Pretreatment Peripheral Blood Mononuclear Cells Research Articles

DOP19 High dimensional profiling of IL-23-responsive cells revealed molecular signatures that predict response to anti-IL-23 therapy in patients with Crohn’s Disease

Abstract Background The interleukin (IL)-23 signaling plays a crucial role in the pathogenesis of Crohn’s disease (CD). IL-23 activates T-helper (Th) 17 cells, IL-17-secreting CD8 T (Tc)17 cells, gd T cells, nature killer (NK) T cells, and group 3 innate lymphoid cells (ILC3) to secrete proinflammatory cytokines including IL-17, IFN-g, and TNF-a. Risankizumab is the first selective IL-23 antagonist approved for moderate-severe CD. However, there is currently no biomarker to predict response to anti-IL-23 antibodies in IBD. Here, we characterized the cell type- and cytokine-specific pathways in IL-23-responsive cells that predict response to risankizumab in CD patients. Methods Adult patients with a history of ileal, colonic, or ileocolonic CD were included. Active CD in terminal ileum and/or colon were confirmed by colonoscopy and/or MRI. Blood samples were collected within 3 months before and 4, 8, 12, and 52 weeks after the initiation of risankizumab. Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved for batch analysis using mass cytometry (CyTOF). Pre-treatment PBMCs were stimulated ex vivo with IL-23, and stained for lineage markers to identify Th17, Tc17, gd T, NKT cells, and ILC3, as well as intracellular cytokines including IL-17, IFN-g, TNF-a, IL-22, IL-6, and GM-CSF, using a pre-optimized and validated antibody panel. The CyTOF data were analyzed using the viSNE tool in Cytobank and FlowJo. Patients’ responses were evaluated 12-40 weeks after the initiation of risankizumab based on clinical symptoms and endoscopic/radiographic assessment. Results Among IL-23-responsive cells, increased frequency of NKT cells was identified in the peripheral blood of non-responders before initiation of risankizumab (Figure 1A). In non-responders, Tc17 cells, gd T cells, and ILC3 demonstrated significant elevations in TNF-a, a key pathogenic mediator in CD. gd T cells from non-responders also showed increased IL-17F before treatment. In contrast, NKT cells from non-responders expressed significantly lower GM-CSF, which was previously shown to alleviate intestinal inflammation in patients and mouse models (Figure 1B). Ongoing study is investigating the cytokine production in IL-23-responsive cells after the initiation of risankizumab, as well as before and after starting ustekinumab, an anti-IL-12/IL-23 antibody. Conclusion High dimensional profiling of pre-treatment PBMCs revealed molecular signatures, e.g., elevated TNF-a in multiple IL-23-responsive cells, which are associated with resistance to risankizumab in CD patients. Those findings may help identify response biomarkers for IL-23 antagonists and provide a rationale for using anti-IL-23/anti-TNF combination therapy in some patients with refractory CD.

Immunologic Signatures of Peripheral Blood T Cells Reveal the Outcome of p53MVA Vaccine and Pembrolizumab Treatment in Patients with Advanced Ovarian Cancer.

Our previous studies indicated that p53-reactive T cells were associated with clinical benefit in patients with advanced ovarian cancer who were treated with p53-expressing modified vaccinia Ankara (p53MVA) vaccine and gemcitabine chemotherapy. To replace chemotherapy with an approach that will enhance vaccine efficacy and antitumor immunity, we treated patients with p53MVA in combination with PD-1 checkpoint blocker, pembrolizumab. We also attempted to further characterize the activation status of T cells prior to vaccination and during treatment. Patients received up to three triweekly vaccinations concurrent with pembrolizumab, followed by pembrolizumab monotherapy at 3-week intervals. Correlative studies analyzed peripheral blood T-cell phenotypes and profiles of immune function gene expression. We observed 6/28 (21%) patients with a clinical benefit to therapy, including 3 partial responses (PR) and 3 patients with stable disease (SD) for 6+ months. The median progression-free survival was 1.8 months (95% confidence interval: 1.7-3.8) and median overall survival was 15.1 months (9.4-30.4). Two patients remain progression-free at 28 and 33 months. Of the 18 patients evaluable in correlative studies, 6 were immunologic responders of whom 5 had clinical benefit (3 PR, 2 SD). Immunologic non-responders expressed in pretreatment peripheral blood mononuclear cell samples high levels of mRNA for multiple molecules associated with terminally differentiated T cells. p53MVA/pembrolizumab immunotherapy showed promising antitumor activity in patients who demonstrated functionally competent peripheral blood T cells. Detection of markers of terminally differentiated T cells before treatment may identify patients unlikely to respond to p53MVA/pembrolizumab. The activity of a combination immunotherapy of p53 vaccine and PD-1 checkpoint blockade in patients with platinum-resistant ovarian cancer was evaluated in a phase II trial. Clinical benefit was correlated with the responsive immune status of patients before and during the treatment, defining potential predictive markers for immune therapy.

