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Supplementary Figure S4 related to Figure 5D-F. (A) Individual tumor volume changes in Detroit 562-xenografted mice treated with vehicle, adavosertib (30 mg/kg, daily, p.o.), alisertib (30 mg/kg, daily, p.o.), or the combinations of alisertib with adavosertib. Tumor volume changes of all mice belonged to this study was shown in bottom panel (n = 6 per group). Significant differences between the combination and single drug alone are shown. (B) Body weight (g) of mice was monitored every other day. Graphs depict Mean {plus minus} SEM.

Supplementary figure legends

Purpose:Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition.Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models.Results:Elevated nuclear AURKA correlated with worse survival among patients with p16(−) HNSCC. Alisertib caused spindle defects, G2–M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment.Conclusions:Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.

Researchers grapple with a challenging and consequential decision each time they choose a journal for manuscript submission. There are several online tools that attempt to identify appropriate journals for a manuscript, but each of these tools has shortcomings in terms of the journal data they provide and the exploration functionality they offer—and not one of these tools is open source. Jot is a free and open-source web application that matches manuscripts in the fields of biomedicine and life sciences with suitable journals, based on a manuscript's title, abstract and (optionally) citations. Jot gathers a wealth of data on journal quality, impact, fit, and open access options that can be explored through a dashboard of linked, interactive visualizations. Visit Jot at https://jot.publichealth.yale.edu, or run your own Jot server using our open-source Python package 'journal_targeter', available from the Python Package Index (PyPI).

Lung squamous-cell carcinoma originates as a consequence of oncogenic molecular variants arising from diverse mutagenic processes such as tobacco, defective homologous recombination, aging, and cytidine deamination by APOBEC proteins. Only some of the many variants generated by these processes actually contribute to tumorigenesis. Therefore, molecular investigation of mutagenic processes such as cytidine deamination by APOBEC should also determine whether the mutations produced by these processes contribute substantially to the growth and survival of cancer. Here, we determine the processes that gave rise to mutations of 681 lung squamous-cell carcinomas, and quantify the probability that each mutation was the product of each process. We then calculate the contribution of each mutation to increases in cellular proliferation and survival. We performed in vitro experiments to determine cytidine deamination activity of APOBEC3B against oligonucleotides corresponding with genomic sequences that give rise to variants of high cancer effect size. The largest APOBEC-related cancer effects are attributable to mutations in PIK3CA and NFE2L2. We demonstrate that APOBEC effectively deaminates NFE2L2 at the locations that confer high cancer effect. Overall, we demonstrate that APOBEC activity can lead to mutations in NFE2L2 that have large contributions to cancer cell growth and survival, and that NFE2L2 is an attractive potential target for therapeutic intervention.

The progression of cancer is an evolutionary process that is challenging to assess between sampling timepoints. However, investigation of cancer evolution over specific time periods is crucial to the elucidation of key events such as the acquisition of therapeutic resistance and subsequent fatal metastatic spread of therapy-resistant cell populations. Here we apply mutational signature analyses within clinically annotated cancer chronograms to detect and describe the shifting mutational processes caused by both endogenous (e.g. mutator gene mutation) and exogenous (e.g. mutagenic therapeutics) factors between tumor sampling timepoints. In one patient, we find that cisplatin therapy can introduce mutations that confer genetic resistance to subsequent targeted therapy with Erlotinib. In another patient, we trace detection of defective mismatch-repair associated mutational signature SBS3 to the emergence of known driver mutation CTNNB1 S37C. In both of these patients, metastatic lineages emerged from a single ancestral lineage that arose during therapy—a finding that argues for the consideration of local consolidative therapy over other therapeutic approaches in EGFR-positive non-small cell lung cancer. Broadly, these results demonstrate the utility of phylogenetic analysis that incorporates clinical time course and mutational signature deconvolution to inform therapeutic decision making and retrospective assessment of disease etiology.

