Doga Gulhan

Abstract Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.

PURPOSE Despite the tissue-agnostic approval of pembrolizumab in mismatch repair deficient (MMRD) solid tumors, important unanswered questions remain about the role of immune checkpoint blockade in mismatch repair–proficient (MMRP) and –deficient endometrial cancer (EC). METHODS This phase II study evaluated the PD-L1 inhibitor avelumab in two cohorts of patients with EC: (1) MMRD/ POLE (polymerase ε) cohort, as defined by immunohistochemical (IHC) loss of expression of one or more mismatch repair (MMR) proteins and/or documented mutation in the exonuclease domain of POLE; and (2) MMRP cohort with normal IHC expression of all MMR proteins. Coprimary end points were objective response (OR) and progression-free survival at 6 months (PFS6). Avelumab 10 mg/kg intravenously was administered every 2 weeks until progression or unacceptable toxicity. RESULTS Thirty-three patients were enrolled. No patient with POLE-mutated tumor was enrolled in the MMRD cohort, and all MMRP tumors were not POLE-mutated. The MMRP cohort was closed at the first stage because of futility: Only one of 16 patients exhibited both OR and PFS6 responses. The MMRD cohort met the predefined primary end point of four ORs after accrual of only 17 patients; of 15 patients who initiated avelumab, four exhibited OR (one complete response, three partial responses; OR rate, 26.7%; 95% CI, 7.8% to 55.1%) and six (including all four ORs) PFS6 responses (PFS6, 40.0%; 95% CI, 16.3% to 66.7%), four of which are ongoing as of data cutoff date. Responses were observed in the absence of PD-L1 expression. IHC captured all cases of MMRD subsequently determined by polymerase chain reaction or genomically via targeted sequencing. CONCLUSION Avelumab exhibited promising activity in MMRD EC regardless of PD-L1 status. IHC for MMR assessment is a useful tool for patient selection. The activity of avelumab in MMRP/non- POLE–mutated ECs was low.

Abstract Mutational activation of KRAS promotes the initiation and progression of cancers, especially in the colorectum, pancreas, lung, and blood plasma, with varying prevalence of specific activating missense mutations. Although epidemiological studies connect specific alleles to clinical outcomes, the mechanisms underlying the distinct clinical characteristics of mutant KRAS alleles are unclear. Here, we analyze 13,492 samples from these four tumor types to examine allele- and tissue-specific genetic properties associated with oncogenic KRAS mutations. The prevalence of known mutagenic mechanisms partially explains the observed spectrum of KRAS activating mutations. However, there are substantial differences between the observed and predicted frequencies for many alleles, suggesting that biological selection underlies the tissue-specific frequencies of mutant alleles. Consistent with experimental studies that have identified distinct signaling properties associated with each mutant form of KRAS, our genetic analysis reveals that each KRAS allele is associated with a distinct tissue-specific comutation network. Moreover, we identify tissue-specific genetic dependencies associated with specific mutant KRAS alleles. Overall, this analysis demonstrates that the genetic interactions of oncogenic KRAS mutations are allele- and tissue-specific, underscoring the complexity that drives their clinical consequences.

We characterize the landscape of somatic mutations-mutations occurring after fertilization-in the human brain using ultra-deep (~250×) whole-genome sequencing of prefrontal cortex from 59 donors with autism spectrum disorder (ASD) and 15 control donors. We observe a mean of 26 somatic single-nucleotide variants per brain present in ≥4% of cells, with enrichment of mutations in coding and putative regulatory regions. Our analysis reveals that the first cell division after fertilization produces ~3.4 mutations, followed by 2-3 mutations in subsequent generations. This suggests that a typical individual possesses ~80 somatic single-nucleotide variants present in ≥2% of cells-comparable to the number of de novo germline mutations per generation-with about half of individuals having at least one potentially function-altering somatic mutation somewhere in the cortex. ASD brains show an excess of somatic mutations in neural enhancer sequences compared with controls, suggesting that mosaic enhancer mutations may contribute to ASD risk.

Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure1-3 and the evolutionary trajectories4 of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.

We propose and explore a new hybrid approach to jet quenching in a strongly coupled medium. The basis of this phenomenological approach is to treat physics processes at different energy scales differently. The high-$Q^2$ processes associated with the QCD evolution of the jet from production as a single hard parton through its fragmentation, up to but not including hadronization, are treated perturbatively. The interactions between the partons in the shower and the deconfined matter within which they find themselves lead to energy loss. The momentum scales associated with the medium (of the order of the temperature) and with typical interactions between partons in the shower and the medium are sufficiently soft that strongly coupled physics plays an important role in energy loss. We model these interactions using qualitative insights from holographic calculations of the energy loss of energetic light quarks and gluons in a strongly coupled plasma, obtained via gauge/gravity duality. We embed this hybrid model into a hydrodynamic description of the spacetime evolution of the hot QCD matter produced in heavy ion collisions and confront its predictions with jet data from the LHC. The holographic expression for the energy loss of a light quark or gluon that we incorporate in our hybrid model is parametrized by a stopping distance. We find very good agreement with all the data as long as we choose a stopping distance that is comparable to but somewhat longer than that in ${\cal N}=4$ supersymmetric Yang-Mills theory. For comparison, we also construct alternative models in which energy loss occurs as it would if the plasma were weakly coupled. We close with suggestions of observables that could provide more incisive evidence for, or against, the importance of strongly coupled physics in jet quenching.

Within the context of a hybrid strong/weak coupling model of jet quenching, we study the modification of the angular distribution of the energy within jets in heavy ion collisions, as partons within jet showers lose energy and get kicked as they traverse the strongly coupled plasma produced in the collision. To describe the dynamics transverse to the jet axis, we add the effects of transverse momentum broadening into our hybrid construction, introducing a parameter $$ K\equiv \widehat{q}/{T}^3 $$ that governs its magnitude. We show that, because of the quenching of the energy of partons within a jet, even when K ≠ 0 the jets that survive with some specified energy in the final state are narrower than jets with that energy in proton-proton collisions. For this reason, many standard observables are rather insensitive to K. We propose a new differential jet shape ratio observable in which the effects of transverse momentum broadening are apparent. We also analyze the response of the medium to the passage of the jet through it, noting that the momentum lost by the jet appears as the momentum of a wake in the medium. After freezeout this wake becomes soft particles with a broad angular distribution but with net momentum in the jet direction, meaning that the wake contributes to what is reconstructed as a jet. This effect must therefore be included in any description of the angular structure of the soft component of a jet. We show that the particles coming from the response of the medium to the momentum and energy deposited in it leads to a correlation between the momentum of soft particles well separated from the jet in angle with the direction of the jet momentum, and find qualitative but not quantitative agreement with experimental data on observables designed to extract such a correlation. More generally, by confronting the results that we obtain upon introducing transverse momentum broadening and the response of the medium to the jet with available jet data, we highlight the importance of these processes for understanding the internal, soft, angular structure of high energy jets.

Whole-genome sequencing of DNA from single cells has the potential to reshape our understanding of mutational heterogeneity in normal and diseased tissues. However, a major difficulty is distinguishing amplification artifacts from biologically derived somatic mutations. Here, we describe linked-read analysis (LiRA), a method that accurately identifies somatic single-nucleotide variants (sSNVs) by using read-level phasing with nearby germline heterozygous polymorphisms, thereby enabling the characterization of mutational signatures and estimation of somatic mutation rates in single cells. Linked-read analysis is a method for analyzing single-cell DNA-sequencing data that accurately identifies somatic single-nucleotide variants by using read-level phasing with nearby germline variants, enabling the characterization of mutational signatures and estimation of somatic mutation rates in single cells.

Accurate somatic mutation detection from single-cell DNA sequencing is challenging due to amplification-related artifacts. To reduce this artifact burden, an improved amplification technique, primary template-directed amplification (PTA), was recently introduced. We analyzed whole-genome sequencing data from 52 PTA-amplified single neurons using SCAN2, a new genotyper we developed to leverage mutation signatures and allele balance in identifying somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) in PTA data. Our analysis confirms an increase in nonclonal somatic mutation in single neurons with age, but revises the estimated rate of this accumulation to 16 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels increase by at least three per year per neuron and are enriched in functional regions of the genome such as enhancers and promoters. Our data suggest that indels in gene-regulatory elements have a considerable effect on genome integrity in human neurons.

