Abstract
Background:
Close proximity between cytotoxic T lymphocytes and tumor cells is required for effective immunotherapy. Three tumor-immune (TI) phenotypes, infiltrated, excluded and desert, ...have been previously described based on the infiltration patterns of CD8+ T cells. However, no quantitative methods exist to define these phenotypes robustly in human solid tumors. Importantly, the molecular features and mechanisms determining these phenotypes are not well understood. Here we report a novel integrated approach to classify and functionally dissect TI phenotypes in human ovarian cancer.
Methods:
CD8 IHC and RNAseq analysis were performed on 370 ovarian tumors from the ICON7 phase III clinical trial, a front-line trial testing the addition of bevacizumab to chemotherapies. A digital image analysis algorithm was developed to quantify the quantity and spatial distribution of CD8+ T cells. Coupling digital pathology with transcriptome analysis, a random forest machine learning algorithm was applied to identify genes associated with these two metrics using a training set (n=155). A gene expression-based classifier was developed for classifying TI phenotypes and validated using testing sets from ICON7 trial and a vendor collection. Functional characterization of key mediators promoting T cell exclusion were carried out by integrating in situ, in vitro and ex vivo analyses on ovarian tumor tissues, cancer associated fibroblasts (CAFs) and ovarian cancer cell lines. Anti-tumor activity of TGFβ blockade in combination with anti-PD-L1 was evaluated in the mouse BrKras ovarian cancer model in FVB background.
Results:
Integrating digital pathology and machine learning on large ovarian tumor cohorts, we developed and validated a 157-gene molecular classifier. We show the TI phenotypes are of biological and clinical importance in ovarian cancer. Two hallmarks of T cell exclusion were identified: 1) loss of MHC I on tumor cells and 2) upregulation of TGFβ/stromal activities. We show that MHC I in ovarian cancer cells is likely regulated by epigenetic mechanisms and TGFβ is a key mediator of T cell exclusion. TGFβ reduced MHC I expression in ovarian cancer cells and induced extracellular matrix and immunosuppressive molecules in human primary fibroblasts. Finally, we demonstrated that combining anti-TGFβ and anti-PD-L1 in the BrKras mouse model improved the anti-tumor efficacy and survival.
Conclusion:
This study provided the first systematic and in-depth characterization of the molecular features and mechanisms underlying the tumor-immune phenotypes in human ovarian cancer. We illuminated a multi-faceted role of TGFβ in mediating crosstalk between tumor cells and CAFs to shape the tumor-immune contexture. Our findings support that targeting the TGFβ pathway represents a promising therapeutic strategy to overcome T cell exclusion and optimize response to cancer immunotherapy.
Citation Format: Melanie Desbois, Akshata Udyavar, Lisa Ryner, Cleopatra Kozlowski, Yinghui Guan, Milena Dürrbaum, Shan Lu, Jean-Philippe Fortin, Hartmut Koeppen, James Ziai, Ching-Wei Chang, Amy Lo, Shilpa Keerthivasan, Marie Plante, Richard Bourgon, Carlos Bais, Priti Hegde, Anneleen Daemen, Shannon Turley, Yulei Wang. Integrated digital pathology and transcriptome analysis identifies molecular mediators of T cell exclusion in ovarian cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 463.
Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the ...generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.
•A humanized bispecific antibody that selectively activates FGFR1/βKlotho complex was generated.•Anti-FGFR1/βKlotho agonist antibody induced sustained thermogenesis in brown fat and induced weight loss.•Anti-FGFR1/βKlotho agonist antibody improved insulin sensitivity even before the onset of weight loss.
McDonough AL, Batavia M, Chen FC, Kwon S, Ziai J. The validity and reliability of the GAITRite system's measurements: a preliminary evaluation. Arch Phys Med Rehabil 2001;82:419-25. Objective: To ...compare the concurrent validity and reliability of the GAITRite™ computerized gait analysis system with validated paper-and-pencil and video-based methods. Design: Within-groups, repeated-measures design. Setting: Research laboratory in a physical therapy education program. Participant: One healthy woman, age 27 years. Interventions: A subject walked across the walkway of the GAITRite system at various walking rates and degrees of step symmetry for 2 of the 3 analyses. Paper placed over the walkway enabled concurrent paper-and-pencil analysis. The subject was concurrently videotaped from the side. For the other analysis, a stride simulator with known step and stride lengths was applied to the walkway to simulate 2 steps and 1 stride. Main Outcome Measures: Cadence, walking speed, right and left step and stride lengths, and right and left step times. Results: Excellent paper-and-pencil and GAITRite correlations (intraclass correlation coefficient ICC > 95) for spatial measures and excellent video-based and GAITRite correlations (ICC > 93) for temporal measures were found. GAITRite measures of step lengths and times were reliable in both walkway center and left-of-center measurements. Conclusions: Based on this data, GAITRite is a valid and reliable tool for measuring selected spatial and temporal parameters of gait.
Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) mutations, ...including G660D, R678Q, E693K and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.
Pahuja et al. show that recurrent HER2 transmembrane domain and juxtamembrane domain mutations enhance HER2 activity by improving the active dimer interface or stabilizing an activating conformation. Importantly, HER2 inhibiting antibodies and small molecules can block the activity of these mutants.
ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform ...expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 1.
Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8
tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data.
ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (K
) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound
Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing
Zr immuno-PET reagents.
Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.
