Asymmetry in the peak in Covid-19 daily cases and the pandemic R-parameter.

Within the context of the standard SIR model of pandemics, we show that the asymmetry in the peak in recorded daily cases during a pandemic can be used to infer the pandemic R-parameter. Using only daily data for symptomatic, confirmed cases, we derive a universal scaling curve that yields: (i) reff, the pandemic R-parameter; (ii) Leff, the effective latency, the average number of days an infected individual is able to infect others and (iii) α, the probability of infection per contact between infected and susceptible individuals. We validate our method using an example and then apply it to estimate these parameters for the first phase of the SARS-Cov-2/Covid-19 pandemic for several countries where there was a well separated peak in identified infected daily cases. The extension of the SIR model developed in this paper differentiates itself from earlier studies in that it provides a simple method to make an a-posteriori estimate of several useful epidemiological parameters, using only data on confirmed, identified cases. Our results are general and can be applied to any pandemic.

Subtype and cell type specific expression of lncRNAs provide insight into breast cancer.

Long non-coding RNAs (lncRNAs) are involved in breast cancer pathogenesis through chromatin remodeling, transcriptional and post-transcriptional gene regulation. We report robust associations between lncRNA expression and breast cancer clinicopathological features in two population-based cohorts: SCAN-B and TCGA. Using co-expression analysis of lncRNAs with protein coding genes, we discovered three distinct clusters of lncRNAs. In silico cell type deconvolution coupled with single-cell RNA-seq analyses revealed that these three clusters were driven by cell type specific expression of lncRNAs. In one cluster lncRNAs were expressed by cancer cells and were mostly associated with the estrogen signaling pathways. In the two other clusters, lncRNAs were expressed either by immune cells or fibroblasts of the tumor microenvironment. To further investigate the cis-regulatory regions driving lncRNA expression in breast cancer, we identified subtype-specific transcription factor (TF) occupancy at lncRNA promoters. We also integrated lncRNA expression with DNA methylation data to identify long-range regulatory regions for lncRNA which were validated using ChiA-Pet-Pol2 loops. lncRNAs play an important role in shaping the gene regulatory landscape in breast cancer. We provide a detailed subtype and cell type-specific expression of lncRNA, which improves the understanding of underlying transcriptional regulation in breast cancer.

Using postal change-of-address data to predict second waves in infections near pandemic epicentres.

We propose that postal Change-of-Address (CoA) data can be used to monitor/predict likely second wave caseloads in viral infections around urban epicentres. To illustrate the idea, we focus on the tri-state area consisting of New York City (NYC) and surrounding counties in New York, New Jersey and Connecticut States. NYC was an early epicentre of the coronavirus disease 2019 (Covid-19) pandemic, with a first peak in daily cases in early April 2020, followed by the second peak in May/June 2020. Using CoA data from the US Postal Service (USPS), we show that, despite a quarantine mandate, there was a large net movement of households from NYC to surrounding counties in the period April-June 2020. This net outward migration of households was strongly correlated with both the timing and the number of cases in the second peaks in Covid-19 cases in the surrounding counties. The timing of the second peak was also correlated with the distance of the county from NYC, suggesting that this was a directed flow and not random diffusion. Our analysis shows that CoA data is a useful method in tracking the spread of an infectious pandemic agent from urban epicentres.

Gene Expression in Barrett's Esophagus Cell Lines Resemble Esophageal Squamous Cell Carcinoma Instead of Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) is strongly associated with Barrett's esophagus (BE), a pre-malignant condition resulting from gastric reflux. Esophageal squamous cell carcinoma (ESCC), the other major subtype of esophageal cancer, shows strong association with smoking and alcohol intake and no association with gastric reflux. In this study, we constructed and validated gene expression signatures of EAC vs. ESCC tumors using publicly available datasets, and subsequently assessed the enrichment levels of these signatures in commonly used EAC and ESCC cell lines, normal esophageal tissues and normal esophageal cell lines, and primary BE tissues and BE cell lines. We found that unlike ESCC cell lines which were quite similar to primary ESCC tumors, EAC cell lines were considerably different from primary EAC tumors but still more similar to EAC tumors than ESCC tumors, as the genes up in EAC vs. ESCC (EAChi) had considerably lower expression in EAC cell lines than EAC tumors. However, more surprisingly, unlike various normal cell lines (EPC2, Het-1A) which were very similar to various tissues from normal esophagus, BE cell lines (BAR-T, CP-A) were extremely different from primary BE tissues, as BE cell lines had substantially lower levels of EAChi and substantially higher levels of ESCChi gene expression. This ESCC-like profile of the BAR-T remained unaltered even after prolonged exposure to an acidic bile mixture in vitro resulting in malignant transformation of this cell line. However, primary BE tissues had EAC-like gene expression profiles as expected. Only one EAC case from the Cancer Genome Atlas resembled BE cell lines, and while it had the clinical profile and some mutational features of EAC, it had some mutational features, the copy number alteration profile, and the gene expression profile of ESCC instead. These incomprehensible changes in gene expression patterns may result in ambiguous changes in the phenotype and warrants careful evaluation to inform selection of appropriate in vitro tools for future studies on esophageal adenocarcinoma.