We analyzed 131 human brains (44 neurotypical, 19 with Tourette syndrome, 9 with schizophrenia, and 59 with autism) for somatic mutations after whole genome sequencing to a depth of more than 200×. Typically, brains had 20 to 60 detectable single-nucleotide mutations, but ~6% of brains harbored hundreds of somatic mutations. Hypermutability was associated with age and damaging mutations in genes implicated in cancers and, in some brains, reflected in vivo clonal expansions. Somatic duplications, likely arising during development, were found in ~5% of normal and diseased brains, reflecting background mutagenesis. Brains with autism were associated with mutations creating putative transcription factor binding motifs in enhancer-like regions in the developing brain. The top-ranked affected motifs corresponded to MEIS (myeloid ectopic viral integration site) transcription factors, suggesting a potential link between their involvement in gene regulation and autism.

The structure of the human neocortex underlies species-specific traits and reflects intricate developmental programs. Here we sought to reconstruct processes that occur during early development by sampling adult human tissues. We analysed neocortical clones in a post-mortem human brain through a comprehensive assessment of brain somatic mosaicism, acting as neutral lineage recorders1,2. We combined the sampling of 25 distinct anatomic locations with deep whole-genome sequencing in a neurotypical deceased individual and confirmed results with 5 samples collected from each of three additional donors. We identified 259 bona fide mosaic variants from the index case, then deconvolved distinct geographical, cell-type and clade organizations across the brain and other organs. We found that clones derived after the accumulation of 90–200 progenitors in the cerebral cortex tended to respect the midline axis, well before the anterior–posterior or ventral–dorsal axes, representing a secondary hierarchy following the overall patterning of forebrain and hindbrain domains. Clones across neocortically derived cells were consistent with a dual origin from both dorsal and ventral cellular populations, similar to rodents, whereas the microglia lineage appeared distinct from other resident brain cells. Our data provide a comprehensive analysis of brain somatic mosaicism across the neocortex and demonstrate cellular origins and progenitor distribution patterns within the human brain. A comprehensive analysis of brain somatic mosaicism across the neocortex demonstrates the origins and distribution patterns of cells within the human brain.

Mutational signature analysis is a recent computational approach for interpreting somatic mutations in the genome. Its application to cancer data has enhanced our understanding of mutational forces driving tumorigenesis and demonstrated its potential to inform prognosis and treatment decisions. However, methodological challenges remain for discovering new signatures and assigning proper weights to existing signatures, thereby hindering broader clinical applications. Here we present Mutational Signature Calculator (MuSiCal), a rigorous analytical framework with algorithms that solve major problems in the standard workflow. Our simulation studies demonstrate that MuSiCal outperforms state-of-the-art algorithms for both signature discovery and assignment. By reanalyzing more than 2,700 cancer genomes, we provide an improved catalog of signatures and their assignments, discover nine indel signatures absent in the current catalog, resolve long-standing issues with the ambiguous 'flat' signatures and give insights into signatures with unknown etiologies. We expect MuSiCal and the improved catalog to be a step towards establishing best practices for mutational signature analysis.

We have previously introduced a hybrid strong/weak coupling model for jet quenching in heavy ion collisions in which we describe the production and fragmentation of jets at weak coupling, using Pythia, and describe the rate at which each parton in the jet shower loses energy as it propagates through the strongly coupled plasma, dE/dx, using an expression computed holographically at strong coupling. The model has a single free parameter that we fit to a single experimental measurement. We then confront our model with experimental data on many other jet observables, focusing in this paper on boson-jet observables, finding that it provides a good description of present jet data. Next, we provide the predictions of our hybrid model for many measurements to come, including those for inclusive jet, dijet, photon-jet and Z-jet observables in heavy ion collisions with energy $$ \sqrt{s}=5:02 $$ ATeV coming soon at the LHC. As the statistical uncertainties on near-future measurements of photon-jet observables are expected to be much smaller than those in present data, with about an order of magnitude more photon-jet events expected, predictions for these observables are particularly important. We find that most of our pre- and post-dictions do not depend sensitively on the form we choose for the rate of energy loss dE/dx of the partons in the shower. This gives our predictions considerable robustness. To better discriminate between possible forms for the rate of energy loss, though, we must turn to intrajet observables. Here, we focus on ratios of fragmentation functions. We close with a suggestion for a particular ratio, between the fragmentation functions of inclusive and associated jets with the same kinematics in the same collisions, which is particularly sensitive to the x- and E-dependence of dE/dx, and hence may be used to learn which mechanism of parton energy loss best describes the quenching of jets.

In a trial of patients with high grade serous ovarian cancer (HGSOC), addition of the ATR inhibitor berzosertib to gemcitabine improved progression free survival (PFS) compared to gemcitabine alone but biomarkers predictive of treatment are lacking. Here we report a candidate biomarker of response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in HGSOC ovarian cancer. Patients with replication stress (RS)-high tumors (n = 27), defined as harboring at least one genomic RS alteration related to loss of RB pathway regulation and/or oncogene-induced replication stress achieve significantly prolonged PFS (HR = 0.38, 90% CI, 0.17-0.86) on gemcitabine monotherapy compared to those with tumors without such alterations (defined as RS-low, n = 30). However, addition of berzosertib to gemcitabine benefits only patients with RS-low tumors (gemcitabine/berzosertib HR 0.34, 90% CI, 0.13-0.86) and not patients with RS-high tumors (HR 1.11, 90% CI, 0.47-2.62). Our findings support the notion that the exacerbation of RS by gemcitabine monotherapy is adequate for lethality in RS-high tumors. Conversely, for RS-low tumors addition of berzosertib-mediated ATR inhibition to gemcitabine is necessary for lethality to occur. Independent prospective validation of this biomarker is required.

Abstract Whole chromosome and arm-level copy number alterations occur at high frequencies in tumors, but their selective advantages, if any, are poorly understood. Here, utilizing unbiased whole chromosome genetic screens combined with in vitro evolution to generate arm- and subarm-level events, we iteratively selected the fittest karyotypes from aneuploidized human renal and mammary epithelial cells. Proliferation-based karyotype selection in these epithelial lines modeled tissue-specific tumor aneuploidy patterns in patient cohorts in the absence of driver mutations. Hi-C-based translocation mapping revealed that arm-level events usually emerged in multiples of two via centromeric translocations and occurred more frequently in tetraploids than diploids, contributing to the increased diversity in evolving tetraploid populations. Isogenic clonal lineages enabled elucidation of pro-tumorigenic mechanisms associated with common copy number alterations, revealing Notch signaling potentiation as a driver of 1q gain in breast cancer. We propose that intrinsic, tissue-specific proliferative effects underlie tumor copy number patterns in cancer.

Retrotransposons can cause somatic genome variation in the human nervous system, which is hypothesized to have relevance to brain development and neuropsychiatric disease. However, the detection of individual somatic mobile element insertions presents a difficult signal-to-noise problem. Using a machine-learning method (RetroSom) and deep whole-genome sequencing, we analyzed L1 and Alu retrotransposition in sorted neurons and glia from human brains. We characterized two brain-specific L1 insertions in neurons and glia from a donor with schizophrenia. There was anatomical distribution of the L1 insertions in neurons and glia across both hemispheres, indicating retrotransposition occurred during early embryogenesis. Both insertions were within the introns of genes (CNNM2 and FRMD4A) inside genomic loci associated with neuropsychiatric disorders. Proof-of-principle experiments revealed these L1 insertions significantly reduced gene expression. These results demonstrate that RetroSom has broad applications for studies of brain development and may provide insight into the possible pathological effects of somatic retrotransposition.

Abstract Purpose: The identification of patients with homologous recombination deficiency (HRD) beyond BRCA1/2 mutations is an urgent task, as they may benefit from PARP inhibitors. We have previously developed a method to detect mutational signature 3 (Sig3), termed SigMA, associated with HRD from clinical panel sequencing data, that is able to reliably detect HRD from the limited sequencing data derived from gene-focused panel sequencing. Experimental Design: We apply this method to patients from two independent datasets: (i) high-grade serous ovarian cancer and triple-negative breast cancer (TNBC) from a phase Ib trial of the PARP inhibitor olaparib in combination with the PI3K inhibitor buparlisib (BKM120; NCT01623349), and (ii) TNBC patients who received neoadjuvant olaparib in the phase II PETREMAC trial (NCT02624973). Results: We find that Sig3 as detected by SigMA is positively associated with improved progression-free survival and objective responses. In addition, comparison of Sig3 detection in panel and exome-sequencing data from the same patient samples demonstrated highly concordant results and superior performance in comparison with the genomic instability score. Conclusions: Our analyses demonstrate that HRD can be detected reliably from panel-sequencing data that are obtained as part of routine clinical care, and that this approach can identify patients beyond those with germline BRCA1/2mut who might benefit from PARP inhibitors. Prospective clinical utility testing is warranted.