Dispersal routes of metastatic cells are not medically detected or even visible. A molecular evolutionary analysis of tumor variation provides a way to retrospectively infer metastatic migration histories and answer questions such as whether the majority of metastases are seeded from clones within primary tumors or seeded from clones within pre-existing metastases, as well as whether the evolution of metastases is generally consistent with any proposed models. We seek answers to these fundamental questions through a systematic patient-centric retrospective analysis that maps the dynamic evolutionary history of tumor cell migrations in many cancers. We analyzed tumor genetic heterogeneity in 51 cancer patients and found that most metastatic migration histories were best described by a hybrid of models of metastatic tumor evolution. Synthesizing across metastatic migration histories, we found new tumor seedings arising from clones of pre-existing metastases as often as they arose from clones from primary tumors. There were also many clone exchanges between the source and recipient tumors. Therefore, a molecular phylogenetic analysis of tumor variation provides a retrospective glimpse into general patterns of metastatic migration histories in cancer patients.

Background The relative importance of genetic and environmental risk factors in gliomagenesis remains uncertain. Methods Using whole-exome sequencing data from 1105 adult gliomas, we evaluate the relative contribution to cancer cell lineage proliferation and survival of single-nucleotide mutations in tumors by IDH mutation subtype and sex. We also quantify the contributions of COSMIC cancer mutational signatures to these tumors, identifying possible risk exposures. Results IDH-mutant tumors exhibited few unique recurrent substitutions—all in coding regions, while IDH wild-type tumors exhibited many substitutions in non-coding regions. The importance of previously reported mutations in IDH1/2, TP53, EGFR, PTEN, PIK3CA, and PIK3R1 was confirmed; however, the largest cancer effect in IDH wild-type tumors was associated with mutations in the low-prevalence BRAF V600E. Males and females exhibited mutations in a similar set of significantly overburdened genes, with some differences in variant sites—notably in the phosphoinositide 3-kinase (PI3K) pathway. In IDH-mutant tumors, PIK3CA mutations were located in the helical domain for females and the kinase domain for males; variants of import also differed by sex for PIK3R1. Endogenous age-related mutagenesis was the primary molecular signature identified; a signature associated with exogenous exposure to haloalkanes was identified and noted more frequently in males. Conclusions Cancer-causing mutations in glioma primarily originated as a consequence of endogenous rather than exogenous factors. Mutations in helical vs kinase domains of genes in the phosphoinositide 3-kinase (PI3K) pathway are differentially selected in males and females. Additionally, a rare environmental risk factor is suggested for some cases of glioma—particularly in males.

The progression of cancer—including the acquisition of therapeutic resistance and the fatal metastatic spread of therapy-resistant cell populations—is an evolutionary process that is challenging to monitor between sampling timepoints. Here we apply mutational signature analysis to clinically correlated cancer chronograms to detect and describe the shifting mutational processes caused by both endogenous (e.g. mutator mutation) and exogenous (e.g. therapeutic) factors between tumor sampling timepoints. In one patient, we find that cisplatin therapy can introduce mutations that increase the likelihood of genetic adaptation to subsequent targeted therapeutics. In another patient, we trace the emergence of known driver mutation CTNNB1 S37C to specific detection of defective mismatch repair associated mutational signature SBS3. Metastatic lineages were found to emerge from a single ancestral lineage arising during therapy—a finding that argues for the consideration of local consolidative therapy over other therapeutic approaches in EGFR-positive non-small cell lung cancer. Broadly, these results demonstrate the utility of phylogenetic analysis that incorporates clinical time course and mutational signature detection to inform clinical decision making and retrospective assessment of disease etiology.

Cancer progression is an evolutionary process. During this process, evolving cancer cell populations encounter restrictive ecological niches within the body, such as the primary tumor, circulatory system, and diverse metastatic sites. Efforts to prevent or delay cancer evolution—and progression—require a deep understanding of the underlying molecular evolutionary processes. Herein we discuss a suite of concepts and tools from evolutionary and ecological theory that can inform cancer biology in new and meaningful ways. We also highlight current challenges to applying these concepts, and propose ways in which incorporating these concepts could identify new therapeutic modes and vulnerabilities in cancer.

