Responding Patients Research Articles

A phase I study of bintrafusp alfa and NHS-IL12 (M9241) alone and in combination with stereotactic body radiation therapy (SBRT) in patients with metastatic genitourinary (GU) malignancies.

4571 Background: Bintrafusp alfa is a novel bifunctional fusion protein targeting PD-L1 and TGF-β which has demonstrated clinical efficacy in some tumor types and a manageable safety profile. M9241 is an immunocytokine that allows targeting of the pro-inflammatory IL-12 cytokine to exposed DNA in necrotic portions of tumors. SBRT can help promote anti-tumor responses and may potentially synergize with immunotherapy by inducing DNA damage. Methods: This is an open label, non-randomized, three arm phase I trial of bintrafusp alfa and M9241 combination therapy administered alone (Arm 1), or with either sequential (Arm 2), or concurrent (Arm 3) SBRT. Eligible patients must have metastatic non-prostate GU cancers with measurable disease. Bintrafusp alfa was used at a flat dose of 1200mg (q2w) for arms 1-3 and a fixed dose of SBRT (24Gy in 3 fractions) was used for arms 2-3. M9241 was administered at 16.8mcg/kg (q4w) for Arm 1 and decreased if side effects occur. Arm 1 used bintrafusp alfa and M9241 alone, with the maximum tolerated dose (MTD) of M9241 being dose level 1 (DL1) for Arm 2. Arm 2 used SBRT followed by bintrafusp alfa and M9241, with the MTD of M9241 being DL1 for Arm 3. Arm 3 used concurrent SBRT with bintrafusp alfa and M9241. The primary objective was to determine the safety and highest tolerable doses of each arm. Secondary objectives include objective response rate (ORR), progression free survival (PFS), and overall survival (OS). Results: Study enrollment began in 12/2019, with interim safety analyses performed quarterly. A total of 16 patients have enrolled, 12 in Arm 1, three in Arm 2, and one in Arm 3 at data cutoff 1/2/23. The first six patients enrolled in Arm 1 had no DLTs allowing Arm 2 to open, Arm 2 had no DLTs after 3 patients enrolled allowing Arm 3 to open. At data cut off four of 16 patients remained on treatment. The median age was 63 and 19% were female. The most frequent TRAEs were fatigue (grade 2, 38%), lipase increase (grade 2+3, 31%), and anemia (grade 2+3, 31%). Grade 3 hematuria occurred in 2 patients (13%); a grade 4 creatinine increase occurred in one (8%). There were no grade 5 TRAEs. Two (13%) patients had treatment discontinued and 5 (31%) had dose reductions due to AEs. Of the 16 patients enrolled on study, 11 were evaluable at data cutoff. Currently three patients (38%) on Arm 1 and one patient (50%) in Arm 2 had objective responses, corresponding to an ORR of 36.4% (4/11). All responses have been partial responses. On Arm 1 the responding patients included one with small cell carcinoma of the renal pelvis, one with Leydig cell tumor, and one with urothelial carcinoma. On Arm 2 the responding patient had a Sertoli cell tumor. Conclusions: The use of bintrafusp alfa and M9241 either alone or in combination with sequential or concurrent SBRT has shown clinical activity in pts with GU tumors and has not been associated with any DLTs. Clinical trial information: NCT04235777 .

Prognostic value of total tumor volume with 68Ga DOTATOC PET/CT in predicting response to 177-Lu-DOTATOC treatment in metastatic well-differentiated neuroendocrine tumors (NETs).

e16248 Background: PET/CT with somatostatin analogues (68 Ga DOTATOC) is essential for decision-making prior to treatment with [177Lu–Dota0–Tyr3]–Octreotate (177Lu-DOTATOC), there are biological characteristics in this type of neoplasms that allow predicting the response to therapy, the tumor burden is one of the most important variables, however its estimation with conventional imaging tools is limited, currently there are reconstruction algorithms based on artificial intelligence that allow estimating the tumor burden using molecular imaging studies with 68 Ga DOTATOC, aim of this work is to determine the value of the total tumor load with this radiotracer as a predictor of overall survival in patients treated with 177Lu DOTATOC. Methods: 45 patients (11 men and 34 women) with an average age of 57.2 (range 47.2 to 66.5) with histopathological and immunohistochemical diagnosis of well-differentiated neuroendocrine tumors (NETs) in the period from 2015 to 2021 who received therapy with (177Lu-DOTATOC) and who had a baseline 68Ga DOTATOC PET to calculate total tumor volume (TVV) and semiquantitative uptake values were retrospectively included. Results: The mean follow-up was 29.3 +/- 6 months. Using ROC curves, a cut-off point of 100 cm3 was determined to differentiate responding and non-responding patients. The difference in the overall survival curves between both groups was statistically significant (470 days vs. 180 days) with p=0.07. The main sites of origin of the primary tumor were pancreas (n=24), lung (n=11), small intestine (n=7) and liver (n=3). The mean tumor volume was 305.8 cm3 (+/- 272). The organs with the highest uptake values were pancreas with SUVmax of 27.9 (range 10.1-42.4), rectum with SUVmax 26.5 (10.7-42.3) and liver with SUVmax of 22.1 (14.4-31.7), these uptake values did not have any type of relation to patient survival. Regarding therapy with 177 Lu DOTATOC, 8 received 14.8 Gbq (400mCi), 12; 22.2 GBq (600mCi) and 25; 29.6 GBq (800 mCi), no serious adverse effect or kidney function impairment was demonstrated after 177Lu DOTATOC therapy. No grade 3 or 4 haematological toxicity was identified. Conclusions: PET/CT with 68 Ga-DOTATOC is routinely used in the management of patients with neuroendocrine tumors and also allows categorizing patients according to their tumor volume to predict overall survival in patients treated with 177 Lu-DOTATOC, thus in such a way that it could be considered as an easily accessible and widely available prognostic imaging biomarker.

Final results of pilot trial to evaluate anti-PD1 and 8 Gy in 1 fx for relapsed refractory multiple myeloma.