Evaluating Tumor-Intrinsic and Patient-Specific Mechanisms of Resistance to Teclistamab in Anti-BCMA Exposed and Naïve Multiple Myeloma

Background: Teclistamab (Tec) is a CD3 x BCMA bispecific antibody granted accelerated FDA approval for treating relapsed/refractory (RR) multiple myeloma (MM) based off the results of the MajesTEC-1 trial (Usmani S, et al. Lancet 2021, Moreau P, et al. N Engl J Med 2022). However, patients with prior exposure to anti-BCMA therapies were excluded from this trial and there is no published data evaluating Tec efficacy in this setting. Additionally, little is known about tumor-intrinsic and patient-specific immunologic predictors of response to Tec. Methods: We performed an IRB-approved analysis of clinical outcomes for 52 commercially treated Tec patients treated as of 07/28/2023 at our center. Results were correlated to pre-treatment BCMA expression measured by immunohistochemistry (IHC). We also profiled peripheral blood T-cells from pre-treatment peripheral blood mononuclear cell (PBMC) samples from a sub cohort of patients via high-dimensional spectral cytometry using a 37-color panel including lineage, exhaustion, and activation markers. Results: Our commercial cohort was older (median age 70 vs 64), more heavily pretreated (median prior lines of therapy 7 vs 5) and had higher incidence of both high-risk cytogenetic abnormalities (33% vs 26%) and extramedullary disease (35% vs 17%) than the MajesTEC-1 population. Additionally, 52% had prior exposure to anti-BCMA therapies, including belantamab mafodotin (31%), anti-BCMA CAR T-cell therapies (37%), and anti-BCMA bispecific antibody therapies (4%), with some having exposure to multiple prior anti-BCMA therapies (17%). Among response evaluable patients (90%), with a median follow-up time of 100 days, the overall response rate (ORR) to Tec was 64% (36% ≥VGPR). Progression free survival (PFS) in patients without prior BCMA therapy was higher than in the anti-BCMA exposed population (median PFS NR vs 102 days, p = 0.032, Figure Panel A). However, the anti-BCMA therapy exposed population was more heavily pretreated (8 vs 5 median prior lines of therapy) and had more patients with high-risk cytogenetic abnormalities (46% vs 19%) and extramedullary disease (42% vs 27%) than the anti-BCMA therapy naïve cohort. No major differences in BCMA expression were noted between responding and non-responding patients or between anti-BCMA exposed and anti-BCMA naïve patients. However, two patients had absent BCMA expression following prior treatment with other BCMA targeting agents and did not respond to Tec. Cytokine release syndrome (CRS) during step-up dosing was strongly associated with Tec efficacy (median PFS NR vs 27 days, p <0.0001), with a 100% ORR for patients with CRS. Immune profiling experiments (Figure Panel B) revealed that Tec responders had peripheral blood enriched for both CD8 + effector memory T-cells (TEM, >6-fold increase, p = 0.0499) and CD8 + effector memory T-cells re-expressing CD45RA (TEMRA, >5-fold increase, p = 0.012) when compared to Tec non-responders. Tec non-responders had peripheral blood enriched for TIGIT + regulatory T-cells (Tregs, 3-fold increase, p = 0.03) when compared to Tec responders. No differences in PD-1, CTLA-4, LAG-3 or TIM-3 expression were observed in Tec responders vs non-responders. Patients with recent relapse from non-BCMA targeting bispecific antibodies (including products targeting FcRH5 or GPRC5D) had peripheral blood enriched for γδ T-cells with high TIGIT expression. Conclusion: Tec is an effective therapy in RRMM with a slightly lower efficacy observed in patients with prior anti-BCMA therapy exposure. PFS reductions among these anti-BCMA exposed patients may be partly due to independent disease associated risk factors. A pre-Tec peripheral blood T-cell population enriched with highly cytotoxic effector T-cells was associated with response to therapy, while suppressive TIGIT +Tregs were associated with nonresponse, suggesting a potential therapeutic role for TIGIT blockade or CD25 + cell depletion to enhance the therapeutic efficacy of bispecific antibodies. Clinical and translational data from additional patients will be presented at the meeting.

