Changes In Tumor-infiltrating Lymphocytes Research Articles

Abstract 6554: Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Repression of the MHC-I and reduced tumor infiltration of cytotoxic T-lymphocytes is often associated with immunotherapy resistance in SCLC. Lurbinectedin is FDA-approved as a second-line treatment for SCLC and we previously showed lurbinectedin as an effective therapeutic strategy in SCLC preclinical models. There are ongoing clinical trials combining immune checkpoint blockade with lurbinectedin in ES-SCLC. However, there is no insight into the effect of lurbinectedin on the immune microenvironment in SCLC. In this study, we evaluated the effect of lurbinectedin on the immune microenvironment and the anti-tumor effect with or without PD-L1 blockade. Results: To determine the effect of lurbinectedin in SCLC models, we treated immunocompetent flank RPP (Rb1, Trp53, and p130) and RPM (Rb1, Trp53, and MYCT58A) tumor-bearing mice with lurbinectedin (0.2 mg/kg, 1/7days) and/or anti-PD-L1 (300µg, 1/7days) for 3 weeks. Mice treated with anti-PD-L1 alone showed no anti-tumor response, and single-agent lurbinectedin caused a delay in tumor growth. However, 6 of the 10 mice treated with the combination of lurbinectedin and anti-PD-L1, had a complete response (100% reduction) and the other 4 had 90% tumor regression. Tumors were resected and analyzed by multicolor flow cytometry for changes in tumor-infiltrating lymphocytes (TILs). Furthermore, analysis of immune cells infiltrating into the tumors post- treatment showed significant induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells in the combination treatment arm. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased, while immunosuppressive M2 type macrophages and MDSC cells were decreased. These effects are consistent with data showing that lurbinectedin treatment leads to an activation of the cGAS/STING pathway, type I/II interferons (IFNα/β), and pro-inflammatory chemokines (CCL5, CXCL10) in tumors. Interestingly, lurbinectedin treatment led to significant upregulation of mRNA and surface expression of MHC class-I genes (HLA-A/B/C) in vitro and in vivo. Finally, bulk RNA sequencing as well as RT-PCR of pre- and post-lurbinectedin treatment demonstrated an increase in DAMPs associated with immunogenic cell death. Conclusion: We provide the first mechanistic insight into the lurbinectedin-induced multimodal immune modulation in SCLC leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Since lurbinectedin is already approved as a second-line agent in SCLC, our preclinical data provide a strong rationale for combining this regimen with inhibitors of the PD-L1 pathway and also highlight the immune subsets that the combination targets. Citation Format: Subhamoy Chakraborty, Yazhini Mahendravarman, Utsav Sen, Avisek Banerjee, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6554.

Characteristics and immunological changes of tumor-infiltrating lymphocytes during neoadjuvant chemo-immunotherapy in epithelial ovarian cancer (251)

<b>Objectives:</b> Epithelial ovarian cancer (EOC) is an aggressive gynecologic malignancy with a poor prognosis. Even after standard-care treatment, most patients eventually relapse and thus show a low survival rate. It is well known that tumor-infiltrating lymphocytes (TILs) are associated with survival outcomes in EOC. However, characteristics and dynamic changes of TILs during treatment courses have not been well investigated in EOC. In this study, we examined the characteristics of TILs from ovarian cancer patients and their dynamic changes during neoadjuvant chemotherapy (NACT) or neoadjuvant chemoimmunotherapy (NACI). <b>Methods:</b> We isolated peripheral blood mononuclear cells (PBMCs) and TILs from treatment-naïve (<i>n</i>=18) or post-NACT treated (<i>n</i>=22) or post-NACI treated EOC patients (<i>n</i>=8). NACT was paclitaxel plus carboplatin, and NACI was NACT with durvalumab plus tremelimumab. Immunologic characteristics and functions of CD8, CD4, and regulatory T (Treg) cells in each group were compared using flow cytometry. <b>Results:</b> The percentages of CD8 TILs of post-NACT and post-NACI were increased, but conventional CD4 and Foxp3⁺ Treg were decreased after NACT and NACI. Therefore, CD8/Treg cells ratio was increased in post-NACT and post-NACI. When we examined the memory differentiation status of CD8 and CD4 TILs, there was no significant difference among treatment-naïve, post-NACT, and post-NACI. The percentages of central memory and effector memory were not significantly different. In addition, comparing expressions of immune checkpoints, including PD-1, CTLA-4, and TIGIT on TILs, there was no significant difference among them. However, 4-1BB⁺ and Ki-67⁺ cells in CD8 and Treg cells were significantly decreased after NACT or NACI. It meant that tumor-reactive proliferating CD8 TILs and Treg cells were decreased after NACT. Next, we examined expressions of TCF-1 and TOX in PD-1+CD8 TIL in post-NACT or post-NACI to investigate changes of exhaustion status, and it was revealed that TCF-1⁺ cells were increased and TOX⁺ cells were decreased after NACT or NACI. However, cytokine production capacity (IFN-γ and TNF-α) of CD8 TILs was not different. In the case of Treg cells, suppressive CD45RA-Foxp3^high Treg cells and CCR8⁺ Treg cells were significantly decreased after NACT or NACI. In addition, comparing post-NACT with post-NACI, CCR8⁺ Treg cells were more significantly decreased in post-NACI. <b>Conclusions:</b> The current study investigated immunologic changes after NACT and NACI in ovarian TILs. According to a decrease in tumor burden during chemotherapy, the immunologic tumor-microenvironment changed to favor antitumor immunity. Furthermore, Treg cells with more suppressive properties (CCR8⁺ or 4-1BB⁺ Treg cells) were more decreased after NACI. Further studies are ongoing on the effect of NACI on Treg cells and their clinical significance in EOC.

Tumor Immune Microenvironment Changes by Multiplex Immunofluorescence Staining in a Pilot Study of Neoadjuvant Talazoparib for Early-Stage Breast Cancer Patients with a Hereditary BRCA Mutation.

