Observations of recurrent somatic mutations in tumors have led to identification and definition of signaling and other pathways that are important for cancer progression and therapeutic targeting. As ...tumor cells contain both an individual's inherited genetic variants and somatic mutations, challenges arise in distinguishing these events in massively parallel sequencing datasets. Typically, both a tumor sample and a "normal" sample from the same individual are sequenced and compared; variants observed only in the tumor are considered to be somatic mutations. However, this approach requires two samples for each individual.
We evaluate a method of detecting somatic mutations in tumor samples for which only a subset of normal samples are available. We describe tuning of the method for detection of mutations in tumors, filtering to remove inherited variants, and comparison of detected mutations to several matched tumor/normal analysis methods. Filtering steps include the use of population variation datasets to remove inherited variants as well a subset of normal samples to remove technical artifacts. We then directly compare mutation detection with tumor-only and tumor-normal approaches using the same sets of samples. Comparisons are performed using an internal targeted gene sequencing dataset (n = 3380) as well as whole exome sequencing data from The Cancer Genome Atlas project (n = 250). Tumor-only mutation detection shows similar recall (43-60%) but lesser precision (20-21%) to current matched tumor/normal approaches (recall 43-73%, precision 30-82%) when compared to a "gold-standard" tumor/normal approach. The inclusion of a small pool of normal samples improves precision, although many variants are still uniquely detected in the tumor-only analysis.
A detailed method for somatic mutation detection without matched normal samples enables study of larger numbers of tumor samples, as well as tumor samples for which a matched normal is not available. As sensitivity/recall is similar to tumor/normal mutation detection but precision is lower, tumor-only detection is more appropriate for classification of samples based on known mutations. Although matched tumor-normal analysis is preferred due to higher precision, we demonstrate that mutation detection without matched normal samples is possible for certain applications.
Multiple myeloma remains an incurable malignancy due to acquisition of intrinsic programs that drive therapy resistance. Here we report that casein kinase-1δ (CK1δ) and CK1ε are therapeutic targets ...in multiple myeloma that are necessary to sustain mitochondrial metabolism. Specifically, the dual CK1δ/CK1ε inhibitor SR-3029 had potent in vivo and ex vivo anti-multiple myeloma activity, including against primary multiple myeloma patient specimens. RNA sequencing (RNA-seq) and metabolic analyses revealed inhibiting CK1δ/CK1ε disables multiple myeloma metabolism by suppressing genes involved in oxidative phosphorylation (OxPhos), reducing citric acid cycle intermediates, and suppressing complexes I and IV of the electron transport chain. Finally, sensitivity of multiple myeloma patient specimens to SR-3029 correlated with elevated expression of mitochondrial genes, and RNA-seq from 687 multiple myeloma patient samples revealed that increased CSNK1D, CSNK1E, and OxPhos genes correlate with disease progression and inferior outcomes. Thus, increases in mitochondrial metabolism are a hallmark of multiple myeloma progression that can be disabled by targeting CK1δ/CK1ε.
CK1δ and CK1ε are attractive therapeutic targets in multiple myeloma whose expression increases with disease progression and connote poor outcomes, and that are necessary to sustain expression of genes directing OxPhos.
Emm55 is a bacterial gene derived from
Streptococcus pyogenes
(
S. pyogenes
) that was cloned into a plasmid DNA vaccine (pAc/emm55). In this study, we investigated the anti-tumor efficacy of pAc/
...emm55
in a B16 murine melanoma model. Intralesional (IL) injections of pAc/
emm55
significantly delayed tumor growth compared to the pAc/Empty group. There was a significant increase in the CD8
+
T cells infiltrating into the tumors after pAc/
emm55
treatment compared to the control group. In addition, we observed that IL injection of pAc/
emm55
increased antigen-specific T cell infiltration into tumors. Depletion of CD4
+
or CD8
+
T cells abrogated the anti-tumor effect of pAc/
emm55
. Combination treatment of IL injection of pAc/
emm55
with anti-PD-1 antibody significantly delayed tumor growth compared to either monotherapy. pAc/
emm55
treatment combined with PD-1 blockade enhanced anti-tumor immune response and improved systemic anti-tumor immunity. Together, these strategies may lead to improvements in the treatment of patients with melanoma.
