William Accomando

DNA methylation arrays as surrogate measures of cell mixture distribution

BackgroundThere has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the labile nature of protein epitopes, requirements for cell processing, and timely cell analysis. In a diverse array of diseases and following numerous immune-toxic exposures, leukocyte composition will critically inform the underlying immuno-biology to most chronic medical conditions. Emerging research demonstrates that DNA methylation is responsible for cellular differentiation, and when measured in whole peripheral blood, serves to distinguish cancer cases from controls.ResultsHere we present a method, similar to regression calibration, for inferring changes in the distribution of white blood cells between different subpopulations (e.g. cases and controls) using DNA methylation signatures, in combination with a previously obtained external validation set consisting of signatures from purified leukocyte samples. We validate the fundamental idea in a cell mixture reconstruction experiment, then demonstrate our method on DNA methylation data sets from several studies, including data from a Head and Neck Squamous Cell Carcinoma (HNSCC) study and an ovarian cancer study. Our method produces results consistent with prior biological findings, thereby validating the approach.ConclusionsOur method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.

Peripheral Blood Immune Cell Methylation Profiles Are Associated with Nonhematopoietic Cancers

Background: Blood leukocytes from patients with solid tumors exhibit complex and distinct cancer-associated patterns of DNA methylation. However, the biologic mechanisms underlying these patterns remain poorly understood. Because epigenetic biomarkers offer significant clinical potential for cancer detection, we sought to address a mechanistic gap in recently published works, hypothesizing that blood-based epigenetic variation may be due to shifts in leukocyte populations. Methods: We identified differentially methylated regions (DMR) among leukocyte subtypes using epigenome-wide DNA methylation profiling of purified peripheral blood leukocyte subtypes from healthy donors. These leukocyte-tagging DMRs were then evaluated using epigenome-wide blood methylation data from three independent case-control studies of different cancers. Results: A substantial proportion of the top 50 leukocyte DMRs were significantly differentially methylated among head and neck squamous cell carcinoma (HNSCC) cases and ovarian cancer cases compared with cancer-free controls (48 and 47 of 50, respectively). Methylation classes derived from leukocyte DMRs were significantly associated cancer case status (P < 0.001, P < 0.03, and P < 0.001) for all three cancer types: HNSCC, bladder cancer, and ovarian cancer, respectively and predicted cancer status with a high degree of accuracy (area under the curve [AUC] = 0.82, 0.83, and 0.67). Conclusions: These results suggest that shifts in leukocyte subpopulations may account for a considerable proportion of variability in peripheral blood DNA methylation patterns of solid tumors. Impact: This illustrates the potential use of DNA methylation profiles for identifying shifts in leukocyte populations representative of disease, and that such profiles may represent powerful new diagnostic tools, applicable to a range of solid tumors. Cancer Epidemiol Biomarkers Prev; 21(8); 1293–302. ©2012 AACR.

Quantitative reconstruction of leukocyte subsets using DNA methylation

BackgroundCell lineage-specific DNA methylation patterns distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. We have developed an approach that uses DNA methylation to simultaneously quantify multiple leukocyte subsets, enabling investigation of immune modulations in virtually any blood sample including archived samples previously precluded from such analysis. Here we assess the performance characteristics and validity of this approach.ResultsUsing Illumina Infinium HumanMethylation27 and VeraCode GoldenGate Methylation Assay microarrays, we measure DNA methylation in leukocyte subsets purified from human whole blood and identify cell lineage-specific DNA methylation signatures that distinguish human T cells, B cells, NK cells, monocytes, eosinophils, basophils and neutrophils. We employ a bioinformatics-based approach to quantify these cell types in complex mixtures, including whole blood, using DNA methylation at as few as 20 CpG loci. A reconstruction experiment confirms that the approach could accurately measure the composition of mixtures of human blood leukocyte subsets. Applying the DNA methylation-based approach to quantify the cellular components of human whole blood, we verify its accuracy by direct comparison to gold standard immune quantification methods that utilize physical, optical and proteomic characteristics of the cells. We also demonstrate that the approach is not affected by storage of blood samples, even under conditions prohibiting the use of gold standard methods.ConclusionsCell mixture distributions within peripheral blood can be assessed accurately and reliably using DNA methylation. Thus, precise immune cell differential estimates can be reconstructed using only DNA rather than whole cells.