Background: Immunotherapies targeting immune checkpoint proteins CTLA-4, PD-1, and PD-L1 have saved lives, but these therapies have only been effective in some patients. Patients positive for expression of immune checkpoint proteins in the tumor microenvironment show better response to immune checkpoint inhibitors. Consequently, knowledge of which genes are consistently expressed in lymphocytes within the tumor microenvironment can convey potentially effective and complementary new immunotherapy targets. Results: We identified 54 genes that have higher co-expression with the pan T-cell marker CD3E than CTLA4 or PDCD1. In a dataset of 26 patients who received anti-PD-1 therapy, we observed that co-expression between CD3E and PDCD1 was higher among responders than non-responders, supporting our correlation-based approach. Conclusions: The genes highlighted in these analyses, which include CD6, TIGIT, CD96, and SLAMF6, warrant further investigation of their therapeutic potential. Methods: We analyzed and ranked genes that were co-expressed with the pan T-cell marker CD3E in 9,601 human tumors, spanning 31 cancer types. To further identify targets that may be complementary to existing PD-1 therapy, we examined and ranked genes with high CD3E co-expression and relatively low PDCD1 co-expression.

Purpose: Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition. Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models. Results: Elevated nuclear AURKA correlated with worse survival among patients with p16(−) HNSCC. Alisertib caused spindle defects, G2–M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment. Conclusions: Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.

Summary Chemsearch is a cross-platform server application for developing and managing a chemical compound library and associated data files, with an interface for browsing and search that allows for easy navigation to a compound of interest, similar compounds or compounds that have desired structural properties. With provisions for access control and centralized document and data storage, Chemsearch supports collaboration by distributed teams. Availability and implementation Chemsearch is a free and open-source Flask web application that can be linked to a Google Workspace account. Source code is available at https://github.com/gem-net/chemsearch (GPLv3 license). A Docker image allowing rapid deployment is available at https://hub.docker.com/r/cgemcci/chemsearch.

Sourcing HIV-1 infection HIV-1 has a multitude of strain variants, but sexual transmission of HIV-1 is assumed to result from productive infection by only one virus particle. Knowing the genetics of the virus strains that are transmitted could be crucial for developing successful vaccine strategies. Using epidemiological and genetic data from 112 pairs of sexual partners, Villabona-Arenas et al. found that individuals with acute infections are more likely to transmit multiple founder virus strains. In a phylodynamic approach that integrated phylogenetic analysis of sequence data with simulation of a transmission chain, the authors showed that multiple variant transmission is doubled during the first 3 months of infection irrespective of whether transmission was heterosexual or by men who have sex with men. Science , this issue p. 103

The Center for Genetically Encoded Materials (C-GEM) is an NSF Phase I Center for Chemical Innovation that comprises six laboratories spread across three university campuses. Our success as a multi-institution research team demanded the development of a software infrastructure, GEM-NET, that allows all C-GEM members to work together seamlessly—as though everyone was in the same room. GEM-NET was designed to support both science and communication by integrating task management, scheduling, data sharing, and collaborative document and code editing with frictionless internal and public communication; it also maintains security over data and internal communications. In this Article, we document the design and implementation of GEM-NET: our objectives and motivating goals, how each component contributes to these goals, and the lessons learned throughout development. We also share open source code for several custom applications and document how GEM-NET can benefit users in multiple fields and teams that are both small and large. We anticipate that this knowledge will guide other multi-institution teams, regardless of discipline, to plan their software infrastructure and utilize it as swiftly and smoothly as possible., Herein we introduce GEM-NET, a set of tools that support the research and broader impact activities of the Center for Genetically Encoded Materials (C-GEM), an NSF Center for Chemical Innovation.

During sexual transmission, the large genetic diversity of HIV-1 within an individual is frequently reduced to one founder variant that initiates infection1. Understanding the drivers of this bottleneck is crucial to develop effective infection control strategies2. Genetic characteristics of the potential founder viruses and events in the recipient partner are both known to contribute to this bottleneck, but little is understood about the importance of the source partner3. To test the hypothesis that the source partner affects the multiplicity of HIV founder variants, we developed a phylodynamic model calibrated using genetic and epidemiological data on all existing transmission pairs for whom the direction of transmission and the infection stage of the source partner are known. Our results demonstrate the importance of infection stage of the source partner, and not exposure route, in determining founder variant multiplicity. Specifically, acquiring infection from someone in the acute (early) stage of infection increases the risk of multiple variant transmission when compared with someone in the chronic (later) stage of infection. This study provides the first direct test of source partner characteristics to explain the low frequency of multiple founder strain infections and can inform clinical intervention study design and interpretation.