The immunological profile of early-stage breast cancer treated with neoadjuvant PARP inhibitors has not been described. The aim of this study was to delineate the changes in the tumor immune microenvironment (TiME) induced by talazoparib. Patients with operable germline BRCA1/2 pathogenic variant (gBRCA1/2+) breast cancer were enrolled in a feasibility study of neoadjuvant talazoparib. Thirteen patients who received 8 weeks of neoadjuvant talazoparib were available for analysis, including 11 paired pre- and post-talazoparib core biopsies. Treatment-related changes in tumor-infiltrating lymphocytes were examined and immune cell phenotypes and their spatial distribution in the TiME were identified and quantified by multiplex immunofluorescence using a panel of 6 biomarkers (CD3, CD8, CD68, PD-1, PD-L1, and CK). Neoadjuvant talazoparib significantly increased infiltrating intratumoral and stromal T-cell and cytotoxic T-cell density. There was no difference in PD-1 or PD-L1 immune cell phenotypes in the pre- and post-talazoparib specimens and PD-L1 expression in tumor cells was rare in this cohort. Spatial analysis demonstrated that pre-talazoparib interactions between macrophages and T cells may correlate with pathologic complete response. This is the first study with phenotyping to characterize the immune response to neoadjuvant talazoparib in patients with gBRCA1/2+ breast cancer. These findings support an emerging role for PARP inhibitors in enhancing tumor immunogenicity. Further investigation of combinatorial strategies is warranted with agents that exploit the immunomodulatory effects of PARP inhibitors on the TiME.

Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer

BackgroundThe H&E stromal tumor-infiltrating lymphocyte (sTIL) score and programmed death ligand 1 (PD-L1) SP142 immunohistochemistry assay are prognostic and predictive in early-stage breast cancer, but are operator-dependent and may have insufficient precision to characterize dynamic changes in sTILs/PD-L1 in the context of clinical research. We illustrate how multiplex immunofluorescence (mIF) combined with statistical modeling can be used to precisely estimate dynamic changes in sTIL score, PD-L1 expression, and other immune variables from a single paraffin-embedded slide, thus enabling comprehensive characterization of activity of novel immunotherapy agents.MethodsSerial tissue was obtained from a recent clinical trial evaluating loco-regional cytokine delivery as a strategy to promote immune cell infiltration and activation in breast tumors. Pre-treatment biopsies and post-treatment tumor resections were analyzed by mIF (PerkinElmer Vectra) using an antibody panel that characterized tumor cells (cytokeratin-positive), immune cells (CD3, CD8, CD163, FoxP3), and PD-L1 expression. mIF estimates of sTIL score and PD-L1 expression were compared to the H&E/SP142 clinical assays. Hierarchical linear modeling was utilized to compare pre- and post-treatment immune cell expression, account for correlation of time-dependent measurement, variation across high-powered magnification views within each subject, and variation between subjects. Simulation methods (Monte Carlo, bootstrapping) were used to evaluate the impact of model and tissue sample size on statistical power.ResultsmIF estimates of sTIL and PD-L1 expression were strongly correlated with their respective clinical assays (p < .001). Hierarchical linear modeling resulted in more precise estimates of treatment-related increases in sTIL, PD-L1, and other metrics such as CD8+ tumor nest infiltration. Statistical precision was dependent on adequate tissue sampling, with at least 15 high-powered fields recommended per specimen. Compared to conventional t-testing of means, hierarchical linear modeling was associated with substantial reductions in enrollment size required (n = 25➔n = 13) to detect the observed increases in sTIL/PD-L1.ConclusionmIF is useful for quantifying treatment-related dynamic changes in sTILs/PD-L1 and is concordant with clinical assays, but with greater precision. Hierarchical linear modeling can mitigate the effects of intratumoral heterogeneity on immune cell count estimations, allowing for more efficient detection of treatment-related pharmocodynamic effects in the context of clinical trials.Trial registrationNCT02950259.

Changes in Tumor-infiltrating Lymphocytes After Neoadjuvant Chemotherapy and Clinical Significance in Triple Negative Breast Cancer.

Neoadjuvant chemotherapy (NAC) can affect tumors and the tumor microenvironment. As changes in tumor-infiltrating lymphocytes (TILs) after NAC and the resulting clinical significance have not been clearly defined, we evaluated both in triple negative breast cancer (TNBC). TIL level was histologically analyzed in pre-NAC biopsy and post-NAC operation specimens from 104 TNBC cases with residual invasive carcinoma after NAC. Forty-three cases (41.3%) showed decreases in TIL level, whereas 29 cases (27.9%) showed increases and no significant changes were found in 32 cases (30.8%). A decrease or increase in TIL levels corresponded to a better disease-free survival (DFS) as compared to unchanged levels. In multivariate analysis, a change in TIL level was an independent prognostic factor for DFS. We identified a prognostic significance of TIL changes after NAC. Assessment of TILs before and after NAC can provide valuable prognostic information for TNBC patients.

Abstract P4-14-01: Modulation of tumor infiltrating lymphocytes (TILs) by palbociclib, trastuzumab, pertuzumab +/- fulvestrant in ER+/HER2+ patients (pts) enrolled in the Michelangelo NA-PHER2 trial

