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ObjectivePancreatic ductal adenocarcinoma (PDAC) still carries a dismal prognosis with an overall 5-year survival rate of 9%. Conventional combination chemotherapies are a clear advance in the treatment of PDAC; however, subtypes of the disease exist, which exhibit extensive resistance to such therapies. Genomic MYC amplifications represent a distinct subset of PDAC with an aggressive tumour biology. It is clear that hyperactivation of MYC generates dependencies that can be exploited therapeutically. The aim of the study was to find and to target MYC-associated dependencies.DesignWe analysed human PDAC gene expression datasets. Results were corroborated by the analysis of the small ubiquitin-like modifier (SUMO) pathway in a large PDAC cohort using immunohistochemistry. A SUMO inhibitor was used and characterised using human and murine two-dimensional, organoid and in vivo models of PDAC.ResultsWe observed that MYC is connected to the SUMOylation machinery in PDAC. Components of the SUMO pathway characterise a PDAC subtype with a dismal prognosis and we provide evidence that hyperactivation of MYC is connected to an increased sensitivity to pharmacological SUMO inhibition.ConclusionSUMO inhibitor-based therapies should be further developed for an aggressive PDAC subtype.

Activated SUMOylation is a hallmark of cancer. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved function of activated SUMOylation, which attenuated the immunogenicity of tumor cells. Activated SUMOylation allowed cancer cells to evade CD8+ T cell-mediated immunosurveillance by suppressing the MHC class I (MHC-I) antigen-processing and presentation machinery (APM). Loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, and the pharmacological inhibition of SUMOylation (SUMOi) resulted in reduced activity of the transcriptional repressor scaffold attachment factor B (SAFB) and induction of the MHC-I APM. Consequently, SUMOi enhanced the presentation of antigens and the susceptibility of tumor cells to CD8+ T cell-mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T cells and thereby drove a feed-forward loop amplifying the specific antitumor immune response. In summary, we showed that activated SUMOylation allowed tumor cells to evade antitumor immunosurveillance, and we have expanded the understanding of SUMOi as a rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.

Background We explored potential predictive biomarkers of immunotherapy response in patients with extensive-stage small-cell lung cancer (ES-SCLC) treated with durvalumab (D) + tremelimumab (T) + etoposide-platinum (EP), D + EP, or EP in the randomized phase 3 CASPIAN trial. Methods 805 treatment-naïve patients with ES-SCLC were randomized (1:1:1) to receive D + T + EP, D + EP, or EP. The primary endpoint was overall survival (OS). Patients were required to provide an archived tumor tissue block (or ≥ 15 newly cut unstained slides) at screening, if these samples existed. After assessment for programmed cell death ligand-1 expression and tissue tumor mutational burden, residual tissue was used for additional molecular profiling including by RNA sequencing and immunohistochemistry. Results In 182 patients with transcriptional molecular subtyping, OS with D ± T + EP was numerically highest in the SCLC-inflamed subtype ( n  = 10, median 24.0 months). Patients derived benefit from immunotherapy across subtypes; thus, additional biomarkers were investigated. OS benefit with D ± T + EP versus EP was greater with high versus low CD8A expression/CD8 cell density by immunohistochemistry, but with no additional benefit with D + T + EP versus D + EP. OS benefit with D + T + EP versus D + EP was associated with high expression of CD4 (median 25.9 vs. 11.4 months) and antigen-presenting and processing machinery (25.9 vs. 14.6 months) and MHC I and II (23.6 vs. 17.3 months) gene signatures, and with higher MHC I expression by immunohistochemistry. Conclusions These findings demonstrate the tumor microenvironment is important in mediating better outcomes with D ± T + EP in ES-SCLC, with canonical immune markers associated with hypothesized immunotherapy mechanisms of action defining patient subsets that respond to D ± T. Trial registration ClinicalTrials.gov, NCT03043872.

