Stephen G. Gaffney

Early and multiple origins of metastatic lineages within primary tumors

Significance The knowledge that cancer is an evolutionary process is old, but only recently can sequencing technology provide data for clinically relevant evolutionary analyses of cancer. Approaches developed for evolutionary biology can reveal the relationship among clonal lineages, the ancestral states of gene sequences, and the timing of evolutionary events. We performed whole exome sequencing of cancer tissues from multiple sites of dozens of subjects, demonstrating nonlinear patterns of tumor progression and early origins of metastatic lineages and quantifying the times of occurrence of driver mutations. These findings direct research attention away from the search for genes that induce metastasis toward genes that are mutated early in tumorigenesis, providing therapeutic targets effective against both primary tumors and metastases. 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.

Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up

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 Fishers 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.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 Fishers 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.

Harnessing Case Isolation and Ring Vaccination to Control Ebola

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.

Mutation profiles in early-stage lung squamous cell carcinoma with clinical follow-up and correlation with markers of immune function.

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.

Characterizing risk of Ebola transmission based on frequency and type of case-contact exposures.

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’.

Heterogeneity and mutation in KRAS and associated oncogenes: evaluating the potential for the evolution of resistance to targeting of KRAS G12C

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 therapies in vivo given the possibilities of existing intratumor heterogeneity or de novo mutation leading to treatment resistance. We performed deep sequencing of 27 KRAS G12-positive lung tumors to determine the prevalence of other oncogenic mutations within KRAS or within commonly mutated downstream genes that could confer resistance at the time of treatment. We also passaged patient-derived xenografts to assess the potential for novel KRAS mutation to arise during subsequent tumor evolution. Furthermore, we estimate the de novo mutation rate in KRAS position 12 and in genes downstream of KRAS. Finally, we present an approach for estimation of the selection intensity for these point mutations that explains their high prevalence in tumors. We find no evidence of heterogeneity that may compromise KRAS G12C targeted therapy within sequenced lung tumors or passaged xenografts. We find that mutations that confer resistance are even less likely to occur downstream of KRAS than to occur within KRAS. Our approach predicts that BRAF V600E would provide the highest fitness advantage for de novo-resistant 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 the de novo mutations likely to confer resistance, mutations in BRAF, a gene with targeted inhibitors presently available, result in subclones with the highest fitness advantage.

Effect Sizes of Somatic Mutations in Cancer

Abstract A major goal of cancer biology is determination of the relative importance of the genetic alterations that confer selective advantage to cancer cells. Tumor sequence surveys have frequently ranked the importance of substitutions to cancer growth by P value or a false-discovery conversion thereof. However, P values are thresholds for belief, not metrics of effect. Their frequent misuse as metrics of effect has often been vociferously decried, even in cases when the only attributable mistake was omission of effect sizes. Here, we propose an appropriate ranking—the cancer effect size, which is the selection intensity for somatic variants in cancer cell lineages. The selection intensity is a metric of the survival and reproductive advantage conferred by mutations in somatic tissue. Thus, they are of fundamental importance to oncology, and have immediate relevance to ongoing decision making in precision medicine tumor boards, to the selection and design of clinical trials, to the targeted development of pharmaceuticals, and to basic research prioritization. Within this commentary, we first discuss the scope of current methods that rank confidence in the overrepresentation of specific mutated genes in cancer genomes. Then we bring to bear recent advances that draw upon an understanding of the development of cancer as an evolutionary process to estimate the effect size of somatic variants leading to cancer. We demonstrate the estimation of the effect sizes of all recurrent single nucleotide variants in 22 cancer types, quantifying relative importance within and between driver genes.

CDKN2A Copy Number Loss Is an Independent Prognostic Factor in HPV-Negative Head and Neck Squamous Cell Carcinoma

Background HPV infection is associated with high p16 expression and good prognosis in head and neck squamous cell carcinomas (HNSCCs). Analysis of CDKN2A, the gene encoding p16, may further elucidate the association between p16 expression and prognosis. We sought to determine whether CDKN2A copy number loss was associated with poor survival in HPV-negative HNSCCs. Methods The Cancer Genome Atlas HNSCC clinical and genomic data were obtained and integrated. Patients <80 years old with a primary tumor in the oral cavity, oropharynx, hypopharynx, or larynx were included. Stratifying by copy number loss status, CDKN2A mRNA and p16 protein expression levels were examined and overall survival (OS) and disease-free survival (DFS) were evaluated. Results 401 patients with HPV-negative HNSCC were identified. 146 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. Median OS for patients with and without CDKN2A copy number loss was 16.5 and 46.6 months, respectively (p = 0.007). Median DFS for both groups was 11.6 and 19.2 months, respectively (p = 0.03). In both univariate and multivariable analyses, stage IV designation, receipt of chemotherapy and CDKN2A copy number loss were predictive of OS. Conclusion CDKN2A copy number loss predicted poor survival independently of other patient and treatment factors and may be a clinically useful prognostic factor.

The likelihood of heterogeneity or additional mutation in KRAS or associated oncogenes to compromise targeting of oncogenic KRAS G12C

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 therapies in vivo given the possibilities of existing intratumor heterogeneity or de novo mutation 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 the de novo mutation rate in KRAS position 12 and in genes downstream of KRAS. 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 for de novo resistant 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 the de novo mutations likely to confer resistance, mutations in BRAF, a gene with targeted inhibitors presently available, result in subclones with the highest fitness advantage. One Sentence Summary Mutations 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.

Abstract 142: Mutation and immune profiles in early-stage lung squamous cell carcinoma

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.