Abstract Background We assessed TILs before and during treatment in patients with ER+/HER2+ breast cancer (BC) enrolled in the NA-PHER2 study, and evaluated the association between TILs and their modulation over treatment with likelihood of achieving a pCR. Methods In the NA-PHER2 study (NCT02530424), patients with centrally confirmed ER+ (&amp;gt;10%) HER2+ BC were treated in two independent, non-randomized, cohorts with neoadjuvant trastuzumab, pertuzumab, palbocilib with (Cohort A, n=30) or without (Cohort B, n=28) fulvestrant (+/-LHRH analogues). We measured TILs by optical evaluation on core-biopsies obtained pre-treatment [pre-CBX, n=56/58 (96.5%)], at 14 days if invasive tumor cells [14d-CBX, n=52/53 (98.1%)], and on residual disease at surgery [SX, n=45/45 (100%)]. We scored TILs as stromal and intratumoral TILs (sTILs, iTILs), and hotspot TILs (hTILs) i.e. the fields of highest TILs density. We investigated the association between baseline and 14 days TILs with pCR, including the early TILs changes (pre vs 14 days) [delta TILs (ΔTILs) was the absolute percentage change). TILs biomarkers were assessed as continuous variable (10% increase). Results The three TILs metrics were significantly correlated at all time-points (all p&amp;lt;1E-04, all corr &amp;gt;0.55). TILs metrics were not associated with T and N stage, PgR status. sTILs were correlated with Ki67 as continuous variable at 14 days (corr 0.41 p=0.003) and surgery (corr 0.34, p=0.025), but not at baseline. At baseline (pre-CBX), only high sTILs were associated with pCR (OR 1.55 [1.01-2.37], p=0.044). At 14 days (on treatment), all TILs assessments were associated with pCR (OR 1.96 [1.16-3.31], p=0.011 for sTILs; OR 5.85 [1.39-24.6], p=0.016 for iTILs; OR 1.58 [1.16-2.14], p=0.004). When combined in multivariate model, they did not provide independent information. Considering early changes of TILs (baseline vs 14 days), only ΔiTILs was associated with pCR (OR 3.09 [1.50-6.38], p=0.002). When baseline, 14 days and dynamic biomarker was assessed in multivariate, only ΔiTILs retained the significance. The predictive value of TILs was similar in the two treatment cohorts. Overall, there was not average difference between any TILs assessment method comparing the three time points. By evaluating only patients with RD and TILs assessment available at all three time points (n=41/45, 91.1%), some intra-patient variability emerged. ΔTIL higher than 10% was observed in 13/41 (31.7%) cases. Seven (17.1%) had an increase, 4 (9.7%) a mixed behavior and 2 (4.9%) had a decrease. Dynamic change of TILs was not associated with change in Ki67. When TILs infiltration in the two treatment cohort was compared at different time points, it was different only at surgery, when sTILs and iTILs were lower in patients treated with Fulvestrant in Cohort A (p=0.19 and p=0.013, respectively). Conclusions In the NA-PHER2 study, we show that in ER+/HER2+ BC the likelihood of achieving pCR using a chemo-free regimen is larger in patients with higher sTILs infiltration. TILs assessment on core biopsies after 14 days was also predictive of pCR, and early changes of iTILs was the most informative predictor suggesting that treatment may facilitate T cell infiltration in agreement with the postulated contribution of CDK4/6 to immune evasion (Goel S Nature 2017, Jerby-Arnon L Cell 2018). No average modulation of immune biomarkers was found. However, individual pts modulation suggests that immune effect might be relevant also for some patients not achieving a pCR. Notably, the presence of endocrine treatment may contribute to dampen immune activation. Changes of Ki67 and TILs were not associated suggesting that they capture different biological phenomena. Supported in part by an unrestricted grant from Pfizer Italia S.r.l. and from Roche S.p.a. Italia Citation Format: Giampaolo Bianchini, Chanel Smart, Giancarlo Bisagni, Marco Colleoni, Mauro Mansutti, Claudio Zamagni, Lucia Del Mastro, Stefania Zambelli, Luca Licata, Antonio Frassoldati, Giuseppe Viale, Pinuccia Valagussa, Luca Gianni. Modulation of tumor infiltrating lymphocytes (TILs) by palbociclib, trastuzumab, pertuzumab +/- fulvestrant in ER+/HER2+ patients (pts) enrolled in the Michelangelo NA-PHER2 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-14-01.

Abstract P1-10-20: Serial TILs: Evaluating the role of mid-treatment tumor infiltrating lymphocytes (TIL) in predicting pathologic complete response (pCR) in early-stage triple negative breast cancer (TNBC)

Abstract Introduction High levels of TIL at baseline are associated with higher pCR rates and better overall survival in TNBC. Recent studies have also indicated that higher TIL in post-NACT residual disease in TNBC are an important independent predictor of improved survival. We evaluated the role of mid-treatment (post-AC; Adriamycin/Cyclophosphamide) TIL in predicting pCR rates in early-stage TNBC. Methods Of 242 patients with stage I-III TNBC enrolled in the ARTEMIS trial (NCT02276443), 156 patients had pre-AC TIL and pCR status available for this analysis. Both pre-and post-AC TIL counts were available in 29 patients. Post-AC TIL counts for the remaining patients were imputed using linear regression with age, race, stage III, vimentin &amp;gt;50% and post-AC tumor volume reduction. Using these imputed TIL counts we evaluated the association of post-AC TIL with pCR. We also evaluated the change in TIL before and after treatment with AC. Results At baseline the median TIL count was 10% (n=156). In the post-AC samples, the median TIL count was 5%. Using imputed TIL counts, we did not conclude that post-AC TIL was associated with pCR (p= 0.28). Using a cut-point of 15% TIL, our analysis showed that baseline TIL is more strongly correlated with pCR than post-AC TIL (Table 1). In our univariable logistic regression, both baseline TIL and the difference in TIL pre-and post- treatment were significantly associated with pCR (p= 0.0015 and p=0.0068, respectively), however in the multivariable analysis only baseline TIL was significant. Our analysis did show that a decrease in TIL from pre- to post-treatment was significantly associated with pCR (p=0.022). However, this measure was not significant in our logistic regression model when pre-TIL was also included. Conclusion Higher pre-treatment TIL correlated more strongly with pCR rate when compared to post-AC TIL. Pre-treatment high TIL was associated with pCR regardless of changes in TIL pre and post treatment. Table 1. Changes in TIL before and after treatmentBaseline TILPost-AC TILN#pCR (%)LowLow6217 (27%)LowHigh4012 (30%)HighLow2513 (52%)HighHigh2916 (55%)TIL; Low: &amp;lt;15, High &amp;gt;15 Citation Format: Nour Abuhadra, Kenneth Hess, Jennifer Litton, Gaiane Rauch, Alastair Thompson, Bora Lim, Beatriz Adrada, Elizabeth Mittendorf, Senthil Damodaran, Rosalind Candelaria, Banu Arun, Wei Tse Yang, Naoto Ueno, Lumarie Santiago, Rashmi Murthy, Nuhad Ibrahim, Sahin Aysegul, William Symmans, Lei Huo, Stacy Moulder. Serial TILs: Evaluating the role of mid-treatment tumor infiltrating lymphocytes (TIL) in predicting pathologic complete response (pCR) in early-stage triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-20.

Changes in Tumor-Infiltrating Lymphocytes and Vascular Normalization in Breast Cancer Patients After Neoadjuvant Chemotherapy and Their Correlations With DFS.