Host immune signatures as predictors of response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC)

Abstract Background Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. However, clinical responses to systemic therapies in mRCC remain largely unpredictable and robust biomarkers are still lacking. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with ICI-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. T-cell receptor sequencing (TCR-seq) was performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-sequencing (RNA-seq) was performed on available pre-treatment PBMCs and primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts (186 VEGF-TT, 126 ICI and 74 ICI+VEGF-TT). Following quality-control, TCR-seq data were available for 367 patients (228 pre-treatment and 139 on-treatment), while RNA-seq data were available for 105 PBMC and 17 tumor-derived pre-treatment samples. In the TCR-seq analysis, responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06) (Fig. 1A), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). Therefore, responders to ICI-based regimens seem to have a more oligoclonal TCR repertoire (increased clonality) at baseline with no treatment-induced changes, whereas non-responders to ICI-based regimens seem to evolve from a more polyclonal to a more oligoclonal TCR repertoire in response to treatment. The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01) (Fig. 1B). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Furthermore, while shared clonotypes were detected between blood and tumor samples, there were no differences in the Jaccard similarity index between responders and non-responders to ICI-based or VEGF-TT regimens. Figure 1: (A) Evaluation of pre-treatment and post-treatment TCR clonality in responders vs. non-responders across treatment cohorts. (B) Differences in the fraction of IGH isotypes between responders and non-responders across treatment cohorts. Conclusions We were successfully able to characterize T-cell receptor and B-cell IgH repertoires in a large cohort of patients with mRCC, and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development. CDMRP DOD Funding: yes

Low Pretreatment CD4+:CD8+ T Cell Ratios and CD39+CD73+CD19+ B Cell Proportions Are Associated with Improved Relapse-Free Survival in Head and Neck Squamous Cell Carcinoma.

The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV+ patients contained increased proportions of CD39-CD73-CD4+ T cells and reduced proportions of CD39-/+CD73+CD4+ T cells compared to the equivalent cells from HPV- patients. Notably, the pretreatment CD4+:CD8+ T cell ratios and CD39+CD73+CD19+ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted.

Alterations in immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients receiving HER2-targeted neo-adjuvant therapy.

The phase II neo-adjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy in combination with trastuzumab, lapatinib or both in patients with HER2+ breast cancer. We studied circulating immune cells looking for alterations in phenotype, genotype and cytotoxic capacity (direct and antibody-dependent cell-mediated cytotoxicity (ADCC)) in the context of treatment response. Peripheral blood mononuclear cells (PBMCs) were isolated from pre- (n = 41) and post- (n = 25) neo-adjuvant treatment blood samples. Direct/trastuzumab-ADCC cytotoxicity of patient-derived PBMCs against K562/SKBR3 cell lines was determined ex vivo. Pembrolizumab was interrogated in 21 pre-treatment PBMC ADCC assays. Thirty-nine pre-treatment and 21 post-treatment PBMC samples were immunophenotyped. Fc receptor genotype, tumour infiltrating lymphocyte (TIL) levels and oestrogen receptor (ER) status were quantified. Treatment attenuated the cytotoxicity/ADCC of PBMCs. CD3+/CD4+/CD8+ T cells increased following therapy, while CD56+ NK cells/CD14+ monocytes/CD19+ B cells decreased with significant post-treatment immune cell changes confined to patients with residual disease. Pembrolizumab-augmented ex vivo PBMC ADCC activity was associated with residual disease, but not pathological complete response. Pembrolizumab-responsive PBMCs were associated with lower baseline TIL levels and ER+ tumours. PBMCs display altered phenotype and function following completion of neo-adjuvant treatment. Anti-PD-1-responsive PBMCs in ex vivo ADCC assays may be a biomarker of treatment response.

Role of CD14+ monocyte-derived oxidised mitochondrial DNA in the inflammatory interferon type 1 signature in juvenile dermatomyositis

ObjectivesTo define the host mechanisms contributing to the pathological interferon (IFN) type 1 signature in Juvenile dermatomyositis (JDM).MethodsRNA-sequencing was performed on CD4+, CD8+, CD14+ and CD19+ cells sorted from pretreatment...