Many targeted cancer therapies rely on biomarkers assessed by scoring of immunohistochemically (IHC)-stained tissue, which is subjective, semiquantitative, and does not account for expression heterogeneity. We describe an image analysis-based method for quantitative continuous scoring (QCS) of digital whole-slide images acquired from baseline human epidermal growth factor receptor 2 (HER2) IHC-stained breast cancer tissue. Candidate signatures for patient stratification using QCS of HER2 expression on subcellular compartments were identified, addressing the spatial distribution of tumor cells and tumor-infiltrating lymphocytes. Using data from trastuzumab deruxtecan-treated patients with HER2-positive and HER2-negative breast cancer from a phase 1 study (NCT02564900; DS8201-A-J101; N = 151), QCS-based patient stratification showed longer progression-free survival (14.8 vs 8.6 months) with higher prevalence of patient selection (76.4 vs 56.9%) and a better cross-validated log-rank p value (0.026 vs 0.26) than manual scoring based on the American Society of Clinical Oncology / College of American Pathologists guidelines. QCS-based features enriched the HER2-negative subgroup by correctly predicting 20 of 26 responders.

SUMOylation is a post-translational modification of proteins that regulates these proteins’ localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma. SUMOylation is a post-translational modification that has been shown to be altered in cancer. Here, the authors show that loss of the SUMO isopeptidase SENP6 leads to unrestricted SUMOylation and genomic instability promoting lymphomagenesis and generating vulnerability to PARP inhibition.

Background Greater understanding of differential therapeutic sensitivity, specifically to immunotherapy, in small-cell lung cancer (SCLC) is required. Methods We explored SCLC heterogeneity through integrated molecular characterization of tumor tissue samples from 159 treatment-naive patients, utilizing genetic, epigenetic, transcriptional, and proteomic profiling, immunohistochemistry staining for multiple biologically relevant markers including transcriptional subtype-defining proteins, and spatial immune profiling using multiplex immunofluorescence. Results Multi-omics analysis confirmed high heterogeneity across/within neuroendocrine and non-neuroendocrine subtypes. Methylomics analysis identified four methylome clusters that may enhance subtype prediction, prognosis, and longitudinal monitoring of subtype evolution. Immunohistochemistry analysis showed high MHC-I expression in non-neuroendocrine subtypes, which have greatest potential benefit from adding immunotherapy to chemotherapy; high DLL3 expression associated with neuroendocrine subtypes and an immune-cold tumor microenvironment. Multiplex immunofluorescence demonstrated associations of MHC-I with spatial arrangement and phenotypic features of immune cells in the tumor microenvironment of high-MHC-I-expressing SCLC, providing mechanistic rationale for MHC-I as a potential biomarker of immunotherapy response. Conclusions This multimodal profiling analysis provides further insights into the biologic complexity of SCLC and highlights potential therapeutic vulnerabilities of distinct disease subtypes. Graphical abstract

The DNA damage response (DDR) acts as a barrier to malignant transformation and is often impaired during tumorigenesis. Exploiting the impaired DDR can be a promising therapeutic strategy; however, the mechanisms of inactivation and corresponding biomarkers are incompletely understood. Starting from an unbiased screening approach, we identified the SMC5‐SMC6 Complex Localization Factor 2 (SLF2) as a regulator of the DDR and biomarker for a B‐cell lymphoma (BCL) patient subgroup with an adverse prognosis. SLF2‐deficiency leads to loss of DDR factors including Claspin (CLSPN) and consequently impairs CHK1 activation. In line with this mechanism, genetic deletion of Slf2 drives lymphomagenesis in vivo . Tumor cells lacking SLF2 are characterized by a high level of DNA damage, which leads to alterations of the post‐translational SUMOylation pathway as a safeguard. The resulting co‐dependency confers synthetic lethality to a clinically applicable SUMOylation inhibitor (SUMOi), and inhibitors of the DDR pathway act highly synergistic with SUMOi. Together, our results identify SLF2 as a DDR regulator and reveal co‐targeting of the DDR and SUMOylation as a promising strategy for treating aggressive lymphoma. Synopsis SLF2 was identified as a tumor suppressor of B‐cell lymphomagenesis and a crucial regulator of CHK1, and its deficiency was associated with defective DNA damage response and synthetic lethality to SUMOylation inhibition. SLF2 is a functionally relevant tumor suppressor in murine and human B‐cell lymphoma. Loss of SLF2 results in CHK1 impairment, transcriptional or post‐transcriptional repression of Claspin, and genomic instability. SLF2 deficiency drives alteration of the SUMO pathway and confers synthetic lethality to pharmacological SUMOylation inhibition. Impaired DNA damage responses, e.g., caused by SLF2 loss or pharmacological inhibition of CHK1, confer synthetic lethality to SUMO inhibition. Graphical Abstract SLF2 was identified as a tumor suppressor of B‐cell lymphomagenesis and a crucial regulator of CHK1, and its deficiency was associated with defective DNA damage response and synthetic lethality to SUMOylation inhibition.