Objective: Changes in the number of various tumor-infiltrating lymphocytes (TILs) and degrees of vascular normalization in breast cancer (BC) patients after neoadjuvant chemotherapy (NAC) were analyzed to screen key factors that can predict the prognosis.Methods: HE-stained sections were used to assess the degree of TILs infiltration; immunohistochemically stained sections were used to assess the infiltration of CD8+, CD4+, FOXP3+ Tregs and the expression of PD-L1; immunofluorescence-stained sections were used to assess the microvessel density (MVD) and microvessel pericyte coverage index (MPI). The expression of them before NAC were compared with those after NAC, and correlations between changes in these parameters and the pathological complete remission (pCR) and DFS of BC patients were analyzed.Results: After NAC, the percentage of patients with enhanced sTILs in the pCR group was significantly higher than that in the Non-pCR group (P < 0.05). Univariate and multivariate analyses showed that the number of FOXP3+ Tregs and MPI before NAC were correlated with pCR (P < 0.05). Survival analysis showed that the DFS of BC patients with reduced FOXP3+ Tregs was significantly better than that of patients with elevated FOXP3+ Tregs (P = 0.029). The sTILs count and MPI were significantly higher in primary tumors than lymph nodes (P < 0.05).Conclusion: After NAC, the reduced infiltration of FOXP3+ Tregs was correlated with an improvement in DFS in BC patients. Changes in the number of FOXP3+ Tregs and the MPI may be used as prognostic markers for BC patients.

The impact of CTLA-4 blockade and interferon-α on clonality of T-cell repertoire in the tumor microenvironment and peripheral blood of metastatic melanoma patients

ABSTRACTPatients with metastatic melanoma were treated with tremelimumab and interferon-α (IFN) in a previously reported clinical trial [NCT00610857]. Responses were assessed by RECIST criteria as complete (CR) or partial (PR), stable disease (SD) or progressive disease (PD). In this study, T-cell receptor (TCR) beta-chain repertoire was immunosequenced in peripheral blood mononuclear cells (PBMC) specimens (N = 33) and tumor samples (N = 18) utilizing the immunoSEQ® Assay to determine repertoire clonality and T cell fractions at pre-treatment (tumor and PBMC), one month (PBMC) and 3 months (PBMC) time points and evaluate its association with clinical outcomes. In the pretreatment tumor microenvironment (TME), T cell clonality was significantly (p = .035) different and greater in patients who achieved disease control (CR, PR, SD) versus those with non-disease control (PD) as best response to treatment. Further, there was significantly (p = .001) increased TCR fraction in tissue of responders (CR, PR) versus non-responders (PD, SD). In examining T cell clonality in the circulation (PBMC), no significant associations were found in the pretreatment samples. However, early on-treatment (4 weeks) there was a significant decrease in T cell clonality that was associated with improved overall survival (p = .01) and progression-free survival (p = .04). In addition, analysis of temporal changes in tumor-infiltrating lymphocytes (TIL) and peripheral TCR repertoire revealed that responders had significantly higher clonal expansion of TIL in the circulation at 4 weeks than non-responders (p = .036). Our study provided interesting mechanistic data related to CTLA-4 Blockade and IFN and potential biomarkers of immunotherapeutic benefit.

On-treatment changes in tumor-infiltrating lymphocytes (TIL) during neoadjuvant HER2 therapy (NAT) and clinical outcome.

574 Background: Higher quantity of pretreatment TIL (PT) is associated with improved pCR and EFS in HER2+ early breast cancer (BC). The value of on-treatment TIL is unknown. Methods: The NeoALTTO trial randomized 455 women with HER2+ BC to 12 weeks NAT with trastuzumab, lapatinib or combination with paclitaxel, followed by FEC after surgery. In the PAMELA trial 151 women received 18 weeks NAT with lapatinib and trastuzumab (±hormonal therapy). TIL were quantified on PT and on-treatment (W2) biopsies using the published method on H&amp;E slides, and tested for associations with pCR (logistic regression), EFS and OS (Cox models) in univariate (UV) and multivariate (MV) analyses. The likelihood ratio test assessed added prognostic value to clinicopathological (CP) variables. pCR associations were validated in PAMELA. We investigated enrichment of immune cell subsets using previously published RNAseq data from NeoALTTO. Results: In NeoALTTO, PT and W2 TIL were evaluable in 277/455 (61%). We defined two groups: immune-poor (L+F) and immune-enriched (II+P), see Table. Immune-enriched (41%; 134) vs poor (59%; 164) patients had significantly higher pCR rates (40% vs 21%; UV OR 2.24; 95%CI 1.31-3.85; P = .003; MV P = .009), and added significant value to CP + PT TIL for prediction of pCR (P = .003). This was further confirmed in PAMELA (N = 94/151) (26% vs 6%; UV P = .021; MV P = .028). In NeoALTTO, the immune-enriched vs poor patients had significantly improved EFS (5 yr est 85% vs 60%; UV HR 0.31; 95%CI 0.18-0.54; P &lt; .001; MV P &lt; .001) and OS (5 yr est 91% vs 77%; UV HR 0.40; 95%CI 0.20-0.82; P = .012; MV P = 0.026), and provided significant added prognostic value beyond CP + pCR + PT TIL (EFS P &lt; .001) In NeoALTTO PT samples, II vs F patients had enrichment of DCs, NKs and CD8+ including tissue resident memory cells (P = .009) suggesting requirement of key immune subsets. Further validation by IHC is ongoing. Conclusions: On-treatment TIL identifies patients more likely to achieve pCR and have improved EFS in early-stage HER2+ BC, beyond CP + PT TIL. This information could aid future trial design. Clinical trial information: NCT00553358, NCT01973660. [Table: see text]

Abstract P6-17-08: Dynamics of tumor-infiltrating lymphocytes (TILs) during neoadjuvant dual HER2 blockade in HER2-positive (HER2+) breast cancer in the absence of chemotherapy