The transmission of SARS-CoV-2 is likely comodulated by temperature and by relative humidity.

Inferring the impact of climate upon the transmission of SARS-CoV-2 has been confounded by variability in testing, unknown disease introduction rates, and changing weather. Here we present a data model that accounts for dynamic testing rates and variations in disease introduction rates. We apply this model to data from Colombia, whose varied and seasonless climate, central port of entry, and swift, centralized response to the COVID-19 pandemic present an opportune environment for assessing the impact of climate factors on the spread of COVID-19. We observe strong attenuation of transmission in climates with sustained daily temperatures above 30 degrees Celsius and simultaneous mean relative humidity below 78%, with outbreaks occurring at high humidity even where the temperature is high. We hypothesize that temperature and relative humidity comodulate the infectivity of SARS-CoV-2 within respiratory droplets.

Migration of households from New York City and the Second Peak in Covid-19 cases in New Jersey, Connecticut and New York Counties.

The five boroughs of New York City (NYC) were early epicenters of the Covid-19 pandemic in the United States, with over 380,000 cases by May 31. High caseloads were also seen in nearby counties in New Jersey (NJ), Connecticut (CT) and New York (NY). The pandemic started in the area in March with an exponential rise in the number of daily cases, peaked in early April, briefly declined, and then, showed clear signs of a second peak in several counties. We will show that despite control measures such as lockdown and restriction of movement during the exponential rise in daily cases, there was a significant net migration of households from NYC boroughs to the neighboring counties in NJ, CT and NY State. We propose that the second peak in daily cases in these counties around NYC was due, in part, to the movement of people from NYC boroughs to these counties. We estimate the movement of people using "Change of Address" (CoA) data from the US Postal Service, provided under the "Freedom of Information Act" of 1967. To identify the timing of the second peak and the number of cases in it, we use a previously proposed SIR model, which accurately describes the early stages of the coronavirus pandemic in European countries. Subtracting the model fits from the data identified, we establish the timing and the number of cases, NCS, in the second peak. We then related the number of cases in the second peak to the county population density, P, and the excess Change of Address, ECoA, into each county using the simple model NCS~PαECoAß which fits the data very well with α = 0.68, ß = 0.31 (R2 = 0.74, p = 1.3e-8). We also find that the time between the first and second peaks was proportional to the distance of the county seat from NY Penn Station, suggesting that this migration of households and disease was a directed flow and not a diffusion process. Our analysis provides a simple method to use change of address data to track the spread of an infectious agent, such as SARS-Cov-2, due to migrations away from epicenters during the initial stages of a pandemic.

Analysis of Covid-19 Data for Eight European Countries and the United Kingdom Using a Simplified SIR Model.