8017 Background: Single agent Immune-Checkpoints, such as Anti-PD1, have not be successful. A pilot phase 1, 2 trial (NCT03267888) was conducted to see if radiotherapy (RT) and anti-PD1 (Pembrolizumab) could provide early signals of safety and response. Methods: Inclusion criteria included patients > 18 years of age, ECOG 0-1, able to give informed consent, have relapsed/refractory myeloma, and have ≥1 osseous and/or extra-osseous lesion that could undergo RT. Patients had to be candidates for anti-PD1 based on organ function testing, have measurable disease per International Myeloma Working Group Criteria (IMWG), and/or have progressive disease on serial staging PET/CT. IRB approval was obtained. RT (8 Gy in 1 fx) was given on day 0, cycle 1 followed by pembrolizumab (200 mg/kg iv on day 2 or 3, then every 3 weeks +/- 7 days) for 2 years or until progression. Primary endpoint was toxicity. Secondary endpoints were IMWG response, abscopal response, overall survival (OS), and immunological changes on serial blood collections. Patients were assessed at 3, 6, and 12 months for progression free survival (PFS) using IMWG and serial PET/CT scans. Patients with stable disease were continued on the trial. Patients that progressed were censored. Standard statistical analysis was performed, and included Kaplan-Meier to estimate OS and PFS. Results: From June, 2018 until October, 2021, 32 patients were screened and 25 were enrolled. Of the enrolled patients, 76% were Caucasian, 64% had ECOG 1, and the mean age was 60 years. Prior to enrollment, the median number of prior lines of therapy that a patient had filed was 5.0 (range: 2 – 11). There was no grade 2 or higher radiation related toxicity within the irradiated volume using CTCAE 4.0. Only one case of ≥ grade 3 (fevers) pembrolizumab-related toxicity was noted. Abscopal response, defined as improvement of a non-targeted lesion, was noted in 5 of 25 patients (20%). IMWG showed robust reduction in the paraproteins and other myeloma labs, suggesting response to radiotherapy and anti-PD1. A total of 8 patients showed response. Of those responding patients, 5 were post CarT cell patients, suggesting that the most benefit may be in post CarT cell patients. None of the post CarT cell patients were noted to have cytokine release syndrome and/or neurotoxicity. Of the responding patients, some of these were associated to have a robust CD 8 T cell activation. The 6 and 12 month PFS for the entire cohort was 31.8% and 22.7%, respectively. The 6 and 12-month OS for the entire cohort was 68% and 64%, respectively. Those that were post CarT, there was a higher 6 month PFS (50%) and OS (71.4%). Conclusions: Combination therapy of single-fraction, low-dose radiation therapy with pembrolizumab appears to be safe and shows early promise of efficacy in MM pts progressing following CarT therapy. Larger trials are warranted in the relapsed/refractory myeloma patients. Clinical trial information: NCT03267888 .

Metastatic sites and lesion numbers cooperated to predict efficacy of PD-1 inhibitor-based combination therapy for patients with metastatic colorectal cancer.

Limited data have been used to evaluate the efficacy of immunotherapy in metastatic colorectal cancer (mCRC). Furthermore, potential markers that can be used to identify responding patients and to further improve efficacy have not been fully explored. In our study, we included a total of 97 patients with mCRC, who each received programmed death-1 (PD-1) inhibitor-based combination therapy at our center. All 12 hypermutated patients benefited from immunotherapy, with median progression-free survival (mPFS) reaching 28.3 months, regardless of liver metastasis. The objective response rate (ORR) of non-hypermutated patients was 16.5% (14/85), with an mPFS of 4.0 months. For non-hypermutated patients, multivariate analysis revealed that the combination of liver metastasis and baseline lesion number significantly stratified response and survival. The lesion-based analysis indicated that the lymph node was the most responsive, followed by the peritoneum and lung, with liver metastasis being the least responsive. None of the patients (0/7) with negative programmed ligand-1 (PD-L1) expression responded, and positive PD-L1 expression may serve as a biomarker (mPFS 5.7 vs. 2.2 months, p = 0.002) that can be used to further guide treatment in non-hypermutated mCRC with liver metastasis (CRLMs). Patients with hypermutated mCRC benefited significantly from immunotherapy, whereas the non-hypermutated cohort with liver metastasis and numerous lesions showed less benefit. The lesion sites reflected varying levels of efficacy, among which PD-L1 potentially cooperated to guide the immunotherapy of CRLMs.

Abstract CT206: A phase I study of binimetinib, a MEK inhibitor, in combination with pembrolizumab in patients with advanced non-small cell lung cancer (NSCLC)

Abstract Introduction: MEK inhibition combined with PD-1 axis inhibition may achieve a greater clinical response than either inhibitor alone due to increased T-cell infiltration of tumours. We combined binimetinib with pembrolizumab in patients with stage IV advanced non-small cell lung cancer (NSCLC) and PD-L1 tumor proportion score>=50%. Methods: A 3 + 3 dose escalation design was used. Binimetinib at a dose level 1 (DL1; 45 mg) or dose level -1 (DL-1; 30 mg) twice daily orally continuously was given with pembrolizumab 200 mg IV q 21 days. The primary objective was to define the recommended phase II dose (RP2D) of the combination. Secondary outcomes included safety of the combination and response (RECIST 1.1) with a planned Phase Ib expansion in patients with RAS/RAF/MEK dysregulated tumours via next generation gene sequencing. Genomic markers are being explored in tissue and plasma. Results: Eleven patients (3 DL1, 8 DL-1) were enrolled with the following mutations: 7 with KRAS (3 G12C, 2 G12D, 1 G12V, 1 G12A), 2 BRAF (1 G409A, 1 V600E) and 1 STK11 which acts as a tumor suppressor gene encoding for LKB1. Two of 3 patients at DL1 experienced dose limiting toxicity (DLT) including grade 3 elevated amylase, grade 3 diarrhea, grade 4 elevated lipase and severe grade 2 rash requiring dose reduction. Of 8 patients treated at DL-1, 1 progressed in cycle 1, another was noncompliant with treatment. Of the remaining 6 patients, 1 experienced DLT with grade 3 rash with inability to administer binimetinib for >75% of cycle 1. The most common toxicities across all cycles (n=11 patients) were: rash (82%), diarrhea (36%) and pruritis (36%). Nine patients were evaluated for response with partial response in 3 (33%), stable disease in 4 (44%) and progressive disease in 2 (22%). All 3 responding patients had RAS or RAF alterations (KRASG12C, KRASG12V, BRAFV600E) while the patient with STK11 mutant disease had early disease progression. Conclusion: The RP2D of the combination in patients with advanced NSCLC is binimetinib 30 mg BID plus pembrolizumab 200 mg IV q21 days. Updated data from cohort expansion including response and molecular correlates of treatment response and resistance will be presented. Citation Format: Christopher F. Theriau, Jamie Feng, Lawson Eng, Frances A. Shepherd, Mary DeCarolis, Vivian Glenns, Swati Kulkarni, Rachel VanderMeer, Urszula Zurawska-Fortin, Desiree Hao, Lisa Le, Isabel Wozniczka, Aida Al-Kindy, Danny Xie, Tong Zhang, Vasanth Subramanian, Tracy Stockley, Penelope A. Bradbury, Geoffrey Liu, Natasha B. Leighl. A phase I study of binimetinib, a MEK inhibitor, in combination with pembrolizumab in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT206.