Single-cell RNA sequencing reveals the potential mechanism of heterogeneity of immunomodulatory properties of foreskin and umbilical cord mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) are heterogeneous populations. Heterogeneity exists within the same tissue and between different tissues. Some studies have found enormous heterogeneity in immunomodulatory function among MSCs derived from different tissues. Moreover, the underlying mechanism of heterogeneity in immunomodulatory abilities is still unclear.MethodsForeskin mesenchymal stromal cells (FSMSCs) and human umbilical cord mesenchymal stromal cells (HuMSCs) were isolated and cultured until the third passage. According to the International Association for Cell Therapy standard, we confirmed the cell type. Then, FSMSCs and HuMSCs were cocultured with human peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) in vitro. Furthermore, the supernatant was sampled for an enzyme-linked immunosorbent assay to investigate the secretion of IL-1β, IL-6, IL-10, TNF-α, and TGF-β1. Finally, we performed single-cell RNA sequencing (scRNA-seq) of FSMSCs and HuMSCs.ResultsWe successfully identified FSMSCs and HuMSCs as MSCs. When cocultured with LPS pretreated PBMCs, FSMSCs and HuMSCs could effectively reduced the secretion of IL-1β and TNF-α. However, FSMSCs stimulated the PBMCs to secrete more IL-10, TGF-β1, and IL-6. Furthermore, 4 cell subsets were identified from integrated scRNA-seq data, including proliferative MSCs (MKI67+, CD146low+, NG2+, PDGFRB−), pericytes (CD146high+, PDGFRB+, MKI67−, CD31−, CD45−, CD34−), immune MSCs (CXCL12high+, PTGIShigh+, PDGFRB+, CD146−, MKI67−) and progenitor proliferative MSCs (CXCL12low+, PTGISlow+, PDGFRB+, CD146−, MKI67−). Among them, we found that immune MSCs with strengthened transcriptional activity were similar to pericytes with regard to the degree of differentiated. Various of immune-related genes, gene sets, and regulons were also enriched in immune MSCs. Moreover, immune MSCs were determined to be close to other cell subsets in cell–cell communication analysis. Finally, we found that the proportion of immune MSCs in foreskin tissue was highest when comparing the subset compositions of MSCs derived from different tissues.ConclusionsFSMSCs show better immunomodulatory capacity than HuMSCs in vitro. Moreover, immune MSCs may play a vital role in the heterogeneity of immunoregulatory properties. This study provides new insights suggesting that immune MSCs can be isolated to exert stable immunoregulatory functions without being limited by the heterogeneity of MSCs derived from different tissues.

T cell repertoire in peripheral blood as a potential biomarker for predicting response to concurrent cetuximab and nivolumab in head and neck squamous cell carcinoma

BackgroundT cell receptor (TCR) signaling profile is a fundamental property that underpins both adaptive and innate immunity in the host. Despite its potential clinical relevance, the TCR repertoire in peripheral...

Circulating Immune Bioenergetic, Metabolic, and Genetic Signatures Predict Melanoma Patients' Response to Anti-PD-1 Immune Checkpoint Blockade.

Immunotherapy with checkpoint inhibitors is improving the outcomes of several cancers. However, only a subset of patients respond. Therefore, predictive biomarkers are critically needed to guide treatment decisions and develop approaches to the treatment of therapeutic resistance. We compared bioenergetics of circulating immune cells and metabolomic profiles of plasma obtained at baseline from patients with melanoma treated with anti-PD-1 therapy. We also performed single-cell RNA sequencing (scRNAseq) to correlate transcriptional changes associated with metabolic changes observed in peripheral blood mononuclear cells (PBMC) and patient plasma. Pretreatment PBMC from responders had a higher reserve respiratory capacity and higher basal glycolytic activity compared with nonresponders. Metabolomic analysis revealed that responder and nonresponder patient samples cluster differently, suggesting differences in metabolic signatures at baseline. Differential levels of specific lipid, amino acid, and glycolytic pathway metabolites were observed by response. Further, scRNAseq analysis revealed upregulation of T-cell genes regulating glycolysis. Our analysis showed that SLC2A14 (Glut-14; a glucose transporter) was the most significant gene upregulated in responder patients' T-cell population. Flow cytometry analysis confirmed significantly elevated cell surface expression of the Glut-14 in CD3+, CD8+, and CD4+ circulating populations in responder patients. Moreover, LDHC was also upregulated in the responder population. Our results suggest a glycolytic signature characterizes checkpoint inhibitor responders; consistently, both ECAR and lactate-to-pyruvate ratio were significantly associated with overall survival. Together, these findings support the use of blood bioenergetics and metabolomics as predictive biomarkers of patient response to immune checkpoint inhibitor therapy.

TP53 Aberrations and Outcomes in MBL and Untreated CLL

TP53 Aberrations and Outcomes in MBL and Untreated CLL

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Single-sample enrichment analysis identified predictive biomarker candidates for nivolumab in patients with non-small cell lung cancer.