Objectives To investigate how T-cell activation interacts with NSUN2 to influence HNSCC patient survival. Materials and Methods The relationships between T-cell activation status (Activation, Intermediate, and Exhaustion), NSUN2 expression, and patient survival were evaluated using Kaplan-Meier survival curves and multivariate Cox regression models in a public dataset with 520 HNSCC patients. HPV status was determined based on a VirusScan analysis of RNA-seq data. Results Among the patients with high NSUN2 expression, the Activation group exhibited longer survival than the Exhaustion group (trend P = 0.056). Adjusted hazards ratios (HRs) were 0.77 (95% CI: 0.49–1.19) for the Intermediate vs Exhaustion, and 0.61 (0.36–1.03) for Activation vs. Exhaustion. In contrast, there is a positive association between T-cell activation score and mortality in the patients with low NSUN2 expression (trend P = 0.016). The adjusted HRs were 1.97 (1.12–3.47) for the Intermediate vs Exhaustion, and 2.06 (1.16–3.68) for the Activation vs Exhaustion. In multivariate cox models with or without HPV status, the interaction between T-cell activation status and NSUN2 expression was statistically significant (P = 0.004 for with HPV status, and P = 0.002 for without, respectively). When not controlling for NSUN2 expression, there was no significant association between T-cell activation score and patient mortality (P = 0.84). Conclusions An interaction between NSUN2 expression and T-cell activation status affects patient survival in HNSCC regardless of HPV status, suggesting that NSUN2 is a potential precision marker for immune-checkpoint blockade, and a potential therapeutic target.

Many genetic and environmental factors have been explored in epidemiological studies of glioma risk but the magnitude of effect of these factors on gliomagenesis is uncertain. A number of constitutional genes associated with glioma risk, many noted to be related to telomere function, have been identified. High-dose ionizing radiation has consistently been associated with an increased glioma risk while a history of allergies and atopy is inversely associated with risk. Results have been inconsistent for most other factors studied but whether this lack of evidence is due to study limitations or a true lack of risk factors is unclear. Using 901 glioma exomes from The Cancer Genome Atlas (TCGA) we estimate the intensity of positive selection on gene variants, and order them by their cancer effect size, quantifying the contribution of gene variants to the somatic evolution of glioma. Examination of subsets of tumor exomes from males and females showed similar patterns in genes mutated but some differences in variant profile for a given gene including PIK3CA suggesting a possible differential response to treatment. Furthermore, these cancer effect sizes enable quantification of the degree to which COSMIC-defined processes associated with genetic pathways and environmental environmental factors within the somatic evolution of tumors contribute mutations that are highly selected, driving gliomagenesis. The results confirm the major roles of previously reported mutations in IDH1, TP53, EGFR, and PIK3CA, but also suggest a large cancer effect for low-prevalence genes including BRAF. Age-related mutagenesis was the primary molecular signature identified for all glioma while homologous recombination disfunction was an important signature for males but not females. Although additional signatures remain to be identified and less common exposures difficult to access, these data suggest a profile of glioma mutations that are caused primarily from endogenous rather than exogenous factors.

Recent studies have revealed the mutational signatures underlying the somatic evolution of cancer, and the prevalences of associated somatic genetic variants. Here we estimate the intensity of positive selection that drives mutations to high frequency in tumors, yielding higher prevalences than expected on the basis of mutation and neutral drift alone. We apply this approach to a sample of 525 head and neck squamous cell carcinoma exomes, producing a rank-ordered list of gene variants by selection intensity. Our results illustrate the complementarity of calculating the intensity of selection on mutations along with tallying the prevalence of individual substitutions in cancer: while many of the most prevalently-altered genes were heavily selected, their relative importance to the cancer phenotype differs from their prevalence and from their P value, with some infrequent variants exhibiting evidence of strong positive selection. Furthermore, we extend our analysis of effect size by quantifying the degree to which mutational processes (such as APOBEC mutagenesis) contributes mutations that are highly selected, driving head and neck squamous cell carcinoma. We calculate the substitutions caused by APOBEC mutagenesis that make the greatest contribution to cancer phenotype among patients. Lastly, we demonstrate via in vitro biochemical experiments that the APOBEC3B protein can deaminate the cytosine bases at two sites whose mutant states are subject to high net realized selection intensities-PIK3CA E545K and E542K. By quantifying the effects of mutations, we deepen the molecular understanding of carcinogenesis in head and neck squamous cell carcinoma.