Abstract Background: TILs in HER2+ breast cancer (BC) predict 1) prognosis in early setting, 2) complete pathological response (pCR) following neoadjuvant antiHER2-based therapy and 3) response to trastuzumab and pembrolizumab in the metastatic setting. However, less is known regarding changes in TILs during antiHER2-based treatment. Methods: Stromal TILs where evaluated centrally using H/E slides in tumor samples from the PAMELA (NCT01973660) neoadjuvant phase II trial. Briefly, 151 women with HER2+ BC were treated with lapatinib and trastuzumab, and hormonal therapy if HR positive, for 18 weeks. TIL levels were determined at baseline (n=148), after 2 weeks of treatment (n=134) and at surgery (n=137). Expression of 560 genes, including immune-related genes (e.g. CD8A, CD4, PD1 and PDL1) was measured at the same timepoints (baseline n=151, 2-weeks n=144, surgery n=144) using the nCounter platform. Intrinsic subtyping at baseline was determined using the PAM50 gene expression predictor. Changes in TILs between 2 time-points were determined by paired t-tests. Correlation of TILs with gene expression was assessed by quantitative SAM analysis using a False Discovery Rate &amp;lt;1%. All statistical tests were two-sided and considered significant when p&amp;lt;0.05. All statistical analyses were carried out using the R software. Results: Compared to baseline, a significant increase in TILs was observed at week 2 in HR- (p&amp;lt;0.001) and HER2-enriched (HER2-E) tumors (p=0.001), but not in HR+ (p=0.133) and non-HER2-E tumors (p=0.067). Within HR- and HER2-E tumors, increase in TILs at week 2 from baseline was observed regardless of pathological response at surgery (pCR and HR- [p=0.008]; RD and HR- [p=0.037]; pCR and HER2-E [p=0.010]; RD and HER2-E [p=0.056]). Compared to week 2, a significant decrease in TILs at surgery was observed in HR- (p=0.002) and HER2-E (p=0.003) tumors, but not in HR+ (p=0.616) and non-HER2-E tumors (p=0.578). Within HR- and HER2-E tumors, a significant decrease in TILs between week 2 and surgery was observed in tumors achieving pCR (p=0.004 and p=0.005), while, in tumors not achieving pCR, no significant tendency was observed (26.4% and 33.0% of tumors showed an increase and a decrease of TILs between week 2 and surgery). Nonetheless, the vast majority of residual tumors (non-pCR) at surgery had TILs above ≥5%: 34.3% 5-10%, 21.0% 10-20%, 15.2% 20-40% and 11.4% ≥40%. Finally, TILs scoring was found highly enriched (FDR&amp;lt;1%) for immune-related genes tracking activated CD8 T-cells (i.e. CD8A, CD3G, LAG3 and PD1). Expression of these immune genes consistently correlated with TIL levels across the 3 time-points. Conclusions: In early HER2+ BC, a general increase in TILs is observed following 2 weeks of dual HER2 blockade. This observation is mostly observed in HR- and HER2-E subtype, but regardless of pathological response at surgery. After 2 weeks of treatment, TILs consistently decrease in patients achieving a pCR, whereas two main patterns of TILs expression are observed in patients with residual disease at surgery. Nonetheless, most residual tumors at surgery are inflamed (i.e. TILs ≥5%) and might be good candidates for clinical trials evaluating adjuvant immune checkpoint inhibitors. Citation Format: Griguolo G, Holgado E, Cortés J, Fasani R, Pascual T, Paré L, Bermejo B, Oliveira M, Morales S, Martinez N, Vidal M, Pernas S, Lopez R, Muñoz M, Galvan P, Garau I, Manso L, Alarcón J, Martínez E, Villagrasa P, LLombart-Cussac A, Prat A, Nuciforo P. Dynamics of tumor-infiltrating lymphocytes (TILs) during neoadjuvant dual HER2 blockade in HER2-positive (HER2+) breast cancer in the absence of chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-08.

Neoadjuvant OX40 therapy in patients with head and neck cancer induces profound changes in tumor-infiltrating lymphocytes

Abstract OX40 is a member of the tumor necrosis factor (TNF) receptor family and, as a co-stimulatory molecule, enhances T-cell proliferation, survival and memory formation. We have also shown that it induces robust antitumor responses in patients with metastatic disease. To examine the effect of OX40 stimulation on the immune response in cancer patients, 16 head and neck squamous cell carcinoma (HNSCC) patients were enrolled in a neoadjuvant phase Ib trial and received anti-OX40 infusion 2 days, 1 week and 2 weeks prior to surgical resection. To evaluate changes in the tumor microenvironment (TME), tissue and peripheral blood samples were obtained prior to infusion and at time of surgery. Assessment of changes in tumor infiltrating lymphocyte (TIL) and peripheral blood populations were performed by flow cytometry and fluorescent multiplex immunohistochemistry (IHC). OX40 administration was well tolerated with low toxicity. Activation and proliferation of CD4+ and CD8+ memory T-cell populations in both the TME and periphery peaked between 12 and 19 days after OX40 infusion, as demonstrated by increased levels of Ki67, CD38 and ICOS. In the TIL, expression of CD39, ICOS and PD-1 was increased on CD4+ T cells in most patients and the frequency of tumor-reactive CD39+CD103+ CD8+ T cells was increased in some patients. IHC analysis revealed striking changes in tissue integrity and increased lymphocyte infiltrates in 4/16 patients. With this study, we have found that neoadjuvant OX40 immunotherapy is feasible and safe in patients with HNSCC and results in robust activation and proliferation of T cells within the tumor. Based on these findings, we are now comparing the transcriptome and changes in TIL repertoire and metabolism before and after treatment.

Early Stage HER2-Positive Breast Cancers Not Achieving a pCR From Neoadjuvant Trastuzumab- or Pertuzumab-Based Regimens Have an Immunosuppressive Phenotype