BackgroundExploratory data from single-arm PhI/II clinical trials provide a unique opportunity for predictive biomarker discovery, but have the limitation to estimate the prognostic value in absence of a control arm. This poses a significant risk to subsequent biomarker-driven PhIII trials. A Digital Twin is the computational description a real patient based on their clinical data,1 including baseline histopathology imaging. In this work, we demonstrate the generation of a virtual randomized PhIII clinical trial (vPhIII) via Digital Twins and their application for predictive biomarker discovery.MethodsEligible baseline tissue samples of NSCLC patients enrolled in the PhI/II trial ‘CP1108’ (NCT01693562) and in the randomized PhIII trial ‘MYSTIC’ (NCT02453282)4 5 were considered for computational pathology analysis. Relying on Quantitative Continuous Scoring for PD-L1 (PD-L1 QCS), the PD-L1 protein expression was digitally quantified and multidimensional whole slide image (WSI) features for PD-L1 expression were obtained.2 3 For each PhI/II patient, its Digital Twin is generated by identifying the most similar matching patient in the PhIII control arm using Euclidean distance and Delaunay triangulation of dimensionality-reduced patient features. This group of Digital Twins comprises the actual observed overall survival (OS) information from the respective study, and therefore could serve as vPhIII. Each PD-L1 QCS feature is evaluated for its median OS time (mOS) benefit in the QCS-positive vPhIII sub-group. The feature providing longest mOS benefit is selected and evaluated for its predictive value validated in the real PhIII cohort.ResultsN=121 Digital Twins were generated as vPhIII cohort (figure 1a), while average mOS benefit analysis for each feature indicated that the 20% quantile of PD-L1 tumor cell expression provides optimal stratification (figure 1b). The mOS comparison of the real PhIII (figure 2a) with the vPhIII (figure 2b) showed the OS benefit from durvalumab treatment has been underestimated by the Digital Twins model. Although the selected QCS feature did not indicate significant treatment benefit in the vPhIII (figure 3a), a retrospective analysis of MYSTIC hints towards beneficial patient stratification (figure 3b).ConclusionsDigital Twins based on imaging data are a promising approach to generate virtual randomized and biomarker stratified PhIII trials based on single-arm PhI/II and historic Standard-of-Care data. This proof-of-concept study demonstrates technical feasibility of this innovative methodology. Although further validation is required, the Digital Twin approach may open new ways towards maximizing the success probability of drug development programs, and faster implementation of Precision Oncology in clinical routine.AcknowledgementsThe authors thank Harald Hessel, Susanne Haneder, Pallavi Sontakke, Karma DaCosta and Regina Alleze (Pathology and Pathology Informatics at AstraZeneca Computational Pathology) for curation and annotation of whole slide image data used in this study.Trial RegistrationNCT01693562, NCT02453282ReferencesChengyue Wu, Guillermo Lorenzo, et al. Integrating mechanism-based modeling with biomedical imaging to build practical digital twins for clinical oncology. Biophys Rev (Melville). 2022 Jun;3(2):021304Schmidt G, Brieu N, Spitzmueller A, Kapil A. A SCORING METHOD FOR AN ANTI-HER2 ANTIBODY-DRUG CONJUGATE THERAPY. Patent application WO2022054009 (A2)Jan Lesniak, Markus Schick, Ross Stewart, et al. Quantitative computational assessment of PD-L1 enables robust patient selection for biomarker-informed anti-PD-L1 treatment of NSCLC patients. Journal for ImmunoTherapy of Cancer 2022;10:doi: 10.1136/jitc-2022-SITC2022.0583Scott J. Antonia, Neil H. Segal, et al. Clinical Activity, Tolerability, and Long-Term Follow-Up of Durvalumab in Patients With Advanced NSCLC. J Thorac Oncol. 2019 Oct;14(10):1794−1806Pages 1794–1806,Rizvi NA, Cho BC, Reinmuth N, et al. Durvalumab With or Without Tremelimumab vs Standard Chemotherapy in First-line Treatment of Metastatic Non-Small Cell Lung Cancer: The MYSTIC Phase 3 Randomized Clinical Trial. JAMA Oncol. 2020 May 1;6(5):661−674Ethics ApprovalClinical studies NCT01693562 and NCT02453282, from which data in this report were obtained, were carried out in accordance with the Declaration of Helsinki and GoodClinical Practice guidelines. The study protocols, amendments, and participant informed consent documents were approved by the appropriate institutional review boards.Abstract 605 Figure 1(A) Delaunay triangulated UMAP dimensionality reduction plot of the PD-L1 QCS feature space used to identify the Digital Twins based on anti-PD-L1 (durvalumab) treated CP1108 patients (blue dots) and MYSTIC control arm (orange dots). Each Twin pair is connected by a red line. (B) Ranking of PD-L1 QCS features by average mOS survival benefit in the vPhIII cohort. The mOS benefits had been calculated using 100 Monte-Carlo runs on a 80% subset of the vPhIII cohort. DT_PDL1_TPS represents the predictive performance of human pathologist PD-L1 tumor proportion score. PD-L1 QCS membrane expression quantiles for each patient’s WSI have been considered in the range from 10% (q10) to 90% (q90)Abstract 605 Figure 2Kaplan Meier analysis of (A) the Ph III MYSTIC study patients (N=303+285) considered in this proof-of-concept work, and (B) vPhIII study Digital Twins (N=121+121) generated by matching considered CP1108 patients with MYSTIC control arm (labelled as CHEMO) patients, using their actually observed OS time and event informationAbstract 605 Figure 3Kaplan Meier analysis of PD-L1 QCS-positive patients in the (A) vPhIII and (B) real PhIII MYSTIC cohort. ‘PD-L1 QCS-positive’ is defined by the condition that the 20% quantile of PD-L1 membrane protien expression on all tumor cells of a patient’s WSI is larger than 9.34 (on a scale from 0 to 255)