Abstract Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53−/− and BRCA1−/− epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. Significance: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.

Homologous recombination DNA-repair deficiency (HRD) is a common driver of genomic instability and confers a therapeutic vulnerability in cancer. The accurate detection of somatic allelic imbalances (AIs) has been limited by methods focused on BRCA1/2 mutations and using mixtures of cancer types. Using pan-cancer data, we revealed distinct patterns of AIs in high-grade serous ovarian cancer (HGSC). We used machine learning and statistics to generate improved criteria to identify HRD in HGSC (ovaHRDscar). ovaHRDscar significantly predicted clinical outcomes in three independent patient cohorts with higher precision than previous methods. Characterization of 98 spatiotemporally distinct metastatic samples revealed low intra-patient variation and indicated the primary tumor as the preferred site for clinical sampling in HGSC. Further, our approach improved the prediction of clinical outcomes in triple-negative breast cancer (tnbcHRDscar), validated in two independent patient cohorts. In conclusion, our tumor-specific, systematic approach has the potential to improve patient selection for HR-targeted therapies.

Centrality selection has been observed to have a large effect on jet observables in pPb collisions at the Large Hadron Collider, stronger than that predicted by the nuclear modification of parton densities. We study to which extent simple considerations of energy–momentum conservation which link the hard process with the underlying event that provides the centrality estimator, affect jets observables in such collisions. We develop a simplistic approach that considers first the production of jets in a pp collision as described by PYTHIA. From each pp collision, the value of the energy of the parton from the proton participating in the hard scattering is extracted. Then, the underlying event is generated simulating a pPb collision through HIJING, but with the energy of the proton decreased according to the value extracted in the previous step, and both collisions are added. This model is able to capture the bulk of the centrality effect for central to semicentral collisions, for the two available sets of data: dijets from the CMS Collaboration and single jets from the ATLAS Collaboration. As expected, the model fails for peripheral collisions where very few nucleons from Pb participate.

5512 Background: The multicenter, open-label, randomized phase 2 NCI-9944 study (NCT02595892) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (HR = 0.57, one-sided log-rank p = 0.044, which met the one-sided significance level of 0.1 used for sample size calculation). Final overall survival (OS) and corresponding biomarker analyses are reported here. Methods: Patients (pts) with platinum-resistant high-grade serous ovarian cancer and unlimited previous lines of cytotoxic therapy in the platinum-sensitive setting but no more than one line of cytotoxic therapy in the platinum-resistant setting, were randomized 1:1 to gemcitabine/berzosertib versus gemcitabine alone. Randomization was stratified based on platinum free interval (PFI), PFI≤3 months versus > 3 months. Crossover from gemcitabine to gemcitabine/berzosertib was allowed upon disease progression by RECIST 1.1. OS was a secondary endpoint while preplanned exploratory correlative studies included assessment of DNA repair pathways and replication stress (RS) alterations by targeted gene sequencing and/or immunohistochemistry (IHC). Results: Seventy pts were randomly assigned to treatment with gemcitabine/berzosertib (34 pts) or gemcitabine alone (36 pts); 15 pts crossed over from gemcitabine to gemcitabine/berzosertib. At the final OS analysis (92.9% maturity), median follow-up was 53.2 weeks in the gemcitabine/berzosertib and 43 weeks in the gemcitabine alone groups. Median OS in the intent-to-treat (ITT) population was 59.4 weeks in the gemcitabine/berzosertib group versus 43.0 weeks in the gemcitabine alone group (HR 0.79, 90% CI 0.52-1.2, one-sided p = 0.18). However, when patients who crossed over to gemcitabine/berzosertib were excluded from analysis, a significant OS benefit was observed with gemcitabine/berzosertib (HR 0.60, 90%CI 0.37−0.97); HR was 0.26 (90% CI 0.1−0.7) in pts with PFI≤3 months and 0.89 (90%CI 0.50−1.59) in pts with PFI > 3 months. Furthermore, significant OS benefit was observed in pts with RS-low tumors (HR 0.39, 90%CI 0.17−0.91, defined as tumors harboring no genomic RS alterations related to loss of RB pathway regulation and/or oncogene-induced RS) but not in pts with RS-high tumors (HR 0.74, 90%CI 0.35−1.56). Additional targeted gene sequencing and IHC analyses as well as analyses adjusting for patient crossover will be reported at the meeting. Conclusions: In the ITT population, gemcitabine/berzosertib did not significantly improve OS versus gemcitabine alone. Pts with PFI≤3 months and pts with RS-low tumors may derive a survival advantage from addition of berzosertib to gemcitabine in the platinum-resistant setting. Clinical trial information: NCT02595892 .

Introduction/BackgroundImmune checkpoint inhibitors (ICIs) have limited efficacy in prostate cancer (PCa). Better biomarkers are needed to predict responses to ICIs. We sought to demonstrate that a panel-based mutational signature identifies mismatch repair (MMR) deficient (MMRd) PCa and is a biomarker of response to pembrolizumab.Patients and MethodsClinico-genomic data was obtained for 2,664 patients with PCa sequenced at Dana-Farber Cancer Institute (DFCI) and Memorial Sloan Kettering (MSK). Clinical outcomes were collected for patients with metastatic castration-resistant PCa (mCRPC) treated with pembrolizumab at DFCI. SigMA was used to characterize tumors as MMRd or MMR proficient (MMRp). The concordance between MMRd with microsatellite instability (MSI-H) was assessed. Radiographic progression-free survival (rPFS) and overall survival (OS) were collected for patients treated with pembrolizumab. Event-time distributions were estimated using Kaplan-Meier methodology.ResultsAcross both cohorts, 100% (DFCI: 12/12; MSK: 43/43) of MSI-H tumors were MMRd. However, 14% (2/14) and 9.1% (6/66) of MMRd tumors in the DFCI and MSK cohorts respectively were microsatellite stable (MSS), and 26% (17/66) were MSI-indeterminate in the MSK cohort. Among patients treated with pembrolizumab, those with MMRd (n = 5) versus MMRp (n = 14) mCRPC experienced markedly improved rPFS (HR = 0.088, 95% CI: 0.011-0.70; P = 0.0064) and OS (HR = 0.11, 95% CI: 0.014-0.80; P = 0.010) from start of treatment. Four patients with MMRd experienced remissions of >=2.5 years.ConclusionSigMA detects additional cases of MMRd as compared to MSI testing in PCa and identifies patients likely to experience durable response to pembrolizumab.Micro-AbstractImmune checkpoint inhibitors (ICIs) have limited efficacy in prostate cancer (PCa) and better biomarkers are needed to predict responses to ICIs. In this study, we found that SigMA detects additional cases of mismatch repair deficiency as compared to microsatellite testing in PCa and identifies patients likely to experience durable response to pembrolizumab.

Despite the overall efficacy of immune checkpoint blockade (ICB) for mismatch repair deficiency (MMRD) across tumor types, a sizable fraction of patients with MMRD still do not respond to ICB. We performed mutational signature analysis of panel sequencing data (n = 95) from MMRD cases treated with ICB. We discover that T>C rich single base substitution (SBS) signatures, SBS26 and SBS54 from the COSMIC Mutational Signatures catalog, identify MMRD patients with significantly shorter overall survival. Tumors with a high burden of SBS26 show over expression and enriched mutations of genes involved in double strand break repair and other DNA repair pathways. They also display chromosomal instability (CIN), likely related to replication fork instability, leading to copy number losses that trigger immune evasion. SBS54 is associated with transcriptional activity and not with CIN, defining a distinct subtype. Consistently, cancer cell lines with a high burden of SBS26 and SBS54 are sensitive to treatments targeting pathways related to their proposed etiology. Together, our analysis offers an explanation for the heterogeneous responses to ICB among MMRD patients and supports an SBS signature based predictor as a prognostic biomarker for differential ICB response.