Background: As the SARS-Cov-2/Covid-19 pandemic continues to ravage the world, it is important to understanding the characteristics of its spread and possible correlates for control to develop strategies of response. Methods: Here we show how a simple Susceptible-Infective-Recovered (SIR) model applied to data for eight European countries and the United Kingdom (UK) can be used to forecast the descending limb (post-peak) of confirmed cases and deaths as a function of time, and predict the duration of the pandemic once it has peaked, by estimating and fixing parameters using only characteristics of the ascending limb and the magnitude of the first peak. Results: The predicted and actual case fatality ratio, or number of deaths per million population from the start of the pandemic to when daily deaths number less than five for the first time, was lowest in Norway (predicted: 44 ± 5 deaths/million; actual: 36 deaths/million) and highest for the United Kingdom (predicted: 578 +/- 65 deaths/million; actual 621 deaths/million). The inferred pandemic characteristics separated into two distinct groups: those that are largely invariant across countries, and those that are highly variable. Among the former is the infective period, TL = 16.3 ± 2.7 days, the average time between contacts, TR = 3.8+/- 0.5 days and the average number of contacts while infective R = 4.4 +/- 0.5. In contrast, there is a highly variable time lag TD between the peak in the daily number of confirmed cases and the peak in the daily number of deaths, ranging from lows of TD = 2,4 days for Denmark and Italy respectively, to highs of TD = 12, 15 for Germany and Norway respectively. The mortality fraction, or ratio of deaths to confirmed cases, was also highly variable, ranging from low values 3%, 5% and 5% for Norway, Denmark and Germany respectively, to high values of 18%, 20% and 21% for Sweden, France, and the UK respectively. The probability of mortality rather than recovery was a significant correlate of the duration of the pandemic, defined as the time from 12/31/2019 to when the number of daily deaths fell below 5. Finally, we observed a small but detectable effect of average temperature on the probability α of infection per contact, with higher temperatures associated with lower infectivity. Conclusions: Our simple model captures the dynamics of the initial stages of the pandemic, from its exponential beginning to the first peak and beyond, with remarkable precision. As with all epidemiological analyses, unanticipated behavioral changes will result in deviations between projection and observation. This is abundantly clear for the current pandemic. Nonetheless, accurate short-term projections are possible, and the methodology we present is a useful addition to the epidemiologist's armamentarium. Our predictions assume that control measures such as lockdown, social distancing, use of masks etc. remain the same post-peak as before peak. Consequently, deviations from our predictions are a measure of the extent to which loosening of control measures have impacted case-loads and deaths since the first peak and initial decline in daily cases and deaths. Our findings suggest that the two key parameters to control and reduce the impact of a developing pandemic are the infective period and the mortality fraction, which are achievable by early case identification, contact tracing and quarantine (which would reduce the former) and improving quality of care for identified cases (which would reduce the latter).

Genomic and immunologic correlates of LAG-3 expression in cancer.

Immune checkpoint blockade leads to unprecedented responses in many cancers. Although currently available agents mostly target the PD-1 and CTLA-4 pathways, agents targeting the immune checkpoint protein LAG-3 are under active clinical development, and early clinical data show that LAG-3 expression is a biomarker of response to LAG-3 blockade. To determine which cancers may benefit most from LAG-3 blockade, we performed a pan-cancer analysis of The Cancer Genome Atlas dataset to identify genomic and immunologic correlates of LAG-3 expression. High mutation burden, and expression of exogenous virus (EBV, HPV) or endogenous retrovirus (ERV3-2), were associated with overexpression of LAG-3 in multiple cancers. Although CD8+ T-cell marker (CD8A) and LAG-3 were strongly co-expressed with each other and with PD-L1 in most cancers, there were three notable exceptions: HPV+ head-neck squamous cell cancer, renal cell cancer, and glioblastoma. These results may have important implications for guiding development clinical trials of LAG-3 blockade.

Analysis of Covid-19 Data for Eight European Countries and the United Kingdom Using a Simplified SIR Model.

