Jeffrey Roskes

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163+PD-L1- myeloid cells and CD8+FoxP3+PD-1low/mid T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution.

The discovery of the Higgs boson in 2012, by the ATLAS and CMS experiments, was a success achieved with only a percent of the entire dataset foreseen for the LHC. It opened a landscape of possibilities in the study of Higgs boson properties, Electroweak Symmetry breaking and the Standard Model in general, as well as new avenues in probing new physics beyond the Standard Model. Six years after the discovery, with a conspicuously larger dataset collected during LHC Run 2 at a 13 TeV centre-of-mass energy, the theory and experimental particle physics communities have started a meticulous exploration of the potential for precision measurements of its properties. This includes studies of Higgs boson production and decays processes, the search for rare decays and production modes, high energy observables, and searches for an extended electroweak symmetry breaking sector. This report summarises the potential reach and opportunities in Higgs physics during the High Luminosity phase of the LHC, with an expected dataset of pp collisions at 14 TeV, corresponding to an integrated luminosity of 3 ab$^{-1}$. These studies are performed in light of the most recent analyses from LHC collaborations and the latest theoretical developments. The potential of an LHC upgrade, colliding protons at a centre-of-mass energy of 27 TeV and producing a dataset corresponding to an integrated luminosity of 15 ab$^{-1}$, is also discussed.

We present an extension of the JHUGen and MELA framework, which includes an event generator and library for the matrix element analysis. It enables simulation, optimal discrimination, reweighting techniques, and analysis of a bosonic resonance and the triple and quartic gauge boson interactions with the most general anomalous couplings. The new features, which become especially relevant at the current stage of LHC data taking, are the simulation of gluon fusion and vector boson fusion in the off-shell region, associated $ZH$ production at NLO QCD including the $gg$ initial state, and the simulation of a second spin-zero resonance. We also quote translations of the anomalous coupling measurements into constraints on dimension-six operators of an effective field theory. Some of the new features are illustrated with projections for experimental measurements with the full LHC and HL-LHC datasets.

We present a study of Higgs boson production in vector boson fusion and in association with a vector boson and its decay to two vector bosons, with a focus on the treatment of virtual loops and virtual photons. Our analysis is performed with the JHU generator framework. Comparisons are made to several other frameworks, and the results are expressed in terms of an effective field theory. New features of this study include a proposal on how to handle singularities involving Higgs boson decays to light fermions via photons, calculation of the partial Higgs boson width in the presence of anomalous couplings to photons, a comparison of the next-to-leading-order electroweak corrections to effects from effective couplings, and phenomenological observations regarding the special role of intermediate photons in analysis of LHC data in the effective field theory framework. Some of these features are illustrated with projections for experimental measurements with the full LHC and HL-LHC datasets.

Multispectral, multiplex immunofluorescence (mIF) microscopy has been used to great effect in research to identify cellular co-expression profiles and spatial relationships within tissue, providing a myriad of diagnostic advantages. As these technologies mature, it is essential that image data from mIF microscopes is reproducible and standardizable across devices. We sought to characterize and correct differences in illumination intensity and spectral sensitivity between three multispectral microscopes. We scanned eight melanoma tissue samples twice on each microscope and calculated their average tissue region flux intensities. We found a baseline average standard deviation of 29.9% across all microscopes, scans, and samples, which was reduced to 13.9% after applying sample-specific corrections accounting for differences in the tissue shown on each slide. We used a basic calibration model to correct sample- and microscope-specific effects on overall brightness and relative brightness as a function of the image layer. We tested the generalizability of the calibration procedure and found that applying corrections to independent validation subsets of the samples reduced the variation to 2.9 ± 0.03%. Variations in the unmixed marker expressions were reduced from 15.8% to 4.4% by correcting the raw images to a single reference microscope. Our findings show that mIF microscopes can be standardized for use in clinical pathology laboratories using a relatively simple correction model.

