A universal molecular control for DNA, mRNA and protein expression.

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

MTOR signaling orchestrates stress-induced mutagenesis, facilitating adaptive evolution in cancer.

In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.

Development and validation of a targeted gene sequencing panel for application to disparate cancers.

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour's molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy.

A quantitative model to predict pathogenicity of missense variants in the TP53 gene.

Germline pathogenic variants in the TP53 gene cause Li-Fraumeni syndrome, a condition that predisposes individuals to a wide range of cancer types. Identification of individuals carrying a TP53 pathogenic variant is linked to clinical management decisions, such as the avoidance of radiotherapy and use of high-intensity screening programs. The aim of this study was to develop an evidence-based quantitative model that integrates independent in silico data (Align-GVGD and BayesDel) and somatic to germline ratio (SGR), to assign pathogenicity to every possible missense variant in the TP53 gene. To do this, a likelihood ratio for pathogenicity (LR) was derived from each component calibrated using reference sets of assumed pathogenic and benign missense variants. A posterior probability of pathogenicity was generated by combining LRs, and algorithm outputs were validated using different approaches. A total of 730 TP53 missense variants could be assigned to a clinically interpretable class. The outputs of the model correlated well with existing clinical information, functional data, and ClinVar classifications. In conclusion, these quantitative outputs provide the basis for individualized assessment of cancer risk useful for clinical interpretation. In addition, we propose the value of the novel SGR approach for use within the ACMG/AMP guidelines for variant classification.

Monogenic and polygenic determinants of sarcoma risk: an international genetic study.

BACKGROUND: Sarcomas are rare, phenotypically heterogeneous cancers that disproportionately affect the young. Outside rare syndromes, the nature, extent, and clinical significance of their genetic origins are not known. We aimed to investigate the genetic basis for bone and soft-tissue sarcoma seen in routine clinical practice. METHODS: In this genetic study, we included 1162 patients with sarcoma from four cohorts (the International Sarcoma Kindred Study [ISKS], 966 probands; Project GENESIS, 48 probands; Asan Bio-Resource Center, 138 probands; and kConFab, ten probands), who were older than 15 years at the time of consent and had a histologically confirmed diagnosis of sarcoma, recruited from specialist sarcoma clinics without regard to family history. Detailed clinical, pathological, and pedigree information was collected, and cancer diagnoses in probands and relatives were independently verified. Targeted exon sequencing using blood (n=1114) or saliva (n=48) samples was done on 72 genes (selected due to associations with increased cancer risk) and rare variants were stratified into classes approximating the International Agency for Research on Cancer (IARC) clinical classification for genetic variation. We did a case-control rare variant burden analysis using 6545 Caucasian controls included from three cohorts (ISKS, 235 controls; LifePool, 2010 controls; and National Heart, Lung, and Blood Institute Exome Sequencing Project [ESP], 4300 controls). FINDINGS: The median age at cancer diagnosis in 1162 sarcoma probands was 46 years (IQR 29-58), 170 (15%) of 1162 probands had multiple primary cancers, and 155 (17%) of 911 families with informative pedigrees fitted recognisable cancer syndromes. Using a case-control rare variant burden analysis, 638 (55%) of 1162 sarcoma probands bore an excess of pathogenic germline variants (combined odds ratio [OR] 1·43, 95% CI 1·24-1·64, p<0·0001), with 227 known or expected pathogenic variants occurring in 217 individuals. All classes of pathogenic variants (known, expected, or predicted) were associated with earlier age of cancer onset. In addition to TP53, ATM, ATR, and BRCA2, an unexpected excess of functionally pathogenic variants was seen in ERCC2. Probands were more likely than controls to have multiple pathogenic variants compared with the combined control cohort group and the LifePool control cohort (OR 2·22, 95% CI 1·57-3·14, p=1·2 × 10(-6)) and the cumulative burden of multiple variants correlated with earlier age at cancer diagnosis (Mantel-Cox log-rank test for trend, p=0·0032). 66 of 1162 probands carried notifiable variants following expert clinical review (those recognised to be clinically significant to health and about which patients should be advised), whereas 293 (25%) probands carried variants with potential therapeutic significance. INTERPRETATION: About half of patients with sarcoma have putatively pathogenic monogenic and polygenic variation in known and novel cancer genes, with implications for risk management and treatment. FUNDING: Rainbows for Kate Foundation, Johanna Sewell Research Foundation, Australian National Health and Medical Research Council, Cancer Australia, Sarcoma UK, National Cancer Institute, Liddy Shriver Sarcoma Initiative.