Understanding the characteristics of the SARS-Cov-2/Covid-19 pandemic is central to developing control strategies. Here we show how a simple Susceptible-Infective-Recovered (SIR) model applied to data for eight European countries and the United Kingdom (UK) can be used to forecast the descending limb (post-peak) of confirmed cases and deaths as a function of time, and predict the duration of the pandemic once it has peaked, by estimating and fixing parameters using only characteristics of the ascending limb and the magnitude of the first peak. As with all epidemiological analyses, unanticipated behavioral changes will result in deviations between projection and observation. This is abundantly clear for the current pandemic. Nonetheless, accurate short-term projections are possible, and the methodology we present is a useful addition to the epidemiologist's armamentarium. Since our predictions assume that control measures such as lockdown, social distancing, use of masks etc. remain the same post-peak as before peak, deviations from our predictions are a measure of the extent to which loosening of control measures have impacted case-loads and deaths since the first peak and initial decline in daily cases and deaths. The predicted and actual case fatality ratio, or number of deaths per million population from the start of the pandemic to when daily deaths number less than five for the first time, was lowest in Norway (pred: 44 ± 5 deaths/million; actual: 36 deaths/million) and highest for the United Kingdom (pred: 578 +/- 65 deaths/million; actual 621 deaths/million). The inferred pandemic characteristics separated into two distinct groups: those that are largely invariant across countries, and those that are highly variable. Among the former is the infective period, T L ( T L ¯ = 16.3 ± 2.7  days ) ; the average time between contacts, T R ( T R ¯ = 3.8 ± 0.5 ) days and the average number of contacts while infective, R ( R ¯ = 4.4 ± 0.5 ) . In contrast, there is a highly variable time lag T D between the peak in the daily number of confirmed cases and the peak in the daily number of deaths, ranging from a low of T D = 2,4 days for Denmark and Italy respectively, to highs of T D = 12, 15 for Germany and Norway respectively. The mortality fraction, or ratio of deaths to confirmed cases, was also highly variable, ranging from low values 3%, 5% and 5% for Norway, Denmark and Germany respectively, to high values of 18%, 20% and 21% for Sweden, France, and the UK respectively. The probability of mortality rather than recovery was a significant correlate of the duration of the pandemic, defined as the time from 12/31/2019 to when the number of daily deaths fell below 5. Finally, we observed a small but detectable effect of average temperature on the probability α of infection per contact, with higher temperatures associated with lower infectivity. Policy implications of our findings are also briefly discussed.

DNA hypomethylation promotes transposable element expression and activation of immune signaling in renal cell cancer.

Recently, we reported that expression of endogenous retroviruses (ERVs) is associated with response to immune checkpoint blockade (ICB) in renal cell carcinoma (RCC). We show that decitabine, a DNA hypomethylating agent, activates transposable element (TE) expression (LINE1 and ERVs ERV3-2 and ERV4700) and antiviral signaling to potentially enhance response to ICB in kidney cancer cell lines and primary cells. KO of RIGI and MDA5 dsRNA sensors attenuated activation of antiviral signaling associated with DNA hypomethylation, and RIGI and MDA5 IPs showed increased ERV binding with decitabine treatment. Bioinformatic analyses showed the decitabine-induced signature could be associated with increased immune infiltration and response to ICB. Cytokine secretion induced by decitabine could modestly improve T cell activation and robustly enhanced T cell migration. In a small retrospective cohort of metastatic clear cell RCC (ccRCC) patients treated with anti-PD1/PDL1 blockade, activation of some antiviral genes was significantly higher in responders. Thus, we identified a potential strategy to induce TE expression through inhibition of DNA methylation in modulating T cell action via regulation of the innate antiviral pathway.

Tissue- and development-stage-specific mRNA and heterogeneous CNV signatures of human ribosomal proteins in normal and cancer samples.

We give results from a detailed analysis of human Ribosomal Protein (RP) levels in normal and cancer samples and cell lines from large mRNA, copy number variation and ribosome profiling datasets. After normalizing total RP mRNA levels per sample, we find highly consistent tissue specific RP mRNA signatures in normal and tumor samples. Multiple RP mRNA-subtypes exist in several cancers, with significant survival and genomic differences. Some RP mRNA variations among subtypes correlate with copy number loss of RP genes. In kidney cancer, RP subtypes map to molecular subtypes related to cell-of-origin. Pan-cancer analysis of TCGA data showed widespread single/double copy loss of RP genes, without significantly affecting survival. In several cancer cell lines, CRISPR-Cas9 knockout of RP genes did not affect cell viability. Matched RP ribosome profiling and mRNA data in humans and rodents stratified by tissue and development stage and were strongly correlated, showing that RP translation rates were proportional to mRNA levels. In a small dataset of human adult and fetal tissues, RP protein levels showed development stage and tissue specific heterogeneity of RP levels. Our results suggest that heterogeneous RP levels play a significant functional role in cellular physiology, in both normal and disease states.

A Quasi Birth-and-Death model for tumor recurrence.