PURPOSE The multicenter, open-label, randomized phase 2 NCI-9944 study ( NCT02595892 ) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (hazard ratio [HR]=0.57, one-sided log-rank P = .044, which met the one-sided significance level of 0.1 used for sample size calculation). METHODS We report here the final overall survival (OS) analysis and biomarker correlations (ATM expression by immunohistochemistry, mutational signature 3 and a genomic biomarker of replication stress) along with post-hoc exploratory analyses to adjust for crossover from gemcitabine to gemcitabine/berzosertib. RESULTS At the data cutoff of January 27, 2023 (>30 months of additional follow-up from the primary analysis), median OS was 59.4 weeks with gemcitabine/berzosertib versus 43.0 weeks with gemcitabine alone (HR 0.79, 90% CI 0.52 to 1.2, one-sided log-rank P = .18). An OS benefit with addition of berzosertib to gemcitabine was suggested in patients stratified into the platinum-free interval ≤3 months (N = 26) subgroup (HR, 0.48, 90% CI 0.22 to 1.01, one-sided log-rank P =.04) and in patients with ATM-negative/low (N = 24) tumors (HR, 0.50, 90% CI 0.23 to 1.08, one-sided log-rank P = .06). CONCLUSION The results of this follow-up analysis continue to support the promise of combined gemcitabine/ATRi therapy in platinum resistant ovarian cancer, an active area of investigation with several ongoing clinical trials.

Immunotherapy has produced disappointing results in recurrent ovarian cancer (OC). However, the prognostic value of tumour-infiltrating lymphocytes (TILs) is largely based on the analysis of treatment-naive tumours. To understand the immunobiology of recurrent cancers, and their evolution, we profiled 170 patient-matched primary-recurrent OC samples from 69 patients of two independent cohorts. By capturing heterogeneous TIL distributions, we identified four immune phenotypes associated with differential prognosis, TILs states and TILs:myeloid networks, which dictate malignant evolution after chemotherapy and recurrence. Notably, recurrent tumours recapitulate the immunogenic patterns of original cancers. Mirroring inflamed human OC, preclinical recurrent Brca1mut tumours maintained activated TILs:dendritic cells (DCs) niches and immunostimulatory tumour-associated macrophages (TAMs). Conversely, recurrent Brca1wt tumours displayed loss of TILs:DCs niches and accumulated immunosuppressive myeloid networks featuring Trem2/ApoEhigh TAMs and Nduf4l2high/Galectin3high malignant states. Our study highlights that persistent immunogenicity in recurrent OC is governed by the crosstalk between dissimilar myeloid cells and TILs, which is BRCA-dependent.

Abstract Although somatic mutations have well-established roles in cancer and certain focal epilepsies, the extent to which mutational mosaicism shapes the developing human brain is poorly understood. Here we characterize the landscape of somatic mutations in the human brain using ultra-deep (~250×) whole-genome sequencing of brains from 59 autism spectrum disorder (ASD) cases and 15 controls. We observe a mean of 26 (±10, range 10-60) somatic single nucleotide variants (sSNVs) per brain present in ≥4% of cells, with enrichment of mutations in coding and putative regulatory regions. Our analysis reveals that the first cell division after fertilization produces ~3.4 mutations, followed by 2-3 mutations in subsequent generations. This rate suggests that a typical individual possesses ~80 sSNVs present in ≥2% of cells—comparable to the number of de novo germline mutations per generation—with about half of individuals having at least one potentially function-altering somatic mutation somewhere in the cortex. Although limited by sample size, ASD brains show an excess of somatic mutations in neural enhancer sequences compared to controls, suggesting that mosaic enhancer mutations may contribute to ASD risk.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

<div>AbstractPurpose:<p>The identification of patients with homologous recombination deficiency (HRD) beyond <i>BRCA1/2</i> mutations is an urgent task, as they may benefit from PARP inhibitors. We have previously developed a method to detect mutational signature 3 (Sig3), termed SigMA, associated with HRD from clinical panel sequencing data, that is able to reliably detect HRD from the limited sequencing data derived from gene-focused panel sequencing.</p>Experimental Design:<p>We apply this method to patients from two independent datasets: (i) high-grade serous ovarian cancer and triple-negative breast cancer (TNBC) from a phase Ib trial of the PARP inhibitor olaparib in combination with the PI3K inhibitor buparlisib (BKM120; NCT01623349), and (ii) TNBC patients who received neoadjuvant olaparib in the phase II PETREMAC trial (NCT02624973).</p>Results:<p>We find that Sig3 as detected by SigMA is positively associated with improved progression-free survival and objective responses. In addition, comparison of Sig3 detection in panel and exome-sequencing data from the same patient samples demonstrated highly concordant results and superior performance in comparison with the genomic instability score.</p>Conclusions:<p>Our analyses demonstrate that HRD can be detected reliably from panel-sequencing data that are obtained as part of routine clinical care, and that this approach can identify patients beyond those with germline <i>BRCA1</i>/<i>2</i>mut who might benefit from PARP inhibitors. Prospective clinical utility testing is warranted.</p></div>

Leiomyosarcoma (LMS) is an aggressive sarcoma for which standard chemotherapies achieve response rates under 30%. There are no effective targeted therapies against LMS. Most LMS are characterized by chromosomal instability (CIN), resulting in part from TP53 and RB1 co-inactivation and DNA damage repair defects. We sought to identify therapeutic targets that could exacerbate intrinsic CIN and DNA damage in LMS, inducing lethal genotoxicity.We performed clinical targeted sequencing in 287 LMS and genome-wide loss-of-function screens in 3 patient-derived LMS cell lines, to identify LMS-specific dependencies. We validated candidate targets by biochemical and cell-response assays in vitro and in seven mouse models.Clinical targeted sequencing revealed a high burden of somatic copy-number alterations (median fraction of the genome altered =0.62) and demonstrated homologous recombination deficiency signatures in 35% of LMS. Genome-wide short hairpin RNA screens demonstrated PRKDC (DNA-PKcs) and RPA2 essentiality, consistent with compensatory nonhomologous end joining (NHEJ) hyper-dependence. DNA-PK inhibitor combinations with unconventionally low-dose doxorubicin had synergistic activity in LMS in vitro models. Combination therapy with peposertib and low-dose doxorubicin (standard or liposomal formulations) inhibited growth of 5 of 7 LMS mouse models without toxicity.Combinations of DNA-PK inhibitors with unconventionally low, sensitizing, doxorubicin dosing showed synergistic effects in LMS in vitro and in vivo models, without discernable toxicity. These findings underscore the relevance of DNA damage repair alterations in LMS pathogenesis and identify dependence on NHEJ as a clinically actionable vulnerability in LMS.

Identifying expressed somatic mutations from single-cell RNA sequencing data de novo is challenging but highly valuable. We propose RESA - Recurrently Expressed SNV Analysis, a computational framework to identify expressed somatic mutations from scRNA-seq data. RESA achieves an average precision of 0.77 on three in silico spike-in datasets. In extensive benchmarking against existing methods using 19 datasets, RESA consistently outperforms them. Furthermore, we applied RESA to analyze intratumor mutational heterogeneity in a melanoma drug resistance dataset. By enabling high precision detection of expressed somatic mutations, RESA substantially enhances the reliability of mutational analysis in scRNA-seq. RESA is available at https://github.com/ShenLab-Genomics/RESA .

Abstract Whole-genome sequencing of DNA from single cells has the potential to reshape our understanding of the mutational heterogeneity in normal and disease tissues. A major difficulty, however, is distinguishing artifactual mutations that arise from DNA isolation and amplification from true mutations. Here, we describe li nked-read a nalysis (LiRA), a method that utilizes phasing of somatic single nucleotide variants with nearby germline variants to identify true mutations, thereby allowing accurate estimation of somatic mutation rates at the single cell level.

We propose a novel hybrid model for jet quenching, including both strong and weak coupling physics where each seems appropriate. Branching in the parton shower is assumed to be perturbative and described by DGLAP evolution, while interactions with the medium result in each parton in the shower losing energy as at strong coupling, as realized holographically. The medium-modified parton shower is embedded into a hydrodynamic evolution of hot QCD plasma and confronted with LHC jet data.