As oropharyngeal cancer (OPC) associated with human papillomavirus (HPV) increases in men, the need for a screening test to diagnose OPC early is crucial. This study agnostically identified ...differentially methylated CpG sites to identify additional biomarkers to improve screening for early OPC.DNA was extracted from oral gargles of 89 early cases and 108 frequency matched healthy controls, and processed for genome-wide methylation using the Illumina Infinium MethylationEPIC BeadChip. Selected sites were combined with our prior methylation data in the EPB41L3 gene (CpG sites 438, 427, and 425) and oral HPV16 and HPV18 status were considered as binary variables (positive/negative). Lasso regression identified CpG sites strongly associated with early OPC. ROC curves with AUC were generated. The panel was validated utilizing bootstrap resampling.Machine learning analyses identified 14 markers that are significantly associated with early OPC, including one EPB41L3 CpG site (438) and oral HPV16 status. A final model was trained on all available samples using the discovered panel and was able to predict early OPC compared with controls with an AUC of 0.970 on the training set. In the bootstrap validation sets, the average AUC was 0.935, indicating adequate internal validity.Our data suggest that this panel can detect OPC early, however external validation of this panel is needed. Further refinement of a panel of biomarkers to diagnose OPC earlier is urgently needed to prevent complex treatment of OPC and associated comorbidities, while reducing risk of recurrence.
This study identified biomarkers using genome-wide methylation to create a panel capable of discerning early oropharyngeal cancer (OPC) from those without OPC. Such a biomarker panel would be an effective tool to detect OPC early and prevent complications of treatment associated with later diagnosis.
BackgroundDespite its common epigenetic suppression in multiple cancers, STING signaling has emerged as a major pathway for augmenting tumor cell antigenicity and initiation of T cell responses.1 2 ...Another aspect of intact activation of STING signaling in tumor cells is downstream induction of T cell-homing chemokines including CXCL10 and CCL5. These chemokines are also among our earlier reported 12-chemokine (12-CK) gene expression signature (GES) predicting the presence of tumor-localized tertiary lymphoid structures (TLSs), which are increasingly shown to correlate with improved survival in certain solid tumor types.3 4 Based on these findings, we hypothesized that epigenetic silencing of STING signaling genes through promoter hypermethylation would be inversely associated with the presence of TLSs.MethodsWe assessed the correlation between the expression of STING signaling genes and the chemokines present in the 12-CK GES across melanomas and urothelial bladder carcinomas using cBioPortal datasets. To extend these studies beyond these tumor types, we performed correlative and survival analyses using the TCGA PanCancer Atlas. Additionally, we determined the correlation between the promoter methylation levels of STING signaling genes and the 12-CK GES score. We also evaluated STING expression in TLS+ and TLS- melanoma samples in situ by immunohistochemistry (IHC).ResultsWe identified a distinct correlation between STING-expressing tumors and each of the twelve chemokines among melanoma and urothelial bladder carcinoma samples. In particular, STING expression was positively correlated with secondary lymphoid organ-associated chemokines, CCL19 (p=0.0077), CCL21 (p=0.0046), and CXCL13 (p=0.0034) in urothelial bladder carcinomas. The presence of TLSs in STING-expressing melanomas was further confirmed by IHC. Using TCGA PanCancer datasets, we observed a strong correlation between the expression of cGAS (Pearson’s r=0.46) and STING (Pearson’s r=0.37) with the 12-CK GES score. In contrast, the methylation levels of cGAS and STING were inversely correlated with the 12-CK GES score (Pearson’s r=-0.37 and -0.41, respectively). Similarly, hypermethylation of STING was correlated with inferior disease-specific survival (DSS) (p<0.0001) in lung adenocarcinomas. Survival analysis on the TCGA skin cutaneous melanoma (SKCM) dataset also indicated significant DSS advantage in 12-CK GES scoreHigh cGASHighpatients (p<0.0001).ConclusionsWe provide evidence that epigenetic state of cGAS and STING cannot only shape tumor antigenicity but is also associated with the 12-CK GES and the presence of TLSs. Considering the well-established prognostic value of TLSs, these findings argue that targeting epigenetic suppression of STING signaling should be considered as a strategy to guide effective immunotherapy-based interventions.AcknowledgementsThis work was supported by the Moffitt Cancer Center Tissue Core and Analytic Microscopy Core Facilities, all comprehensive cancer center facilities designated by the National Cancer Institute (P30 CA076292). This work was funded by: NCI-NIH (1R01 CA148995, 1R01 CA184845, P30 CA076292, P50 CA168536), CJG Fund, Chris Sullivan Fund, V Foundation, Melanoma Research Foundation, and Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferencesFalahat R, Berglund A, Putney RM, Perez-Villarroel P, Aoyama S, Pilon- Thomas S, Barber GN, Mulé JJ. Epigenetic reprogramming of tumor cell-intrinsic STING function sculpts antigenicity and T cell recognition of melanoma. PNAS. 2021;118(15).Falahat R, Berglund A, Perez-Villarroel P, Putney RM, Hamaidi I, Kim S, Pilon-Thomas S, Barber GN, Mulé JJ. Epigenetic state determines the in vivo efficacy of STING agonist therapy. Nature Communications. 2023;14(1):1573.Messina JL, Fenstermacher DA, Eschrich S, Qu X, Berglund AE, Lloyd MC, Schell MJ, Sondak VK, Weber JS, Mulé JJ. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy?. Scientific reports. 2012;2(1):765.Schumacher TN, Thommen DS. Tertiary lymphoid structures in cancer. Science. 2022;375(6576):eabf9419.