Decreased NK Cells in Patients with Head and Neck Cancer Determined in Archival DNA

Purpose: Natural killer (NK) cells are a key element of the innate immune system implicated in human cancer. To examine NK cell levels in archived bloods from a study of human head and neck squamous cell carcinoma (HNSCC), a new DNA-based quantification method was developed. Experimental Design: NK cell-specific DNA methylation was identified by analyzing DNA methylation and mRNA array data from purified blood leukocyte subtypes (NK, T, B, monocytes, granulocytes), and confirmed via pyrosequencing and quantitative methylation specific PCR (qMSP). NK cell levels in archived whole blood DNA from 122 HNSCC patients and 122 controls were assessed by qMSP. Results: Pyrosequencing and qMSP confirmed that a demethylated DNA region in NKp46 distinguishes NK cells from other leukocytes, and serves as a quantitative NK cell marker. Demethylation of NKp46 was significantly lower in HNSCC patient bloods compared with controls (P < 0.001). Individuals in the lowest NK tertile had over 5-fold risk of being a HNSCC case, controlling for age, gender, HPV16 status, cigarette smoking, alcohol consumption, and BMI (OR = 5.6, 95% CI, 2.0 to 17.4). Cases did not show differences in NKp46 demethylation based on tumor site or stage. Conclusions: The results of this study indicate a significant depression in NK cells in HNSCC patients that is unrelated to exposures associated with the disease. DNA methylation biomarkers of NK cells represent an alternative to conventional flow cytometry that can be applied in a wide variety of clinical and epidemiologic settings including archival blood specimens. Clin Cancer Res; 18(22); 6147–54. ©2012 AACR.

Leukocyte-adjusted epigenome-wide association studies of blood from solid tumor patients.

Epigenome-wide studies of DNA methylation using blood-derived DNA from cancer patients are complicated by the heterogeneity of cell types within blood and the associated cell lineage specification of DNA methylation signatures. Here, we applied a novel set of analytic approaches to assess the association between cancer case-status and DNA methylation adjusted for leukocyte variation using blood specimens from three case-control cancer studies (bladder: 223 cases, 205 controls; head and neck: 92 cases, 92 controls; and ovarian: 131 cases, 274 controls). Using previously published data on leukocyte-specific CpG loci and a recently described approach to deconvolute subject-specific blood composition, we performed an epigenome-wide analysis to examine the association between blood-based DNA methylation patterns and each of the three aforementioned solid tumor types adjusted for cellular heterogeneity in blood. After adjusting for leukocyte profile in our epigenome-wide analysis, the omnibus association between case-status and methylation was significant for all three studies (bladder cancer: P = 0.047; HNSCC: P = 0.013; ovarian cancer: P = 0.0002). Subsequent analyses revealed that CpG sites associated with cancer were enriched for transcription factor binding motifs involved with cancer-associated pathways. These results support the existence of cancer-associated DNA methylation profiles in the blood of solid tumor patients that are independent of alterations in normal leukocyte distributions. Adoption of the methods developed here will make it feasible to rigorously assess the influence of variability of normal leukocyte profiles when investigating cancer related changes in blood-based epigenome-wide association studies.

Epigenetic biomarkers of T-cells in human glioma

Immune factors are thought to influence glioma risk and outcomes, but immune profiling studies to further our understanding of the immune response are limited by current immunodiagnostic methods. We developed a new assay to capture glioma immune biology based on quantitative methylation specific PCR (qMSP) of two T-cell genes (CD3Z: T-cells, and FOXP3: Tregs). Flow cytometry of T-cells correlated well with the CD3Z demethylation assay (r = 0.93; p < 2.2 × 10−16), demonstrating the validity of the assay. Furthermore, there was a high correlation between qMSP and immunohistochemistry (IHC) in quantifying tumor infiltrating T-cells (r = 0.85; p = 3.4 × 10−11). Applying our qMSP methods to archival whole blood from 65 glioblastoma multiforme (GBM) cases and 94 non-diseased controls, GBM cases had highly statistically significantly lower T-cells (p = 1.7 × 10−9) as well as Tregs (p = 5.2 × 10−11) and a modestly lower ratio of Tregs/T-cells (p = 0.024). Applying the methods to 120 excised glioma tumors, we observed that tumor infiltrating CD3+ T-cells were positively correlated with glioma tumor grade (p = 5.7 × 10−7), and that Tregs were enriched in tumors compared with peripheral blood indicating active chemoattraction of suppressive Tregs into the tumor compartment. Poorer patient survival was correlated with higher levels of tumor infiltrating T-cells (p = 0.01) and Tregs (p = 0.04). DNA methylation based immunodiagnostics represent a new generation of powerful laboratory tools offering many advantages over conventional methods that will facilitate large clinical epidemiologic studies and capitalize on stored archival blood and tissue banks.

