High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we ...sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a 'core' module of antiviral genes is expressed very early by a few 'precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced 'peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.
Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context ...to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS-FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS-FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.
Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and ...phenotypic output, with important functional consequences. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs or proteins simultaneously, because genomic profiling methods could not be applied to single cells until very recently. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.
How the immune system readies for battleAlthough gene expression is tightly controlled at both the RNA and protein levels, the quantitative contribution of each step, especially during dynamic ...responses, remains largely unknown. Indeed, there has been much debate whether changes in RNA level contribute substantially to protein-level regulation. Jovanovic et al. built a genome-scale model of the temporal dynamics of differential protein expression during the stimulation of immunological dendritic cells (see the Perspective by Li and Biggin). Newly stimulated functions involved the up-regulation of specific RNAs and concomitant increases in the levels of the proteins they encode, whereas housekeeping functions were regulated posttranscriptionally at the protein level.Science, this issue 10.1126/science.1259038; see also p. 1066 Protein expression is regulated by the production and degradation of messenger RNAs (mRNAs) and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics so as to build a quantitative genomic model of the differential regulation of gene expression in lipopolysaccharide-stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for more than half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction for newly activated cellular functions and by protein life-cycle changes for remodeling of preexisting functions.
In response to the COVID-19 pandemic, there has been a rapid growth in the use of telehealth/telemedicine that will likely be sustained in the postpandemic setting. Mobile health applications (apps) ...can be used as part of the telehealth encounter to monitor patient-reported outcomes (PROs) and enhance patient-provider communication.
A systematic review was performed of mobile health apps with symptom trackers. We searched the iOS App Store and Android Google Play using the words cancer, oncology, and symptom tracker. Apps were included if they incorporated a symptom tracking function that could allow patients with cancer to record symptoms and PROs. Apps were evaluated using the mobile apps rating scale, which includes engagement, functionality, aesthetics, information, and app subjective quality.
The initial search yielded 1189 apps, with 101 apps eligible after title and description screening. A total of 41 apps met eligibility criteria and were included in this study. The majority of apps (73%, n = 30) were general health/pain symptom trackers, and 27% (n = 11) were cancer-specific. The app quality mean scores assessed using the mobile apps rating scale ranged from 2.43 to 4.23 (out of 5.00). Only 1 app has been trialed for usability among patients with cancer.
Although various symptom tracking apps are available, cancer-specific apps remain limited. Future collaboration between oncologists, app developers, and patients to optimize PRO assessment and integration with telehealth/telemedicine encounters to increase symptom recognition and enhance patient-provider communication is urgently needed.
The emergence of human papillomavirus (HPV)-positive oropharyngeal cancer and evolving tobacco use patterns have changed the landscape of head and neck cancer epidemiology internationally. We ...investigated updated trends in oropharyngeal cancer incidence worldwide.
We analyzed cancer incidence data between 1993 and 2012 from 42 countries using the Cancer Incidence in Five Continents database volumes V through XI. Trends in oropharyngeal cancer incidence were compared with oral cavity cancers and lung squamous cell carcinomas using log-linear regression and age period-cohort modeling.
In total, 156 567 oropharyngeal cancer, 146 693 oral cavity cancer, and 621 947 lung squamous cell carcinoma patients were included. Oropharyngeal cancer incidence increased (P < .05) in 19 and 23 countries in men and women, respectively. In countries with increasing male oropharyngeal cancer incidence, all but 1 had statistically significant decreases in lung squamous cell carcinoma incidence, and all but 2 had decreasing or nonsignificant net drifts for oral cavity cancer. Increased oropharyngeal cancer incidence was observed both in middle-aged (40-59 years) and older (≥60 years) male cohorts, with strong nonlinear birth cohort effects. In 20 countries where oropharyngeal cancer incidence increased for women and age period-cohort analysis was possible, 13 had negative or nonsignificant lung squamous cell carcinoma net drifts, including 4 countries with higher oropharyngeal cancer net drifts vs both lung squamous cell carcinoma and oral cavity cancer (P < .05 for all comparisons).
Increasing oropharyngeal cancer incidence is seen among an expanding array of countries worldwide. In men, increased oropharyngeal cancer is extending to older age groups, likely driven by human papillomavirus-related birth cohort effects. In women, more diverse patterns were observed, suggesting a complex interplay of risks factors varying by country, including several countries where female oropharyngeal cancer increases may be driven by HPV.
Lymph node (LN) involvement is an important factor in guiding adjuvant treatment for patients with endometrial cancer. Risk factors for LN involvement are fairly well-established for endometrial ...adenocarcinoma, but it is not as well defined whether these factors similarly predict LN positivity in less common histologies.