A Correction to this paper has been published: https://doi.org/10.1038/s41593-021-00830-8.

Abstract Mutational signature analysis is a recent computational approach for interpreting somatic mutations in the genome. Its application to cancer data has enhanced our understanding of mutational forces driving tumorigenesis and demonstrated its potential to inform prognosis and treatment decisions. However, methodological challenges remain for discovering new signatures and assigning proper weights to existing signatures, thereby hindering broader clinical applications. Here we present MuSiCal (Mutational Signature Calculator), a rigorous analytical framework with novel algorithms that solves major problems in the standard workflow. Our simulation studies demonstrate that MuSiCal outperforms state-of-the-art algorithms for both signature discovery and assignment. By reanalyzing over 2,700 cancer genomes, we provide an improved catalog of signatures and their assignments, discover nine indel signatures absent in the current catalog, resolve long-standing issues with the ambiguous ‘flat’ signatures, and give insights into signatures with unknown etiologies. We expect MuSiCal and the improved catalog to be a step towards establishing best practices for mutational signature analysis.

Centrality selection has been observed to have a large effect on jet observables in pPb collisions at the Large Hadron Collider, stronger than that predicted by the nuclear modification of parton densities. We study to which extent simple considerations of energy-momentum conservation between the hard process and the underlying event affect jets observables in such collisions. We develop a simplistic approach that considers first the production of jets in a pp collision as described by PYTHIA. From each pp collision, the value of the energy of the parton from the proton participating in the hard scattering is extracted. Then, the underlying event is generated simulating a pPb collision through HIJING, but with the energy of the proton decreased according to the value extracted in the previous step, and both collisions are superimposed. This model is able to capture the bulk of the centrality effect for central to semicentral collisions, for the two available sets of data: dijets from the CMS Collaboration and single jets from the ATLAS Collaboration. As expected, the model fails for peripheral collisions where very few nucleons from Pb participate.

Abstract Introduction: Identification of individuals at risk of developing cancer is key to prevention, particularly among carriers of germline BRCA1/2 mutations. Cancers that develop in this population display loss of the wildtype copy BRCA1/2 and a reproducible pattern of somatic passenger mutations manifesting homologous recombination deficiency (HRD) (Davies et al. 2017). It is not known how much time elapses between the bi-allelic loss of genes responsible for homologous recombination deficiency and the development of cancer. A better understanding of this process and estimating the time between cancer initiation to diagnosis in patients with germline BRCA1/2 mutations would inform early detection strategies in the high-risk group. Methods: Passenger mutations in the cancer genome capture the life history of each cancer. We analyzed patterns of somatic mutations in whole genomes of 27 cancers from patients with germline BRCA1/2 mutations who developed cancers of different tissues with whole genome duplications (WGD). We conducted a meta-analysis of data from the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium (n=14) and the SCANB breast cancer biobank (n=13) (Staaf et al. 2019). Whole genome duplications in tumors allow for approximate timing of onset of HRD. The “molecular clock” mutations in characteristic CpG context accumulate with age and are used to time genomic events. Following bi-allelic BRCA1/2 loss, additional types of somatic mutations accumulate, notably the single point mutation signature SBS3 and HRD-associated indel and rearrangement signatures. Somatic mutations are assigned to the most likely signature which generated them. In tumors that carry it, whole genome duplication enables relative timing of other genomic events. Mutations that occur before and after the duplication are distinguished using allele frequencies (Gerstung et al. 2020). Results: Using this methodology, we observe that HRD-associated SBS3 accounts for as much as 40-60% of the duplicated mutations, suggesting that HRD developed prior to WGD. We use the “molecular clock” to time the intervals before and after WGD, and we estimate that WGD occurs six to thirteen years (interquartile range) prior to the most recent common ancestor of diagnosed tumor. Next, using the relative proportions of SBS3 and clock signatures deduced from post-WGD mutations, we infer how long before WGD SBS3 started to accumulate, providing an even closer bound for HRD development time point than the WGD timing alone. Conclusions: We establish a lower bound on the dormancy period of homologous recombination deficiency prior to cancer diagnosis which would inform early detection strategies in the high-risk group of BRCA1/2 carriers. Citation Format: Dominik Glodzik, Doga Gulhan, Peter J. Park. Timing onset of homologous recombination deficiency before cancer diagnosis in BRCA1/2 mutation carriers. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P002.

Abstract Background Breast cancers in women with a germline BRCA1/2 mutation (gBRCAm) have homologous recombination deficiency (HRD) and are sensitive to therapies causing double-strand DNA breaks. In TBCRC 031 (INFORM), both neoadjuvant cisplatin and doxorubicin/cyclophosphamide (“AC”) in gBRCAm carriers resulted in a complete pathologic response in 18% and 26% respectively in patients with newly diagnosed HER2–negative breast cancer. Herein, we describe molecular features from tumor whole exome (WES) and transcriptome sequencing (RNAseq) associated with response. Methods TBCRC 031 (the INFORM Trial - NCT01670500) was a randomized phase II neoadjuvant trial comparing the efficacy of cisplatin versus AC in gBRCAm carriers with stage I-III HER2–negative breast cancer. Of 118 patients enrolled, 92 patients provided fresh frozen research biopsies, collected prior to chemotherapy initiation, which were subjected to WES and RNAseq. Variants were called using GATK best practices and intratumoral heterogeneity was inferred from mutations and variant allele frequencies using Mutant Allele Tumor Heterogeneity (MATH) scores. Mutational Signature 3 (Sig3) and Genomic Instability Score (GIS) were calculated with SigMA and scarHRD, respectively. RNAseq data were utilized to perform differential gene expression and functional analyses, while cellular deconvolution was performed with CIBERSORTx trained against breast cancer single cell data. Patients were grouped according to their residual cancer burden (RCB) as responders (RCB-0 or 1), or non-responders (RCB-2 or 3). P-values ≤ 0.05 were considered statistically significant and when appropriate, adjustment for multiple testing was performed using a false discovery rate ≤ 5%. Results Of the 92 patients with gBRCAm, 59 (64%) had triple-negative and 33 (36%) were hormone-receptor positive HER2-negative breast cancer, 40 (43%) were classified as responders and 52 (57%) as non-responders. WES analysis revealed that indices of HRD were high (Sig3 exposure predominant and GIS ≥ 42) across most samples irrespective of receptor status and not significantly different among responders and non-responders. In contrast, responders exhibited lower levels of intratumor heterogeneity than non-responders (median MATH 42.9 vs. 33.5, p = 0.01). 223 genes were differentially expressed between responders and non-responders following control for tumor hormone receptor status, BRCA1/2 mutation, and menopausal status. Pathways identified as significantly enriched in upregulated genes were indicative of intrinsic immune activation in responders (e.g., T-cell activation, immune response-signaling, and regulation of leukocyte activation). Cellular deconvolution of the tumor microenvironment confirmed that responders presented a higher proportion of T-cells (p = 0.025) and myeloid cells (p = 0.003) in the tumor samples, while perivascular-like cells were enriched in non-responders (p = 0.034). Conclusion In this analysis of the largest cohort of treatment-naïve gBRCAm breast cancer to date, we found that response to cytotoxic chemotherapy is associated with a transcriptional program of intrinsic immune activation and an increased population of intratumoral T-cells and myeloid cells. Lower levels of intratumor heterogeneity and higher immune activation were associated with response to chemotherapy gBRCAm carriers while HRD scores were not. Citation Format: Felipe Batalini, Doga C. Gulhan, Xanthi Lida Katopodi, Nikolas Kalavros, Antuan Tran, Dimitra Karagkouni, Emily Stern-Gatof, Stuart Schnitt, Judy E. Garber, Gerburg M. Wulf, Peter J. Park, Ioannis Vlachos, Nadine Tung. Intratumor heterogeneity and intrinsic immune activation are associated with response to chemotherapy in BRCA-related breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-12-03.