BackgroundStromal tumor-infiltrating lymphocytes (TILs) might predict pathologic complete response (pCR) in patients with HER2-positive (HER2+) breast cancer treated with trastuzumab (H). Docetaxel (T), carboplatin (C), H, and pertuzumab (P) have immune-modulating effects. Pre- and post-treatment immune biomarkers in cancers treated with neoadjuvant TCH with or without P are lacking. In this study we quantified baseline and changes in TILs, cluster of differentiation (CD) 4+, CD8+, FoxP3+, and PD-L1+ cells using immunohistochemistry (IHC) and quantified productive T-cell receptor β (TCRβ) rearrangements and TCRβ clonality using next-generation sequencing (NGS) in 30 HER2+ breast cancer tissues treated with neoadjuvant H with or without P regimens. Materials and MethodsThirty pre- and post-neoadjuvant TCH (n = 4) or TCHP (n = 26) breast cancer tissues were identified. TILs were quantified manually using hematoxylin and eosin. CD4, CD8, FoxP3, and PD-L1 were stained using IHC. TCRβ was evaluated using NGS. Immune infiltrates were compared between pCR and non-pCR groups using the Wilcoxon rank sum test. ResultsA pCR occurred in 15 (n = 15; 50%) cancers (TCH n = 2; TCHP, n = 13). Pretreatment TILs, CD4+, CD8+, FoxP3+, and PD-L1+ cells were not associated with response (P = .42, P = .55, P = .19, P = .66, P = .87, respectively. Pretreatment productive TCRβ and TCRβ clonality did not predict response, P = .84 and P = .40, respectively). However, post-treatment CD4+ and FoxP3+ cells (T-regulatory cells) were elevated in the non-pCR cohort (P = .042 and P = .082, respectively). ConclusionAn increase in regulatory T cells in non-pCR tissues suggests the development of an immunosuppressive phenotype. Further investigation in a larger cohort of samples is warranted to validate these findings.

Abstract PD6-09: The immune microenvironment in hormone receptor-positive breast cancer patients and relationship to treatment outcome following preoperative chemotherapy plus bevacizumab

Abstract Background: Hormone receptor-positive (HR+) tumors have fewer tumor-infiltrating lymphocytes (TILs) and lower response rates to immune checkpoint inhibitors (ICI), either as single agents or in combination with chemotherapy, than triple negative cancers. However, some HR+ cancers do respond to ICI and biomarkers that accurately reflect the immune microenvironment may help guide the use of ICI therapy. Prior evidence suggests that macrophage-related immune pathways may be relevant to the pathophysiology of HR+ BC. Methods: HR+/HER2- patients were identified from a prospective trial of preoperative bevacizumab (preop bev) followed by bev with adriamycin/cyclophosphamide/paclitaxel dose-dense chemotherapy (chemo). Tumor samples were collected at diagnosis and surgery (pre-tx and post-tx), and PD-L1 expression (by immunohistochemistry), TILs, and Nanostring PanCancer Immune Profiling Panel were evaluated on both pre-tx and post-tx specimens. Pre-tx whole transcriptome sequencing was performed. Pathologic response at surgery was centrally assessed by Miller-Payne (MP) and residual cancer burden (RCB) scores. An immune score was calculated for each pre-tx specimen by integrating 10 published immune signatures. Immune cell subsets were inferred from bulk transcriptional data using CIBERSORT and immune cell-specific signatures from MSigDB. Results: 55 patients who received trial therapy and had at least 1 evaluable specimen were included for analysis. Pre-tx TILs and tumor PD-L1 (tPD-L1) scores are shown in the table. 18% of pre-tx tumors had “high” (≥10%) TILs and “high” TILs were associated with significantly higher immune signature score (p=0.004). Immune score correlated highly with proportion of CIBERSORT anti-tumor M1 macrophages as well as CD8 T-cell signatures (r&amp;gt;0.65 and p&amp;lt;0.001). Higher pre-tx TILs, tPD-L1, or immune score were each significantly associated with more favorable RCB and MP in unadjusted analyses (all Spearman p&amp;lt;0.01 for pathologic markers; ANOVA p&amp;lt;0.04 for immune score). After adjustment for age and tumor grade, higher pre-tx TILs and tPD-L1 were associated with favorable RCB (p&amp;lt;0.01 for both), and higher pre-tx tPD-L1 correlated with favorable MP (p=0.03). Pathologic complete response occurred in 4 pts; all 4 had high pre-tx TILs, pre-tx tPD-L1, or both. Among patients with residual disease, large changes (&amp;gt;5%) in TILs or tPD-L1 from pre-tx to post-tx were rare: 2 pts each had large changes in TIL or tPD-L1 score (N=38/N=31 pairs, respectively). Conclusions: High levels of tumor-lymphocyte interaction were seen in only a minority of untreated HR+ breast tumors, and did not typically change with chemo plus bev. An immune score derived from bulk RNAseq correlated with histological observations in these specimens. Nonetheless, TILs, tPD-L1, and signature-derived immune score were significantly associated with pathologic response to preop treatment in HR+ disease. Early data suggest that the role of M1 macrophages in HR+ tumors warrants further investigation. ScoreTILs (N=50 evaluable)Tumor PD-L1 (N=51)0%0 pts (0%)28 pts (55%)&amp;gt;0-5% (low)19 (38%)18 (35%)&amp;gt;5-10% (intermediate)22 (44%)3 (6%)&amp;gt;10% (high)9 (18%)2 (4%) Citation Format: Waks AG, Stover DG, Barry W, Dillon D, Gjini E, Rodig SJ, Brock J, Baltay M, Savoie J, Winer EP, Krop I, Tolaney SM. The immune microenvironment in hormone receptor-positive breast cancer patients and relationship to treatment outcome following preoperative chemotherapy plus bevacizumab [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-09.

Stromal lymphocyte infiltration after neoadjuvant chemotherapy is associated with aggressive residual disease and lower disease-free survival in HER2-positive breast cancer

The role of tumor-infiltrating lymphocytes (TILs) in breast cancer has been extensively studied over the last decade. High TILs levels have been associated with pathological response rate in the neoadjuvant setting and with better outcomes in the adjuvant setting. However, little attention has been paid to changes in TILs and residual TIL levels after neoadjuvant chemotherapy (NAC). We investigated TIL levels before, after chemotherapy, and their dynamics during treatment; and we assessed the correlation of these levels with response to NAC and prognosis. We identified 175 patients with primary HER2-positive breast cancers receiving NAC+/- trastuzumab between 2002 and 2011. Microbiopsy specimens and paired surgical samples were evaluated for stromal lymphocyte infiltration. Univariate and multivariate analyses were carried out to assess the association of clinical and pathological factors with pathological complete response (pCR) and disease-free survival. Baseline TIL levels were not significantly associated with pCR. TIL levels decreased during treatment in 78% of the patients. The magnitude of the decrease was strongly associated with pCR. After chemotherapy, TIL levels were high in tumors displaying aggressive patterns (high residual cancer burden score, mitotic index >22, tumor cellularity >5%). In the population with residual disease, TIL levels >25% at the end of NAC were significantly associated with an adverse outcome (TILs >25%, HR = 7.98, P = 0.009) after multivariate analyses including BMI, post-NAC mitotic index and tumor grade. A decrease in TIL levels during chemotherapy was positively associated with response to treatment. In tumor failing to achieve pCR, post-NAC lymphocytic infiltration was associated with higher residual tumor burden and adverse clinical outcome. Further studies are required to characterize immune infiltration in residual disease to identify candidates who could benefit from second-line therapy trials including immune checkpoint inhibitors.