A major cause of chemoresistance and recurrence in tumors is the presence of dormant tumor foci that survive chemotherapy and can eventually transition to active growth to regenerate the cancer. In this paper, we propose a Quasi Birth-and-Death (QBD) model for the dynamics of tumor growth and recurrence/remission of the cancer. Starting from a discrete-state master equation that describes the time-dependent transition probabilities between states with different numbers of dormant and active tumor foci, we develop a framework based on a continuum-limit approach to determine the time-dependent probability that an undetectable residual tumor will become large enough to be detectable. We derive an exact formula for the probability of recurrence at large times and show that it displays a phase transition as a function of the ratio of the death rate µA of an active tumor focus to its doubling rate λ. We also derive forward and backward Kolmogorov equations for the transition probability density in the continuum limit and, using a first-passage time formalism, we obtain a drift-diffusion equation for the mean recurrence time and solve it analytically to leading order for a large detectable tumor size N. We show that simulations of the discrete-state model agree with the analytical results, except for O(1/N) corrections. As an example of the use of our model in a clinical setting, we show that a range of model parameters can fit Kaplan-Meier recurrence-free survival data for ovarian cancer. Finally, we show in simulations that extending the duration of chemotherapy increases both the mean recurrence time and the asymptotic (large-time) probability of no recurrence. Our results should be useful in planning optimized chemotherapy dosing and duration for cancer treatment, especially in cancer types for which no targeted therapy is available.

TuBA: Tunable biclustering algorithm reveals clinically relevant tumor transcriptional profiles in breast cancer.

BACKGROUND: Traditional clustering approaches for gene expression data are not well adapted to address the complexity and heterogeneity of tumors, where small sets of genes may be aberrantly co-expressed in specific subsets of tumors. Biclustering algorithms that perform local clustering on subsets of genes and conditions help address this problem. We propose a graph-based Tunable Biclustering Algorithm (TuBA) based on a novel pairwise proximity measure, examining the relationship of samples at the extremes of genes' expression profiles to identify similarly altered signatures. RESULTS: TuBA's predictions are consistent in 3,940 breast invasive carcinoma samples from 3 independent sources, using different technologies for measuring gene expression (RNA sequencing and Microarray). More than 60% of biclusters identified independently in each dataset had significant agreement in their gene sets, as well as similar clinical implications. Approximately 50% of biclusters were enriched in the estrogen receptor-negative/HER2-negative (or basal-like) subtype, while >50% were associated with transcriptionally active copy number changes. Biclusters representing gene co-expression patterns in stromal tissue were also identified in tumor specimens. CONCLUSIONS: TuBA offers a simple biclustering method that can identify biologically relevant gene co-expression signatures not captured by traditional unsupervised clustering approaches. It complements biclustering approaches that are designed to identify constant or coherent submatrices in gene expression datasets, and outperforms them in identifying a multitude of altered transcriptional profiles that are associated with observed genomic heterogeneity of diseased states in breast cancer, both within and across tumor subtypes, a promising step in understanding disease heterogeneity, and a necessary first step in individualized therapy.

Endogenous retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma.

Human endogenous retroviruses (hERVs) are remnants of exogenous retroviruses that have integrated into the genome throughout evolution. We developed a computational workflow, hervQuant, which identified more than 3,000 transcriptionally active hERVs within The Cancer Genome Atlas (TCGA) pan-cancer RNA-Seq database. hERV expression was associated with clinical prognosis in several tumor types, most significantly clear cell renal cell carcinoma (ccRCC). We explored two mechanisms by which hERV expression may influence the tumor immune microenvironment in ccRCC: (i) RIG-I-like signaling and (ii) retroviral antigen activation of adaptive immunity. We demonstrated the ability of hERV signatures associated with these immune mechanisms to predict patient survival in ccRCC, independent of clinical staging and molecular subtyping. We identified potential tumor-specific hERV epitopes with evidence of translational activity through the use of a ccRCC ribosome profiling (Ribo-Seq) dataset, validated their ability to bind HLA in vitro, and identified the presence of MHC tetramer-positive T cells against predicted epitopes. hERV sequences identified through this screening approach were significantly more highly expressed in ccRCC tumors responsive to treatment with programmed death receptor 1 (PD-1) inhibition. hervQuant provides insights into the role of hERVs within the tumor immune microenvironment, as well as evidence that hERV expression could serve as a biomarker for patient prognosis and response to immunotherapy.