<div>Abstract<p>Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive <i>TP53</i><sup>−/−</sup> and <i>BRCA1</i><sup>−/−</sup> epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance.</p>Significance:<p>This study shows that <i>BRCA1</i>-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.</p></div>

Supplementary Figure from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

Supplementary Table from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

Supplementary Table from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

<p>Supplementary Figures</p>

<p>Supplementary Materials and methods showing details of the in vitro and genomic analyses.</p>

<p>Supplementary Table 2 showing the positive and negative GSEA results for each cell line.</p>

<p>Supplementary Table 1 showing reagents and antibodies used in the study.</p>

Supplementary Figure from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

Supplementary Table from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

Supplementary Table from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

<div>AbstractPurpose:<p>The identification of patients with homologous recombination deficiency (HRD) beyond <i>BRCA1/2</i> mutations is an urgent task, as they may benefit from PARP inhibitors. We have previously developed a method to detect mutational signature 3 (Sig3), termed SigMA, associated with HRD from clinical panel sequencing data, that is able to reliably detect HRD from the limited sequencing data derived from gene-focused panel sequencing.</p>Experimental Design:<p>We apply this method to patients from two independent datasets: (i) high-grade serous ovarian cancer and triple-negative breast cancer (TNBC) from a phase Ib trial of the PARP inhibitor olaparib in combination with the PI3K inhibitor buparlisib (BKM120; NCT01623349), and (ii) TNBC patients who received neoadjuvant olaparib in the phase II PETREMAC trial (NCT02624973).</p>Results:<p>We find that Sig3 as detected by SigMA is positively associated with improved progression-free survival and objective responses. In addition, comparison of Sig3 detection in panel and exome-sequencing data from the same patient samples demonstrated highly concordant results and superior performance in comparison with the genomic instability score.</p>Conclusions:<p>Our analyses demonstrate that HRD can be detected reliably from panel-sequencing data that are obtained as part of routine clinical care, and that this approach can identify patients beyond those with germline <i>BRCA1</i>/<i>2</i>mut who might benefit from PARP inhibitors. Prospective clinical utility testing is warranted.</p></div>

<p>Supplementary Figures</p>

<p>Supplementary Table 2 showing the positive and negative GSEA results for each cell line.</p>

<p>Supplementary Table 1 showing reagents and antibodies used in the study.</p>

<p>Supplementary Materials and methods showing details of the in vitro and genomic analyses.</p>

e13003 Background: The emergence of somatic KMT2C mutations has been associated with resistance to various treatments, including chemotherapy and endocrine therapy in patients with estrogen receptor-positive breast cancers. The impact of these mutations in patients treated with poly-ADP ribose polymerase inhibitors (PARPi) and phosphatidylinositol-3-kinase inhibitors (PI3Ki) is unknown. Methods: Circulating tumor DNA (ctDNA) was obtained from plasma and peripheral blood at different time points from 38 patients prior to treatment with the combination of the PARPi olaparib with the alpha-specific PI3Ki alpelisib (NCT01623349). CtDNA was also collected immediately before the first cycle and at the time of disease progression. CtDNA underwent deep targeted sequencing (tumor-normal pairs, 397 genes (2Mb) at 25,000x somatic and 10,000x germline) at the Broad Institute of MIT and Harvard. Variants that were exonic, non-synonymous, and predicted to lead to loss-of-function were considered mutations. Cohorts were stratified according to the presence or absence of the genomic alteration, and progression-free survival was compared with the Kaplan-Meier method and log-rank test. A Cox regression was performed using age, tumor type, germline BRCA1/2 mutation status, performance status, and number of previous lines of treatment as covariates. Results: The KMT2C gene was one of the 3 genes most frequently mutated in our cohort, affecting 5 patients (15%) after the exclusion of those with variants with non-deleterious alterations. The other 2 commonly mutated genes were TP53 (76%) and PIK3CA (15%). In the comparison of patients based on these most common mutations, we identified that patients with somatic KMT2C mutations (n=5) have longer mPFS than those that were wild type (n=28): mPFS 386 vs. 151 days, log-rank p = 0.025. Cox proportional hazards model accounting for age, tumor type, germline BRCA1/2 mutation status, performance status, and number of previous lines revealed a hazard ratio of 0.16; p = 0.007. All somatic KMT2C mutations were proximal to the SET domain critical for methyltransferase activity. Upon progression, the KMT2C-mutated clones were suppressed to below the detection level of mutation calling algorithms. Analysis of the raw files revealed that the clones were suppressed but not eliminated. Conclusions: While the emergence of KMT2C has been reported to be associated with cancer progression and resistance to different therapies, patients with deleterious KMT2C mutations in the ctDNA may benefit from therapy with PARPi and PI3Ki.

Abstract Oncogenic mutations in otherwise normal tissue are common in many adult tissues 1,2 . This suggests multiple events need to converge to drive tumourigenesis and that many processes such as tissue differentiation may be protective against carcinogenesis. Within the liver Wnt/β-catenin signalling maintains zonal differentiation during liver homeostasis 3,4 . However, the CTNNB1 oncogene—encoding β-catenin—is also frequently mutated in hepatocellular carcinoma, resulting in aberrant Wnt signalling that promotes cell growth 5,6 . Here we investigated the antagonistic interplay between Wnt-driven growth and differentiation in zonal hepatocyte populations during liver tumorigenesis. We found that β-catenin mutants transiently stimulated growth in zone-1 and -2 hepatocytes within the Wnt-low region of the liver lobule – before inducing a differentiated zone-3 fate. One key feature lacking within the differentiated zone-3 hepatocytes that is often upregulated in human cancer was MYC which can synergise with β-catenin to initiate HCC in mice. When mutant β-catenin and MYC were expressed cell-agnostically across the liver lobule, a subset of mutant hepatocytes were proliferative and tumorigenic. These exhibited a selective upregulation of the zone-2 factors IGFBP2, cyclin D1, and mTOR and a reduction in the levels of Wnt signalling when compared to non-proliferative Wnt-high single cell clones. Inhibition of mTOR signalling with Rapamycin blocked tumourigenesis. Furthermore, restricting β-catenin and MYC mutations with Lgr5-Cre to zone-3 hepatocytes suppressed tumourigenesis. This was associated with high Wnt signalling and the lack of zone-2 factors permissive to growth. Importantly other oncogenic-drivers could promote tumourigenesis from zone-3 hepatocytes but tumours that arose had all lost their zone 3 features. Therefore we propose that zonal identity dictates hepatocyte susceptibility to Wnt-driven tumorigenesis and that escaping Wnt-induced differentiation is essential for liver cancer.

Abstract Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Abstract Background. The PARP inhibitor (PARPi) Olaparib is FDA-approved for treatment of metastatic castration-resistant prostate cancer (mCRPC) harboring a deleterious alteration in specific homologous recombination (HR) genes based on results of the PROfound trial. Qualifying HR gene alterations include BRCA1, BRCA2, ATM (Cohort A), and BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51C, RAD51D (Cohort B). However, there is heterogeneity in the degree to which different HR genes associate with response to PARPi. Better clinical biomarkers are needed to accurately identify patients likely to benefit from PARPi. Methods. Patients with prostate adenocarcinoma who underwent panel sequencing (OncoPanel V3) at the Dana-Farber Cancer Institute were identified. Patients were classified on the presence of pathogenic alterations in Cohort A or B genes from PROfound. The computational tool SigMA was applied to detect a mutational signature of HR deficiency (sHRD). Clinical outcomes in patients treated with Olaparib were collected. Differences between groups were compared using Fisher's exact test and survival analyses were performed using the Kaplan-Meier and Cox Proportional Hazards methods. P<0.05 was considered significant. Results. 492 patients were identified, of which 33% were sHRD. A significantly higher proportion of patients were sHRD in those with Cohort A alterations vs no HR alteration (47% vs 30%, P = 0.0020); however, there was no difference in the rates of sHRD between patients with Cohort B alterations vs no HR alterations. When stratifying patients by individual HR alterations, only those with BRCA1/2 two-copy loss (2CL) were significantly more likely to be sHRD compared to patients with no HR alteration (79% vs 30%; P = 9.1 x 10-8). 31 patients with mCRPC were treated with Olaparib, of which 12 (39%) were sHRD. Of those 12, 8 had BRCA2 2CL, 1 had an ATM 2CL and three had no HR alteration. There was no difference in the rate of PSA50 responses between patients with vs without Cohort A+B alterations (37% vs 8.3%; P = 0.11), while patients with vs without sHRD were significantly more likely to achieve PSA50 (67% vs 0%; P = 6.3 x 10-5). Patients with Cohort A+B alterations (HR = 0.31, P = 0.0046) and sHRD (HR = 0.088; P = 1.9 x 10-5) exhibited significantly improved radiographic progression-free survival (rPFS) to Olaparib compared to those lacking the biomarker. However, on multivariate analysis, including clinical variables associated with improved rPFS to Olaparib, the presence of BRCA2 2CL (the alteration most strongly associated with PARPi response), and sHRD, sHRD was the only variable independently associated with improved rPFS to Olaparib (HR = 0.15, P = 0.012). The potential value of sHRD was exemplified by an sHRD patient without a HR alteration who exhibited a 64% decline in PSA from baseline and an rPFS of 11 months on Olaparib. Conclusion. SigMA-predicted HRD outperformed the current FDA-approved biomarker (deleterious alterations in one of 14 HR genes) for identifying patients with mCRPC likely to benefit from Olaparib. Citation Format: Daniel Boiarsky, Alok Tewari, Doga Gulhan, Ziad Bakouny, Guruprasad Ananda, Hunter Savignano, Gitanjali Lakshminarayana, Toni K Choueiri, Mary-Ellen Taplin, Jacob E Berchuk. A panel-based mutational signature of homologous recombination deficiency associates with response to PARP inhibition in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B009.