Patients diagnosed with pathologic T1-T2 carcinosarcoma, clear cell, uterine papillary serous carcinoma (UPSC), and mixed histologic type endometrial cancer between 2004 and 2016 undergoing primary surgery with at least 1 lymph node sampled in the National Cancer Data Base were identified. Logistic regression was performed to identify primary pathologic tumor predictors of LN positivity. Nomograms were created to predict overall, pelvic only, and paraaortic with or without pelvic LN involvement.
Among 11,390 patients included, 1950 (18%) were node positive. On multivariable analysis, increasing pathologic tumor stage (pT2 versus pT1a, odds ratio OR 3.63, 95% confidence interval CI 3.15–4.18, p < 0.001), increase in tumor size per centimeter (OR 1.08, 95% CI 1.06–1.10, p < 0.001), and the presence of lymphovascular invasion (LVI) (OR 4.97, 95% CI 4.43–5.57, p < 0.001) were predictive of overall LN positivity. Relative to carcinosarcoma, both clear cell (OR 1.54, 95% CI 1.22–1.95, p < 0.001) and UPSC (OR 1.73, 95% CI 1.48–2.02, p < 0.001) histology were significantly associated with a higher risk of LN positivity while mixed histology was not (OR 1.07, 95% CI 0.92–1.24, p = 0.42).
Among patients with non-endometrioid endometrial cancer, predictors of LN positivity are similar to endometrial adenocarcinoma. The nomograms provided could be helpful in making adjuvant treatment decisions for these less common histologies.
•Risk of positive lymph nodes (LN+) is variable between patients with different endometrial non-endometrioid histologies.•Lymphovascular invasion (LVI) predicts a 5-fold increased risk of (LN+) in endometrial non- endometrioid histologies.•Risk of regional LN+ correlated with pathologic tumor stage, specific non-endometrioid tumor histology, and tumor size.•A predictive nomogram was generated to estimate individualized patient risk of LN+ based upon pathologic factors.
Studies have observed that women have better outcomes than men in melanoma, but less is known about the influence of sex differences on outcomes for other aggressive cutaneous malignancies.
To ...investigate whether women and men have disparate outcomes in Merkel cell carcinoma (MCC).
Patients with nonmetastatic MCC undergoing surgery and lymph node evaluation were identified from the National Cancer Database (NCDB) and the Surveillance, Epidemiology, and End Results (SEER) database. Kaplan-Meier analysis and Cox proportional hazards regression models were used for overall survival, and competing-risks analysis and Fine-Gray models were used for cause-specific and other-cause mortality.
The NCDB cohort (n = 4178) included 1516 (36%) women. Women had a consistent survival advantage compared with men in propensity score–matched analysis (66.0% vs 56.8% at 5 years, P < .001) and multivariable Cox regression (hazard ratio, 0.68; 95% confidence interval, 0.61-0.75; P < .001). Similarly, women had a survival advantage in the SEER validation cohort (n = 1202) with 457 (38.0%) women, which was entirely due to differences in MCC-specific mortality (5-year cumulative incidence: 16.4% vs 26.7%, P = .002), with no difference in other-cause mortality (16.8% vs 17.8%, P = .43) observed in propensity score–matched patients.
Potential selection bias from a retrospective data set.
In MCC, women have improved survival compared with men, driven by MCC-related mortality.
For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. ...With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. This unit describes a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second "template-switch" primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.
Current lymph node (LN) staging for Merkel cell carcinoma (MCC) does not account for the number of metastatic LNs, which is a primary driver of survival in multiple cancers.
To determine the impact ...of the number of metastatic LNs on survival in MCC.
Patients with MCC undergoing surgery were identified from the National Cancer Database (NCDB). The association between metastatic LN number and survival was modeled with restricted cubic splines. A novel nodal classification system was derived by using recursive partitioning analysis. MCC patients undergoing surgery in the Surveillance, Epidemiology, and End Results (SEER) Program were used as validation cohort.
Among 3670 patients in the NCDB, increasing metastatic LN number was associated with decreased survival (P < .001). Mortality risk increased continuously with each additional positive LN when using multivariable, nonlinear modeling. According to a novel staging system derived via recursive partitioning analysis, the hazard ratio for death in multivariable regression compared with patients without LN involvement was 1.24 (P = .049), 2.08 (P < .001), 3.24 (P < .001), and 6.13 (P < .001) for the proposed N1a (1-3 metastatic LNs with microscopic detection), N1b (1-3 metastatic LNs with macroscopic detection), N2 (4-8 metastatic LNs), and N3 (≥9 metastatic LNs), respectively. This system was validated in the SEER cohort and showed improved concordance compared with the American Joint Committee on Cancer, Eighth Edition.
Retrospective design.
Number of metastatic LNs is the dominant nodal factor driving survival in patients with MCC.
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