<h3>Background</h3> PD-1/PD-L1 pathway blockade has improved survival in patients with advanced NSCLC. Neoadjuvant (pre-operative) anti-PD-1 plus chemotherapy was also recently approved for patients with resectable stage II/III NSCLC. However, among patients receiving neoadjuvant anti-PD-1-based therapy, only 33–45% achieved a major pathologic response (MPR, ≤10% of residual viable tumor), highlighting the need for biomarkers predicting response.^1,3 Based upon recent results in advanced melanoma showing that CD8+FoxP3+ cells were strongly associated with therapeutic response,⁴ we hypothesized that these cells would also be predictive of response in resectable NSCLC. Additionally, we performed single-cell RNA sequencing (ssRNAseq) to define the phenotype of CD8+FoxP3+ cells, given reports suggesting an immunosuppressive role. <h3>Methods</h3> Pre-treatment formalin-fixed paraffin-embedded tumor specimens from the first-in-human clinical trial of neoadjuvant anti-PD-1 (nivolumab) +/- anti-CTLA-4 (ipilimumab) in NSCLC (NCT02259621)^1,3 were stained with a 6-marker multiplex immunofluorescence mIF panel (PD-1, PD-L1, CD8, CD163, FoxP3, and cytokeratin). Eight specimens were from patients demonstrating MPRs and 17 were from patients with non-MPRs. The densities of immune cell populations within the tumor microenvironment (TME) were analyzed using the AstroPath platform and the area under the receiver operating characteristic curve (AUC) for each possible cell phenotype was calculated for predicting MPR.⁴ The association for cell phenotypes with event-free survival (EFS) and overall survival (OS) was determined using the log-rank test. scRNAseq analyses were performed on freshly collected CD3+ TIL from 15 of the same NSCLC patients. T-cells were clustered by UMAP and were queried for co-expression of CD8 and FoxP3. <h3>Results</h3> The density of CD8+FoxP3+ T-cells was significantly elevated in patients achieving MPR (AUC=0.78, p=0.014, N=25). This association was strongest in the PD-1(+) (AUC=0.83, p=0.004) and PD-L1(-) (AUC=0.81, p=0.007) subsets. The AUCs for CD8+FoxP3+ cells were stronger than any other cell phenotype labeled by this 6-plex mIF assay. Patients whose TMEs contained CD8+FoxP3+ cells (n=18) when compared to those lacking this phenotype (n=7) had improved EFS and OS (41 vs. 8 months, p=0.041; and 26 vs. 8 months, p=0.074, respectively). scRNAseq studies of the CD8+FoxP3+ T-cell subset revealed a transcriptome compatible with a highly-activated, cytotoxic phenotype (<i>CCL5, CD8A, GZMB, NKG7, CTSW, CD8B</i>, <i>LINC02446</i>, <i>GZMK</i> all highly expressed). <h3>Conclusions</h3> CD8+FoxP3+ T-cells in the NSCLC TME do not represent immunosuppressive cells, as has been previously reported, but instead represent highly-potent early, effector T-cells. When detected by mIF in pre-treatment NSCLC tumor specimens, these cells associate with major pathologic response and improved survival outcomes following neoadjuvant anti-PD-1. <h3>References</h3> Forde, P. M., Chaft, J. E., Smith, K. N., Anagnostou, V., Cottrell, T. R., Hellmann, M. D., Zahurak, M., Yang, S. C., Jones, D. R., Broderick, S., Battafarano, R. J., Velez, M. J., Rekhtman, N., Olah, Z., Naidoo, J., Marrone, K. A., Verde, F., Guo, H., Zhang, J., Caushi, J. X., … Pardoll, D. M. Neoadjuvant PD-1 Blockade in Resectable Lung Cancer. <i>N Engl J Med</i>. 2018; <b>378</b>, 1976–1986. Forde, P. M., Spicer, J., Lu, S., Provencio, M., Mitsudomi, T., Awad, M. M., Felip, E., Broderick, S. R., Brahmer, J. R., Swanson, S. J., Kerr, K., Wang, C., Ciuleanu, T.-E., Saylors, G. B., Tanaka, F., Ito, H., Chen, K.-N., Liberman, M., Vokes, E. E., … Girard, N. Neoadjuvant Nivolumab plus Chemotherapy in Resectable Lung Cancer. <i>N Engl J Med</i>. 2022; <b>386</b>, 1973–1985. Reuss, J. E., Anagnostou, V., Cottrell, T. R., Smith, K. N., Verde, F., Zahurak, M., Lanis, M., Murray, J. C., Chan, H. Y., McCarthy, C., Wang, D., White, J. R., Yang, S., Battafarano, R., Broderick, S., Bush, E., Brock, M., Ha, J., Jones, D., Merghoub, T., … Forde, P. M. (2020). Neoadjuvant nivolumab plus ipilimumab in resectable non-small cell lung cancer. <i>J Immunother Cancer</i>. 2020; <b>8</b>, e001282. Berry, S., Giraldo, N. A., Green, B. F., Cottrell, T. R., Stein, J. E., Engle, E. L., Xu, H., Ogurtsova, A., Roberts, C., Wang, D., Nguyen, P., Zhu, Q., Soto-Diaz, S., Loyola, J., Sander, I. B., Wong, P. F., Jessel, S., Doyle, J., Signer, D., … Taube, J. M. Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD1 blockade. <i>Science</i>. 2021; <b>372</b>, eaba2609. <h3>Ethics Approval</h3> This study was conducted in accordance with the Declaration of Helsinki and was performed following Johns Hopkins University IRB approval (#NA_00085595). This protocol allows for the retrieval of tissue from archives from patients who signed an informed written consent or with waiver of consent.