<p>Supplementary Figure S7 Computationally assisted analysis of cell culture confluence of representative cell culture images of LMS04 cultures untreated or treated with 2nM doxorubicin, 400nM peposertib, or the combination of both. Segmentation was performed with the generalist deep learning-based segmentation algorithm Cellpose v0.6 (34). Multiple segmentation runs with increasing cell size diameter parameter (30-120px) were required to capture all cells.</p>

<p>Supplementary Figure S11: A-G. Relative body weight change of mice in cohorts treated with low-dose doxorubicin or pegylated liposomal doxorubicin, peposertib, and the combination of both, in the drug efficacy studies illustrated in Figure 5 and Supplementary Figure S9. H. Experimental design and drug doses utilized</p>

<p>Supplementary Figure S3: Mutated genes, fraction and counts of non-structural mutations in each gene (right) in clinical samples from 251 LMS tumors sequenced with OncoPanel. A. Alterations in TP53, RB1 and ATRX were detected in 73%, 73% and 31% of LMS, respectively, including single copy deletions and low amplitude gains; B. Alterations in genes mutated in >4% of samples, excluding single copy deletions and low amplitude gains. Deleterious mutations impacting BRCA2 or BRCA1 were demonstrated in 49% of LMS and were most often heterozygous deletions, whereas 8% of LMS had identifiable BRCA1/BRCA2 intragenic mutations or homozygous deletions. As with other targeted NGS platforms, these findings underestimate the true frequency of TP53 and RB1 mutations, given challenges detecting inactivating structural variants, particularly in large genes such as RB1.</p>

<p>Supplementary Figure S7 Computationally assisted analysis of cell culture confluence of representative cell culture images of LMS04 cultures untreated or treated with 2nM doxorubicin, 400nM peposertib, or the combination of both. Segmentation was performed with the generalist deep learning-based segmentation algorithm Cellpose v0.6 (34). Multiple segmentation runs with increasing cell size diameter parameter (30-120px) were required to capture all cells.</p>

<p>Supplementary Figure S1: A. Fraction of genome altered (FGA) in non-LMS cancers compared to LMS from GENIE v9.0 (37). B. FGA in LMS and in other tumor types in the GENIE dataset, including high grade sarcomas.</p>

<p>Supplementary Figure S6 Representative cell culture images of LMS04 cultures untreated or treated with 2nM doxorubicin, 400 nM peposertib, or the combination of both, utilized for computationally assisted cell culture confluence analysis (See Supplementary Figure S7).</p>

<p>Supplementary Figure S4: PRKDC and RPA2 are essential for LMS cells, as demonstrated by cell morphology after shRNAmediated knockdown under puromycin selection at day 7 in LMS04 and LMS05 cells (quantification is provided in Figure 2C).</p>

<div>AbstractPurpose:<p>Leiomyosarcoma (LMS) is an aggressive sarcoma for which standard chemotherapies achieve response rates under 30%. There are no effective targeted therapies against LMS. Most LMS are characterized by chromosomal instability (CIN), resulting in part from <i>TP53</i> and <i>RB1</i> co-inactivation and DNA damage repair defects. We sought to identify therapeutic targets that could exacerbate intrinsic CIN and DNA damage in LMS, inducing lethal genotoxicity.</p>Experimental Design:<p>We performed clinical targeted sequencing in 287 LMS and genome-wide loss-of-function screens in 3 patient-derived LMS cell lines, to identify LMS-specific dependencies. We validated candidate targets by biochemical and cell-response assays <i>in vitro</i> and in seven mouse models.</p>Results:<p>Clinical targeted sequencing revealed a high burden of somatic copy-number alterations (median fraction of the genome altered =0.62) and demonstrated homologous recombination deficiency signatures in 35% of LMS. Genome-wide short hairpin RNA screens demonstrated <i>PRKDC</i> (DNA-PKcs) and <i>RPA2</i> essentiality, consistent with compensatory nonhomologous end joining (NHEJ) hyper-dependence. DNA-PK inhibitor combinations with unconventionally low-dose doxorubicin had synergistic activity in LMS <i>in vitro</i> models. Combination therapy with peposertib and low-dose doxorubicin (standard or liposomal formulations) inhibited growth of 5 of 7 LMS mouse models without toxicity.</p>Conclusions:<p>Combinations of DNA-PK inhibitors with unconventionally low, sensitizing, doxorubicin dosing showed synergistic effects in LMS <i>in vitro</i> and <i>in vivo</i> models, without discernable toxicity. These findings underscore the relevance of DNA damage repair alterations in LMS pathogenesis and identify dependence on NHEJ as a clinically actionable vulnerability in LMS.</p></div>

<div>AbstractPurpose:<p>Leiomyosarcoma (LMS) is an aggressive sarcoma for which standard chemotherapies achieve response rates under 30%. There are no effective targeted therapies against LMS. Most LMS are characterized by chromosomal instability (CIN), resulting in part from <i>TP53</i> and <i>RB1</i> co-inactivation and DNA damage repair defects. We sought to identify therapeutic targets that could exacerbate intrinsic CIN and DNA damage in LMS, inducing lethal genotoxicity.</p>Experimental Design:<p>We performed clinical targeted sequencing in 287 LMS and genome-wide loss-of-function screens in 3 patient-derived LMS cell lines, to identify LMS-specific dependencies. We validated candidate targets by biochemical and cell-response assays <i>in vitro</i> and in seven mouse models.</p>Results:<p>Clinical targeted sequencing revealed a high burden of somatic copy-number alterations (median fraction of the genome altered =0.62) and demonstrated homologous recombination deficiency signatures in 35% of LMS. Genome-wide short hairpin RNA screens demonstrated <i>PRKDC</i> (DNA-PKcs) and <i>RPA2</i> essentiality, consistent with compensatory nonhomologous end joining (NHEJ) hyper-dependence. DNA-PK inhibitor combinations with unconventionally low-dose doxorubicin had synergistic activity in LMS <i>in vitro</i> models. Combination therapy with peposertib and low-dose doxorubicin (standard or liposomal formulations) inhibited growth of 5 of 7 LMS mouse models without toxicity.</p>Conclusions:<p>Combinations of DNA-PK inhibitors with unconventionally low, sensitizing, doxorubicin dosing showed synergistic effects in LMS <i>in vitro</i> and <i>in vivo</i> models, without discernable toxicity. These findings underscore the relevance of DNA damage repair alterations in LMS pathogenesis and identify dependence on NHEJ as a clinically actionable vulnerability in LMS.</p></div>

<p>Supplementary Table S3: List of pathogenic or likely pathogenic mutations in LMS based on 1) ClinVar (20); 2) functional classification as deleterious by both SIFT (21) and PolyPhen (22); and 3) truncating nature of the mutation.</p>

<p>Supplementary Figure S2: A. Counts of small deletions (<30kb) are compared in Sig3+ and Sig3- LMS tumors (analyzed by OncoPanel v3 and v 3.1, n=166). B. Counts of small deletions (<30kb) are significantly higher in tumors with deleterious alterations in POLH, POLD1, FANCM or XRCC1, all of which are implicated in DNA double strand break repair during different phases of the cell cycle (**** = p < 0.0001; n.s.= not significant)</p>