Abstract CT040: A first-in-human phase 1 trial of IO-108, an antagonist antibody targeting LILRB2 (ILT4), as monotherapy and in combination with pembrolizumab in adult patients with advanced relapsed or refractory solid tumors: Dose escalation study

Abstract Background: IO-108 is a fully human IgG4 monoclonal antibody with high affinity and specificity towards LILRB2 (also known as ILT4). It blocks the interaction of LILRB2 with the known ligands for LILRB2, including HLA-G, ANGPTLs, SEMA4A and CD1d. Methods: IO-108-CL-001 is a first-in-human, Phase 1a/1b study in patients with relapsed/refractory solid tumors (NCT05054348). The dose escalation phase enrolled patients into escalating dose cohorts of IO-108 (60-1800 mg, Q3W IV) as monotherapy or in combination with pembrolizumab. Patients with disease progression on monotherapy could crossover to the combination arm. Primary objectives were safety and tolerability, and identification of the recommended phase 2 dose (RP2D). Secondary and exploratory objectives include evaluation of pharmacokinetics (PK), immunogenicity, pharmacodynamic (PD) biomarker effects and anti-tumor activity as measured by objective response rate (RECIST 1.1). Results: Twenty-five relapsed/refractory solid tumor patients (median 4.5 prior lines of therapy for monotherapy and 3.6 for combination therapy) were treated with IO-108 monotherapy (n=12) or IO-108 + pembrolizumab (n=13). IO-108 was well-tolerated up to the maximal administered dose of 1800 mg Q3W as monotherapy and in combination with pembrolizumab, with no SAEs related to IO-108 and no DLTs observed. Maximum tolerated dose (MTD) was not reached. Full receptor occupancy through 21 days was achieved at ≥600 mg. The preliminary RP2D is 1200 mg Q3W. Dose-expansion cohorts of IO-108 monotherapy and IO-108 + anti-PD-1 are ongoing.Twenty-three patients were efficacy evaluable (11 monotherapy, 12 combination therapy plus 1 crossover). Overall response rate was 9% in monotherapy cohort (1 Merkel cell carcinoma, prior pembrolizumab followed by nivolumab/ipilimumab) and 23% in combination therapy (2 cholangiocarcinoma, 1 MSS CRC with neuroendocrine features)​. The best overall response was 1 CR, 4 SD among monotherapy, and was 3 PR, 4 SD among combination therapy.​ The 4 responding patients remain on study with an on-going treatment duration of 8 to 12 months as of abstract submission.Consistent with the MOA, clinical benefit correlated with baseline characteristics and post-treatment changes in PD biomarkers including reprogramming of myeloid cells and activation of T cells. Conclusion: The initial encouraging data support further development of IO-108, both as monotherapy and in combination with anti-PD-(L)1 for patients with advanced solid tumors. Citation Format: Matthew H. Taylor, Manish R. Patel, John D. Powderly, Paul Woodard, Luke Chung, Hongyu Tian, Xiang Hong, Kyu Hong, Donna Valencia, Tao Huang, Xiao Min Schebye, Charlene Liao, Aung Naing. A first-in-human phase 1 trial of IO-108, an antagonist antibody targeting LILRB2 (ILT4), as monotherapy and in combination with pembrolizumab in adult patients with advanced relapsed or refractory solid tumors: Dose escalation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT040.

Assessment of Individual Radiosensitivity in Breast Cancer Patients Using a Combination of Biomolecular Markers

About 5% of patients undergoing radiotherapy (RT) develop RT-related side effects. To assess individual radiosensitivity, we collected peripheral blood from breast cancer patients before, during and after the RT, and γH2AX/53BP1 foci, apoptosis, chromosomal aberrations (CAs) and micronuclei (MN) were analyzed and correlated with the healthy tissue side effects assessed by the RTOG/EORTC criteria. The results showed a significantly higher level of γH2AX/53BP1 foci before the RT in radiosensitive (RS) patients in comparison to normal responding patients (NOR). Analysis of apoptosis did not reveal any correlation with side effects. CA and MN assays displayed an increase in genomic instability during and after RT and a higher frequency of MN in the lymphocytes of RS patients. We also studied time kinetics of γH2AX/53BP1 foci and apoptosis after in vitro irradiation of lymphocytes. Higher levels of primary 53BP1 and co-localizing γH2AX/53BP1 foci were detected in cells from RS patients as compared to NOR patients, while no difference in the residual foci or apoptotic response was found. The data suggested impaired DNA damage response in cells from RS patients. We suggest γH2AX/53BP1 foci and MN as potential biomarkers of individual radiosensitivity, but they need to be evaluated with a larger cohort of patients for clinics.

Abstract 2281: Dual immune checkpoint blockade induces analogous alterations in the dysfunctional CD8+ T cell and activated Treg compartment

Abstract Immune checkpoint blockade has shown clinical activity in a range of cancer types. To dissect the effect of neoadjuvant PD-1 and CTLA4 blockade on intratumoral T cells in head and neck squamous cell carcinoma, we analyzed immune infiltrates in tumor biopsies from responding and non-responding patients. At baseline, a higher ratio between active (4-1BB+) and inactive regulatory CD4+ T cells was associated with response to therapy. Furthermore, upon therapy, this active Treg population showed a profound decrease in responding patients. In an analogous process, intratumoral dysfunctional CD8+ T cells transitioned to a state of reduced activity and dysfunction in responding patients, while in non-responding patients, NK cells showed an increased cytotoxic transcriptional profile upon treatment. These data reveal the immunological changes in response to dual PD-1 and CTLA4 blockade, including a parallel remodeling of presumed tumor-reactive T cell compartments in responding patients, and indicate that the presence of an activated Treg compartment at baseline may predict response. Citation Format: Anne M. van der Leun, Joleen J. Traets, Joris L. Vos, Joris B. Elbers, Sanne Patiwael, Xiaohang Qiao, Mercedes Machuca-Ostos, Daniela S. Thommen, John B. Haanen, Ton N. Schumacher, Charlotte L. Zuur. Dual immune checkpoint blockade induces analogous alterations in the dysfunctional CD8+ T cell and activated Treg compartment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2281.

Abstract 2119: Intravenous vitamin C therapy for cisplatin ineligible bladder cancer patients (CI-MIBC): Early investigation into potential pharmacodynamic biomarkers

Abstract Neoadjuvant cisplatin based chemotherapy is considered standard of care for patients with locally advanced bladder cancer (BCa). However, upwards of 50% of patients are ineligible due to poor performance status, underlying kidney disease, peripheral neuropathy, hearing loss and/or cardiac disease. There are currently no accepted alternative regimens with significant activity, and many cisplatin ineligible patients usually proceed directly to surgery. Prior research in other cancers has indicated intravenous vitamin C (IVC), or ascorbate, may serve as a beneficial adjuvant to less toxic therapeutic regimens. We initiated a single arm, Simon 2-stage, Window of Opportunity trial using high dose IVC with single cycle gemcitabine/carboplatin (GCa) (NCT04046094). The primary outcome was pathologic downstaging. Twelve patients with newly diagnosed CI-MIBC were enrolled. Patients received single cycle GCa and IVC titrated to 350-400 mg/dL for ~21 days followed by cystectomy after ~4-6 weeks. As reported, pathological downstaging (<ypT2N0Mx) was present in 4 (33%) of patients. Three of the four patients had a complete response (ypT0NoMx) including one patient with plasmacytoid variant histology. Treatment was well tolerated with no attributable AE/SAEs. Given the discrepancy in response levels, we began identifying potential markers of pharmacodynamic efficacy to inform future trials. Novel data evaluating Ki67+ cells by immunohistochemistry indicate widespread proliferation in non-responders to IVC/GCa, whereas responding patients had visibly fewer positive cells, suggesting a direct effect on cell death and proliferation. TUNEL staining was present in all samples, although often minimally, and thus TUNEL may not yield enough signal to reliably make determinations about treatment outcomes. Urinary cytokine values were assessed using multiplex ELISA and reported as pg/mL urine. Multiple cytokines including IL-1RA, CCL2, IL-8, CXCL10 were present at markedly high concentrations in patients with BCa. Cytokine levels generally increased between diagnostic resection and radical cystectomy indicating potential immune activation by the IVC/GCa treatment. Cytokine levels generally fell between radical cystectomy and follow-up samples suggesting resolution of inflammation after cystectomy. In conclusion, IVC/GCa may be a viable alternative neoadjuvant treatment strategy for patients with BCa that are ineligible for cisplatin-based chemotherapy. Identifying biomarkers of response remains a major area of interest to refine study designs and identify patients most likely to benefit from therapy. Citation Format: Benjamin L. Woolbright, Ganeshkumar Rajendran, Erika Abbott, Zeb Zacharias, Jon Houtman, Rahul Parikh, Qi Chen, Ameer Hamza, Elizabeth Wulff-Burchfield, Jeffrey M. Holzbeierlein, Jeanne Drisko, Michael D. Henry, John A. Taylor. Intravenous vitamin C therapy for cisplatin ineligible bladder cancer patients (CI-MIBC): Early investigation into potential pharmacodynamic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2119.