ERBB3 is a pseudokinase domain-containing member of the ERBB family of receptor tyrosine kinases (RTKs). Following ligand binding, ERBB receptors homo- or hetero-dimerize, leading to a head-to-tail ...arrangement of the intracellular kinase domains, where the "receiver" kinase domain of one ERBB is activated by the "activator" domain of the other ERBB in the dimer. In ERBB3, a conserved valine at codon 943 (V943) in the kinase C-terminal domain has been shown to be important for its function as an "activator" kinase
. Here we report a knock-in mouse model where we have modified the endogenous
allele to allow for tissue-specific conditional expression of
(
). Additionally, we generated an
(
) conditional knock-in mouse model where the conserved aspartate in the DFG motif of the pseudokinase domain was mutated to abolish any potential residual kinase activity. While
animals developed normally, homozygous
expression during development resulted in embryonic lethality. Further, tissue specific expression of
in the mammary gland epithelium following its activation using
resulted in delayed elongation of the ductal network during puberty. Single-cell RNA-seq analysis of
mammary glands showed a reduction in a specific subset of fibrinogen-producing luminal epithelial cells.
BackgroundThe prognostic value of intra-tumoral cytotoxic T cells has been demonstrated in multiple tumor types. However, immune infiltrate heterogeneity can lead to potentially significant ...misrepresentations of marker prevalence in routine histological sections. We examined step sections of breast and colorectal cancer samples for CD8+ T cell prevalence by standard chromogenic immunohistochemistry to determine marker variability and inform practice of T cell biomarker assessment in formalin-fixed, paraffin-embedded (FFPE) tissue samples.MethodsSerial sections were cut at approximately 25 um intervals from 13 primary colorectal and 12 breast carcinoma (8 ductal adenocarcinoma, 4 medullary) FFPE samples until blocks were exhausted and stained for CD8 using DAB-based chromogenic immunohistochemistry. Stained sections were digitally imaged and CD8+ lymphocytes within defined regions of interest (ROI) including the tumor and surrounding stroma were enumerated. Using a linear model/ANOVA framework, statistical analyses of CD8+ cell count variance between patients as well as between levels within a patient sample were performed. Means, medians, and dispersion values of percent CD8 positive cells (CD8 cells/total ROI cells) were calculated. Similarity of CD8 prevalence between slices of a given sample was measured by percent of variability accounted for by slice as a factor in a linear model and an intra-class correlation coefficient (ICC) ranging from zero (no similarity) to 1 (identical).ResultsThe mean and median CD8+ cell percentages varied between breast ductal adenocarcinoma (mean 11.32%, median 6.35) and colonic adenocarcinoma samples (mean 3.91%, median 2.88%) and were highest in medullary breast carcinoma (mean 25.32%, median 18.55%). However, minimal variation in cytotoxic T cell counts between levels within any given tumor block was observed. Percent variability (%) in CD8 counts between step sections from colonic adenocarcinoma (0.1%), medullary breast carcinoma (0.1%) and ductal adenocarcinoma (0.1%) blocks were minor. ICC calculated for percent CD8 positive cells between levels of a sample was 0.99 for both tumor types. Resampling-based methods show CD8 as reliable marker for classifying patients as CD8-positive or negative over a range of cut-offs.ConclusionsOur results show that CD8+ T cell distribution is highly homogeneous within a standard tissue sample in both colorectal and breast carcinomas. As such, cytotoxic CD8 T cell prevalence by immunohistochemistry on a single level can be considered representative of cytotoxic CD8 T cell infiltration for the entire tumor section within the block. These findings support the technical validity of biomarker strategies relying on CD8 immunohistochemistry in FFPE tumors.
BackgroundIncreased numbers of OX40+ T cells within the primary tumor of colorectal cancer (CRC) patients has been associated with improved survival. Similarly, OX40 expression in sentinel lymph ...nodes is inversely associated with tumor grade and clinical stage in melanoma patients. However, the prevalence of OX40+ tumor-associated lymphocytes remains underreported in many human solid tumor types. In this study, we report prevalence of OX40 by chromogenic immunohistochemistry in melanoma, colorectal, bladder, renal cell (RCC), triple-negative breast (TNBC), non-small cell lung (NCSCLC) and ovarian cancers and its association with available clinical parameters.MethodsTumor-associated OX40+ cells in melanoma (n=40), bladder (n=51), CRC (n=40), NSCLC (n=40), TNBC (n=45), ovarian (n=40) and RCC (n=42) patient samples including primary site and a subset of matched metastases have been stained for OX40 using a DAB-based chromogenic immunohistochemistry assay. Stained sections have been independently scored for OX40+ lymphocyte area percentage by two pathologists and a subset will be digitally imaged and analyzed. Image analysis includes OX40+ lymphocyte enumeration and area calculation within pathologist-defined regions of interest (ROI) to include the tumor and surrounding stroma. OX40 prevalence (OX40-positive cells/mm2 and % OX40+ cell area) were compared with patient age, clinical parameters, and outcome, where available, to determine potential associations.ResultsResults to be reported include a.) pathologist scoring of OX40+ cell prevalence b.) image analysis of OX40+ cell number and area percentage in a subset of indications c.) concordance between image analysis and pathologists' scores d.) OX40 prevalence by tumor type e.) OX40 prevalence and association with clinical features, where available, and statistical significance.ConclusionsPrevalence of OX40 expression and association with clinical features are first reported in some solid tumor types.
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