e21097 Background: Although anti-PD-1/PD-L1 monotherapy has achieved clinical success in non-small cell lung cancer (NSCLC), definitive predictive biomarkers remain to be elucidated. We assumed that by combining gene expression signatures with patient clinical data, we could identify a novel promising biomarker to predict response to anti-PD-1/PD-L1 monotherapy in NSCLC patients. Moreover, the characterization of these signatures will help us to decipher the complexity of tumor-immune interactions and better understand the tumor microenvironment (TME) that favors clinical response to nivolumab monotherapy. Methods: From clinically annotated NSCLC patients (n = 40) with nivolumab monotherapy in the second- or later-line settings, we prospectively collected tumor tissues and peripheral blood mononuclear cells (PBMCs) before first dose of nivolumab and PBMCs after first 4 or 5 doses of nivolumab. All tumor tissue and PBMC samples obtained were applied to whole-transcriptome sequencing (RNA-seq). We extracted transcriptomic datasets of lung adenocarcinoma (LUAD) (n = 20) and lung squamous cell carcinoma (LUSC) (n = 18) from the results, separately analyzed each histological subtype. To elucidate biological processes associated with clinical outcomes, we performed a supervised gene set enrichment analysis (GSEA) approach and an unsupervised single sample scoring approach. Results: In LUAD, we observed that gene sets related to interferon (type I and II) signaling (‘IFN signatures’) and antigen processing and presentation (‘APP signatures’) were significantly enriched in pre-treatment PBMCs of responders. IFN and APP signatures, which are closely related to each other, functionally cooperate to activate anti-tumor immune response. The enrichment of IFN and APP signatures provides the possibility that responders have a pre-existing anti-tumor immunity prior to nivolumab monotherapy. In LUSC, neither IFN nor APP signatures were enriched in pre-treatment tumor tissues and PBMCs of responders. Instead, gene sets related to the regulation of the TME (‘TME signatures’) are significantly enriched in pre-treatment tumor tissues of non-responders. The enrichment of TME signatures suggested that non-responders have an extremely immunosuppressive TME. These findings highlighted that responsive LUAD inherently have a high immunogenicity to elicit effective anti-tumor responses, whereas responsive LUSC have a similar level of immunogenicity as non-responsive LUSC but are free from an extremely immunosuppressive TME. Conclusions: We found that nivolumab enhanced anti-tumor immunity in patients with LUAD in a quite different way from patients with LUSC. Our study provides a blueprint for innovating combinational immunotherapy and supporting patient selection and treatment strategies on long-term clinical outcomes.

Association of DNA-Methylation Profiles With Immune Responses Elicited in Breast Cancer Patients Immunized With a Carbohydrate-Mimicking Peptide: A Pilot Study.

Immune response to a given antigen, particularly in cancer patients, is complex and is controlled by various genetic and environmental factors. Identifying biomarkers that can predict robust response to immunization is an urgent need in clinical cancer vaccine development. Given the involvement of DNA methylation in the development of lymphocytes, tumorigenicity and tumor progression, we aimed to analyze pre-vaccination DNA methylation profiles of peripheral blood mononuclear cells (PBMCs) from breast cancer subjects vaccinated with a novel peptide-based vaccine referred to as P10s-PADRE. This pilot study was performed to evaluate whether signatures of differentially methylated (DM) loci can be developed as potential predictive biomarkers for prescreening subjects with cancer who will most likely generate an immune response to the vaccine. Genomic DNA was isolated from PBMCs of eight vaccinated subjects, and their DNA methylation profiles were determined using Infinium® MethylationEPIC BeadChip array from Illumina. A linear regression model was applied to identify loci that were differentially methylated with respect to anti-peptide antibody titers and with IFN-γ production. The data were summarized using unsupervised-learning methods: hierarchical clustering and principal-component analysis. Pathways and networks involved were predicted by Ingenuity Pathway Analysis. We observed that the profile of DM loci separated subjects in regards to the levels of immune responses. Canonical pathways and networks related to metabolic and immunological functions were found to be involved. The data suggest that it is feasible to correlate methylation signatures in pre-treatment PBMCs with immune responses post-treatment in cancer patients going through standard-of-care chemotherapy. Larger and prospective studies that focus on DM loci in PBMCs is warranted to develop pre-screening biomarkers before BC vaccination.Clinical Trial Registration: www.ClinicalTrials.gov, Identifier: NCT02229084.

Anti-PD-1 responsive circulating immune cells as biomarkers of response to neoadjuvant chemotherapy in HER2+ breast cancer (BC).