Abstract 3279: Systemic immune response invoked by short-course oxaliplatin-based chemotherapy (FLOX) for efficacy of sequential immune checkpoint blockade (ICB; nivolumab) in microsatellite-stable (MSS) metastatic colorectal cancer (mCRC)

Abstract Purpose: Most mCRC patients harbor MSS disease without inherent immunogenicity. The METIMMOX study (NCT03388190) explored an unconventional concept, that patients with previously untreated, unresectable abdominal metastases from MSS-CRC may achieve therapeutic efficacy from short-course oxaliplatin-based chemotherapy (FLOX) and sequential ICB (nivolumab). Among study patients who were assigned this highly experimental treatment, 15% obtained long-lasting complete response and more than a half met an early occurring radiologic signal of ICB responsiveness predicting significantly extended progression-free survival (PFS) compared to study control arm patients given standard FLOX chemotherapy [PMID: 36229579]. The aim of the present study was to verify if systemic tumor-defeating immunity had been invoked by the short-course FLOX treatment in the responding patients. The fms-related tyrosine kinase-3 ligand (Flt3L) is a circulating factor that reflects the direct cytotoxic effects of chemotherapy and also activates dendritic cells that present the shed tumor antigens to tumor-targeting T-cells. Procedures: Study patients were randomly assigned to the control arm of FLOX (oxaliplatin 85 mg/m2 on day 1 and bolus 5-fluorouracil 500 mg/m2 and folinic acid 100 mg on days 1 and 2) Q2W or the experimental arm of repeat sequential cycles of 2 FLOX Q2W and 2 nivolumab (240 mg) Q2W. Radiologic response assessment was done every 8 weeks with PFS as the primary endpoint. Patients were categorized into those with target lesion (TL) reduction of ≥10% or <10% at the first post-baseline assessment (8 weeks) [PMID: 36229579]. Serum Flt3L was measured at baseline and following the initial 2 FLOX cycles (at 4 weeks) using a Luminex assay. Results: For all subjects, median Flt3L was 103.8 pg/ml (range 24.6-306.1) at baseline and 162.4 pg/ml (range 53.3-503.8) at 4 weeks. For the experimental arm patients (n = 34), the higher 4-week Flt3L level, the deeper overall radiologic response (rho = -0.39, p = 0.022, Spearman correlation). The 4-week Flt3L level was higher in patients who obtained ≥10% TL reduction (n = 18; median 233.5 pg/ml, range 94.9-466.7) than in those who did not meet this 8-week predictive signal of extended PFS (n = 16; median 161.9 pg/ml, range 53.3-374.8; p = 0.022, Mann-Whitney U test). Selecting a 160 pg/ml cut-off, patients with 4-week Flt3L values above (n = 21) obtained longer PFS (median of 13.6 months, 95% CI 7.6-19.7) than those with serum values below (n = 13; median PFS of 5.9 months, 95% CI 2.4-9.3; p = 0.046, log-rank test). Conclusions: High circulating Flt3L after initial short-course FLOX chemotherapy, as a proxy of invoked tumor-defeating immunity, was associated with an early radiologic signal of ICB responsiveness and improved PFS in patients with MSS-mCRC. Citation Format: Sebastian Meltzer, Kjersti Flatmark, Anniken J. Fuglestad, Hanne M. Hamre, Christian Kersten, Eva Hofsli, Marianne G. Guren, Halfdan Sorbye, Anne Negård, Anne Hansen Ree. Systemic immune response invoked by short-course oxaliplatin-based chemotherapy (FLOX) for efficacy of sequential immune checkpoint blockade (ICB; nivolumab) in microsatellite-stable (MSS) metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3279.

Abstract 4539: Tumor evolution and immune microenvironment dynamics define response to neoadjuvant treatment of esophageal adenocarcinoma

Abstract Introduction: Locally advanced esophageal adenocarcinoma (EAC) remains difficult to treat, and resistance is common. The ecological and evolutionary dynamics responsible for treatment failure are incompletely understood. Aim & Methods: We performed a multi-omic study with a multi-timepoint strategy to examine neoadjuvant treatment response at clonal resolution and in the surrounding tumor microenvironment. EAC samples from chemotherapy responding (REs) and chemotherapy non-responding (NRs) patients with locally advanced EAC were collected at three time points (prior, during, and after neoadjuvant therapy) within the multicenter MEMORI trial. Whole exome sequencing (mean depth 300x) was performed on 47 samples from 17 REs and on 24 samples from 10 NRs. Matched RNA sequencing was performed on 53 samples from 17 REs and on 26 samples from 10 NRs. To characterize immune response we performed imaging mass cytometry (IMC) with a 18-marker panel on 26 samples from 9 REs and 16 samples from 6 NRs and T-cell receptor sequencing in 18 RE-samples and 9 NR-samples. Results: We observed profound changes in mutation signatures over time, characterized by C>A transitions after exposure to neoadjuvant chemotherapy (FOLFOX), which is consistent with earlier observed oxaliplatin induced mutational signatures. Phylogenetic analysis showed no major changes in the clonal make-up during treatment, suggesting phenotypic plasticity rather than clonal evolution caused treatment resistance. EAC samples displayed widespread copy number alterations (CNAs), as previously described. CNAs arising during treatment were significantly more likely to be small, focal alterations rather than large alterations. At the transcriptome level neoadjuvant treatment led to significant changes with significant upregulation of immune and stromal pathways and oncogenic pathways such as KRAS, Hedgehog and WNT. The presence of immune escape mechanisms (defined as LOH or mutations in HLA, B2M mutations or PDL-1 overexpression) and a lack of clonal T-cell expansions were linked to poor clinical treatment response. Moreover, IMC analyses showed a less activated T-cell phenotype in NRs than in REs throughout treatment. Conclusion: Using a multi-timepoint approach, we integrated genetic analyses with transcriptomic analyses and analyses of the tumor immune microenvironment for a holistic understanding of treatment resistance. This study identifies profound transcriptional changes during treatment with limited evidence that clonal replacement is the cause, suggesting phenotypic plasticity as a mechanism for therapy resistance. The presence of immune escape, a less activated T-cell phenotype and a lack of clonal T-cell expansions in patients with poor clinical treatment response has high pharmacological relevance and could be exploited via combined immune-chemotherapy treatments. Citation Format: Melissa Barroux, Jacob Househam, Eszter Lakatos, Tahel Ronel, Ann-Marie Baker, Henrike Salié, Max Mossner, Kane Smith, Chris Kimberley, Salpie Nowinski, Alison Berner, Vinaya Gunasri, Marnix Jansen, Giulio Caravagna, Julia Slotta-Huspenina, Wilko Weichert, Markus Alberstmeier, Benny Chain, Helmut Friess, Bertram Bengsch, Roland M. Schmid, Jens T. Siveke, Michael Quante, Trevor A. Graham. Tumor evolution and immune microenvironment dynamics define response to neoadjuvant treatment of esophageal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4539.