We present an extension of the JHUGen and MELA framework, which includes an event generator and library for the matrix element analysis. It enables simulation, optimal discrimination, reweighting techniques, and analysis of a bosonic resonance and the triple and quartic gauge boson interactions with the most general anomalous couplings. The new features, which become especially relevant at the current stage of LHC data taking, are the simulation of gluon fusion and vector boson fusion in the off-shell region, associated $ZH$ production at NLO QCD including the $gg$ initial state, and the simulation of a second spin-zero resonance. We also quote translations of the anomalous coupling measurements into constraints on dimension-six operators of an effective field theory. Some of the new features are illustrated with projections for experimental measurements with the full LHC and HL-LHC datasets.

This thesis develops ideas and advanced techniques for studying the Higgs boson’s interactions with known matter and force particles, including simultaneous measurement of the Higgs boson’s associated production and decay, while allowing for multiple parameters sensitive to new phenomena.

This chapter provides a detailed description of the alignment and calibration procedure for the CMS detector’s silicon tracker. The algorithms used to align the detector are presented and the plots used to validate the results of that alignment are shown. The chapter also explains methods for detecting systematic misalignments of the tracker and concludes with a summary of the tracker alignment performance for the 2015–2018 run period.

This chapter describes the Higgs boson’s interactions with other particles and provides a theoretical basis for the experimental analyses discussed in the next chapter. Special attention is given to matrix element calculations and their uses in producing Monte Carlo simulations of Higgs boson events, reweighting those simulations, and distinguishing between hypotheses.

This chapter presents a series of analyses, increasing in complexity and precision, to measure anomalous couplings of the Higgs boson. The later measurements in the sequence use the Higgs boson’s decay to four leptons onshell and offshell, its production in association with jets or top quarks, to measure its interactions with vector bosons and fermions. Advanced statistical methods are employed to measure multiple anomalous couplings simultaneously, taking into account the complicated contributions of quantum mechanical interference.

This chapter describes the experimental setup for the analyses described in the thesis. The Large Hadron Collider and the various parts of the Compact Muon Solenoid experiment, as well as the data collection procedure, are presented.

This chapter gives an overview of the Standard Model of particle physics, and in particular the Higgs boson, from a historical perspective. The key discoveries that led to the formulation of the Standard Model are presented, concluding with the limitations of the Standard Model and the motivations for the analyses described in later chapters of this thesis are described.

This chapter summarizes the results obtained in the previous chapters and presents several extensions of this analysis that can be performed in the future. Projections of an analysis identical to the current one using the data to be collected over the next few years are shown, and possible improvements to the analysis, which will become feasible with more data, are described.

We present a study of Higgs boson production in vector boson fusion and in association with a vector boson and its decay to two vector bosons, with a focus on the treatment of virtual loops and virtual photons. Our analysis is performed with the JHU generator framework. Comparisons are made to several other frameworks, and the results are expressed in terms of an effective field theory. New features of this study include a proposal on how to handle singularities involving Higgs boson decays to light fermions via photons, calculation of the partial Higgs boson width in the presence of anomalous couplings to photons, a comparison of the next-to-leading-order electroweak corrections to effects from effective couplings, and phenomenological observations regarding the special role of intermediate photons in analysis of LHC data in the effective field theory framework. Some of these features are illustrated with projections for experimental measurements with the full LHC and HL-LHC datasets.