Durable complete responses in some recurrent high-grade glioma patients treated with Toca 511 + Toca FC

Background Vocimagene amiretrorepvec (Toca 511) is an investigational gamma-retroviral replicating vector encoding cytosine deaminase that, when used in combination with extended-release 5-fluorocytosine (Toca FC), results preclinically in local production of 5-fluorouracil, depletion of immune-suppressive myeloid cells, and subsequent induction of antitumor immunity. Recurrent high-grade glioma (rHGG) patients have a high unmet need for effective therapies that produce durable responses lasting more than 6 months. In this setting, relapse is nearly universal and most responses are transient. Methods In this Toca 511 ascending-dose phase I trial (NCT01470794), HGG patients who recurred after standard of care underwent surgical resection and received Toca 511 injected into the resection cavity wall, followed by orally administered cycles of Toca FC. Results Among 56 patients, durable complete responses were observed. A subgroup was identified based on Toca 511 dose and entry requirements for the follow-up phase III study. In this subgroup, which included both isocitrate dehydrogenase 1 (IDH1) mutant and wild-type tumors, the durable response rate is 21.7%. Median duration of follow-up for responders is 35.7+ months. As of August 25, 2017, all responders remain in response and are alive 33.9+ to 52.2+ months after Toca 511 administration, suggesting a positive association of durable response with overall survival. Conclusions Multiyear durable responses have been observed in rHGG patients treated with Toca 511 + Toca FC in a phase I trial, and the treatment will be further evaluated in a randomized phase III trial. Among IDH1 mutant patients treated at first recurrence, there may be an enrichment of complete responders.

Abstract 5630: Immune profile of tumor microenvironment helps predict response in patients treated with an investigational immunotherapeutic consisting of a retroviral replicating vector (Toca 511) and an extended-release formulation of 5-fluorocytosine (Toca FC)

Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector (RRV). RRVs selectively infect cancer cells due to defects in innate and adaptive immune responses found in cancers that support virus replication, and cell division requirements for virus integration into the genome. Toca 511 spreads through cancer cells, stably delivering an optimized cytosine deaminase (CD) gene that converts the prodrug Toca FC (investigational, extended-release 5-fluorocytosine) into 5-fluorouracil (5-FU), a canonical chemotherapeutic. In preclinical tumor models, as infected cancer cells are killed, diffusible 5-FU also kills nearby susceptible cells, including uninfected cancer cells, and myeloid derived suppressor cells (MDSC) that contribute to immune-suppression in the tumor microenvironment. This action by Toca 511 and Toca FC has been shown in animal models to generate a durable anti-tumor immune response that can be transferred to naive, untreated animals. The Toca 511 and Toca FC immunotherapeutic are proposed to remodel the tumor microenvironment to break tumor tolerance resulting in induction of antitumor activity by the patient9s immune system and durable complete responses. In a phase 1 clinical study for recurrent high grade glioma (NCT01470794), Toca 511 was injected into resection cavity walls at time of resection followed by multiple courses of oral Toca FC. We observed multi-year durable and objective responses; including 5 ongoing complete responses in a group of 23 patients in the higher dose single agent treatment cohorts given approximately the same Toca 511 doses and having the same entry criteria as an ongoing Phase 3 study in recurrent high grade glioma (NCT02414165). Patient tumors at time of resection were analyzed by exome sequencing, RNA sequencing, IHC, and TCR sequencing, before Toca 511 treatment. In this study, we report higher levels of tumor infiltrating T cells by TCR sequencing, before the start of treatment, were significantly associated with responding patients compared to patients whose disease progressed. The significance of this data is supported by the preclinical mechanism of action reported previously. Additionally, we plan to report on T cell, B cell, and myeloid populations in the tumor as measured by IHC and RNA sequencing and their relationship to clinical response. Data reported here will provide mechanistic context to the immunotherapeutic mode of action proposed to account for durable responses seen in treatment of brain tumors with Toca 511 and Toca FC. Citation Format: Derek Ostertag, William Accomando, Leah Mitchell, Maria Rodriguez-Aguirre, Daniel Hogan, Oscar Diago, Dawn Gammon, Ali Haghighi, Harry Gruber, Asha Das, Douglas Jolly. Immune profile of tumor microenvironment helps predict response in patients treated with an investigational immunotherapeutic consisting of a retroviral replicating vector (Toca 511) and an extended-release formulation of 5-fluorocytosine (Toca FC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5630.