Activating mutations in RAS genes are associated with approximately 20% of all human cancers. New targeted therapies show preclinical promise in inhibiting the KRAS G12C variant, however, concerns exist regarding the effectiveness of such therapiesin vivogiven the possibilities of existing intratumor heterogeneity orde novomutation leading to treatment resistance. We performed deep sequencing of 27 KRAS G12 positive lung tumors and found no evidence of other oncogenic mutations within KRAS or within commonly mutated downstream genes that could confer resistance at the time of treatment. Furthermore, we estimate thede novomutation rate in KRAS position 12 and in genes downstream ofKRAS.We find that mutations that confer resistance are about as likely to occur downstream of KRAS as within KRAS. Moreover, we present an approach for estimation of the selection intensity for these point mutations that explains their high prevalence in tumors. Our approach predicts that BRAF V600E would provide the highest fitness advantage forde novoresistant subclones. Overall, our findings suggest that resistance to targeted therapy of KRAS G12C positive tumors is unlikely to be present at the time of treatment and, among thede novomutations likely to confer resistance, mutations in BRAF, a gene with targeted inhibitors presently available, result in subclones with the highest fitness advantage.One Sentence SummaryMutations conferring resistance to KRAS G12C targeted therapy are unlikely to be present at the time of resection, and the likely mechanisms of evolved resistance are predicted be ones that are responsive to therapies that are in development or that are already available.

Meningioma is the most commonly diagnosed primary brain tumor and is associated with significant morbidity. Children treated with therapeutic radiation to the head are at almost ten-fold risk of radiation-associated meningioma (RAM). RAM tumors are associated with aggressive histological features and reduced survival but treatment options remain limited. To discover genes associated with RAM we performed whole-exome sequencing on matched constitutional and tumor materials from 20 patients treated in childhood with ionizing radiation to the head and subsequently diagnosed with cranial meningioma. The mean number of protein-altering somatic mutations was 139.2 (median=94, range=49-541) and did not vary significantly by sex, grade, age at initial or meningioma diagnosis, time since radiation, dose, meningioma location or initial diagnosis (Acute Lymphoblastic Leukemia (ALL) versus other). The average proportion of the genome with copy number variation (CNV) was 6.4 (range 1.2-15.4). Males had twice the CNV rate of females (8.4 vs 4.8, respectively, p=0.05). Although no other variable was significantly associated with difference in CNV rate, of the 5 patients with a CNV value of 10% or more, 4 (80%) had a prior diagnosis of ALL. Eleven genes with significantly more somatic mutations than expected by chance were identified (NF2, VKORC1, GAB1, METTL25, AK3, IL15RA, MIPOL1, MetAP2, AWAT1, ERO1L, ATP6V0D2). The most common were NF2 (n=4 samples) and AK3 (n=4 samples). Two samples had mutations in MetAP2, a gene that encodes the protein methionine aminopeptidase (MetAP2). MetAP2 inhibitors are currently in use for the treatment of a number of cancers suggesting a possible new treatment for some patients with RAM. Our findings suggest a high rate of mutations in RAM; with the exception of NF2 these changes are dissimilar from those reported in non-radiation associated meningioma.

Background Lung squamous cell carcinoma (LUSC) accounts for 20–30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. Methods Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher’s exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. Results This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. Conclusion(s) Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

Many aspects of the evolutionary process of tumorigenesis that are fundamental to cancer biology and targeted treatment have been challenging to reveal, such as the divergence times and genetic clonality of metastatic lineages. To address these challenges, we performed tumor phylogenetics using molecular evolutionary models, reconstructed ancestral states of somatic mutations, and inferred cancer chronograms to yield three conclusions. First, in contrast to a linear model of cancer progression, metastases can originate from divergent lineages within primary tumors. Evolved genetic changes in cancer lineages likely affect only the proclivity toward metastasis. Single genetic changes are unlikely to be necessary or sufficient for metastasis. Second, metastatic lineages can arise early in tumor development, sometimes long before diagnosis. The early genetic divergence of some metastatic lineages directs attention toward research on driver genes that are mutated early in cancer evolution. Last, the temporal order of occurrence of driver mutations can be inferred from phylogenetic analysis of cancer chronograms, guiding development of targeted therapeutics effective against primary tumors and metastases.