The reliable authentication of cell lines is a prerequisite for the reproducibility and replicability of experiments. A common method of cell line authentication is the fragment length analysis (FLA) of short-tandem repeats (STR) by capillary electrophoresis. However, this technique is not always accessible and is often costly. Using a microfluidic electrophoresis system, we analyzed the quality and integrity of different murine cell lines by STR profiling. As a proof of concept, we isolated and immortalized hematopoietic progenitor cells (HPC) of various genotypes through retroviral transduction of the fusion of the estrogen receptor hormone-binding domain with the coding sequence of HoxB8. Cell lines were maintained in the HPC state with Flt3 ligand (FL) and estrogen treatment and could be characterized upon differentiation. In a validation cohort, we applied this technique on primary mutant Kras-driven pancreatic cancer cell lines, which again allowed for clear discrimination. In summary, our study provides evidence that FLA of STR-amplicons by microfluidic electrophoresis allows for stringent quality control and the tracking of cross-contaminations in both genetically stable HPC lines and cancer cell lines, making it a simple and cost-efficient alternative to traditional capillary electrophoresis.

BackgroundSmall cell lung cancer (SCLC) is an aggressive and largely immune-cold cancer type, for which chemotherapy combined with Immuno-oncology (IO) therapies is providing benefit only in a subgroup of patients. SCLC is a highly heterogeneous cancer with at least four major subtypes.1 Among them, the ‘inflamed’ subtype is characterized by an inflamed immune gene signature and high expression of MHC class I (MHC-I) antigen presentation and shows the greatest benefit from the addition of IO treatment to chemotherapy,2 suggesting that MHC-I could serve as a biomarker for IO therapies. Here, we aimed to assess the spatial characteristics of immune cells in MHC-I high SCLC cases to investigate and support its role as a potential biomarker for IO therapies.MethodsWe combined a computational pathology approach with multiplex immunofluorescence (mIF) to profile the SCLC tumor microenvironment (TME). To this end, 126 SCLC formalin-fixed, paraffin-embedded tissue samples were stained with two mIF panels consisting of six markers each: (A) PanCK, CD8, CD68, PD-1, PD-L1, and Ki67; (B) CD20, NKp46, CD1c, CD66b, ICOS, and FOXP3. Based on these panels, we investigated the location and phenotype of each cell in the tumor center and within the stroma and tumor parenchyma. Additional slides from the same tissue blocks were immunohistochemically stained with MHC-I and scored by pathologists. Starting from the observation that high MHC-I expression was associated with higher densities of CD8+ T-cells,3 we further explored the TME characteristics of MHC-I SCLC cases.ResultsBeyond higher densities of CD8+ cytotoxic T-cells, we observed higher densities of FOXP3+ regulatory T-cells, and ICOS+ T-cells in the tumor center of MHC-I high cases. Considering the role of MHC-I in antigen presentation and T-cell