Stress-induced cellular adaptive strategies: ancient evolutionarily conserved programs as new anticancer therapeutic targets.

Despite the remarkable achievements of novel targeted anti-cancer drugs, most therapies only produce remission for a limited time, resistance to treatment, and relapse, often being the ultimate outcome. Drug resistance is due to highly efficient adaptive strategies utilized by cancer cells. Exogenous and endogenous stress stimuli are known to induce first-line responses, capable of re-establishing cellular homeostasis and determining cell fate decisions. Cancer cells may also mount second-line adaptive strategies, such as the mutator response. Hypermutable subpopulations of cells may expand under severe selective stress, thereby accelerating the emergence of adapted clones. As with first-line protective responses, these strategies appear highly conserved, and are found in yeasts and bacteria. We hypothesize that evolutionarily conserved programs rheostatically regulate mutability in fluctuating environments, and contribute to drug resistance in cancer cells. Elucidating the conserved genetic and molecular mechanisms may present novel opportunities to increase the effectiveness of cancer therapies.

High frequency of germline TP53 mutations in a prospective adult-onset sarcoma cohort.

Sarcomas are a key feature of Li-Fraumeni and related syndromes (LFS/LFL), associated with germline TP53 mutations. Current penetrance estimates for TP53 mutations are subject to significant ascertainment bias. The International Sarcoma Kindred Study is a clinic-based, prospective cohort of adult-onset sarcoma cases, without regard to family history. The entire cohort was screened for mutations in TP53 using high-resolution melting analysis and Sanger sequencing, and multiplex-ligation-dependent probe amplification and targeted massively parallel sequencing for copy number changes. Pathogenic TP53 mutations were detected in blood DNA of 20/559 sarcoma probands (3.6%); 17 were germline and 3 appeared to be somatically acquired. Of the germline carriers, one appeared to be mosaic, detectable in the tumor and blood, but not epithelial tissues. Germline mutation carriers were more likely to have multiple cancers (47% vs 15% for non-carriers, P = 3.0×10(-3)), and earlier cancer onset (33 vs 48 years, P = 1.19×10(-3)). The median survival of mutation carriers following first cancer diagnosis was not significantly different from non-carriers. Only 10/17 (59%) pedigrees met classical or Chompret criteria for LFS. In summary, germline TP53 mutations are not rare in adult patients with sarcoma, with implications for screening, surveillance, treatment and genetic counselling of carriers and family members.

Neogenesis of lymphoid structures and antibody responses occur in human melanoma metastases.

Lymphoid neogenesis, or the development of lymphoid structures in nonlymphoid organs, is frequently observed in chronically inflamed tissues, during the course of autoimmune, infectious, and chronic graft rejection diseases, in which a sustained lymphocyte activation occurs in the presence of persistent antigenic stimuli. The presence of such ectopic lymphoid structures has also been reported in primary lung, breast, and germline cancers, but not yet in melanoma. In this study, we observed ectopic lymphoid structures, defined as lymphoid follicles comprising clusters of B lymphocytes and follicular dendritic cells (DC), associated with high endothelial venules (HEV) and clusters of T cells and mature DCs, in 7 of 29 cutaneous metastases from melanoma patients. Some follicles contained germinal centers. In contrast to metastatic lesions, primary melanomas did not host follicles, but many contained HEVs, suggesting an incomplete lymphoid neogenesis. Analysis of the repertoire of rearranged immunoglobulin genes in the B cells of microdissected follicles revealed clonal amplification, somatic mutation and isotype switching, indicating a local antigen-driven B-cell response. Surprisingly, IgA responses were observed despite the nonmucosal location of the follicles. Taken together, our findings show the existence of lymphoid neogenesis in melanoma and suggest that the presence of functional ectopic lymphoid structures in direct contact with the tumor makes the local development of antimelanoma B- and T-cell responses possible.