Background Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers. Methods We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fisher’s exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome. Results LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration. Conclusion(s) Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.

SummaryPathScore quantifies the level of enrichment of somatic mutations within curated pathways, applying a novel approach that identifies pathways enriched across patients. The application provides several user-friendly, interactive graphic interfaces for data exploration, including tools for comparing pathway effect sizes, significance, gene-set overlap and enrichment differences between projects.Availability and ImplementationWeb application available at pathscore.publichealth.yale.edu. Site implemented in Python and MySQL, with all major browsers supported. Source code available at github.com/sggaffney/pathscore with a GPLv3 license.Contactstephen.gaffney@yale.eduSupplementary InformationAdditional documentation can be found at http://pathscore.publichealth.yale.edu/faq.

As a devastating Ebola outbreak in West Africa continues, non-pharmaceutical control measures including contact tracing, quarantine, and case isolation are being implemented. In addition, public health agencies are scaling up efforts to test and deploy candidate vaccines. Given the experimental nature and limited initial supplies of vaccines, a mass vaccination campaign might not be feasible. However, ring vaccination of likely case contacts could provide an effective alternative in distributing the vaccine. To evaluate ring vaccination as a strategy for eliminating Ebola, we developed a pair approximation model of Ebola transmission, parameterized by confirmed incidence data from June 2014 to January 2015 in Liberia and Sierra Leone. Our results suggest that if a combined intervention of case isolation and ring vaccination had been initiated in the early fall of 2014, up to an additional 126 cases in Liberia and 560 cases in Sierra Leone could have been averted beyond case isolation alone. The marginal benefit of ring vaccination is predicted to be greatest in settings where there are more contacts per individual, greater clustering among individuals, when contact tracing has low efficacy or vaccination confers post-exposure protection. In such settings, ring vaccination can avert up to an additional 8% of Ebola cases. Accordingly, ring vaccination is predicted to offer a moderately beneficial supplement to ongoing non-pharmaceutical Ebola control efforts., Author Summary Public health efforts for controlling the 2014–2015 Ebola outbreak in West Africa have focused on contact tracing and isolation of symptomatic individuals. In addition, substantial resources have been committed to scaling up the production of experimental vaccines. Ring vaccination—the vaccination of the contacts of an infected individual—was successfully implemented to achieve smallpox eradication. Ring vaccination is particularly feasible and effective in settings where the supply of vaccines is limited and disease incidence is low. Using a disease transmission model, we evaluated the benefit of adding ring vaccination to case isolation in Liberia and Sierra Leone. We found that ring vaccination could have averted up to 126 cases in Liberia and 560 cases in Sierra Leone, thereby saving lives and intervention resources.

6060 Background: HPV infection is associated with high p16 expression and relatively good prognosis in head and neck cancers. Analysis of CDKN2A, the gene that encodes the p16 tumor suppressor protein, may further elucidate the association between HPV status and prognosis in head and neck squamous cell carcinomas (HNSCCs). We aimed to identify whether CDKN2A copy number loss was associated with poor survival in HNSCCs stratified by HPV status. Methods: We analyzed The Cancer Genome Atlas (TCGA) head and neck cancer data, integrating genomic measurements with clinical metadata. Patients 85 years old or younger with a primary tumor in the oral cavity, oropharynx, hypopharynx, or larynx were included. Defining CDKN2A copy number loss as a relative log2 copy number ratio < −0.6, CDKN2A mRNA and p16 protein expression levels were compared to confirm significant differences in gene transcription and translation between the copy number loss and non-copy number loss patient groups. Overall survival (OS) and disease-free survival (DFS) were evaluated to characterize prognostic differences between genomic groups. Results: 397 patients negative for HPV (HPV−) and 91 patients positive for HPV (HPV+) HNSCC were identified. 139 HPV− patients and 9 HPV+ patients demonstrated CDKN2A copy number loss. The CDKN2A copy number loss group expressed significantly lower levels of CDKN2A mRNA and p16 protein than did the non-copy number loss group in both HPV+ and HPV− disease. Median OS for HPV− patients with and without CDKN2A copy number loss was 21.8 months and 46.0 months (P = 0.02). Median DFS was 12.0 and 19.4 months respectively (P < 0.05). Median OS for HPV+ patients with and without CDKN2A copy number loss was 12.7 months and 57.4 months (P = 0.004) and median DFS was 7.0 and 36.6 months respectively (P = 0.02). Conclusions: CDKN2A copy number loss was associated with low CDKN2A mRNA and p16 protein expression, with poor prognosis in terms of disease-free and overall survival.