activation, we investigated the proportion of CD8;PD-1;Ki67+ T-cells out of all CD8+ cells. Of note, we observed a compelling association of a high proportion of CD8;PD-1;Ki67+ T-cells with high MHC-I. This effect was particularly prominent in the tumor parenchyma and absent in the stroma, revealing an association with functionally relevant presentation of tumor antigens by MHC-I on SCLC tumor cells. Interestingly, we did not observe alterations in other immune cell populations like myeloid dendritic cells, macrophages, and granulocytes.ConclusionsWe utilized computational pathology to comprehensively profile the composition and spatial arrangement of the TME in inflamed SCLC cases defined by high MHC-I expression. Our findings provide the functional rationale for MHC-I as a biomarker for a potentially increased response to IO therapies.4 ReferencesGay CM, Stewart CA, Park EM, Diao L, Groves SM, Heeke S, Nabet BY, Fujimoto J, Solis LM, Lu W, Xi Y, Cardnell RJ, Wang Q, Fabbri G, Cargill KR, Vokes NI, Ramkumar K, Zhang B, Della Corte CM, Robson P, Swisher SG, Roth JA, Glisson BS, Shames DS, Wistuba II, Wang J, Quaranta V, Minna J, Heymach JV, Byers LA. Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities. Cancer Cell. 2021 Mar 8;39(3):346–360.e7. doi: 10.1016/j.ccell.2020.12.014. Epub 2021 Jan 21. PMID: 33482121; PMCID: PMC8143037.Mahadevan NR, Knelson EH, Wolff JO, Vajdi A, Saigí M, Campisi M, Hong D, Thai TC, Piel B, Han S, Reinhold BB, Duke-Cohan JS, Poitras MJ, Taus LJ, Lizotte PH, Portell A, Quadros V, Santucci AD, Murayama T, Cañadas I, Kitajima S, Akitsu A, Fridrikh M, Watanabe H, Reardon B, Gokhale PC, Paweletz CP, Awad MM, Van Allen EM, Lako A, Wang XT, Chen B, Hong F, Sholl LM, Tolstorukov MY, Pfaff K, Jänne PA, Gjini E, Edwards R, Rodig S, Reinherz EL, Oser MG, Barbie DA. Intrinsic Immunogenicity of Small Cell Lung Carcinoma Revealed by Its Cellular Plasticity. Cancer Discov. 2021 Aug;11(8):1952–1969. doi: 10.1158/2159–8290.CD-20–0913. Epub 2021 Mar 11. PMID: 33707236; PMCID: PMC8338750.Vuko M, Xie M, Gavaldon MA, Segerer F, Spitzmueller A, Hessel H, Testori M, Zimmermann J, Surace M, Heininen-Brown M, Canales JR, Saran S, Angell H, Schmidt G, Sade H, Barrett C, Schick M, Fabbri G. 155 MHC class I antigen presentation is associated with an inflamed SCLC tumor microenvironment characterized by a higher density of cytotoxic T-cells in closer proximity to tumor cells. Journal for ImmunoTherapy of Cancer 2022;10:doi: 10.1136/jitc-2022-SITC2022.0155Rudin CM, Balli D, Lai WV, Richards AL, Nguyen E, Egger JV, Choudhury NJ, Sen T, Chow A, Poirier JT, Geese WJ, Hellmann MD, Forslund A. Clinical benefit from immunotherapy in patients with small cell lung cancer is associated with tumor capacity for antigen presentation. J Thorac Oncol. 2023 May 18:S1556–0864(23)00554–3. doi: 10.1016/j.jtho.2023.05.008. Epub ahead of print. PMID: 37210008.Ethics ApprovalAll samples from which data in this report were generated, were obtained from an internal repository. All protocols, amendments, and participant informed consent documents were approved by the appropriate institutional review boards.