Serial Peripheral Blood Samples Research Articles

Baseline immune state and T-cell clonal kinetics are associated with durable response to CAR-T therapy in large B-cell lymphoma

AbstractEngineered cellular therapy with CD19-targeting chimeric antigen receptor T cells (CAR-Ts) has revolutionized outcomes for patients with relapsed/refractory large B-cell lymphoma (LBCL), but the cellular and molecular features associated with response remain largely unresolved. We analyzed serial peripheral blood samples ranging from the day of apheresis (day –28/baseline) to 28 days after CAR-T infusion from 50 patients with LBCL treated with axicabtagene ciloleucel by integrating single-cell RNA and T-cell receptor sequencing, flow cytometry, and mass cytometry to characterize features associated with response to CAR-T. Pretreatment patient characteristics associated with response included the presence of B cells and increased absolute lymphocyte count to absolute monocyte count ratio (ALC/AMC). Infusion products from responders were enriched for clonally expanded, highly activated CD8+ T cells. We expanded these observations to 99 patients from the ZUMA-1 cohort and identified a subset of patients with elevated baseline B cells, 80% of whom were complete responders. We integrated B-cell proportion ≥0.5% and ALC/AMC ≥1.2 into a 2-factor predictive model and applied this model to the ZUMA-1 cohort. Estimated progression-free survival at 1 year in patients meeting 1 or both criteria was 65% vs 31% for patients meeting neither criterion. Our results suggest that patients’ immunologic state at baseline affects the likelihood of response to CAR-T through both modulation of the T-cell apheresis product composition and promoting a more favorable circulating immune compartment before therapy. These baseline immunologic features, measured readily in the clinical setting before CAR-T, can be applied to predict response to therapy.

Investigation of the effects of IL-13 and IL-22 cytokine levels on disease activity, prognosis, and treatment response in multiple sclerosis patients treated with fingolimod and glatiramer acetate

Background: Multiple Sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease. In this study, we investigated serum protein levels of Interleukin-13 (IL-13) and Interleukin-22 (IL-22) cytokines. These cytokines play an important role in the generation and regulation of the inflammatory response, which is the pathogenesis of MS, and are potential biomarkers for monitoring therapeutic response. cytokines may play a role in the development of MS lesions. Methods: The study included 66 MS patients and 22 healthy individuals. IL-13 and IL-22 cytokine protein levels were measured by ELISA from peripheral blood serum samples collected from the participants. Patient demographics and treatment history data were also collected. Results: IL-13 and IL-22 parameters were lower in MS patients compared to the control group. There was a significant difference between the patient and control groups in terms of IL-13 (p<0.001). Although the mean IL-22 level of the control group was higher than the patient group, the difference did not reach a significant level (p: 0.257). Conclusion: The results of the study suggest that IL-13 and IL-22 cytokines play an important role in the pathogenesis of MS and are affected by fingolimod and glatiramer acetate treatment.

Repurposing ibrutinib for chemo-immunotherapy in a phase 1b study of ibrutinib with indoximod plus metronomic cyclophosphamide and etoposide for patients with childhood brain cancer.

TPS2703 Background: In children, brain cancer is the leading cause of cancer related death. The indoleamine 2,3-dioxygenase (IDO) pathway is a metabolic checkpoint, expressed by host myeloid and dendritic cells, that suppresses anti-tumor immune responses following chemotherapy. We recently published results of a first-in-children phase 1 trial (NCT02502708) that showed the oral IDO-pathway inhibitor indoximod was well tolerated and provided meaningful clinical benefit for many patients with progressive childhood brain tumors, when given in combination with oral chemotherapy regimens (1). Using preclinical models, we have also reported the importance of the Bruton’s Tyrosine Kinase (BTK) pathway as a key upstream driver of IDO during chemotherapy (2). Thus, we hypothesize that adding the BTK-inhibitor ibrutinib to the previously studied regimen of investigational indoximod IDO-inhibitor plus oral metronomic cyclophosphamide and etoposide will synergistically enhance anti-tumor immune responses, leading to improvement in Objective Response Rate (ORR) with manageable overlapping toxicity. Methods: The current study (NCT05106296) is an Investigator-Sponsored, open label, single-arm phase 1b trial comprised of a Dose-escalation Cohort (n=18) using a 3+3 dose escalation design to establish safety and dosing of ibrutinib in the combined regimen; followed by an Expansion Cohort (n=19) at the MTD to evaluate efficacy. Patients are treated with the all-oral 4-drug chemo-immunotherapy regimen in 28-day cycles using: (i) ibrutinib [Study Dose once daily on days 1-21]; (ii) indoximod [38.4 mg/kg/day divided twice daily throughout the cycle]; (iii) cyclophosphamide [2.5 mg/kg/dose, maximum dose 100 mg, once daily on days 1-21]; and (iv) etoposide [50 mg/m2/dose once daily on days 1-21]. Eligible patients are age 12 to 25 years with relapsed or refractory brain cancer, MRI evidence of current active disease not recently treated with radiation/proton therapy, ECOG performance score 0-2, and meet standard organ function requirements. Patients with systemic infection, autoimmune disease, recent live-virus vaccination, comorbid conditions that may overlap with expected regimen toxicities, or chronic treatment with anticoagulants or strong CYP3A inhibitors are excluded. Primary Objectives are to: (i) determine the pediatric recommended phase 2 dose (RP2D) of ibrutinib for the combined regimen (Dose-escalation Cohort), and (ii) evaluate preliminary evidence of efficacy for the combined regimen in terms of ORR (Expansion Cohort). Exploratory analyses use single-cell RNA and TCR sequencing (scRNAseq/TCRseq) of cryopreserved serial peripheral blood samples to monitor for treatment-expanded TCR clonotypes and study their phenotype. 1. Neuro-Oncology 26:348-361, 2024. 2. Immunity54:2354-2371, 2021. Clinical trial information: NCT05106296 .

Seroprevalence of Trypanosoma cruzi among children from Veracruz, Mexico: Epidemiological baseline for a control model based on Chagas disease active transmission

Introduction. In 2021, the Secretaría de Salud de México and the Pan American Health Organization launched an initiative to interrupt intra-domiciliary vector transmission of Trypanosoma cruzi based on the prevalence of Chagas disease in children. The Mexican State of Veracruz was leading this initiative. Objective. To estimate the seroprevalence of T. cruzi infection among children under 15 years of age from rural areas of Veracruz, México. Materials and methods. We identified eight localities of high priority from the Municipality of Tempoal, Veracruz, for baseline serology. Blood samples were collected on filter paper from 817 individuals between June and August 2017, for screening with a third-generation enzyme immunoassay. Reactive cases were confirmed by indirect hemagglutination, enzyme-linked immunosorbent assay, and indirect immunofluorescence tests on peripheral blood serum samples. We calculated seroprevalence and 95% confidence intervals (CI). Results. We confirmed Chagas disease cases in children under 15 years of age with a seroprevalence of 1,9% (95 % CI = 1,12-3,16) in the localities of Citlaltepetl, Cornizuelo, Cruz de Palma and Rancho Nuevo. Conclusions. These results indicate recent transmission of T. cruzi in these communities and allow to establish an epidemiological baseline for the design and implementation of a model focused on geographical areas with active transmission to advance toward the elimination of intra-domiciliary vector transmission of this parasite in Mexico.

Abstract 1184: Combination pembrolizumab and radiotherapy induces anti-tumor immune responses in immunologically-cold non-small cell lung cancer

Abstract Introduction: Primary resistance to immune checkpoint inhibition (ICI) is in part driven by a “cold” tumor microenvironment in the context of low PD-L1 expression, low tumor mutation burden or oncogenic mutations that drive tumor immune exclusion. Radiation has the potential to sensitize tumors to ICI through extra-tumoral T cell activation in response to tumor antigens released from irradiated cancer cells (abscopal effect). Here, we investigate tumor microenvironment and T cell repertoire reshaping leveraging serial biospecimens from a phase 2 randomized clinical trial of stereotactic body radiation therapy followed by pembrolizumab (SBRT arm) vs. pembrolizumab alone (control arm) in non-small cell lung cancer (NSCLC; NCT02492568). Methods: We employed whole exome sequencing, RNA sequencing and T cell receptor (TCR) sequencing to analyze 293 serial tumor and peripheral blood samples collected at baseline and after 8 weeks of treatment from 72 patients in the control (n=35) and SBRT (n=37) arms. Immunologically-cold tumors were defined as low TMB (<300 mutations per exome, n=43), PD-L1 null (0% expression on immunohistochemistry, n=41), or WNT-activated (presence of mutations affecting genes in the WNT pathway, n=10). TCR clonotypic differential abundance analyses and RNA sequencing deconvolution and gene set enrichment analyses were performed on baseline and on-therapy blood and tumor samples from the control and SBRT arms. Results: Patients with immunologically-cold tumors in the SBRT arm had significantly longer progression free survival compared to the pembrolizumab monotherapy arm (log rank p=0.029 for TMB-low, p=0.022 for PD-L1-null, p=0.037 for the WNT-activated subsets). Induction of interferon-gamma, interferon-alpha, antigen processing and presentation, and inflammatory response gene sets was significantly enriched on-therapy in the SBRT arm (FDR adjusted p<0.01 for TMB-low, PD-L1-null, and WNT-activated tumors). We observed greater enrichment in newly expanded TCR clones on-therapy intratumorally and in peripheral blood in the SBRT arm compared to the control arm (mean 16.17 vs 7.54 clonotypes, p=0.022 in tumor; mean 4.56 vs 1.00, p=0.039 in blood; Mann-Whitney U test). Similarly, we noted greater expansion of pre-existing TCR clones in the SBRT arm than within the control arm (mean 34.83 vs 18.69, p=0.021 in tumor; mean 21.61 vs 8.15, p=0.009 in blood; Mann-Whitney U test). Within the SBRT arm, there was no significant difference in T cell expansion between TMB-stratified (<300 mutations vs >=300 mutations) or PD-L1-stratified (0% vs >= 1% expression) groups. Conclusions: SBRT followed by pembrolizumab was associated with priming and augmenting anti-tumor immune responses in immunologically-cold NSCLC, which opens a therapeutic window of opportunity for radiotherapy to overcome primary resistance to immune checkpoint inhibition. Citation Format: Justin Huang, Willemijn S. Theelen, Zineb Belcaid, Mimi Najjar, Christopher Cherry, Archana Balan, James R. White, Noushin Niknafs, Jaime Wehr, M.M. van den Heuvel, Rachel Karchin, Paul Baas, Victor E. Velculescu, Valsamo Anagnostou. Combination pembrolizumab and radiotherapy induces anti-tumor immune responses in immunologically-cold non-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 1184.

Checkpoint Immunotherapy Is Associated with Preferential Activation of Tumor-Antigen Specific CD4 + T Cells in MDS

Background: Patients with intermediate and higher-risk MDS are generally treated with the DNA hypomethylating agents (HMAs) azacytidine and decitabine. These agents improve survival and cytopenias in ~50% of those treated; but only responders derive clinical benefit, and no pre-treatment response prediction has proven efficacy. There is thus an urgent need to develop new approaches to complement or enhance the response to standard-of-care HMA therapies. Studies suggest that HMAs might act in part via activation of anti-tumor immune mechanisms. Based upon this hypothesis a series of early phase trials combining immune checkpoint inhibitors (ICI: both anti-PD1/PDL1 and CTLA4) with azacitidine in MDS/AML patients were undertaken but clinical and immunological efficacy have proven disappointing. In these studies, the lack of antigen specificity made systematic assessment of the anti-MDS immune response and milieu challenging. Antigen specific assessments are particularly important in MDS patients treated with ICIs because distinct from solid tumors, myeloid stem cell cancers involve the populations of antigen presenting cells that modify the capacity for T-cell response, such as dendritic cells. Our group has shown that MDS/AML patients treated with decitabine show increased expression of NY-ESO-1, a well-characterized tumor antigen whose expression is normally suppressed through dense promoter hypermethylation. In our prior Phase I study, MDS patients received vaccination against NY-ESO-1 in combination with standard decitabine. This combination was not only safe, but it also induced NY-ESO-1 specific CD4 + and CD8 + T cell responses in most patients. We hypothesized that combining anti-NY-ESO-1 vaccination (an antigen specific approach) with decitabine and an ICI would allow us to understand the immune synapse in patients with MDS. Methods: We developed an investigator-initiated Phase 1 trial in transplant-ineligible patients with MDS/low blast count AML adding an anti-PD-1 ICI to our established combination of decitabine + NY-ESO-1 vaccination (#NCT 0335871). This open-label, non-randomized single center Phase 1 study used an HLA unrestricted NY-ESO-1 vaccine (CDX-1401 (1 mg) + poly-ICLC (1.8 mg)) in combination with standard dose decitabine (20mg/m2/d x 5 days) and nivolumab (3 mg/kg every 2 weeks), 4 cycles of combination therapy were planned on study; patients deriving clinical benefit could continue treatment at the discretion of the treating physician. The study was conducted in accordance with the Declaration of Helsinki and approved by the Roswell Park Cancer Institute (RPCI) Internal Review Board. Six patients were enrolled, treated, and completed > 4 cycles of decitabine; serial bone marrow and peripheral blood samples were collected for analysis. The combination was safe. Results: Two patients developed CR and the remainder had stable disease as the best response to therapy (Table 1). The majority of patients mounted NY-ESO-1 specific CD4 + T cell responses associated with up-regulation of anti-PD-1 immunotherapy gene signatures in the CD4 + memory T cell compartment. No responses in CD8 + T cells were seen. In seeking explanation for the disparate findings in CD4 + and CD8 + T-cell compartments, we examined conventional dendritic cell populations associated with activation of CD8 + T cells and identified that our patients had markedly reduced numbers of conventional dendritic cells marked by high expression of CD141 (cDC1). This population is critical for a successful response to immunotherapy. cDC1 from MDS patients also showed reduced expression of genes that are key for optimal T-cell activation and expansion. Conclusions: These results suggest that the use of immunotherapy to induce effective, cytotoxic anti-tumor T cell responses depends upon the myeloid immunologic milieu in MDS patients. The critical importance of cDC1 function has recently been described and appreciated in the context of solid tumor immunotherapy. The observation of both numerical and functional defects of this cell population in patients with myeloid neoplasia provides a possible explanation for the disappointing efficacy of immunotherapies in this disease state. Approaches to augment the number and function of specific cDC1 populations in myeloid disease might overcome this defect and thereby enhance the efficacy of immunotherapy for patients with MDS.

The Impact of Epstein-Barr Virus Infection on Second Cancer Development in Newly Diagnosed Myeloma Patients Treated with Lenalidomide in the UK NCRI Myeloma XI Trial

Background Several factors have been linked to second primary malignancy (SPM) development in myeloma patients, including advanced age, immunocompromise and therapy, such as melphalan and lenalidomide (len). The impact of infection, including EBV is yet to be elucidated. Here we address this in the context of the UK NCRI Myeloma XI Trial for newly diagnosed transplant eligible (TE) and transplant non-eligible (TNE) patients. Methods Myeloma XI was a phase III, randomised, multi-centre, parallel group design, open-label trial comparing cyclophosphamide, thalidomide, len, carfilzomib and bortezomib induction combinations and len maintenance treatment in TE and TNE patients. TE patients received high dose melphalan supported by autologous stem cell transplantation (ASCT). Patients in both pathways were randomised to maintenance with len or observation. In total, 4358 patients entered. Planned SPM analysis was conducted in May 2019, with 318 patients developing an SPM prior to that point. Median follow-up from trial entry was 60 months (IQ range 47-76). A nested cohort analysis was performed to determine EBV infection and immunity status in 170 SPM patients for which serum samples were available. Forty-two trial patients who did not develop an SPM were used as a control group. EBV assessment time-points included pre-induction, post induction, 4 months post ASCT (TE only) and 1 year post induction. Peripheral blood serum samples were tested for EBV EBNA IgG Antibody/index (LIAISON®), EBV VCA IgM Antibody/Index (LIAISON®) and EBV positivity was determined using the artus® EBV RG PCR Kit from Qiagen. Active or recent EBV infection was evidenced by a positive EBV IgM antibody and/or EBV PCR. Results Of the 170 patients, 208 SPM were noted with 147 patients developing 1 SPM and 23 patients developing ≥2. One hundred and five patients developed an SPM following maintenance randomisation, with 76 receiving len and 29 being observed. The remaining 65 patients developed an SPM prior to maintenance. Eighty-four SPMs were diagnosed in 68 TE patients and 124 SPMs in 102 TNE patients. Of the 170 SPM patients, 8 had evidence of recent or active infection at the time of testing, evidenced by either IgM EBV antibodies (n=5), positive EBV PCR (n=2) or both (n=1). The median EBV DNA copies/ml was 353 (range 337-428). The median IgM antibody titre was 71.5U/ml (26 - >160). No patients in the control group had evidence of recent EBV infection (p=0.36). Of the 8 patients with evidence of EBV infection, 6 developed >1 SPM, Table 1. Five had been treated in the TE pathway, with all developing post maintenance randomisation, and 4 of the 5 receiving len maintenance. None of the 3 TNE patients received len maintenance, with 2 developing an SPM prior to maintenance randomisation. The median time from EBV testing to SPM diagnosis was 17·9 months (0.5-51.7). Two of the EBV positive patients developed a haematological SPM: myelodysplastic syndrome and Hodgkin lymphoma. Four patients developed a solid malignancy: lung, breast, ovary, and colon. The remaining cases were non-melanoma skin cancers, Table 1. Immune status (EBV IgG) was also determined. In TE patients, the median EBV IgG titre was 65.3U/ml at diagnosis (<3 - 600, n=48), falling to 28.1U/ml following induction (<3 - 326, n=41) and recovering to 44.2U/ml one year post ASCT (<3 - 600, n=22). In TNE patients, the median IgG titre at diagnosis was 39.1U/ml (<3 to 517, n=72), falling to 31.2U/ml 4 months post induction (<3 - 287, n=42) and recovering to 51.3U/ml after 1 year (<3 - 554, n=41). In the SPM patients the IgG EBV titres were noted to be <20U/ml in 5 of the 8 patients. Three of the eight patients had no detectable EBV IgG level (<3U/ml) Conclusions These data suggest that EBV infection is unlikely to be a risk factor for SPM development in myeloma patients. This is supported by the finding that EBV IgG titres were consistent with immunity in most patients (>20U/ml) and even in the patients with possible EBV infection, EBV DNA was only identified in 3 patients, and at a low level. In addition, the SPM developed are not classically associated with EBV infection or reactivation. It is noted that EBV infection status was not always determined at the time of SPM development. Despite this, the data does show that EBV immunity recovers following therapy, even in the post ASCT setting. EBV monitoring is therefore not supported by this work, although in patients who develop ≥2 SPM, further clarification is required.

Personalized Residual Disease Detection in Acute Myeloid Leukemia with a Multiplexed Genomic Fingerprint Predicts Patient Outcomes

Background: Acute myeloid leukemia (AML) is the most common leukemia in adults. While therapeutic options for AML have continued to improve, nearly half of all patients who achieve clinical remission will relapse within several years after diagnosis. Risk factors for relapse include age, cytogenetics, specific gene mutations, and the detection of residual disease after treatment. Current molecular approaches for measuring residual disease are designed primarily around a single gene. This limits both the sensitivity and the applicability of the assay to all patients, as AML is a highly heterogenous genetic disease. In this study, we demonstrate the utility of a comprehensive, personalized, multiplexed genomic assay that exhibits high sensitivity, excellent quantitation, and promising performance across tissue types. This approach enables the serial, non-invasive monitoring of patients with AML and the prediction of patient outcome. Methods: Serial samples of bone marrow, peripheral blood, and urine were collected at multiple time points from 12 patients with AML who were treated with standard-of-care therapy. Time points range from presentation to relapse, with a median of 4 time points per patient (range of 2-10). A median of 7 samples were collected per patient (range of 2 to 16). DNA was extracted from the peripheral blood mononuclear cells (PBMCs), the cell-free fraction of peripheral blood, bone marrow mononuclear cells, and the cell-free fraction of urine. Deep whole genome sequencing of paired presentation and remission samples identified hundreds of candidate leukemia-associated somatic single-nucleotide variants comprising the personalized genomic fingerprint of each patient's disease. A representative set of variants (n=30-50) was selected for each patient, and a personalized set of primers was designed for the Multiplex Accurate Sensitive Quantification (MASQ) next-generation sequencing protocol. The sensitivity of this assay allows for single variants to be quantified down to at least 1 in 100,000, resulting in an overall sensitivity below 1 in 1 million for residual disease detection. Quantification of the individual variants from the fingerprint was evaluated in samples of all tissue types collected (bone marrow, blood cells, blood cell-free DNA, and urine cell-free DNA) and all time points collected (presentation, post-induction, post-consolidation, and follow-up visits). Results: Serial monitoring of the leukemia genomic fingerprint enables risk assessment after each stage of treatment. Using Multiplex Accurate Sensitive Quantification (MASQ), we detected leukemia-associated variants in the blood and bone marrow of all patients during remission, at levels ranging from less than 10^-6 to 10^-2. We find consistent low signal, less than 10^-4, in both bone marrow and blood in patients with good prognosis. In contrast, we find high signal in remission, greater than 10^-3, in patients with poor prognosis. In the analysis of serial samples, frequencies drop over time with additional treatment and increase preceding clinical identification of relapse. Figure 1A shows the trajectory of 50 variants over 5 time points in PBMCs, preceding relapse. The overall signal increases at 7 months prior to clinical relapse and again at 5 months prior to clinical relapse. Figure 1B shows a comparison of the aggregate estimates of the leukemia genomic fingerprint in the cellular and cell-free components of the blood. While total DNA yields from the cell-free fraction of blood are lower than from PBMCs, we find that the frequency of the leukemia genomic fingerprint in the two sources of DNA is highly similar. Bone marrow and peripheral blood also show highly similar measurements of residual disease, suggesting that non-invasive monitoring with only a blood draw is a promising approach. Signal of the leukemia fingerprint is also detectable in urine, but at reduced levels and much more variable across patients. Conclusion: Our study shows the capability and utility of a personalized genomic residual disease detection method using MASQ to monitor patients and predict patient outcome. The high sensitivity and accurate quantitation of MASQ are demonstrated in a case study where relapse could be detected molecularly 7 months prior to clinical identification. Collection and analysis of a larger case series is ongoing.

Fluorescence lifetime imaging microscopy for early diagnosis and severity prediction of preeclampsia with Nile Blue probe

Preeclampsia (PE) is a common pregnancy complication and the leading cause of maternal and perinatal mortality. Unfortunately, the early diagnostic methods for PE are still rare. Fluorescence lifetime imaging (FLIM) technology has proven to be applicable for diagnosis of various diseases. Here, we explore the possibility of the FLIM technique for PE early diagnosis and severity prediction with Nile Blue probe as biosensor. 23 placental slices and 162 third-trimester-collected maternal peripheral blood serum samples were stained with Nile blue and imaged by FLIM system. Fluorescence lifetimes of the probe increased significantly as the disease worsened (p < 0.0001). Characterization of the probe showed an increasing tendency in lifetimes under lower polarity conditions and revealed that the reason for the lifetime differences in serum sample was polarity changes caused by abnormal lipid metabolism in serum. For early diagnosis, we investigated 42 12th-week-collected chronic hypertension (CH) serum samples and successfully distinguished PE patients from pregnant women. With the functions of measuring fluorescence lifetime and detecting polarity changes caused by an abnormal lipid microenvironment in maternal peripheral blood, FLIM technology, together with Nile Blue probe, presents a feasible and advantageous approach for PE early noninvasive diagnosis and severity prediction.

SABRSeq: A Randomized Phase Ib Trial of SABR Sequencing with Pembrolizumab in Metastatic Non-Small Cell Lung Cancer (NSCLC)

The optimal sequencing of stereotactic ablative body radiotherapy (SABR) with anti-PD1 checkpoint blockade is unknown. The purpose of SABRseq was to assess the toxicity of the combination of SABR delivered either before or after the commencement of pembrolizumab. The central hypothesis is that the treatment combination will have a safety profile that is clinically acceptable and demonstrate anti-tumor efficacy. Furthermore, we hypothesize that differences in systemic immune activation will be observed between treatment arms. This was a single institutional phase Ib randomized clinical trial (Trial ID NCT03307759). Eligible patients had metastatic NSCLC, ECOG performance 0-1, and had not received prior checkpoint immunotherapy, high-dose radiation (>36 Gy) within 6 months, or either systemic or radiation therapy within 4 weeks of randomization. Eligible patients had either TPS≥50% PD-L1 expression in the first-line or TPS≥1% PD-L1 expression with prior chemotherapy exposure. Patients were randomized to SABR before the commencement of pembrolizumab [ARM1] or SABR commencing after pembrolizumab [ARM2]. SABR was delivered in a single fraction of 18-20 Gy to 1-3 lesions. The primary endpoint was treatment-related adverse events (AE's) related to SABR and/or pembrolizumab. Secondary endpoints included best overall response (BOR), overall survival (OS), and progression-free survival (PFS). Translational objectives included the evaluation of longitudinal changes in immunological cellular subsets within peripheral blood to explore changes in systemic immunity and circulating tumor DNA (ctDNA) dynamics. Between December 2017 and December 2019, 13 patients were randomized. The median follow-up was 37 months. The study was closed early due to poor accrual. Median age was 66 years, with 11 patients (84%) having adenocarcinoma. Nine (69%) were enrolled in the first-line setting. The median [range] number of lesions was 6 [3-11]. The median [range] cycles of pembrolizumab delivered in ARM 1 was 13 [12-32] and ARM 2 was 9 [3-34]. Grade 3 treatment-related AEs were experienced in 0 of 5 patients in ARM1 and in 1 of 8 patients in ARM 2 (hyperglycemia). There were no grade 4 or 5 adverse events reported. The BOR by both RECIST and iRECIST criteria was CR in one patient, PR in seven patients, SD in five patients. Median (95% Cis) PFS was 12.4 months (6.3-21.0), and median (95% Cis) OS was 47.1 months (12.6-not reached; 2-year point estimate 62% [31-82]). Mass cytometry was used on serial peripheral blood samples to examine changes in the frequency of immune cells, changes in T cell activation, differentiation and functional polarization state. Targeted sequencing was performed to assess ctDNA. Translational outcomes will be presented. There was no evidence of a concerning safety signal from either SABR before or after start of pembrolizumab. The combination demonstrated activity with promising PFS and OS and is worthy of evaluation in larger randomized trials.

HGG-28. INTERIM ANALYSIS OF THE GCC1949 PHASE 2 TRIAL USING INDOXIMOD-BASED CHEMO-IMMUNOTHERAPY FOR PATIENTS WITH PEDIATRIC BRAIN TUMORS

Abstract Brain tumors are the leading cause of cancer-related death in children. We report interim results of an ongoing multi-institutional phase 2 trial (NCT04049669) of the IDO pathway-inhibitor indoximod utilized in a chemo-immunotherapy regimen for patients 3-21 years of age with either recurrent malignant brain cancer or newly-diagnosed diffuse intrinsic pontine glioma (DIPG). Patients are treated with oral indoximod (38.4 mg/kg/day, divided BID) plus oral temozolomide (200 mg/m2/day for 5 days) in 28-day cycles. Patients for whom radiation therapy is planned as standard-of-care receive indoximod during the up-front radiation regimen. During treatment, palliative radiation, surgery, or dexamethasone are allowed as needed for clinical management. To date, 53 patients have been treated. Estimated median follow-up time was 23 months (range 0.2-35.5 months), and indoximod-based therapy was well tolerated. Estimated median overall survival (OS) was 15 months for the entire mixed population (n= 53, 95%-C.I. 11.5-23.2 months); 23.8 months for recurrent ependymoma (n=26); 13.5 months for recurrent medulloblastoma (n=12); 5.8 months for recurrent glioblastoma/diffuse midline glioma (GBM/DMG, n=9, excludes DIPG); and 9.6 months for newly-diagnosed DIPG (n=6). For the 39 response-evaluable patients to date (those with measurable disease and at least one follow-up MRI on-therapy), objective response in any single lesion by pediatric RANO criteria occurred in 20/39 patients (9/17 ependymoma, 9/11 medulloblastoma, 0/6 GBM/DMG, 2/5 DIPG). Lesion response was associated with significantly better overall survival (estimated median OS 23.2 months, n=20) compared to patients without lesion response (median OS 6.6 months, n=19) (p=0.0005). Single-cell RNA sequencing of serial peripheral blood samples allowed identification of expanded T cell clones, which was correlated with survival. Updated results from the first 53 patients will be presented. These data are replicating results of the preceding phase 1 study (NCT02502708) and provide preliminary evidence of anti-tumor activity in pediatric brain tumors.

Effect of Sodium Hypochlorite 0.05% on MMP-9 Extracellular Release in Chronic Wounds.

In chronic wounds, high concentrations of matrix metalloproteinases (MMPs) can cause excessive proteolysis and slow wound healing. Consequently, restoring a proper MMP balance can help reduce the risk of a chronic wound. An antiseptic solution containing 0.05% sodium hypochlorite (Amukine Med 0.05%, Angelini S.p.A.; hereafter termed NaClO solution) is available on the market. The NaClO solution was proven effective and safe in managing infected skin wounds. To further characterize its activity, this study evaluated the in vitro activity of the NaClO solution on the monocyte release of MMPs. Human monocytic THP-1 (ATCC® TIB-202™) cell lines were differentiated into macrophages and treated with different concentrations of NaClO (from 0.05% to 5 × 10-7%). In addition, the THP-1 cell line was stimulated with wound fluid (WF) from patients with active venous leg ulcers in the inflammatory phase. The effect of NaClO (0.025-0.0062%) was also evaluated on healthy human peripheral blood serum samples. The effects of treatments on the gelatinolytic activity of MMP-9 were evaluated by gelatin zymography. The effects on MMPs release were evaluated through the Pro™ Human MMP 9-plex Assay. An exploratory scratch wound healing assay was also performed. The NaClO solution reduced the gelatinolytic activity of MMP-9 and its activated form. The downregulation of MMP-9 gelatinolytic activity was also observed in peripheral blood serum. The MMPs profile showed a reduction in MMP-1 release (p < 0.05) and a slight reduction of the release of MMP-9 and MMP-12 after the treatment with LPS and the NaClO solution. A slight improvement in wound healing was observed after macrophage activation and treatment with the NaClO solution. The results obtained suggest a possible ability of the NaClO solution to modulate the proteolytic pathways in the wound microenvironment, further characterizing its activity and use in clinical practice during wound care.

Abstract 2268: Single cell RNA sequencing defines dynamic immune cell subsets in serial colon and peripheral blood samples in a patient with checkpoint inhibitor-induced colitis

Abstract Background: Immune checkpoint inhibition is an increasingly popular strategy for the management of numerous malignancies. Unfortunately, severe inflammatory toxicities affect up to 60% of patients, often leading to treatment discontinuation in settings where few alternative therapies are available to control disease. Checkpoint inhibitor induced colitis is a common toxicity for which the underlying mechanisms are poorly defined. Methods: To better understand the changing colon-specific and peripheral immune environments over the course of disease progression and treatment, we collected blood and colon tissue from a Merkel Cell carcinoma patient who developed colitis after treatment with pembrolizumab. We performed 10X Genomics single cell RNA sequencing on samples collected before, during and after checkpoint inhibition and various interventions to mitigate toxicity. Results: We identified both shared and colon-exclusive clonal expansion of T cells defined by distinct transcriptional programs, including cytotoxicity, memory, and proliferation markers, at various stages of the disease process. These clones decreased dramatically in the colon after infliximab and vedolizumab therapies. Conclusions: We identified several distinct T cell subsets as potential drivers of colitis in the colon. T cell clones that potentially recognize colonic tissue with a high degree of specificity can be harnessed for therapeutic intervention. Our findings emphasize the need for a more detailed understanding of the immune environment in the colon and suggest the potential for peripheral blood as a readout for immune activity during CPI treatment. Citation Format: Jacqueline E. Mann, Liliana Lucca, Matthew Austin, Ross D. Merkin, Lilach Aizenbud, Kevan Herold, Marie Robert, Harriet M. Kluger. Single cell RNA sequencing defines dynamic immune cell subsets in serial colon and peripheral blood samples in a patient with checkpoint inhibitor-induced colitis [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 2268.

A phase II study of hypofractionated radiation therapy to augment immune response in patients with metastatic gastrointestinal malignancies progressing on immune therapy (ARM-GI).

TPS817 Background: Metastatic disease remains a consistent challenge for patients with gastrointestinal (GI) malignancies. Multiple immune checkpoint inhibitors (ICI) have been FDA approved for several GI indications. Still, patients will progress on these therapies, either immediately or after initial response. Palliative radiation therapy (RT) is an effective treatment that can be delivered to symptomatic metastases. It also has been reported to lead to an abscopal effect, which is an induction of response in a distant tumor. This is thought to result from increased tumor immunogenicity, tumor microenvironment modulation, and immune cell recruitment. Thus, palliative RT could not only relieve symptoms, but also potentially reinvigorate the immune response, prolong ICI efficacy, and delay next-line systemic therapy that carries risks of unknown efficacy and tolerability. Ongoing studies seek to elucidate prognostic and predictive biomarkers of the abscopal effect. However, cohorts of patients with GI malignancies are limited, so there remains a need for research in combining RT and immunotherapy in the context of metastatic GI cancer. ARM-GI aims to study the radiation-augmented immune response in patients with metastatic GI cancers progressing on immunotherapy. Methods: ARM-GI is a phase 2, single arm study. Key eligibility criteria include confirmed metastatic GI malignancy, progressive disease per RECIST v1.1 on any ICI, and at least 2 metastases, one of which can be safely left unirradiated. The target prescription dose is 30 Gy in 5 fractions to symptomatic sites through intensity modulated RT or stereotactic body RT. Patients will continue the same immunotherapy after RT and be evaluated per RECIST v1.1 longitudinally. The primary endpoint is overall response rate (ORR) per RECIST v1.1. Secondary endpoints include ORR per iRECIST, progression free survival, overall survival, local control in radiated lesion(s), effect of distant radiation on unirradiated target lesions, incidence of new metastatic lesions, safety by CTCAE v5.0, and time to new systemic therapy. The study will enroll 28 patients, with recruitment ongoing. Serial peripheral blood samples are collected for exploratory studies that will analyze for association with disease response and quantification of changes in circulating immune cell subsets after RT compared to baseline. Clinical trial information: NCT04221893 .

Longitudinal analysis of mucosa-associated invariant T cells in sepsis reveals their early numerical decline with prognostic implications and a progressive loss of antimicrobial functions.

Sepsis-elicited immunosuppression elevates the risk of secondary infections. We used a clinically relevant mouse model and serial peripheral blood samples from patients to assess the antimicrobial activities of mucosa-associated invariant T (MAIT) cells in sepsis. Hepatic and splenic MAIT cells from B6-MAITCAST mice displayed increased CD69 expression and a robust interferon-γ (IFNγ) production capacity shortly after sublethal cecal ligation and puncture, but not at a late timepoint. Peripheral blood MAIT cell frequencies were reduced in septic patients at the time of intensive care unit (ICU) admission, and more dramatically so among nonsurvivors, suggesting the predictive usefulness of early MAIT cell enumeration. In addition, at ICU admission, MAIT cells from sepsis survivors launched stronger IFNγ responses to several bacterial species compared with those from patients who subsequently died of sepsis. Of note, while low human leukocyte antigen (HLA)-DR+ monocyte frequencies, widely regarded as a surrogate indicator of sepsis-induced immunosuppression, were gradually corrected, the numerical insufficiency of MAIT cells was not resolved over time, and their CD69 expression continued to decline. MAIT cell responses to bacterial pathogens, a major histocompatibility complex-related protein 1 (MR1) ligand, and interleukin (IL)-12 and IL-18 were also progressively lost during sepsis and did not recover by the time of ICU/hospital discharge. We propose that MAIT cell dysfunctions contribute to post-sepsis immunosuppression.

Durvalumab (MEDI 4736) with extended neoadjuvant regimens in rectal cancer: A randomised phase II trial (PRIME-RT).

TPS282 Background: Advances in multi-modality treatment of locally advanced rectal cancer (LARC) have resulted in low local recurrence rates, but many patients still die from distant disease. There is increasing recognition that with neoadjuvant treatment some patients achieve a complete response and may avoid surgical resection. PRIME-RT tests the inclusion of neoadjuvant immunotherapy with the aim of enhancing complete response rates, improving stoma-free survival and reducing distant relapse. Methods: PRIME-RT is a multi-centre, open label, phase II, randomised trial for patients with newly diagnosed LARC. Eligible patients are randomised to Arm A: short course radiotherapy (25 Gray in 5 fractions) with concomitant durvalumab, followed by durvalumab and FOLFOX chemotherapy, or Arm B: long course chemoradiotherapy (50 Gray to primary tumour, 45 Gray to elective nodes, in 25 fractions with capecitabine) with concomitant durvalumab followed by FOLFOX and durvalumab. The primary endpoint is complete response rate in each arm. Bio-specimens including serial tumour biopsies and peripheral blood samples are collected prior to, during, and following treatment to explore the molecular and immunological factors underpinning treatment response. The main trial will recruit up to 42 patients and commence after a safety run-in which is recruiting patients with metastatic disease. After opening in January 2021, the four patients completed the safety run in and the main trial commenced in March 2022. The trial is currently open across 5 UK sites. Up to date recruitment details will be provided at the time of presentation but at the time of writing this is ahead of target. Early recruitment to PRIME-RT has shown that adding immunotherapy in the neoadjuvant setting for LARC is feasible. Furthermore, on treatment biospecimen collection is also feasible across multiple sites. The expectation is that the trial will provide efficacy and safety information which allows the optimal treatment approach to be tested within a larger phase clinical trial. Core funding (Glasgow CRUK CTU) and trial-specific funding (Astrazeneca). Clinical trial: NCT04621370; ISRCTN18138369. Clinical trial information: ISRCTN18138369 .

Biomarker Development Trial of Satraplatin in Patients with Metastatic Castration-Resistant Prostate Cancer.

In the phase III SPARC trial, satraplatin, an oral platinum analogue, demonstrated anticancer activity in men with metastatic castration-resistant prostate cancer (mCRPC). Repeat biopsies are uncommon in mCRPC, limiting the feasibility of tissue-based biomarkers. This phase II study sought to evaluate the feasibility and utility of blood-based biomarkers to identify platinum-sensitive mCRPC. Patients with mCRPC who had progressed on docetaxel were enrolled at a single center from 2011 to 2013. Subjects received satraplatin 80 mg/m2 by mouth daily on days 1-5 and prednisone 5 mg PO twice daily, on a 35-day cycle. Serial peripheral blood samples were collected for biomarker assessment. Thirteen docetaxel-refractory mCRPC patients were enrolled, with a median age of 69 years (range 54-77 years) and median PSA of 71.7 ng/mL (range 0.04-3057). Four of 13 patients (31%) responded to satraplatin (defined as a PSA decline of ≥30%). Responders demonstrated improved time to disease progression (206 vs. 35 days, HR 0.26, 95% CI, 0.02-0.24, P = .003). A 6-gene peripheral blood RNA signature and serum tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were assessed as biomarkers, but neither was significantly associated with response to satraplatin. In this small series, one-third of mCRPC patients responded to platinum-based chemotherapy. Peripheral blood biomarker measurement is feasible in mCRPC, though the biomarkers we investigated were not associated with platinum response. Other biomarkers, such as DNA damage repair mutations, should be evaluated.

Diversity of Circulating NKT Cells in Defense against Carbapenem-Resistant Klebsiella Pneumoniae Infection.

Nosocomial infection caused by carbapenem-resistant Klebsiella pneumonia (CRKP) infection has become a global public health problem. Human NK and NKT cells in peripheral immune responses are recognized as occupying a critical role in anti-bacterial immunity. Through performed scRNA-seq on serial peripheral blood samples from 3 patients with CRKP undergoing colonization, infection, and recovery conditions, we were able to described the immune responses of NK and NKT cells during CRKP infection and identified a mechanism that could contribute to CRKP clearance. The central player of CRKP infection process appears to be the NKT subset and CD56hiNKT subset which maintained immune competence during CRKP colonization. With time, CRKP leads to the loss of NK and CD160hiNKT cells in peripheral blood, resulting in suppressed immune responses and increased susceptibility to opportunistic infection. In summary, our study identified a possible mechanism for the CRKP invasion and to decipher the clues behind the host immune response that influences CRKP infection pathogenesis.

Elucidating the Heterogeneity of Serum Metabolism in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia by Raman Spectroscopy.

Myelodysplastic syndrome (MDS) is difficult to diagnose and classify because it has the potential to evolve into acute myeloid leukemia (AML). Raman spectroscopy and orthogonal partial least squares discrimination analysis (OPLS-DA) are used to systematically analyze peripheral blood serum samples from 33 patients with MDS, 25 patients with AML, and 29 control volunteers to gain insight into the heterogeneity of serum metabolism in patients with MDS and AML. AML patients show unique serum spectral data compared to MDS patients with considerably greater peak intensities of collagen (859 and 1345 cm-1) and carbohydrate (920 and 1123 cm-1) compared to MDS patients. Screening and bioinformatics analysis of MDS- and AML-related genes based on the Gene Expression Omnibus (GEO) database shows that 1459 genes are differentially expressed, and the main signaling pathways are related to Th17 cell differentiation, pertussis, and cytokine receptor interaction. Statistical analysis of serological indexes related to glucose and lipid metabolism shows that patients with AML have increased serum triglyceride (TG) levels and decreased total protein levels. This study provides a spectral basis for the relationship between the massive serological data of patients and the typing of MDS and AML and provides important information for the rapid and early identification of MDS and AML.

The role of herpesviruses and pneumocysts in infectious complications in children during liver transplantation

Infections occupy one of the central places among the complications of transplants. The frequency of fatal infectious complications during the first twelve months after transplantation ranges from 2.6 to 51.7%. Identifying markers of opportunistic infections before transplantation will help reduce the likelihood of developing these infections after induced immunosuppression. The aim of the study was to study the role of herpesviruses and pneumocysts in the occurrence of infectious complications in children before and after liver transplantation based on the detection of markers of a number of herpesvirus infections and pneumocystosis. The article presents the results of a comprehensive examination for markers of herpesvirus infections and pneumocystosis of 70 children who were treated at the Shumakov Transplantation Research Center. It should be noted that 55 patients (78.6%) were diagnosed with infectious complications, of which 46 people (65.7%) had pneumonia. To detect IgM and IgG antibodies to herpesviruses and pneumocysts, peripheral blood serum samples were examined by enzyme immunoassay (ELISA). Detection of common HBV antigens was carried out by indirect immunofluorescence reaction (NRIF). Early antigens and reproduction of herpesviruses were detected by rapid culture method (BCM) on Vero and M-19 cell cultures for CMVI. In the event of infectious complications (pneumonia) in children who underwent related liver transplantation, the number of patients with active EBI markers increased almost 7 times and active HCV-6 markers increased 3.5 times. The paper shows the need for a broader laboratory screening of opportunistic infections, which will serve to achieve better clinical results, contribute to the formation of more advanced diagnostic algorithms, as well as improve epidemiological surveillance of these infections.