<p>Supplementary Figure S10: Effect of peposertib (100 mg/kg bid), low-dose pegylated liposomal doxorubicin (3 mg/kg qw) and combination on additional LMS PDX. A. CTG-1182. B. CTG-1005. C. CTG-1079.</p>

<p>Supplementary Table S1: Cancer-associated genes interrogated by Oncopanel. Samples in this study were analyzed with three versions of the assay (v1, v2, v3 and v3.1) – the gene content of each panel is indicated</p>

<p>Supplementary Figure S8: Synergistic effects of peposertib and low-dose doxorubicin combinations in LMS cells. Cell proliferation assay (BrdU incorporation) demonstrates that addition of low-dose doxorubicin to DNA-PKi treatment results in synergistic effects on LMS03, LMS04 and LMS05 cell proliferation. Cells were treated for 7 days with the drug combinations at the indicated doses; BrdU incorporation over 24h was measured at day 7 with a luminescence-based ELISA assay (Roche). Synergy scores and heatmap / surface plots were generated using SynergyFinder (RRID:SCR_019318)(35).</p>

<p>Supplementary Figure S6 Representative cell culture images of LMS04 cultures untreated or treated with 2nM doxorubicin, 400 nM peposertib, or the combination of both, utilized for computationally assisted cell culture confluence analysis (See Supplementary Figure S7).</p>

<p>Supplementary Table S4: Molecular features of patient-derived LMS models used in these studies (three LMS cells lines and one LMS PDX), including clinical features of LMS tumors from Dana-Farber/Brigham and Women’s Hospital from which they were derived</p>

<p>Supplementary Table S5: Molecular features of patient-derived LMS models from Champions Oncology used in in vivo validation studies (five LMS PDX), including clinical and morphological features of LMS tumors from which they were derived.</p>

<p>Supplementary Figure S9: Cell proliferation assay (BrdU incorporation) demonstrates that combination of DNA-PK inhibition with low-dose doxorubicin reduces LMS03 and LMS05 cell proliferation compared to either drug alone. Peposertib and AZD7648 are structurally unrelated DNA-PK inhibitors. Cells were treated for 7 days with each drug and the combinations at the indicated doses; BrdU incorporation over 24h was measured at day 7 with a luminescence-based ELISA assay (Roche)</p>

<p>Supplementary Table S1: Cancer-associated genes interrogated by Oncopanel. Samples in this study were analyzed with three versions of the assay (v1, v2, v3 and v3.1) – the gene content of each panel is indicated</p>

<p>Supplementary Figure S5: Biochemical effects of increasing doses of doxorubicin in LMS04 cells, starting at unconventionally low doses.</p>

<p>Supplementary Table S4: Molecular features of patient-derived LMS models used in these studies (three LMS cells lines and one LMS PDX), including clinical features of LMS tumors from Dana-Farber/Brigham and Women’s Hospital from which they were derived</p>

<p>Supplementary Figure S3: Mutated genes, fraction and counts of non-structural mutations in each gene (right) in clinical samples from 251 LMS tumors sequenced with OncoPanel. A. Alterations in TP53, RB1 and ATRX were detected in 73%, 73% and 31% of LMS, respectively, including single copy deletions and low amplitude gains; B. Alterations in genes mutated in >4% of samples, excluding single copy deletions and low amplitude gains. Deleterious mutations impacting BRCA2 or BRCA1 were demonstrated in 49% of LMS and were most often heterozygous deletions, whereas 8% of LMS had identifiable BRCA1/BRCA2 intragenic mutations or homozygous deletions. As with other targeted NGS platforms, these findings underestimate the true frequency of TP53 and RB1 mutations, given challenges detecting inactivating structural variants, particularly in large genes such as RB1.</p>

<p>Supplementary Figure S11: A-G. Relative body weight change of mice in cohorts treated with low-dose doxorubicin or pegylated liposomal doxorubicin, peposertib, and the combination of both, in the drug efficacy studies illustrated in Figure 5 and Supplementary Figure S9. H. Experimental design and drug doses utilized</p>

<p>Supplementary Figure S10: Effect of peposertib (100 mg/kg bid), low-dose pegylated liposomal doxorubicin (3 mg/kg qw) and combination on additional LMS PDX. A. CTG-1182. B. CTG-1005. C. CTG-1079.</p>

<p>Supplementary Table S5: Molecular features of patient-derived LMS models from Champions Oncology used in in vivo validation studies (five LMS PDX), including clinical and morphological features of LMS tumors from which they were derived.</p>

<p>Supplementary Table S3: List of pathogenic or likely pathogenic mutations in LMS based on 1) ClinVar (20); 2) functional classification as deleterious by both SIFT (21) and PolyPhen (22); and 3) truncating nature of the mutation.</p>

<p>Supplementary Figure S1: A. Fraction of genome altered (FGA) in non-LMS cancers compared to LMS from GENIE v9.0 (37). B. FGA in LMS and in other tumor types in the GENIE dataset, including high grade sarcomas.</p>

<p>Supplementary Figure S8: Synergistic effects of peposertib and low-dose doxorubicin combinations in LMS cells. Cell proliferation assay (BrdU incorporation) demonstrates that addition of low-dose doxorubicin to DNA-PKi treatment results in synergistic effects on LMS03, LMS04 and LMS05 cell proliferation. Cells were treated for 7 days with the drug combinations at the indicated doses; BrdU incorporation over 24h was measured at day 7 with a luminescence-based ELISA assay (Roche). Synergy scores and heatmap / surface plots were generated using SynergyFinder (RRID:SCR_019318)(35).</p>

<p>Supplementary Figure S2: A. Counts of small deletions (<30kb) are compared in Sig3+ and Sig3- LMS tumors (analyzed by OncoPanel v3 and v 3.1, n=166). B. Counts of small deletions (<30kb) are significantly higher in tumors with deleterious alterations in POLH, POLD1, FANCM or XRCC1, all of which are implicated in DNA double strand break repair during different phases of the cell cycle (**** = p < 0.0001; n.s.= not significant)</p>

<p>Supplementary Table S2: Sig3 scores and FGA values in LMS tumor samples. Sig3+ samples are predicted with a classifier trained in WGS LMS data. FGA values are calculated on CNVkit-based copy number profiles (see methods).</p>

<p>Supplementary Figure S4: PRKDC and RPA2 are essential for LMS cells, as demonstrated by cell morphology after shRNAmediated knockdown under puromycin selection at day 7 in LMS04 and LMS05 cells (quantification is provided in Figure 2C).</p>

<p>Supplementary Table S2: Sig3 scores and FGA values in LMS tumor samples. Sig3+ samples are predicted with a classifier trained in WGS LMS data. FGA values are calculated on CNVkit-based copy number profiles (see methods).</p>

<p>Supplementary Figure S5: Biochemical effects of increasing doses of doxorubicin in LMS04 cells, starting at unconventionally low doses.</p>

<p>Supplementary Figure S9: Cell proliferation assay (BrdU incorporation) demonstrates that combination of DNA-PK inhibition with low-dose doxorubicin reduces LMS03 and LMS05 cell proliferation compared to either drug alone. Peposertib and AZD7648 are structurally unrelated DNA-PK inhibitors. Cells were treated for 7 days with each drug and the combinations at the indicated doses; BrdU incorporation over 24h was measured at day 7 with a luminescence-based ELISA assay (Roche)</p>

<div>Abstract<p>Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive <i>TP53</i><sup>−/−</sup> and <i>BRCA1</i><sup>−/−</sup> epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance.</p>Significance:<p>This study shows that <i>BRCA1</i>-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.</p></div>

Building upon the hybrid strong/weak coupling model for jet quenching, we incorporate and study the effects of transverse momentum broadening and medium response of the plasma to jets on a variety of observables. For inclusive jet observables, we find little sensitivity to the strength of broadening. To constrain those dynamics, we propose new observables constructed from ratios of differential jet shapes, in which particles are binned in momentum, which are sensitive to the in-medium broadening parameter. We also investigate the effect of the back-reaction of the medium on the angular structure of jets as reconstructed with different cone radii R. Finally we provide results for the so called “missing-pt”, finding a qualitative agreement between our model calculations and data in many respects, although a quantitative agreement is beyond our simplified treatment of the hadrons originating from the hydrodynamic wake.