e12646 Background: HER2+ BC patients (pts) achieving a pathological complete response (pCR) to neo-adjuvant therapy have better long-term survival outcomes. Alternative treatment strategies may be more beneficial for pts that will not achieve a pCR but there are no predictive biomarkers for stratification of pts to appropriate clinical studies. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by immune cells like NK cells can be elicited by therapeutic antibody therapies including the HER2-targeting trastuzumab (T). We hypothesised that the presence of immune checkpoint-inhibited ADCC-mediating immune cells in the blood may identify pts with an immune-suppressed tumour microenvironment who will not achieve a pCR. We used an in vitro functional assay to assess the PD-1-inhibited ADCC response of pre-treatment peripheral blood mononuclear cells (PBMCs) from HER2+ BC pts as a potential biomarker of pCR. Methods: Pre-treatment PBMC samples (n = 21) were obtained from HER2+ BC pts who received neo-adjuvant chemotherapy (docetaxel, carboplatin) with a HER2-targeted therapy (T n = 5, T and lapatinib (L) n = 13, or L alone n = 3), ICORG 10-05 (NCT01485926). Pt response was determined as pCR (n = 7), partial response (PR, n = 10) or non-response (NR, n = 4). The immune function assay used a flow cytometry-based method to determine the T (10µg/µl)-mediated ADCC (T-ADCC) capacity of the PBMCs +/- the anti-PD-1 therapy pembrolizumab (P) (10µg/µl). The HER2+ BC cell line SKBR3 was used as the target cell line. Direct PBMC-mediated cytotoxicity against the non-MHC Class I-restricted leukemic K562 cell line was also determined. Statistical analysis was carried out using the MedCalc program. Results: There was no significant difference in direct cytotoxicity against K562 (pCR vs NR p = 0.13, pCR vs PR p = 0.52, PR vs NR p = 0.40), or in T-ADCC against SKBR3 (pCR vs NR p = 0.71, pCR vs PR p = 0.52, PR vs NR p = 0.32), between response cohorts in the absence of P. When T-ADCC was examined in individual pt samples, the addition of P resulted in significant increases in T-ADCC vs T-ADCC in the absence of P in 3/4 NR (p = 0.03, p = 0.01, p = 0.002) and 4/10 PR (p = 0.003, p = 0.007, p = 0.009, p = 0.01). No significant increase in T-ADCC was observed in the pCR cohort (0/7) as a result of the addition of P. Using pre-treatment PBMCs, the assay detected 7/14 (50%) of HER2+ BC pts who did not achieve a pCR. Conclusions: PD-1-inhibited ADCC-capable immune cells may identify a subset of HER2+ BC pts who will not achieve a pCR. Further investigation is warranted using a larger patient cohort.

Abstract C009: Clinical pharmacokinetic and pharmacodynamic analyses support biological effectivity of the PARP inhibitor olaparib as radiosensitizer at low doses

Abstract Introduction: PARP inhibitors are promising radiosensitizers. Currently identified recommended phase II doses of PARP inhibitors in combination with radiotherapy are 10-fold lower than the EMA and FDA approved dose as single agent. The purpose of this study is to investigate the biologically effective dose range of olaparib for its use as radiosensitizer by assessing pharmacokinetics (PK) and pharmacodynamics (PD). Both the inhibition of pre-treatment PAR levels and the inhibition of radiation induced PARylation therefore have to be determined. Methods: We included PK and PD samples of all NSCLC and breast cancer patients treated at olaparib doses up to 50mg bi-daily (BID) in two phase 1 trials combining radical radiotherapy with olaparib. Plasma samples were collected for PK analysis (ranging from steady state pre-dose olaparib until +12/24 hrs after intake) and peripheral blood mononuclear cells (PBMCs) for PD analysis (pre-treatment, +3 hrs and +12/24 hrs after olaparib intake). Breast cancer patients with the primary tumor in situ had biopsies taken pre-treatment and during olaparib treatment (+3 hrs after intake) before the first fraction of radiotherapy. Olaparib concentrations were determined by HPLC-MS/MS. PAR levels were determined by commercially available ELISA, following both the NCI protocol and REP assay (de Haan et al, 2017) that includes ex vivo irradiation of intact cells to activate PARylation by PARP. PAR levels determined by the REP assay were used to calculate PAR level inhibition during treatment. Results: PK/PD data from blood samples were available from 28 NSCLC patients and 7 breast cancer patients treated with radical radiotherapy and olaparib (17 at 25mg once daily (QD), 14 at 25mg BID and 4 at 50mg BID). Repeat biopsies were available from six out of seven breast cancer patients. The plasma olaparib concentrations increased with the dose levels and showed an expected wide intra-patient variation. In tumors the olaparib concentration varied between 178 and 1441 ng/g with a median tumor to plasma ratio of 0.47. PD analyses demonstrate a significant reduction in PAR levels in PBMCs during olaparib treatment (>95% at +3 hrs after olaparib intake for all dose levels, >90% at +12 hrs in the 25mg BID dose level and 66-99% at +24 hrs in the 25mg QD dose level; all p<0.0001). In pre-treatment samples, ex vivo radiation induced PAR levels by 66-fold (range 23 to 174). This radiation induced PARylation was abolished during olaparib treatment. Compared to corresponding pre-treatment PBMC samples, PAR levels in pre-treatment tumor biopsy samples were higher in all but one patient (median 7-fold higher, range 0.6-60), and increased by only 1.5-fold after ex vivo radiation. Also in tumors, olaparib abolished radiation induced PARylation. PAR levels were reduced by 89% (range 83-99.7%, p<0.0001). Conclusion: Olaparib doses as low as 25mg once and twice daily inhibit PARP activation by irradiation and reduce PAR levels >95% in PBMCs and >83% in tumors, thereby showing biological effectivity. The tumor olaparib concentrations determined in this study were all within a range that has been shown to radiosensitize in preclinical models. Together this supports further development of PARP inhibitors as radiosensitizer at low doses. Citation Format: Rosemarie de Haan, Dick Pluim, Manon Verwijs, Gabe Sonke, Michel van den Heuvel, Baukelien van Triest, Marcel Verheij, Conchita Vens. Clinical pharmacokinetic and pharmacodynamic analyses support biological effectivity of the PARP inhibitor olaparib as radiosensitizer at low doses [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C009. doi:10.1158/1535-7163.TARG-19-C009