During the initial months of the 2013–2016 Ebola epidemic, rapid geographical dissemination and intense transmission challenged response efforts across West Africa. Contextual behaviours associated with increased risk of exposure included travel to high-transmission settings, caring for sick and preparing the deceased for traditional funerals. Although such behaviours are widespread in West Africa, high-transmission pockets were observed. Superspreading and clustering are typical phenomena in infectious disease outbreaks, as a relatively small number of transmission chains are often responsible for the majority of events. Determining the characteristics of contacts at greatest risk of developing disease and of cases with greatest transmission potential could therefore help curb propagation of infection. Our analysis of contact tracing data from Montserrado County, Liberia, suggested that the probability of transmission was 4.5 times higher for individuals who were reported as having contact with multiple cases. The probability of individuals developing disease was not significantly associated with age or sex of their source case but was higher when they were in the same household as the infectious case. Surveillance efforts for rapidly identifying symptomatic individuals and effectively messaged campaigns encouraging household members to bring the sick to designated treatment centres without administration of home care could mitigate transmission. This article is part of the themed issue ‘The 2013–2016 West African Ebola epidemic: data, decision-making and disease control’.

PURPOSE: Lung squamous cell carcinoma (LUSC) accounts for 20-30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We sought to perform whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. METHODS: Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers was done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using ANOVA and Fisher tests. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. RESULTS: This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. CONCLUSION: Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC. Citation Format: Murim Choi, Humam Kadara, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen G. Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Roy S. Herbst, Ignacio I. Wistuba. Mutation and immune profiles in early-stage lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 142.

PURPOSE: Lung adenocarcinomas (LUADs) lead to the preponderance of deaths attributable to lung cancer. We performed whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of LUADs to better understand the molecular pathogenesis of this disease and identify clinically relevant molecular markers. METHODS: We performed WES of 108 paired surgically resected stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Additionally, ten immune related markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers Exact tests. Cox proportional hazards regression models were employed for multivariate analysis of clinical outcome. RESULTS: LUADs in this cohort exhibited an average of 243 coding mutations per tumor. We identified 28 genes with significant enrichment for mutation. SETD2-mutant LUADS exhibited relatively poor recurrence-free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS in KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis demonstrated that PD-L1 expression was increased in smoker compared to non-smoker LUADs and, along with other immune markers, was positively correlated with somatic mutation burden. Moreover, immune marker levels including PD-L1 were elevated in TP53-mutant LUADs. In contrast, STK11 and U2AF1 mutant tumors exhibited a suppressed immune response and LUADs with PIK3CA mutations exhibited markedly decreased tumoral PD-L1 expression. CONCLUSION: Our study highlights mutations that may impact clinical outcome and personalized strategies for immune-based therapy of early-stage LUAD patients. Citation Format: Humam Kadara, Murim Choi, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen G. Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Ignacio I. Wistuba, Roy S. Herbst. Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 89.

There are two errors in Fig 1. In Fig 1, the label for the edge connecting the latent state (E) to the removed state (R) should be τχ. The label for the edge connecting the latent state (E) to the observed state (TE) should be τ(1-χ). Please see the corrected Fig 1 here. Fig 1 A) Our dynamic model is driven by the spatial correlation of individuals in the population. New latent infections depend on the connections between susceptible and infectious individuals (red). Case isolation and ring vaccination depend on the connections ...