three miRNAs demonstrated a sensitivity and specificity of 83% and 51%, respectively. CONCLUSION: Plasma miRNAs demonstrated modest sensitivity and specificity as diagnostic tests or triage tools for endometriosis. Other groups' findings were not replicated and accorded poorly with our results. Circulating miRNAs demonstrate diagnostic potential, but stringent, standardized methodological approaches are required for the development of a clinically applicable tool.

Imprinted and ancient gene: a potential mediator of cancer cell survival during tryptophan deprivation.

BACKGROUND: Depletion of tryptophan and the accumulation of tryptophan metabolites mediated by the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1), trigger immune cells to undergo apoptosis. However, cancer cells in the same microenvironment appear not to be affected. Mechanisms whereby cancer cells resist accelerated tryptophan degradation are not completely understood. We hypothesize that cancer cells co-opt IMPACT (the product of IMPrinted and AnCienT gene), to withstand periods of tryptophan deficiency. METHODS: A range of bioinformatic techniques including correlation and gene set variation analyses was applied to genomic datasets of cancer (The Cancer Genome Atlas) and normal (Genotype Tissue Expression Project) tissues to investigate IMPACT's role in cancer. Survival of IMPACT-overexpressing GL261 glioma cells and their wild type counterparts cultured in low tryptophan media was assessed using fluorescence microscopy and MTT bio-reduction assay. Expression of the Integrated Stress Response proteins was measured using Western blotting. RESULTS: We found IMPACT to be upregulated and frequently amplified in a broad range of clinical cancers relative to their non-malignant tissue counterparts. In a subset of clinical cancers, high IMPACT expression associated with decreased activity of pathways and genes involved in stress response and with increased activity of translational regulation such as the mTOR pathway. Experimental studies using the GL261 glioma line showed that cells engineered to overexpress IMPACT, gained a survival advantage over wild-type lines when cultured under limiting tryptophan concentrations. No significant difference in the expression of proteins in the Integrated Stress Response pathway was detected in tryptophan-deprived GL261 IMPACT-overexpressors compared to that in wild-type cells. IMPACT-overexpressing GL261 cells but not their wild-type counterparts, showed marked enlargement of their nuclei and cytoplasmic area when stressed by tryptophan deprivation. CONCLUSIONS: The bioinformatics data together with our laboratory studies, support the hypothesis that IMPACT mediates a protective mechanism allowing cancer cells to overcome microenvironmental stresses such as tryptophan deficiency.