Endogenous retrovirus expression is associated with response to immune checkpoint blockade in clear cell renal cell carcinoma.

Although a subset of clear cell renal cell carcinoma (ccRCC) patients respond to immune checkpoint blockade (ICB), predictors of response remain uncertain. We investigated whether abnormal expression of endogenous retroviruses (ERVs) in tumors is associated with local immune checkpoint activation (ICA) and response to ICB. Twenty potentially immunogenic ERVs (πERVs) were identified in ccRCC in The Cancer Genome Atlas data set, and tumors were stratified into 3 groups based on their expression levels. πERV-high ccRCC tumors showed increased immune infiltration, checkpoint pathway upregulation, and higher CD8+ T cell fraction in infiltrating leukocytes compared with πERV-low ccRCC tumors. Similar results were observed in ER+/HER2- breast, colon, and head and neck squamous cell cancers. ERV expression correlated with expression of genes associated with histone methylation and chromatin regulation, and πERV-high ccRCC was enriched in BAP1 mutant tumors. ERV3-2 expression correlated with ICA in 11 solid cancers, including the 4 named above. In a small retrospective cohort of 24 metastatic ccRCC patients treated with single-agent PD-1/PD-L1 blockade, ERV3-2 expression in tumors was significantly higher in responders compared with nonresponders. Thus, abnormal expression of πERVs is associated with ICA in several solid cancers, including ccRCC, and ERV3-2 expression is associated with response to ICB in ccRCC.

On statistical modeling of sequencing noise in high depth data to assess tumor evolution.

One cause of cancer mortality is tumor evolution to therapy-resistant disease. First line therapy often targets the dominant clone, and drug resistance can emerge from preexisting clones that gain fitness through therapy-induced natural selection. Such mutations may be identified using targeted sequencing assays by analysis of noise in high-depth data. Here, we develop a comprehensive, unbiased model for sequencing error background. We find that noise in sufficiently deep DNA sequencing data can be approximated by aggregating negative binomial distributions. Mutations with frequencies above noise may have prognostic value. We evaluate our model with simulated exponentially expanded populations as well as data from cell line and patient sample dilution experiments, demonstrating its utility in prognosticating tumor progression. Our results may have the potential to identify significant mutations that can cause recurrence. These results are relevant in the pre-treatment clinical setting to determine appropriate therapy and prepare for potential recurrence pretreatment.

Using Cluster Analysis to Group Countries for Cost-effectiveness Analysis: An Application to Sub-Saharan Africa.

OBJECTIVE: To explore the use of cluster analysis to define groups of similar countries for the purpose of evaluating the cost-effectiveness of a public health intervention-maternal immunization-within the constraints of a project budget originally meant for an overall regional analysis. METHODS: We used the most common cluster analysis algorithm, K-means, and the most common measure of distance, Euclidean distance, to group 37 low-income, sub-Saharan African countries on the basis of 24 measures of economic development, general health resources, and past success in public health programs. The groups were tested for robustness and reviewed by regional disease experts. RESULTS: We explored 2-, 3- and 4-group clustering. Public health performance was consistently important in determining the groups. For the 2-group clustering, for example, infant mortality in Group 1 was 81 per 1,000 live births compared with 51 per 1,000 in Group 2, and 67% of children in Group 1 received DPT immunization compared with 87% in Group 2. The experts preferred four groups to fewer, on the ground that national decision makers would more readily recognize their country among four groups. CONCLUSIONS: Clusters defined by K-means clustering made sense to subject experts and allowed a more detailed evaluation of the cost-effectiveness of maternal immunization within the constraint of the project budget. The method may be useful for other evaluations that, without having the resources to conduct separate analyses for each unit, seek to inform decision makers in numerous countries or subdivisions within countries, such as states or counties.

Immune Activation and Benefit From Avelumab in EBV-Positive Gastric Cancer.