Abstract 4334: Clever-1/PD-L1 ratio predicts response to Bexmarilimab, a novel macrophage-guided immunotherapy, in immune deprived cancers

Abstract Introduction: Clever-1 is an immunosuppressive scavenger receptor expressed on tumor associated macrophages. High levels of Clever-1 are associated with poor survival, T-cell exclusion and dysfunction, and immunotherapy resistance. Bexmarilimab (FP-1305) is a novel humanized anti-Clever-1 IgG4-antibody capable of promoting an immune switch, potentially leading to intratumoral proinflammatory responses in patients. In a first-in-human all-comer single agent Phase I/II study in advanced solid tumors (NCT03733990) approximately 30% of patients in hepatocellular carcinoma, cutaneous melanoma and cholangiocarcinoma achieved immune activation, characterized by an IFNg response, which led to a survival benefit (ASCO 2022). Here, we show that patients getting a therapy benefit have immunologically cold tumors with low PD-L1 staining and high Clever-1 staining. Methods: Pre-treatment tumor samples were immunohistochemically stained for Clever-1 (4G9, Santa Cruz) using Ventana platform and scored by % of intratumoral positive cells out of all viable cells and PD-L1 (22C3, Agilent) with combined positivity scoring (CPS). Ratio (PD-L1/Clever-1) at baseline was compared using non-parametric Wilcoxon-test according to clinical benefit. Results: 23 successful baseline biopsies and stainings were obtained, 6 per cutaneous melanoma, 11 per cholangiocarcinoma and 1 per hepatocellular carcinoma, from patients treated with bexmarilimab. Patients had received an average of three previous lines of therapy. At baseline, patients that saw a clinical benefit (CBR) from bexmarilimab had average PD-L1/Clever-1 ratio of 0.2 compared to 4.4 (p=0.042, Wilcoxon nonparametric test) in patients that did not respond to the treatment. Conclusion: PD-L1/Clever-1 IHC staining ratio may be used to predict bexmarilimab responding patients. These patients have a low PD-L1 staining and low IFNg levels as reported previously, and are often refractory to checkpoint inhibitors and other T cell activating agents. Bexmarilimab provides a novel therapy option for a tumor group that is otherwise poorly responsive to immune therapies. Citation Format: Mari L. Björkman, Elisa M. Vuorinen, Juho Jalkanen, Marie-Louise Fjällskog, Jussi Koivunen, Teppo Huttunen, Maija Hollmén, Petri Bono. Clever-1/PD-L1 ratio predicts response to Bexmarilimab, a novel macrophage-guided immunotherapy, in immune deprived cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4334.

Comprehensive biomarker analysis of long-term response to trastuzumab in patients with HER2-positive advanced gastric or gastroesophageal adenocarcinoma

BackgroundA subgroup of patients with HER2-positive metastatic gastric and gastroesophageal junction cancers shows long-term response under trastuzumab maintenance monotherapy. Obviously, HER2 status alone is not able to identify these patients. We performed this study to identify potential new prognostic biomarkers for this long-term responding patient group. Patients and methodsTumour samples of 19 patients with HER2-positive metastatic gastric and gastroesophageal junction cancer who underwent trastuzumab treatment were retrospectively collected from multiple centres. Patients were divided into long-term responding (n = 7) or short-term responding group (n = 12) according to progression-free survival (PFS≥12 months vs. PFS < 12 months). Next-generation sequencing and microarray-based gene expression analysis were performed along with HER2 and PD-L1 immunohistochemistry. ResultsLong-term responding patients had significantly higher PD-L1 combined positive scores (CPS) and CPS correlated with longer progression-free survival. PD-L1 positivity (CPS ≥ 1) was further associated with an increased CD4+ memory T-cell score. The ERBB2 copy number as well as the tumour mutational burden could not discriminate between short-term and long-term responding patients. Genetic alterations and coamplifications in HER2 pathway associated genes such as EGFR, which were connected to trastuzumab resistance, were present in 10% of the patients and equally distributed between the groups. ConclusionThe study highlights the clinical relevance of PD-L1 testing also in the context of trastuzumab treatment and offers a biological rational by demonstrating elevated CD4+ memory T-cells scores in the PD-L1-positive group.

Efficacy and safety of the MDM2–p53 antagonist BI 907828 in patients with advanced biliary tract cancer: Data from two phase Ia/Ib dose-escalation/expansion trials.

543 Background: Standard-of-care treatment for advanced biliary tract cancer (BTC) is chemotherapy and outcomes remain poor; hence, there is a clinical need for effective targeted treatments. As MDM2 is a negative regulator of the tumor suppressor p53, blocking the MDM2–p53 interaction is a potential antitumor strategy. Further, preliminary data indicate that MDM2 amplification is a negative prognostic marker in BTC. The MDM2–p53 antagonist BI 907828 has shown preclinical antitumor activity in a range of malignancies and is currently being assessed in two phase Ia/Ib dose-escalation/expansion trials in patients with advanced solid tumors: as monotherapy (NCT03449381) and in combination with an anti-PD-1 antibody, ezabenlimab (NCT03964233). Here, we present data for patients with advanced BTCs in these trials. Methods: Patients in the monotherapy trial received escalating doses of BI 907828 on day 1 of 21-day cycles (q3w). Patients in the combination trial received escalating doses of BI 907828 and 240 mg ezabenlimab q3w (doublet); one patient also received the anti-LAG-3 antibody BI 754111 (a drug that has since been discontinued; triplet). Results: A total of 8 patients with BTC were enrolled in the 2 trials, 4 in the monotherapy trial and 4 in the combination trial. In the monotherapy trial, 2 patients had ampullary carcinoma (both received BI 907828 45 mg q3w), and 2 had cholangiocarcinoma (CC; 1 received BI 907828 45 mg q3w and 1 with intrahepatic CC [iCC] received 80 mg q3w). In the combination trial, 3 patients with iCC received 30 mg/45 mg BI 907828 doublet or 45 mg triplet, and 1 with gallbladder carcinoma (GBC) received BI 907828 45 mg doublet. Across both trials, 5 patients achieved PR, 2/4 in the monotherapy trial and 3/4 in the combination trial. In the monotherapy trial, the responding patients had iCC (80 mg q3w; MDM2-amplified; 73% tumor shrinkage; PFS event at 404 days) and ampullary adenocarcinoma (45 mg q3w; MDM2-amplified; 51% tumor shrinkage; ongoing, PFS censored at 255 days). In the combination trial, all 3 responding patients had MDM2-amplified biliary tract adenocarcinoma (2 iCC, 1 GBC); tumor shrinkage was 49–54%; PFS was 162–241 days. A further 2 patients (1 in each trial) achieved stable disease (SD). In the monotherapy trial, most common grade ≥3 treatment-related AEs (TRAEs) were thrombocytopenia, decreased white blood cell (WBC) count and neutropenia (2 patients each). In the combination trial, most common grade ≥3 TRAEs were anemia and decreased WBC count (3 patients each), neutropenia and thrombocytopenia (2 patients each). Conclusions: The MDM2–p53 antagonist BI 907828 has shown a manageable safety profile and encouraging preliminary efficacy in patients with BTC, with 5 PRs and 2 SD in 8 patients. A phase IIa/IIb trial of BI 907828 (45 mg q3w) in patients with BTC is planned to start recruitment at the end of 2022 (NCT05512377). Clinical trial information: NCT03449381 and NCT03964233 .