Epigenetic regulation of O
-alkylguanine DNA alkyltransferase (MGMT) is surrogate of intrinsic resistance to temozolomide (TMZ). However, mechanisms associated with adaptive resistance evolution of ...glioblastoma (GBM) relative to MGMT methylation remain unclear. We hereby report a paradoxical yet translational epigenetic regulation of plasticity towards adaptive resistance in GBM. Based on an adaptive resistance model of GBM cells with differential MGMT methylation profiles, MGMT-hypermethylation enhanced genetic and phenotypic plasticity towards adaptive resistance to TMZ while MGMT hypomethylation limited plasticity. The resulting model-associated adaptive resistance gene signature negatively correlated with GBM patient survival. XAF1, a tumor suppressor protein, paradoxically emerged as a mediator of differential plasticities towards adaptive resistance to TMZ through epigenetic regulation. XAF1 promoted resistance both in-vitro and in-vivo. Furthermore, XAF1 expression negatively correlated with XAF1 promoter methylation status, and negatively correlate with GBM patient survival. Collectively, XAF1 appears to have a pradoxical yet translational role in GBM.
Previous studies on dose-escalated radiotherapy in head and neck cancer have shown mixed results, and it is not established which patients would benefit from dose escalation. Further, while dose ...escalation does not appear to increase late toxicity, this needs to be confirmed with longer follow-up. In this study, we analysed treatment outcome and toxicity in 215 patients with oropharyngeal cancer treated with dose-escalated radiotherapy (>72 Gy, EQD2, α/β = 10 Gy, boost by brachytherapy or simultaneous integrated boost) and a matched cohort of 215 patients treated with standard dose external-beam radiotherapy (68 Gy) between 2011 and 2018 at our institution. The 5-year overall survival (OS) was 77.8% (72.4-83.6) and 73.7% (67.8-80.1) in the dose-escalated and standard dose group, respectively (
= 0.24). Median follow-up was 78.1 (49.2-98.4) and 60.2 (38.9-89.4) months in the dose-escalated and standard dose groups, respectively. Grade ≥3 osteoradionecrosis (ORN) and late dysphagia were more common in the dose-escalated group compared to the standard dose group, with 19 (8.8%) vs. 4 (1.9%) patients developing grade ≥3 ORN (
= 0.001), and 39 (18.1%) vs. 21 (9.8%) patients developing grade ≥3 dysphagia (
= 0.01). No predictive factors to help select patients for dose-escalated radiotherapy were found. However, the remarkably good OS in the dose-escalated cohort, despite a predominance of advanced tumour stages, encourages further attempts to identify such factors.
Plant growth is the result of cell proliferation in meristems, which requires a careful balance between the formation of new tissue and the maintenance of a set of undifferentiated stem cells. Recent ...studies have provided important information on several genetic networks responsible for stem cell maintenance and regulation of cell differentiation in the apical meristems of shoots and roots. Nothing, however, is known about the regulatory networks in secondary meristems like the vascular cambium of trees. We have made use of the large size and highly regular layered organization of the cambial meristem to create a high-resolution transcriptional map covering 220 micrometer of the cambial region of aspen (Populus tremula). Clusters of differentially expressed genes revealed substantial differences in the transcriptomes of the six anatomically homogenous cell layers in the meristem zone. Based on transcriptional and anatomical data, we present a model for the position of the stem cells and the proliferating mother cells in the cambial zone. We also provide sets of marker genes for different stages of xylem and phloem differentiation and identify potential regulators of cambial meristem activity. Interestingly, analysis of known regulators of apical meristem development indicates substantial similarity in regulatory networks between primary and secondary meristems.
Bioinformatic scientists are often asked to do widespread analyses of publicly available datasets in order to identify genetic alterations in cancer for genes of interest; therefore, we sought to ...create a set of tools to conduct common statistical analyses of The Cancer Genome Atlas (TCGA) data. These tools have been developed in response to requests from our collaborators to ask questions, validate findings, and better understand the function of their gene of interest. We describe here what data we have used, how to obtain it, and what figures we have found useful.
PDF