Patients with BRCA mutated ovarian cancer may have fewer circulating MDSC and more peripheral CD8+ T cells compared with women with BRCA wild-type disease during the early disease course.

Immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are associated with immunologic tolerance and poor prognosis in ovarian cancer (OvCa). We hypothesized that women with germline BRCA1 and BRCA2 mutation-associated (gBRCAm) OvCa would have fewer circulating immunosuppressive immune cells compared to those with BRCA wild-type (BRCAwt) disease during their early disease course (<5 years post-diagnosis) where gBRCAm is a favorable prognostic factor. We collected and viably froze peripheral blood mononuclear cells (PBMCs) from patients with recurrent OvCa olaparib clinical trials (NCT01445418/NCT01237067). Immune subset analyses were performed using flow cytometry for Tregs, exhausted CD8+ T cells, monocytes and MDSCs. Functional marker expression, including cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3) and programmed cell death protein 1 (PD-1) was evaluated. Data were analyzed using FlowJo. Pretreatment PBMCs were collected from 41 patients (16 gBRCAm/25 BRCAwt). The percentage of MDSCs among viable CD45+ PBMC was lower in gBRCAm OvCa compared with BRCAwt OvCa (median 0.565 vs. 0.93%, P=0.0086) but this difference was not seen in those women >5 years post-diagnosis. CD8+ T cells among viable CD45+ PBMCs and CTLA-4+/CD8+ T cells were higher in gBRCAm carriers than patients with BRCAwt, in particular for those <5 years post-diagnosis (median 20.4 vs. 9.78%, P=0.031 and median MFI 0.19 vs. 0.22, P=0.0074, respectively). TIM-3 expression on Tregs was associated with poor progression-free survival, independent of gBRCAm status (P<0.001). Our pilot data suggested that patients with gBRCAm OvCa may have fewer circulating MDSCs but higher CD8+ T cells in PBMCs during their early disease course. This may contribute to the observed survival benefit for these women in their first post-diagnosis decade.

Abstract 4094: The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo

Abstract Background: G100, which contains the synthetic TLR4 agonist Glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water emulsion (SE) for intratumoral therapy, is currently in clinical development in combination with pembrolizumab for the treatment of B-cell follicular lymphoma (FL). The current study was undertaken to investigate the interaction of G100 with rituximab, the anti-CD20 mAb that is standard-of-care treatment for FL. Methods: In vitro studies were performed using peripheral blood mononuclear cells (PBMC) from healthy human donors to investigate the effect of GLA-AF (an aqueous formulation of GLA) on natural killer (NK) cell activation. IFNγ production in NK cells after activation with plate-bound anti-CD16 or target K562 leukemia cells was measured by intracellular cytokine staining (ICS). Rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) was measured using pretreated or untreated PBMC and rituximab-coated Raji Burkitt’s lymphoma cells as target cells. In vivo studies were carried out in Balb/c mice with bilateral A20 lymphoma. Mice received G100 alone (10μg, intratumoral, 3 injections/week), anti-CD20 alone (clone 5D2, provided by Genentech, 200μg, intraperitoneal, 1 injection/week), or G100+anti-CD20 for a total of 3 weeks. Results: Incubation of PBMC from 3 different donors with GLA-AF (1 μg/mL) resulted in NK cell activation as demonstrated by increased IFNγ production, as well as enhanced response to the K562 target cells and anti-CD16. When PBMC from 5 normal donors were used in rituximab-mediated ADCC assay, pretreated PBMCs had significantly enhanced lysis of target Raji cells compared to untreated PBMC (p&amp;lt;0.05). In mice with bilateral A20 tumors, the combination of G100 with anti-CD20 resulted in significantly better tumor control and prolonged survival (n=5 per group, p&amp;lt;0.05). Conclusions: G100 enhances NK cell activity and rituximab-mediated ADCC in human PBMC. Combination of G100 and anti-CD20 mAb has synergistic anti-tumor effects in a mouse lymphoma model. These data support clinical testing of G100 with rituximab in patients with FL. Citation Format: Hailing Lu, Alec Betancur, Jan ter Meulen. The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo [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 4094.

