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Supplementary Data from Prediction of Immunotherapy Response in Melanoma through Combined Modeling of Neoantigen Burden and Immune-Related Resistance Mechanisms

Supplementary Table from Prediction of Immunotherapy Response in Melanoma through Combined Modeling of Neoantigen Burden and Immune-Related Resistance Mechanisms

Purpose:While immune checkpoint blockade (ICB) has become a pillar of cancer treatment, biomarkers that consistently predict patient response remain elusive due to the complex mechanisms driving immune response to tumors. We hypothesized that a multi-dimensional approach modeling both tumor and immune-related molecular mechanisms would better predict ICB response than simpler mutation-focused biomarkers, such as tumor mutational burden (TMB).Experimental Design:Tumors from a cohort of patients with late-stage melanoma (n = 51) were profiled using an immune-enhanced exome and transcriptome platform. We demonstrate increasing predictive power with deeper modeling of neoantigens and immune-related resistance mechanisms to ICB.Results:Our neoantigen burden score, which integrates both exome and transcriptome features, more significantly stratified responders and nonresponders (P = 0.016) than TMB alone (P = 0.049). Extension of this model to include immune-related resistance mechanisms affecting the antigen presentation machinery, such as HLA allele-specific LOH, resulted in a composite neoantigen presentation score (NEOPS) that demonstrated further increased association with therapy response (P = 0.002).Conclusions:NEOPS proved the statistically strongest biomarker compared with all single-gene biomarkers, expression signatures, and TMB biomarkers evaluated in this cohort. Subsequent confirmation of these findings in an independent cohort of patients (n = 110) suggests that NEOPS is a robust, novel biomarker of ICB response in melanoma.

Supplementary Figure from Prediction of Immunotherapy Response in Melanoma through Combined Modeling of Neoantigen Burden and Immune-Related Resistance Mechanisms

Purpose: While immune checkpoint blockade (ICB) has become a pillar of cancer treatment, biomarkers that consistently predict patient response remain elusive due to the complex mechanisms driving immune response to tumors. We hypothesized that a multi-dimensional approach modeling both tumor and immune-related molecular mechanisms would better predict ICB response than simpler mutation-focused biomarkers, such as tumor mutational burden (TMB). Experimental Design: Tumors from a cohort of patients with late-stage melanoma (n = 51) were profiled using an immune-enhanced exome and transcriptome platform. We demonstrate increasing predictive power with deeper modeling of neoantigens and immune-related resistance mechanisms to ICB. Results: Our neoantigen burden score, which integrates both exome and transcriptome features, more significantly stratified responders and nonresponders (P = 0.016) than TMB alone (P = 0.049). Extension of this model to include immune-related resistance mechanisms affecting the antigen presentation machinery, such as HLA allele-specific LOH, resulted in a composite neoantigen presentation score (NEOPS) that demonstrated further increased association with therapy response (P = 0.002). Conclusions: NEOPS proved the statistically strongest biomarker compared with all single-gene biomarkers, expression signatures, and TMB biomarkers evaluated in this cohort. Subsequent confirmation of these findings in an independent cohort of patients (n = 110) suggests that NEOPS is a robust, novel biomarker of ICB response in melanoma.

Multicellular organisms employ concurrent gene regulatory programs to control development and physiology of cells and tissues. The Drosophila melanogaster model system has a remarkable history of revealing the genes and mechanisms underlying fundamental biology yet much remains unclear. In particular, brain xenobiotic protection and endobiotic regulatory systems that require transcriptional coordination across different cell types, operating in parallel with the primary nervous system and metabolic functions of each cell type, are still poorly understood. Here we use the unsupervised machine learning method independent component analysis (ICA) on majority fresh-frozen, bulk tissue microarrays to define biologically pertinent gene expression signatures which are sparse, i.e. each involving only a fraction of all fly genes. We optimize the gene expression signature definitions partly through repeated application of a stochastic ICA algorithm to a compendium of 3,346 microarrays from 221 experiments provided by the Drosophila research community. Our optimized ICA model of pan fly gene expression consists of 850 modules of co-regulated genes that map to tissue developmental stages, disease states, cell-autonomous pathways and presumably novel processes. Importantly, we show biologically relevant gene modules expressed at varying amplitudes in whole brain and isolated adult blood-brain barrier cell levels. Thus, whole tissue derived ICA transcriptional signatures that transcend single cell type boundaries provide a window into the transcriptional states of difficult to isolate cell ensembles maintaining delicate brain physiologies. We believe the fly ICA gene expression signatures set, by virtue of the success of ICA at inferring robust often low amplitude patterns across large datasets and the quality of the input samples, to be an important asset for analyzing compendium and newly generated microarray or RNA-seq expression datasets.

Intractable epilepsies, that is, seizure disorders that do not respond to currently available therapies, are difficult, often tragic, neurological disorders. Na+ channelopathies have been implicated in some intractable epilepsies, including Dravet syndrome (Dravet 1978), but little progress has been forthcoming in therapeutics. Here we examine a Drosophila model for intractable epilepsy, the Na+ channel gain-of-function mutant parabss1 that resembles Dravet syndrome in some aspects (parker et al. 2011a). In particular, we identify second-site mutations that interact with parabss1, seizure enhancers, and seizure suppressors. We describe one seizure-enhancer mutation named charlatan (chn). The chn gene normally encodes an Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription factor transcriptional repressor of neuronal-specific genes. We identify a second-site seizure-suppressor mutation, gilgamesh (gish), that reduces the severity of several seizure-like phenotypes of parabss1/+ heterozygotes. The gish gene normally encodes the Drosophila ortholog of casein kinase CK1g3, a member of the CK1 family of serine-threonine kinases. We suggest that CK1g3 is an unexpected but promising new target for seizure therapeutics.