Immune biomarkers and treatment (tx) outcome in hormone receptor-positive (HR+) breast cancer (BC) patients (pts) treated with preoperative chemotherapy (preop chemo) plus bevacizumab (bev).

e12134 Background: Though preliminary study of the BC immune microenvironment suggests that HR+ tumors are less immune-active than other subtypes, immune biomarkers may have important clinical implications in HR+ BC pts. Methods: 78 HR+/HER2- BC pts were enrolled on a prospective trial of preop bev followed by bev with adriamycin/cyclophosphamide/paclitaxel dose-dense chemo. Tumor samples were collected at diagnosis and surgery (pre-tx and post-tx). PD-L1 expression (by immunohistochemistry) and tumor-infiltrating lymphocytes (TILs) were scored. Whole transcriptome sequencing and Nanostring PanCancer Immune Profiling Panel were performed. Pathologic response at surgery was assessed by Miller-Payne (MP) and residual cancer burden (RCB) scores. We calculated adjusted correlations by linear/logistic regression for RCB/dichotomized MP, respectively. Results: 55 pts who received trial tx and had &gt;1 analyzable specimen are included. Pre-tx TILs and tumor PD-L1 (tPD-L1) scores (see table) were slightly positively correlated (Spearman rho 0.23, p=0.1). Large changes (&gt;5%) in TILs or tPD-L1 from pre-tx to post-tx were rare: 2 pts each had large changes in TIL or tPD-L1 score (N=38 and N=31 pairs, respectively). Higher pre-tx TILs or tPD-L1 were significantly associated with more favorable RCB and MP (all Spearman p&lt;0.01) in unadjusted analyses. After adjustment for age and tumor grade, higher pre-tx TILs and tPD-L1 were associated with more favorable RCB (p&lt;0.01 for both), and higher pre-tx tPD-L1 correlated with more favorable MP (p=0.03). Pathologic complete response occurred in 4 pts; all 4 had high pre-tx TILs, pre-tx tPD-L1, or both. Analysis of immune-related RNA signatures is ongoing. Conclusions: High levels of tumor-immune interaction were seen in only a minority of untreated HR+ breast tumors, and did not typically change after tx with chemo plus bev. Nonetheless, TILs and tPD-L1 are significantly associated with pathologic response to preop tx in HR+ disease. [Table: see text]

Abstract P2-04-24: Changes of tumor-infiltrating lymphocytes and programmed death-ligand1 positivity after neoadjuvant chemotherapy in patients with locally advanced breast cancer

Abstract Background Recently, the balance in immune system between immune surveillance and tolerance is known to be associated with the prognosis of breast cancer patients. The aim of this study was to investigate changes of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand1 (PD-L1) status after neoadjuvant chemotherapy (NAC) and their impact on recurrence in patients with locally advanced breast cancer. Methods Paired samples (before and after NAC) of triple negative or HER2+ breast cancer tissue were obtained from clinical stage II or III patients (n=39) undergoing NAC and subsequent breast resection. The assessment of immunohistochemical (IHC) staining for PD-L1 were performed. Immunostaining of forkhead box P3 (Foxp3) and CD4/CD8 were performed for subtyping of TILs in triple negative breast cancer (TNBC) only. Clinicopathologic data including baseline characteristics, tumor response and recurrence were reviewed. Results Proportion of PD-L1 (+) tumor cells in pre-chemotherapy tissue was 20% (5/25) in HER2+ and 28.6% (4/14) in TNBC. It could not predict pathologic complete response. Most post-chemotherapy tissue (21/24) showed same PD-L1 positivity with pre-chemotherapy tissue. The rest showed the decreased PD-L1 positivity after NAC. In cases of TNBC, the increased number of CD8+ T cells was significantly associated with PD-L1 positivity in pre-chemotherapy tissue. (p=0.001) Foxp3+ T cell proportions decreased significantly (p=0.046) and CD8+/Foxp3+ T-cell ratio increased significantly (p=0.023) after NAC. The patients with increased number of CD8+ T cells after NAC had a tendency to live longer without recurrence compared to patients with decreased CD8+ T cells (62.3 vs 38.1 months, p=0.158). Conclusion Our data provides the clinical evidence that PD-L1 positivity are associated with CD8+ T cell proportion and increased CD8+ T cells after NAC might be good prognostic marker. The role of immunologic balance as a prognostic marker for recurrence must be evaluated in future study. Citation Format: Choi YJ, Lee JH, Moon SY, Choi JY, Jung SP, Bae JW, Park KH. Changes of tumor-infiltrating lymphocytes and programmed death-ligand1 positivity after neoadjuvant chemotherapy in patients with locally advanced breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-24.

SD-101, a Novel Class C CpG-Oligodeoxynucleotide (ODN) Toll-like Receptor 9 (TLR9) Agonist, Given with Low Dose Radiation for Untreated Low Grade B-Cell Lymphoma: Interim Results of a Phase 1/2 Trial