Abstract 4466: Somatic mutations within chronic lymphocytic leukemia (CLL) putative driver genes are associated with outcomes beyond the CLL international prognostic index (CLL-IPI)

Abstract CLL is a clinically heterogeneous disease with wide ranging disease course. A novel CLL-IPI based on Rai/Binet stage, IGHV-mutation status, TP53 mutation/deletion, B2M level, and age was developed to stratify patients into 4 risk groups, with a c-statistic of 0.75. Next-generation sequencing has identified ~60 genes recurrently mutated in CLL, some of which are associated with poor overall survival, whereas the clinical effect of most genes is still unknown. Herein, we examine whether somatic mutations in these putative driver genes are associated with time to first treatment (TTT), and whether they add prognostic value beyond CLL-IPI. Based on the 2008 International Workshop CLL criteria, we identified 100 CLL and 96 high-count monoclonal B-cell lymphocytosis (MBL) newly diagnosed from the Mayo Clinic CLL biobank. Pre-treatment peripheral blood mononuclear cells were collected &amp;lt;2 years of diagnosis and tumor DNA was extracted from sorted CD5+/CD19+. We sequenced the coding regions of 61 recurrently mutated CLL driver genes using a custom SureSelect panel, with 24 samples per flow cell in Illumina HiSeq 4000. The average coverage depth was &amp;gt;1000X. Somatic mutations were called using MuTect2 in tumor-only mode. To remove germline variants, variants were eliminated based on minor allele frequencies &amp;gt;0.01%, identified in 1000 Genomes Project, ExAC and/or ESP6500 databases, unless present in known mutation hotspots or COSMIC. After filtering, high/moderate impact mutations were analyzed using Cox regression, to estimate hazard ratios (HR) and 95% confidence intervals (CI) to test for associations with TTT. Among 196 patients the most commonly mutated genes were TP53 (11%), ATM (10%), SF3B1 (10%), NOTCH1 (9%), CHD2 (8%), and BIRC3 (7%). The median follow-up was 8.7 years, and 73 patients were subsequently treated. ATM (HR=3.27, CI:1.8-6.1, P=0.0002) and NOTCH1 (HR=2.41, CI:1.3-4.6, P=0.008) were associated with TTT. When evaluating the total number of mutated genes, we found 32%, 29%, and 39% patients had ≥2, 1, or 0 genes mutated, respectively, and this was associated with shorter TTT (HR=1.74, CI:1.3-2.4, P=0.0005) adjusting for sex and CLL-IPI with a c-statistic=0.8 (CI: 0.75-0.84). When stratified by CLL-IPI, the association held for low (N=99, HR=1.88, CI:1.1-3.4, P=0.03) and intermediate risk (N=54, HR=1.87, CI:1.1-3.2, P=0.03) but not high/very high risk (N=35, HR=1.07, CI:0.6-1.9, P=0.83). We demonstrated that the total number of CLL putative driver genes with high or moderate impact mutations provided prognostic information in newly diagnosed CLL/MBL beyond CLL-IPI. Moreover, even among those with low or intermediate CLL-IPI risk, the total number of somatic mutations separated those patients who progressed. Sequencing the CLL driver genes at time of diagnosis could be a potential biomarker for outcome prediction. Citation Format: Geffen Kleinstern, Daniel R. O’Brien, Brian F. Kabat, Kari G. Chaffee, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, James R. Cerhan, Neil E. Kay, Susan L. Slager, Esteban Braggio. Somatic mutations within chronic lymphocytic leukemia (CLL) putative driver genes are associated with outcomes beyond the CLL international prognostic index (CLL-IPI) [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 4466.

Peripheral Blood TCR Repertoire Profiling May Facilitate Patient Stratification for Immunotherapy against Melanoma.

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4; however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE50, with low values corresponding to more clonality and lack of TCR diversity) in pretreatment peripheral blood mononuclear cells from melanoma patients treated with anti-CTLA4 (n = 42) or anti-PD1 (n = 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A receiver operating characteristic curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but, on the other hand, predicted a poor response to CTLA4 inhibition. Multivariate logistic regression models identified DE50 as the only independent predictive factor for response to anti-CTLA4 therapy (P = 0.03) and anti-PD1 therapy (P = 0.001). Fisher exact tests confirmed the association of low DE50 with response in the anti-CTLA4 (P = 0.041) and the anti-PD1 cohort (P = 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCR-based method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.