Panobinostat Induced Spatial In Situ Biomarkers Predictive of Anti-PD-1 Efficacy in Mouse Mammary Carcinoma.

Immunotherapies, including anti-PD-1 immune checkpoint blocking (ICB) antibodies, have revolutionized the treatment of many solid malignancies. However, their efficacy in breast cancer has been limited to a subset of patients with triple-negative breast cancer, where ICBs are routinely combined with a range of cytotoxic and targeted agents. Reliable biomarkers predictive of the therapeutic response to ICB in breast cancer are critically missing, though a combination response has been associated with immunogenic cell death (ICD). Here, we utilized a recently developed integrated analytical platform, the multiplex implantable microdevice assay (MIMA), to evaluate the presence and spatial cell relations of literature-based candidate markers predictive of ICB efficacy in luminal mouse mammary carcinoma. MIMA integrates (i) an implantable microdevice for the localized delivery of small amounts of drugs inside the tumor bed with (ii) sequential multiplex immunohistochemistry (mIHC) and spatial cell analysis pipelines to rapidly (within days) describe drug mechanisms of action and find predictive biomarkers in complex tumor tissue. We show that the expression of cleaved caspase-3, ICAM-1, neuropilin-1, myeloperoxidase, calreticulin, galectin-3, and PD-L1 were spatially associated with the efficacy of panobinostat, a pan-HDAC inhibitor that was previously shown to induce immunogenic cell death and synergize with anti-PD-1 in breast cancer. PD-L1 by itself, however, was not a reliable predictor. Instead, ICB efficacy was robustly identified through the in situ hotspot detection of galectin-3-positive non-proliferating tumor zones enriched in cell death and infiltrated by anti-tumor cytotoxic neutrophils positive for ICAM-1 and neuropilin-1. Such hotspots can be specifically detected using distance-based cluster analyses. Single-cell measurements of the functional states in the tumor microenvironment suggest that both qualitative and quantitative effects might drive effective therapy responses. Overall, the presented study provides (i) complementary biological knowledge about the earliest cell events of induced anti-tumor immunity in breast cancer, including the emergence of resistant cancer stem cells, and (ii) newly identified biomarkers in form of specific spatial cell associations. The approach used standard cell-type-, IHC-, and FFPE-based techniques, and therefore the identified spatial clustering of in situ biomarkers can be readily integrated into existing clinical or research workflows, including in luminal breast cancer. Since early drug responses were detected, the biomarkers could be especially applicable to window-of-opportunity clinical trials to rapidly discriminate between responding and resistant patients, thus limiting unnecessary treatment-associated toxicities.

Brushing Characteristics and Dietary Patterns of Orthodontic Patients with Fixed Appliances

Background: Proper brushing practices are required to maintain good oral hygiene. This is of utmost importance in patients with fixed orthodontic appliances as these appliances can facilitate plaque accumulation and make cleaning process difficult. Hence if good oral hygiene is not maintained with these appliances then deterioration of gingival and periodontal health will result. Frequent intake of acidic drinks and sweet food can further harm the teeth. Aim: To determine tooth brushing characteristics of orthodontic patients with fixed appliances. Determine dietary patterns of such patients. Determine other oral hygiene measures used by them. Study Design: Cross sectional study Duration of the study: 6 months Methodology: Questionnaires were conveniently distributed among 104 orthodontic patients who met the inclusion criteria and provided consent to provide the information. Data collected was analyzed using SPSS 2020. Results: Most (70%) of the responding patients brush two times daily. Correct brush type (soft) is in use by 77% of the individuals. Approximately 64-66% of individuals combine vertical and horizontal technique of tooth brushing, use interdental toothbrush and report that they hardly intake fizzy drinks. However, only 55% patients have reported brushing for total duration of 2 minutes. Around 30% of the respondents have frequent intake of junk food and sugary food. Practical Implication: According to our knowledge, this study is the first study to date to evaluate the brushing habits and dietary patterns in Pakistani orthodontic patients with fixed appliances. Conclusions: The present study shows that majority of the patients show good brushing practices but need to improve their total duration of brushing. Frequent intake of junk food, sweets and fizzy drinks are also avoided by most patients. Use of fluoride mouthwashes and floss was seen in lesser proportion of surveyed patients. Keywords: Brushing characteristics, oral hygiene measures, dietary patterns, orthodontic patients with fixed appliances.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated with Expansion and Maturation of NK Cells