Response to immune checkpoint therapy can be associated with a high mutation burden, but other mechanisms are also likely to be important. We identified a patient with metastatic gastric cancer with meaningful clinical benefit from treatment with the anti-programmed death-ligand 1 (PD-L1) antibody avelumab. This tumor showed no evidence of high mutation burden or mismatch repair defect but was strongly positive for presence of Epstein-Barr virus (EBV) encoded RNA. Analysis of The Cancer Genome Atlas gastric cancer data (25 EBV+, 80 microsatellite-instable [MSI], 310 microsatellite-stable [MSS]) showed that EBV-positive tumors were MSS. Two-sided Wilcoxon rank-sum tests showed that: 1) EBV-positive tumors had low mutation burden (median = 2.07 vs 3.13 in log10 scale, P < 10-12) but stronger evidence of immune infiltration (median ImmuneScore 2212 vs 1295, P < 10-4; log2 fold-change of CD8A = 1.85, P < 10-6) compared with MSI tumors, and 2) EBV-positive tumors had higher expression of immune checkpoint pathway (PD-1, CTLA-4 pathway) genes in RNA-seq data (log2 fold-changes: PD-1 = 1.85, PD-L1 = 1.93, PD-L2 = 1.50, CTLA-4 = 1.31, CD80 = 0.89, CD86 = 1.31, P < 10-4 each), and higher lymphocytic infiltration by histology (median tumor-infiltrating lymphocyte score = 3 vs 2, P < .001) compared with MSS tumors. These data suggest that EBV-positive low-mutation burden gastric cancers are a subset of MSS gastric cancers that may respond to immune checkpoint therapy.

Widespread alternative exon usage in clinically distinct subtypes of Invasive Ductal Carcinoma.

Cancer cells can have different patterns of exon usage of individual genes when compared to normal tissue, suggesting that alternative splicing may play a role in shaping the tumor phenotype. The discovery and identification of gene variants has increased dramatically with the introduction of RNA-sequencing technology, which enables whole transcriptome analysis of known, as well as novel isoforms. Here we report alternative splicing and transcriptional events among subtypes of invasive ductal carcinoma in The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) cohort. Alternative exon usage was widespread, and although common events were shared among three subtypes, ER+ HER2-, ER- HER2-, and HER2+, many events on the exon level were subtype specific. Additional RNA-seq analysis was carried out in an independent cohort of 43 ER+ HER2- and ER- HER2- primary breast tumors, confirming many of the exon events identified in the TCGA cohort. Alternative splicing and transcriptional events detected in five genes, MYO6, EPB41L1, TPD52, IQCG, and ACOX2 were validated by qRT-PCR in a third cohort of 40 ER+ HER2- and ER- HER2- patients, showing that these events were truly subtype specific.

Identifying a Clinically Applicable Mutational Burden Threshold as a Potential Biomarker of Response to Immune Checkpoint Therapy in Solid Tumors.

PURPOSE: An association between mutational burden and response to immune checkpoint therapy has been documented in several cancer types. The potential for such a mutational burden threshold to predict response to immune checkpoint therapy was evaluated in several clinical datasets, where mutational burden was measured either by whole-exome sequencing (WXS) or using commercially available sequencing panels. METHODS: WXS and RNA-seq data of 33 solid cancer types from TCGA were analyzed to determine whether a robust immune checkpoint activating mutation (iCAM) burden threshold associated with evidence of immune checkpoint activation exists in these cancers that may serve as a biomarker for response to immune checkpoint blockade therapy. RESULTS: We find that a robust iCAM threshold, associated with signatures of immune checkpoint activation, exists in 8 of 33 solid cancers: melanoma, lung adenocarcinoma, colon adenocarcinoma, endometrial cancer, stomach adenocarcinoma, cervical cancer, ER+HER2- breast cancer, and bladder-urothelial cancer. Tumors with mutational burden higher than the threshold (iCAM+) also had clear histologic evidence of lymphocytic infiltration. In published datasets of melanoma, lung adenocarcinoma and colon cancer, patients with iCAM+ tumors had significantly better response to immune checkpoint therapy compared to those with iCAM- tumors. ROC analysis using TCGA predictions as gold standard showed that iCAM+ tumors are accurately identifiable using clinical sequencing assays, such as FoundationOne or StrandAdvantage. Using the FoundationOne derived threshold, analysis of 113 melanoma tumors, showed that iCAM+ patients have significantly better response to immune checkpoint therapy. iCAM+ and iCAM- tumors have distinct mutation patterns and different immune microenvironments. CONCLUSION: In 8 solid cancers, a mutational burden threshold exists that may predict response to immune checkpoint blockade. This threshold is identifiable using available clinical sequencing assays.