Tumor-infiltrating lymphocytes: apparently good for melanoma patients. But why?

Tumor-infiltrating T lymphocytes (TILs) are observed in a number of human primary or metastatic tumors. Recently, gene expression profiling experiments suggested that the presence of T cells in metastatic melanomas before vaccinating the patients with tumor antigens could be a biomarker for clinical benefit from the vaccines. In this context, we review results pertaining to TILs in human melanomas, their prognostic value, and some possible reasons why their presence could help in selecting melanoma patients for vaccination against tumor-specific antigens.

Peripheral blood lymphocytes genetically modified to express the self/tumor antigen MAGE-A3 induce antitumor immune responses in cancer patients.

Dendritic cell (DC) targeting in vivo has recently been shown to confer strong and protective cytotoxic T lymphocyte (CTL)-based immunity in tumor murine models. Our group has recently demonstrated in preclinical models that the infusion of genetically modified lymphocytes (GMLs) expressing the self/tumor antigen TRP-2 is able to elicit functional TRP-2-specific effectors with antitumor activity by targeting DCs in vivo. Here we have analyzed vaccine- and tumor-specific immune responses of 10 melanoma patients treated with autologous GMLs expressing the cancer germline gene MAGE-A3. Three of 10 patients treated with MAGE-A3-GML showed an increase of circulating anti-MAGE-A3 T cells, and developed skin delayed-type hypersensitivity to MAGE-A3. Interestingly, in 2 of these patients, with progressive and measurable tumors at study entry, anti-MAGE-A3 T cells were detected not only in the blood but also within tumors resected after vaccination. These results demonstrate that the infusion of MAGE-A3-GML elicits antitumor T cells, which are capable of trafficking to inflamed tissues and of infiltrating tumors. Clinical studies on a larger group of patients are needed to evaluate the clinical efficacy of such a strategy.

Lymphocytes genetically modified to express tumor antigens target DCs in vivo and induce antitumor immunity.

The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2-transduced (TRP-2-transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c(+)CD8alpha(+) DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ-resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.

Direct effects of polymyxin B on human dendritic cells maturation. The role of IkappaB-alpha/NF-kappaB and ERK1/2 pathways and adhesion.

Polymyxin B is a lipopolysaccharide binding antibiotic used to inactivate potential lipopolysaccharide contaminations when evaluating the activity of different agents on innate immune cells. We report that polymyxin B is able to induce directly in monocyte-derived human dendritic cells (DCs) several functional and molecular modifications characteristic of DCs undergoing a maturation process. DCs incubated with polymyxin B up-regulate the expression of HLA class I and II, the co-stimulatory CD86 molecule, and show an increase in the fraction of adherent cells at short time, which persist at 48 h of incubation. Adhesion to the plate was required for the polymyxin B-induced DCs maturation. A transient activation of IkappaB-alpha/NF-kappaB and ERK1/2 pathways at short time and a further ERK1/2 activation at long term were also detected. Neither up-regulation of the maturation marker CD83 nor activation of p38 nor induction of cytokines secretion was observed in DCs treated with polymyxin B. We demonstrated that inhibition of IkappaB-alpha/NF-kappaB pathway abolishes polymyxin B effects. ERK1/2 inhibition instead allowed DCs treated with polymyxin B to progress in their maturation process as revealed by the increased up-regulation of the CD83 co-stimulatory molecules, the activation of p38, and the reduced adhesion to culture plates at 48 h of incubation. Our results indicate that polymyxin B induces a partial maturation of human DCs through increased adhesion to a substrate and activation of the IkappaB-alpha/NF-kappaB pathway. The increased ERK1/2 activation observed, even though correlating with the initial phases of the maturation process, actually inhibits the occurrence of full maturation.