ABSTRACT Background. Hodgkin Lymphoma (HL) Hodgkin Lymphoma (HL) is a B-Cell neoplasia characterized by a high degree of response to chemotherapy and an overall favourable outcome in responding patients. Despite the efficacy of first line therapy, however, about 30% of patients affected by HL are either refractory or eventually relapse (R/R) after first or second line therapy followed by autologous hemopoietic stem cell transplantation (ASCT). Immune checkpoint inhibitors (CI) have demonstrated clinical activity in HL patients relapsing after ASCT, although only 20% complete response (CR) rate are usually observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Aims. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI) and to investigate the lymphocyte subpopulation involved in the mechanism of response to CI in HL. Results. Seventeen patients with relapse/refractory (R/R) HL (median age 29 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 12 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 5 responding patients received ALI alone, as a control cohort. Most patients showed CMV seropositivity at the time of enrolment, without signs of active infection (positive anti-CMV IgG, negative IgM and undetectable CMV DNA). No adverse events were recorded, specifically, no immune-related toxicity was observed and none of the patients showed CMV reactivation. All patients receiving ALI + CI (treated patients) achieved negative PET scan CR and 11/12 are alive and disease-free after a median follow-up of 33 months. The only relapse occured 23 months after last ALI. Six of the responding patients underwent subsequent allogeneic SCT in a CR status and 1 patients are currently waiting for transplantation. Two of the patients in the control arm relapsed after a median of 6 months (4-8 months) Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients (Fig.1). In detail, different patterns of NK cell development could be identified. Indeed, starting from one month after transplantation, in the group of patients undergoing ALI plus nivolumab, a relevant fraction of NK cells displayed a mature phenotype characterized by a CD56dimCD16bright expression (Fig. 1). On the contrary, in control patients, NK cells were characterized by a more immature phenotype (high frequencies of CD56brightCD16neg/dim NK cells) even at late time points (three months) after transplantation (Fig. 1). This event occurred in all the treated (ALI + nivolumab) patients analyzed (Fig. 1). Finally, PB from patients undergoing ALI + nivolumab was enriched in unconventional CD56dimCD16dim NK cells (Fig. 1). One month after transplantation, CD94/NKG2A+ KIR- NK cells were largely predominant in all patients, whereas CD94/NKG2A- KIR+ NK cells were present in low proportions. An increase of CD94/NKG2A- KIR+ subset occurred primarily in treated patients starting from t2. At t3, the significant increase of CD94/NKG2A- KIR+ NK cells occurred in the treated group but not in the control group . This event occurred in all the treated (ALI + nivolumab) patients analyzed, regardless of their serological status for HCMV, but was more evident in treated patients who were seropositive for HCMV. Remarkably, PB from HCMV seropositive patients undergoing ALI + nivolumab became enriched over time in differentiated NK cells characterized by high expression of NKG2C receptor (the activating counterpart of NKG2A), as compared to HCMV+ control patients. This subset represents the so-called "adaptive" NK cell population that usually increases during HCMV infection/reactivation. In this case, however, this event occurred in the absence of HCMV reactivation, and only in HCMV seropositive patients treated with ALI + nivolumab. Conclusions. Our data show anti-tumour activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favour the expansion of the "adaptive" NK cell compartment, especially in patients with CMV seropositivity, in the absence of CMV reactivation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Changes of Transcriptomic Landscape in Blasts of Responding and Refractory Acute Myeloid Leukemia Patients after Chemotherapy

Despite many ongoing clinical drug trials and advances in acute myeloid leukemia (AML) therapy, the 3+7 induction regimen with anthracyclines and cytarabine (+/- midostaurin) remains the golden standard of AML treatment. Approximately 40 % of AML patients do not achieve a complete hematological remission (CR) after the first cycle of 3+7 chemotherapy and face an adverse prognosis. One of the main causes of treatment failure is chemoresistance of leukemia cells acquired by various mechanisms, all of which have not been identified as yet. We present here a unique study of transcriptome landscape mapping of separated AML blasts from paired samples collected before and after chemotherapy administration, focusing on differences between responding (RES) and refractory (REF) AML. The aim of this study was to describe transcriptional changes in leukemic blasts between RES and REF AML patients after chemotherapy administration, and to identify molecular pathways involved in reduced sensitivity to chemotherapy. Leukemic blasts were sorted from peripheral blood of AML patients collected at diagnosis (day 0, n=30) and on the third day (day 3, n=16) of 3+7 chemotherapy. CD34+ and CD117+ antibodies were used for blast separation on autoMACS (Miltenyi Biotec). 100 ng of high quality DNAse-treated RNA (RIN>8) was ribodepleted via the RiboCop rRNA Depletion kit (Lexogen) and the library was prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB). Transcriptome was sequenced on NovaSeq (Illumina). For data analysis, we divided patients by primary response status into RES and REF groups based on CR achievement after the first cycle of 3+7 therapy. Quantification of gene expression was performed using StringTie2 software (version 1.3.6). Resulting data were subsequently analyzed and visualized in R software 4.0.0 using edgeR and pheatmap packages. The String and GO databases were used to annotate genes into molecular pathways and biological processes. We identified 75 differentially expressed genes (FDR<0.05) at day 0 between RES (n=15) and REF (n=15) AML patients (Fig.1a). Among genes upregulated (n=62) in REF AML, those previously associated with inferior prognosis such as MN1 (transcriptional regulator), ABCA6 (membrane transporter) and SETBP1 (transcriptional regulator) were identified. On the contrary, PRF1 (involved in killing transformed cells) and TLE1 (negative regulator of NF-κB) were among genes downregulated (n=13) in REF AML. In general, genes upregulated in REF AML were assigned to Golgi membrane, carbohydrate transport and interferon signalling and genes upregulated in RES AML to immune response and the β-catenin/TCF transcriptional complex. In the pairwise analysis (day 0 vs. day 3), we identified 82 differentially expressed genes in RES (Fig.1b, n=6) and 121 differentially expressed genes in REF AML (Fig.1c, n=10). There were 42 genes identically deregulated in both groups and they were annotated into DNA damage/stress response, DNA repair and apoptotic pathways. Next, we compared genes that were deregulated only in RES or REF AML. In RES AML, there was a big cluster of downregulated histone and chromatin remodelling genes that was missing in REF AML. Upregulated genes in RES AML included those with antiproliferative, proapoptotic and p53 stabilizing functions. In REF AML, the principal deregulated genes were those affecting cytoskeleton remodelling (downregulated), p53 signalling network (upregulated) and genotoxicity pathways (upregulated). We identified differences in leukemic blast transcriptome between RES and REF AML before and after chemotherapy administration. The role of some differentially expressed genes has already been elucidated in AML pathogenesis, others deserve a closer look to evaluate their relevance as potential targets for emerging therapies. Instant downregulation of histone genes after chemotherapy administration seems to be a strong sign of RES AML and requires further attention for its use as a biomarker for early stratification into good versus poor responder AML. This project was supported by Ministry of Health of the Czech Republic (00023736, IHBT) and by GA CR (No. 20-19162S). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting CNS Lymphoma with Intravenous Axicabtagene Ciloleucel: Evidence for Transcriptional Evolution Towards a Prominent Interferon Signature in CAR-T Cells Trafficking to the Tumor Site