Abstract 4240: A novel approach to adjust for immune cell distribution in blood-based epigenome-wide association studies of cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epigenome-wide association studies (EWAS) promise to advance our understanding of epigenetic variation in cancer and are made possible by the recent emergence of cost-effective high-throughput technologies. Blood is the most widely available source of genomic DNA that can be used for study of cancer-associated alterations in DNA methylation (DNAm). These analyses are complicated by the heterogeneity of blood, which represents multiple cell types with unique DNAm signatures. Shifts in immune profile could confound results since altered leukocyte numbers are common in response to exposures or disease. We applied a novel approach to blood-based EWAS, adjusting for leukocyte composition estimated by epigenetic deconvolution of blood. Normal human peripheral blood leukocytes (n = 47) were isolated by magnetic activated cell sorting and purity was confirmed by fluorescence activated cell sorting. DNAm was interrogated using the Infinium HumanMethylation27 BeadArray (Illumina) on the sorted leukocyte samples and peripheral blood samples from 3 independent case-control studies of bladder cancer (223 cases, 205 controls), head and neck squamous cell carcinoma (HNSCC; 92 cases, 92 controls), and ovarian cancer (131 cases, 274 controls). Differentially methylated loci associated with leukocyte lineage were identified using a series of linear mixed effects models fit to the DNAm data for each of the 26,486 autosomal CpGs for the sorted leukocytes, yielding an F-statistic for each locus. The subject-specific leukocyte distribution (vector ω) was estimated using constrained projections, as previously described by Houseman et al (2012). Several regression parameters were estimated: βj, representing the association of case-status and DNAm at CpG j, unadjusted for ω; αj, representing the corresponding association adjusted for ω, and Γ, the association of case-status and ω. Γ, β and α were each adjusted for age, sex and smoking for bladder cancer and HNSCC, and age for ovarian cancer. Statistical inference was achieved by permutation, where null distributions were obtained by permuting case-status with respect to DNAm values and other covariates, using an omnibus test adjusted for multiple-comparisons, constructed by comparing the observed average F-statistic across all CpGs to the corresponding quantity obtained from the permutation distribution. After adjusting for leukocyte composition (α), the association between DNAm and case-status was significant for all 3 studies (bladder cancer: p = 0.047; HNSCC: p = 0.013; ovarian cancer: p = 0.0002). Subsequent analyses revealed that cancer-associated pathways were overrepresented among significant loci. These results indicate cancer-specific variation in DNAm of peripheral blood, independent of immune cell shifts. Further research is indicated for elucidation of mechanisms driving these observations. Citation Format: Scott M. Langevin, E. A. Houseman, William P. Accomando, Devin C. Koestler, Brock C. Christensen, Heather H. Nelson, Margaret R. Karagas, Carmen J. Marsit, John K. Wiencke, Karl T. Kelsey. A novel approach to adjust for immune cell distribution in blood-based epigenome-wide association studies of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4240. doi:10.1158/1538-7445.AM2013-4240

Abstract LB-374: A novel method for the detection of circulating NK cells in archived blood reveals a decrease in NK cells in head and neck squamous cell carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Patients with head and neck squamous cell carcinoma (HNSCC) frequently exhibit abnormal immune cell levels and activities, leading many to believe that progression of the disease is linked to evasion of immune surveillance. A more complete understanding of immune cell alterations could be obtained from analysis of archival samples, but this type of study is not possible using current immunodiagnostic methods. Combining information on cell lineage specific differentially methylated regions (DMRs) with methylation specific quantitative PCR (MS-qPCR) enables highly sensitive and accurate counts of individual cell types in archival blood and tissues. DNA methylation and mRNA expression in magnetic microbead (MACS) isolated, flow cytometry (FACS) validated normal human peripheral blood leukocytes were assessed using the Infinium HumanMethylation27 and Whole-Genome DASL HT (Illumina) microarrays. Cell-specific DMRs were identified by comparative analysis of DNA methylation and mRNA expression patterns between different cell lineages, and validated by MS-qPCR and pyrosequencing. Analysis of the microarray data revealed CpG loci significantly demethylated in NK cells compared to all other leukocyte lineages (q < 0.1) within genes exhibiting significantly increased mRNA expression in NK cells (q < 0.1). Pyrosequencing and MS-qPCR assays confirmed a NK cell specific demethylated region. The MS-qPCR assay was calibrated using known quantities of NK cell DNA to facilitate quantification of these cells in biological samples, and was then applied to archival peripheral blood DNA samples from 122 control donors and 122 HNSCC patients. Demethylation of the candidate region was significantly lower in HNSCC patient bloods compared with control bloods (p < 0.001), but was not significantly associated with age, gender, HPV16 (E6 and/or E7) serology, cigarette smoking, alcohol consumption, BMI, or disease treatment. Measurements from control samples were used to establish normal demethylation tertiles. Proportion of total HNSCC cases decreased significantly with increasing tertile (p < 0.001). Multivariate logistic regression controlling for age, gender, cigarette smoking, alcohol consumption, BMI, and HPV16 serology confirmed increased HNSCC risk for individuals in the lowest tertile (OR = 5.6, 95% CI: 2.0, 17.4) and second lowest tertile (OR = 4.9, 95% CI: 1.8, 16.1). The advent of DNA methylation based immunodiagnostic methods will make assessment of immune cell profiles in archived samples that have not undergone rigorous special handling requirements possible. Using a novel NK cell specific demethylated region to detect and quantify NK cells in the blood, we have detected a HNSCC associated decrease in circulating NK cells that is independent of disease treatments, known exposures and other putative risk factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-374. doi:1538-7445.AM2012-LB-374