Introduction: Prior studies have shown preliminary clinical efficacy in combining CpG-ODN with radiation therapy (XRT) to patients with indolent B-cell lymphoma. We report interim Phase 1/2 data of combination XRT and SD-101, a synthetic class C CpG-ODN TLR9 agonist, selected for the strong induction of type I interferon.Methods: This dose-escalation Phase 1/2 trial enrolled patients with untreated indolent B-cell lymphoma having at least 2 measurable lesions (one palpable). The primary endpoints were safety and alpha-interferon-gene induction. Secondary endpoints included efficacy assessment using Cheson (1999) criteria and quantification of changes in tumor-infiltrating lymphocytes. A single lesion was treated with XRT (2 Gy daily X 2 days) followed by a single intratumoral dose of SD-101 (same lesion). 4 additional doses of intratumoral SD-101 were given weekly over next 4 weeks. The treated lesion and distal lesions were monitored during the study. Pharmacodynamic assessment included flow cytometry analysis of immune infiltrates in an FNA sample of the treated tumor and RT-PCR RNA assay of whole blood to assess induction of alpha-interferon genes. Efficacy assessment included imaging (CT at 3, 6, and every 6 months thereafter).Results: As of 01 Aug 2016, 13 patients were treated with escalating doses of SD-101 at 1, 2, 4 or 8 mg/dose. There were no dose limiting toxicities. The majority of related adverse events (AEs) were Grade 1 (mild). The most common were flu-like symptoms (chills, headache, malaise, myalgia), typically resolving within ≤ 48 hours without requiring intervention (e.g., acetaminophen). An induction of alpha-interferon genes occurred at all dose levels with a similar level of induction.For the dose expansion phase, 15 patients were enrolled at 1 mg (6) and 8 mg dose group (9). Similar to dose escalation phase, the most common related events were again flu-like symptoms, typically resolving within ≤ 48 hours mitigated with acetaminophen. There was one dose delay for Grade 3 neutropenia in one patient (1 mg) that resolved following drug interruption. Twenty-nine Grade 3 transient flu-like symptoms were reported for four patients (44%) who received the 8 mg dose (e.g., chills headache, malaise, myalgia, and fatigue). Only one of the four patients experienced all 5 Grade 3 flu-like symptoms after intratumoral injection. There were no grade 4 or serious events reported, and no patient discontinued due to an adverse event.A reduction in tumor sizes was observed in the study over time (see Figure 1). At Day 90 the median changes in the product of diameters from baseline were -46.1% and -10.2% for treated tumor and distal tumors, respectively. At Day 540, the median changes were -68.6% and -24.1%, respectively.In patients with follicular lymphoma (largest subgroup), preliminary data suggest a higher percentage of CD8+ T cells in the treated lesion (FNA at day 8) correlated with an increased abscopal response.Conclusions: Intratumoral SD-101 following radiation therapy has been well tolerated and preliminary efficacy, promising. Abscopal anti-tumor activity was observed with preliminary data suggesting that a higher percentage of CD8+ T cells in the treated lesion correlated with an increased abscopal response seen in follicular lymphoma. Enrollment is ongoing with an option for cycle 2 retreatment. [Display omitted] DisclosuresLevy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Reagan:Seattle Genetics: Research Funding. Friedberg:Bayer: Other: Data Safety Monitoring Committee. Bartlett:Gilead: Consultancy. Leung:Dynavax: Employment. Peterkin:Dynavax: Consultancy. Xing:Dynavax: Employment. Coffman:Dynavax: Employment. Janssen:Dynavax: Employment. Candia:Dynavax: Employment.

Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients.

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2+, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8+ and CD4+ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2+ and TN breast cancers have a lower percentage of TILs and CD8+ and CD4+ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.

Abstract CT047: SD-101, a novel intratumoral class C CpG-ODN, given with low-dose radiation in patients with untreated low-grade B-cell lymphoma: interim results of a phase I trial

Abstract Prior studies have shown preliminary clinical efficacy in combining CPG-ODN with radiation therapy (XRT) to patients with indolent B-cell lymphoma. We report Phase 1 data of combination XRT and SD-101, a synthetic class C CPG-ODN, selected for strong induction of type I interferon. Methods: This dose-escalation Phase I trial (3+3 design) enrolled patients with untreated indolent B-cell lymphoma. The primary endpoints were safety and alpha-interferon-gene induction. Secondary endpoints included efficacy assessment using Cheson (1999) criteria and quantification of changes in tumor-infiltrating lymphocytes. Patients had a single lesion treated with XRT (2 Gy daily X 2 days) followed by weekly intratumoral SD-101 × 5 doses. Pharmacodynamic assessment included flow cytometry assessment of T-cell infiltrates in a FNA sample of the treated tumor and RT-PCR RNA assay of whole blood to assess induction of alpha-interferon genes. Efficacy assessment included imaging (CT at 3, 6, and every 6 months thereafter). Results: As of 23 Dec 2015, 13 patients total were enrolled and treated with SD-101 doses of 1, 2, 4 or 8 mg/dose. There were no dose limiting toxicities. Twelve patients received all planned doses of XRT and SD-101. One patient (8 mg) withdrew consent for treatment after receiving 1 SD-101 dose due to Grade 2 confusion and flu-like symptoms. The most common adverse events (AEs) (decreasing frequency) were chills, malaise, myalgia, fatigue, headache, pyrexia, and nausea, typically resolving within 48 hours. One serious AE occurred (Grade 3 Pulmonary Embolism (4 mg)) which was incidental and asymptomatic. There were 2 dose delays due to an AE (Grade 3 neutropenia) in the same patient (8 mg) that resolved without treatment. The other AE was Grade 3 malaise reported in 2 patients (8 mg). At study Day 90, a reduction of the product of diameters in treated tumors occurred in 12 patients (median -45.3%; range [-87, +100]) and in non-treated tumors occurred in 11 patients (median -8.1%; range [-48, +45]). An induction of alpha-interferon genes occurred at all dose levels with similar level of induction. At the treated site, regulatory T-cells (T Regs) were reduced in 8 of 10 patients (average decrease 22.3 + 9.5%) at Day 8. There was an average reduction of 83.3 + 9.9% in follicular T helper cells (Tfh) at Day 8 (n = 9 with baseline Tfh). Conclusions: SD-101 combined with radiation therapy showed preliminary abscopal anti-tumor activity and was well tolerated. A maximum tolerated dose has not yet been reached at the doses explored. Target engagement of the TLR-9 receptor (induction of interferon-alpha genes) was observed across all dose groups. A decrease in the proportion of T Regs and Tfh cells suggests modulation of their inhibitory effects and tumor growth promoting effects, respectively. The study is currently enrolling 2 expansion cohorts at 1 mg and 8 mg. Citation Format: Ronald Levy, Nancy Bartlett, Jonathan Friedberg, Patrick Reagan, Leo Gordon, Craig Bergman, Robert Coffman, Robert Janssen, Albert Candia, Michael Khodadoust, Matthew Frank, Steven Long, Debra Czerwinski, Michael Chu, Holbrook Kohrt. SD-101, a novel intratumoral class C CpG-ODN, given with low-dose radiation in patients with untreated low-grade B-cell lymphoma: interim results of a phase I trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT047.