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013

Despite the remarkable response of some melanoma patients to checkpoint inhibitor therapy, significant numbers of patients do not achieve complete response. To understand this differential response, there is an increasing interest in identifying biomarkers and mechanisms that influence immunotherapy effectiveness. In this study, we characterize the immuno-genomics of tumors from a series of melanoma patients that have received anti-PD-1 checkpoint inhibitors to assess potential factors influencing response. To better understand mechanisms of anti-PD-1 response, we sequenced and genomically profiled tumors from 19 stage III and IV melanoma patients where response was evaluated using RECIST criteria. Of the 19 patients, there were 5 complete responders (CR), 8 partial responders (PR), and 6 progressive disease (PD) patients. Immuno-genomic profiling was performed using Personalis' ACE ImmunoID platform, an augmented exome/transcriptome platform and analysis pipeline that allows for assessment of tumor mutations, neoantigens, HLA typing, gene expression quantification, tumor micro-environment, and tumor escape mechanisms. The molecular information for each of the 19 melanoma patient samples was then analyzed together with the corresponding clinical response to anti-PD-1 therapy. We identified 3 outlier patients, which, while having very high neoantigen burden, did not achieve complete response (2 PR & 1 PD). One of these patients had extremely high expression of IDO1, which may facilitate immune escape in a PD-1 independent manner. Two independent HLA mutations in HLA-A and HLA-B (stop-gain mutation and splice site mutation, respectively) were found in the second patient, leading to the likely loss of surface expression of two classes of HLA-A and HLA-B proteins. If these three high neoantigen burden individuals with proposed tumor escape mechanisms are removed from consideration, we found a highly significant association between neoantigen burden and response to anti-PD-1 therapy (PD + PR vs CR, P = 0.00046). We also observed that, in our cohort, response to anti-PD-1 therapy was more accurately predicted by neoantigen burden than mutational burden. In conclusion, we observed a strong correlation between response to anti-PD-1 therapy in melanoma patients and neoantigen burden when tumor escape mechanisms are considered. In our patients, we saw highly suggestive resistance mechanisms that involve perturbations to elements of the antigen presenting machinery and checkpoint blockade. This highlights the potential importance of broad immuno-genomic profiling of patients that are candidates for receiving immunotherapy. We are continuing to increase our cohort size to observe both how well the neoantigen burden holds to anti-PD-1 response and to identify additional mechanisms for immune evasion. Citation Format: Jie Wang, Sean Michael Boyle, Christina Lee, Eric Levy, Zeid Rusan, Sekwon Jang, Richard Chen. Molecular profiling of anti-PD-1 treated melanoma patients reveals importance of assessing neoantigen burden and tumor escape mechanisms for clinical treatment [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 5710.

Flies carrying a kcc loss-of-function mutation are more seizure-susceptible than wild-type flies. The kcc gene is the highly conserved Drosophila melanogaster ortholog of K+/Cl− cotransporter genes thought to be expressed in all animal cell types. Here, we examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNA interference (RNAi) of kcc in various sets of neurons was sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was particularly effective in causing seizure-sensitivity. Knockdown of kcc in glia or neurons during development caused a reduction in seizure induction threshold, cell swelling, and brain volume increase in 24–48 hour old adult flies. Third instar larval peripheral nerves were enlarged when kcc RNAi was expressed in neurons or glia. Results suggest that a threshold of K+/Cl− cotransport dysfunction in the nervous system during development is an important determinant of seizure-susceptibility in Drosophila. The findings presented are the first attributing a causative role for glial cation-chloride cotransporters in seizures and epileptogenesis. The importance of elucidating glial cell contributions to seizure disorders and the utility of Drosophila models is discussed.

Despite the frequency of seizure disorders in the human population, the genetic and physiological basis for these defects has been difficult to resolve. Although many genetic contributions to seizure susceptibility have been identified, these involve disparate biological processes, many of which are not neural specific. The large number and heterogeneous nature of the genes involved makes it difficult to understand the complex factors underlying the etiology of seizure disorders. Examining the effect known genetic mutations have on seizure susceptibility is one approach that may prove fruitful. This approach may be helpful in both understanding how different physiological processes affect seizure susceptibility and identifying novel therapeutic treatments. We review here factors contributing to seizure susceptibility in Drosophila, a genetically tractable system that provides a model for human seizure disorders. Seizure-like neuronal activities and behaviors in the fruit fly are described, as well as a set of mutations that exhibit features resembling some human epilepsies and render the fly sensitive to seizures. Especially interesting are descriptions of a novel class of mutations that are second-site mutations that act as seizure suppressors. These mutations revert epilepsy phenotypes back to the wild-type range of seizure susceptibility. The genes responsible for seizure suppression are cloned with the goal of identifying targets for lead compounds that may be developed into new antiepileptic drugs.