Background: Chemorefractory primary and secondary central nervous system lymphomas (CNSL) have a dismal prognosis, and represent a major unmet need in oncology. To address this, we conducted a pilot trial of axi-cel in patients with relapsed/refractory CNSL. All patients had Ommaya reservoirs, such that after CAR-T infusion, paired CSF and blood could be collected and analyzed daily, allowing the interrogation of single-cell CAR-T transcriptional profiles at an unprecedented level of detail. This analysis enabled us to address key mechanistic questions relevant to CAR-T therapy for CNS lymphoma, including determining the impact of tumor site (brain vs lymphoid tissue) on CAR-T efficacy and toxicity, and discovering the drivers of CAR-T cell transcriptional evolution as these cells move between their sites of action. Methods: Adult patients with either primary or secondary relapsed/refractory CNSL were enrolled on the 'Axi-cel In CNS Lymphoma' Trial (Clinicaltrials.gov# NCT04608487). Patients received fludarabine/cyclophosphamide lymphodepletion followed by axi-cel infusion. Blood and CSF were sampled daily between D0-14 after infusion, with data from CAR-T maximum expansion (Days 5-9) reported here. 5' 10x Single-cell RNA-Seq (scRNA-Seq) and TCR-Seq were performed on peripheral blood mononuclear cells (PBMC), enriched T cells, and CSF-derived cells. scRNA-Seq data was analyzed with the Python packages 'scanpy' and 'scVI' to identify and characterize high-quality CD4+ and CD8+ T cells, and to conduct differential expression tests. Gene set enrichment analysis (GSEA) was used to compare CSF and blood CD4+ and CD8+ CAR-T cells using the 'clusterProfiler' package in R. Results: Detailed clinical results from this trial are reported in an accompanying abstract. In brief, data from the first 7 patients on-study demonstrated no treatment-limiting toxicities and an ORR of 86%, with 6 of 6 responding patients achieving a CR by 3 months. 2 of the responding patients have progressed, 1 at 6 months and 1 at 15 months. To interrogate the mechanisms driving axi-cel efficacy in inducing CNS responses, we analyzed 129,088 CAR+ and non-CAR T cells from the peripheral blood (64,337 cells), the CSF (37,070 cells), and the axi-cel product (27,681 cells) from 5 enrolled patients, with blood/CSF collected around the time of maximum CAR-T expansion (Days 5-9 post-infusion). Enumeration of CAR-T cells in the blood vs CSF revealed a mean 13% CAR-Ts of total blood T cells (range 7-24%), and a mean 18% (range 13-28%) in the CSF. CD4+ CAR-T cells predominated in the infused product (mean 79% CAR-T cells were CD4+, range 68-95%), with the CD4:CD8 balance maintained at peak expansion (with 77% (range 60-83%) CAR-T cells in the blood being CD4+, and 95% (range 91-97%) CAR-T cells in the CSF being CD4+). Sc-TCR sequencing enabled an evaluation of the T cell repertoire, revealing a similarly broad CAR-T repertoire in both the blood (mean 1,375 clones (range 313-2,563) and CSF (mean 1,090 clones, range 268-1,965). scRNA-Seq gene expression and clustering analysis revealed a striking distinction between blood and CSF CAR-Ts: While blood CAR-Ts exhibited a prominent proliferation gene expression signature (analyzed by single cell GSEA), CSF CAR-Ts, obtained on the same days as those from the blood, exhibited strong enrichment for interferon-pathway associated genes (Panel 1). Although the majority of CAR-T clones identified were unique to either the blood or CSF, we were able to identify 54 shared clones between these compartments. Analysis of these clones further substantiated the transcriptional evolution of CAR-Ts between the blood and CSF. Thus, individual shared clones tracked in blood and CSF from multiple patients exhibited an evolution from a more diverse proliferation/IFN pathway profile in the blood, to predominantly IFN pathways enriched in CSF CAR-Ts (Panel 2). Conclusions: Daily examination of the blood and CSF by scRNA-Seq and scTCR-Seq has identified a major distinction between blood and CSF CAR-Ts at the level of individual CAR-T clones, revealing the evolution of a prominent interferon pathway transcriptomic signature of CAR-Ts in the CSF. These results suggest that IFN pathway enrichment may play a major role in CAR-T efficacy for the eradication of CNS disease in patients with CNSL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase II Study of Pembrolizumab and Fractionated External Beam Radiotherapy in Patients with Relapsed and Refractory Large B-Cell Lymphoma

Background: Relapsed/refractory (R/R) large B-cell lymphoma (LBCL) poses a therapeutic challenge, however immune based interventions are changing the landscape of salvage therapy. Manipulation of immune checkpoint pathways involving programmed cell death protein 1 (PD-1) and its ligands PD-L1/PD-L2 can trigger anti-tumor effects in LBCL. However, results with checkpoint inhibitor monotherapy for LBCL have been disappointing with reported overall response rates (ORR) of 10% for patients who experienced relapse after autologous stem cell transplantation (ASCT) and 3% for those ineligible for ASCT (Lesokhin AM et al J Clin Oncol. 2016). Radiation therapy (RT) is highly effective in R/R LBCL and may enhance response when coupled with immune based therapy. We sought to evaluate the efficacy of RT combined with pembrolizumab among patients with R/R non-Hodgkin Lymphoma (NHL) treated with RT to 1-2 sites of refractory disease with at least one index lesion excluded from the RT field for evaluation of abscopal effect. Methods: We conducted a single center, open label, phase II trial of patients with R/R NHL with progressive lymphoma after 2 or more lines of therapy. Eligible patients had PET avid disease amenable to RT and at least one distinct lesion outside of the planned RT field that would serve as the index (abscopal) lesion(s) by which the response to therapy would be defined. Patients were administered a fixed dose of 200 mg of pembrolizumab intravenously every 21 days beginning on day 2 of RT planned for 24 months or until progression. External beam RT was planned to dose of 40-50 Gy in ≤3 Gy fractions. The primary endpoint was ORR (defined as complete and partial response) of the unirradiated lesion(s) at 3 months (after completion of RT and 3 cycles of pembrolizumab). Secondary endpoints included safety, progression free survival (PFS) and overall survival (OS). ORR and dose limiting toxicity (DLT) were monitored simultaneously using Bayesian stopping boundaries based on an ORR of at least 35% at 3 months and DLT rate at cycle one below 30%. A sample size of 20 was planned. Results: Between February 2018 and November 2020, 17 patients were enrolled on the study and received at least 1 cycle of pembrolizumab and at least 1 fraction of RT. The median age was 66 years (range 24 - 83); 29% (n=5) of patients were female. Most patients had LBCL (88%, n=15, double hit, n=5), one patient had PMBCL (6%) and one patient had NK T-cell lymphoma (6%). The median number of prior therapies was 3 (range 2-13) with 53% (n=9) of patients having received ≥3 prior lines of treatment. Eighteen percent (n=3) of patients received prior ASCT and 53% (n=9) received CAR-T cell therapy prior to enrollment. The median number of unirradiated lesions excluded from the RT field was 2 (range 1-24). The median diameter of the largest unirradiated lesion was 2.4 cm (range 1.1 - 8.0 cm). The median RT dose delivered was 45 Gy (range 22.5 -50). The median follow up for the 17 patients was 34.75 months. Sixteen patients had a response assessment, one patient suffered from a stroke and response was not assessed. The ORR was 28% (CR 23% [n=3] and PR 15% [n=2]). Local control within the radiated field was achieved in 11 of the 16 patients (69%). For all 17 patients, the median PFS and OS were 2.7 months and 6.3 months respectively. The 2 year PFS and OS rates were 11.8% and 23.5% respectively (Figure 1). There were 13 deaths, 12 due to lymphoma and 1 due to stroke. For the five responding patients, the median duration of response was 13.3 months (range 5.0 - 50.5 months). Two patients remain in CR 37.6 (PMBCL) and 50.5 (double hit LBCL) months after protocol therapy. The trial was terminated early due to poor accrual. Treatment was generally well tolerated. One of the long term survivors with a history of chronic steroid use experienced grade 4 avascular necrosis in the RT field. Conclusion: In this single center, phase II trial of pembrolizumab and concurrent RT among patients with highly refractory LBCL, local control within the radiation field was encouraging however abscopal responses were minimal. Further exploration of the mechanism behind the exceptional responses in two patients treated with RT and pembrolizumab in this high-risk population is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal