Serial Peripheral Blood Mononuclear Cell Research Articles

A phase Ib/II study of pacritinib, an interleukin 1 receptor associated kinase 1 (IRAK1) inhibitor, in patients (pts) with solid tumors harboring the 1q21.3 copy number amplification (CNA).

43 Background: The role of IRAK1 pathway in inflammatory and immune response is established in autoimmune diseases but less well-understood in cancer. Chromosome 1q21.3 CNA detected in plasma cell-free DNA was observed in pts with advanced solid tumors, and associated with IRAK1 upregulation. In preclinical animal models, IRAK1 upregulation promoted tumorigenesis, while its inhibition led to decreased tumor growth. Pacritinib is a JAK2/FLT3 inhibitor that is FDA approved for treatment in myelofibrosis, but also have known activity against IRAK1. We hypothesize that pacritinib may be effective in disease control of pts with plasma 1q21.3 CNA. Methods: A phase Ib/II clinical trial was designed to investigate the efficacy of pacritinib in pts with treatment (rx) refractory solid tumors harboring 1q21.3 CNA. Serial pt tumor and blood samples were collected and analyzed to elucidate the effect of IRAK1 inhibition on tumor microenvironment and systemic immune modulation. Results: 330 patients were screened, 1q21.3 CNA was detected in 30.1% of pts. Highest incidence of 1q21.3 CNA positivity was detected in breast cancer (39.8%), colorectal cancer (39.7%) and lung cancer (36.1%). 12 pts were commenced on rx over 3 dose levels (DL) (DL1: n=6, DL2: n=3, DL3: n=3). At DL1 (200mg BID), no DLTs were observed, but 2 pts had G3 transaminitis attributed to drug/disease, and 1 pt had intolerable G2 rash; decision was made for dose reduction. At DL2 (200mg OM, 100mg ON), no DLTs were observed. Reescalation to 200mg BID (DL3) was carried out with no DLT observed. Pacritinib at 200mg BD was declared the recommended phase II dose in pts with solid tumors. In the dose expansion cohort, 8 pts (4 colorectal cancer, 3 hepatobiliary [HPB] cancer, 1 lung cancer) have been enrolled thus far. No objective responses were observed. Within pts with HPB tumors, 1 pt with pancreatic cancer had a progression-free survival (PFS) of 25.0 weeks, and 1 pt with cholangiocarcinoma had a PFS of 15.9 weeks. Preliminary analysis of serial tumor biopsy samples suggest an expansion of CD8+ T cell population with pacritinib rx. Serial peripheral blood mononuclear cells analysis showed no significant trend in change in CD8+ T cell population, but a predominance of PD-1+ T cells with rx, and also an increase in CD4+ T cell population. In the myeloid cell population, there appears to be decrease in dendritic cell population, but no significant change in trend of macrophage was observed. Conclusions: Pacritinib at 200mg BID is safe and tolerable in pts with solid tumors. IRAK1 inhibition appears to modulate immune cell populations both in the tumor microenvironment and systemic circulation. Dose expansion is underway, with potential signal of disease control in HPB tumors. Clinical trial information: NCT04520269 .

Alloreactive T cells temporarily increased in the peripheral blood of patients before liver allograft rejection.

T cells are key mediators of alloresponse during liver transplantation (LTx). However, the dynamics of donor-reactive T-cell clones in peripheral blood during a clinical T-cell-mediated rejection (TCMR) episode remain unknown. Here, we collected serial peripheral blood mononuclear cell samples spanning from pre-LTx to 1 year after LTx and available biopsies during the TCMR episodes from 26 rejecting patients, and serial peripheral blood mononuclear cell samples were collected from 96 nonrejectors. Immunophenotypic and repertoire analyses were integrated on T cells from rejectors, and they were longitudinally compared to nonrejected patients. Donor-reactive T-cell clone was identified and tracked by cross-matching with the mappable donor-reactive T-cell receptor repertoire of each donor-recipient pair in 9 rejectors and 5 nonrejectors. Before transplantation, the naive T-cell percentage and T-cell receptor repertoire diversity of rejectors was comparable to that of healthy control, but it was reduced in nonrejectors. After transplantation, the naïve T-cell percentages decreased, and T-cell receptor repertoires were skewed in rejectors; the phenomenon was not observed in nonrejectors. Alloreactive clones increased in proportion in the peripheral blood of rejectors before TCMR for weeks. The increase was accompanied by the naïve T-cell decline and memory T-cell increase and acquired an activated phenotype. Intragraft alloreactive clone tracking in pre-LTx and post-LTx peripheral blood mononuclear cell samples revealed that the pretransplant naïve T cells were significant contributors to the donor-reactive clones, and they temporarily increased in proportion and subsequently reduced in blood at the beginning of TCMR. Together, our findings offer an insight into the dynamic and origin of alloreactive T cells in clinical LTx TCMR cases and may facilitate disease prediction and management.

Comparative single-cell RNA sequencing analysis of immune response to inactivated vaccine and natural SARS-CoV-2 infection.

Uncovering the immune response to an inactivated SARS-CoV-2 vaccine (In-Vac) and natural infection is crucial for comprehending COVID-19 immunology. Here we conducted an integrated analysis of single-cell RNA sequencing (scRNA-seq) data from serial peripheral blood mononuclear cell (PBMC) samples derived from 12 individuals receiving In-Vac compared with those from COVID-19 patients. Our study reveals that In-Vac induces subtle immunological changes in PBMC, including cell proportions and transcriptomes, compared with profound changes for natural infection. In-Vac modestly upregulates IFN-α but downregulates NF-κB pathways, while natural infection triggers hyperactive IFN-α and NF-κB pathways. Both In-Vac and natural infection alter T/B cell receptor repertoires, but COVID-19 has more significant change in preferential VJ gene, indicating a vigorous immune response. Our study reveals distinct patterns of cellular communications, including a selective activation of IL-15RA/IL-15 receptor pathway after In-Vac boost, suggesting its potential role in enhancing In-Vac-induced immunity. Collectively, our study illuminates multifaceted immune responses to In-Vac and natural infection, providing insights for optimizing SARS-CoV-2 vaccine efficacy.

Four-Color ImmunoSpot® Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Specific B Cells-Secreting Immunoglobulins of All Four Classes and Subclasses.

The B lymphocyte response can encompass four immunoglobulin (Ig) classes and four IgG subclasses, each contributing fundamentally different effector functions. Production of the appropriate Ig class/subclass is critical for both successful host defense and avoidance of immunopathology. The assessment of an antigen-specific B cell response, including its magnitude and Ig class/subclass composition, is most often confined to the antibodies present in serum and other biological fluids and neglects monitoring of the memory Bcell (Bmem) compartment capable of mounting a faster and more efficient antibody response following antigen reencounter. Here, we describe how the frequency and Ig class and IgG subclass use of an antigen-specific Bmem repertoire can be determined with relatively little labor and cost, requiring only 8×105 freshly isolated peripheral blood mononuclear cells (PBMC), or if additional cryopreservation and polyclonal stimulation is necessary, 3 × 106 PBMC per antigen. To experimentally validate such cell saving assays, we have documented that frequency measurements of antibody-secreting cells (ASC) yield results indistinguishable from those of enzymatic (ELISPOT) or fluorescent (FluoroSpot) versions of the ImmunoSpot® assay, including when the latter are detected in alternative fluorescent channels. Moreover, we have shown that frequency calculations that are based on linear regression analysis of serial PBMC dilutions using a single well per dilution step are as accurate as those performed using replicate wells. Collectively, our data highlight the capacity of multiplexed Bcell FluoroSpot assays in conjunction with serial dilutions to significantly reduce the PBMC requirement for detailed assessment of antigen-specific B cells. The protocols presented here allow GLP-compliant high-throughput measurements which should help to introduce high-dimensional Bmem characterization into the standard immune monitoring repertoire.

Abstract A003: High-throughput TCR sequencing demonstrates induction of long-lasting HPV16-specific T cell responses in VB10.16 vaccinated advanced cervical cancer patients

Abstract Background: Assessment of vaccine-induced T cell responses is a primary pharmacodynamic readout in cancer vaccine clinical trials. High-throughput sequencing of T cell receptors (TCRs) is emerging as a rapid and scalable method, suitable for late-stage clinical trials, and offers sensitive and accurate quantification of the full T cell repertoire. Here we performed ELISpot and TCR sequencing on serial peripheral blood mononuclear cell (PBMC) samples from a clinical phase 2 trial investigating the therapeutic HPV16 cancer vaccine VB10.16 in combination with atezolizumab in advanced cervical cancer patients (NCT04405349). Methods: T cell responses were assessed by ex vivo IFN-g ELISpot (n=36). Immunosequencing of the TCRB locus was performed in 10 patients with available PBMCs, representing clinical response, stable disease, and progressive disease as best overall response. Immunosequencing of PBMC-derived gDNA was performed on up to five timepoints per patient, ranging from week 10 to 52. Novel and expanded clones from baseline to on-treatment timepoints were determined by a differential abundance framework, using binomial models in pair-wise comparisons, enabling longitudinal tracking of significantly expanded T cell clones. The sequences were matched and annotated to HPV16-specific TCRs present in a proprietary database of confirmed HLA class I HPV16-specific T cell clones; the database was constructed from querying the T cell repertoires of 92 healthy donors with 146 peptides derived from HPV16 E6 and E7 in MIRA assay. Results: An increased T cell response was significantly associated with disease control assessed by RECIST1.1 (n=24 patients with disease control vs n=12 patients with progressive disease, p=0.0113) and patients with a >2-fold increase measured by ex vivo IFN-g ELISpot showed a numerically improved progression-free survival (8 vs 3.7 months median PFS). The longitudinal tracking of TCRs provided a detailed insight into the dynamics of the overall TCR repertoire during treatment. Expansion of both pre-existing and newly expanded T cell clones was observed from week 10 and persisted until the end of treatment. Despite the non-exhaustive database, at least one verified HPV16-specific CD8 T cell clone was expanded in 8 out of 10 patients, supporting that the clonotypic expansion is caused by VB10.16. In 5 out of 7 patients with disease control, the breadth of HPV16-specific TCRs increased after vaccination, demonstrating induction of clinically relevant T cell responses. Conclusions: We demonstrate induction of strong and long-lasting HPV16-specific T cell responses after treatment with VB10.16 and atezolizumab in advanced cervical cancer patients. Induction of HPV16-specific T cell responses was significantly correlated with clinical efficacy. Immunosequencing and HPV16-specific annotation allowed longitudinal tracking of expanded TCRs and demonstrated a potential clinical relevance of increased repertoire diversity of HPV16-specific CD8 T cells in patients. Citation Format: Kaja C G Berg, Paula Bousquet, Milena Blaga, Thomas Bello, Mohammad Arabpour, Mikkel W Pedersen, Karoline Schjetne. High-throughput TCR sequencing demonstrates induction of long-lasting HPV16-specific T cell responses in VB10.16 vaccinated advanced cervical cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A003.

Kinetics of Neoantigen Specific T Cells in Post-Transplant Relapse

Introduction: In allogeneic stem cell transplantation (alloSCT), donor-derived T cells mediate the graft-versus-leukemia effect (GVL) by recognizing antigens expressed by leukemia cells. These antigens may include either major- or minor-histocompatibility alloantigens, leukemia over-expressed antigens (leukemia-associated antigens), or neoantigens derived from leukemic somatic mutations. In solid tumors, higher somatic mutation burdens are associated with better response to checkpoint inhibitors, which underscores the importance of neoantigen-specific T-cells in cancer immunotherapy. However, mutation burdens are relatively low in acute leukemia, and the role of neoantigens in GVL remains unknown. Here, we screened potential neoantigens and identified neoantigen-reactive T cells in the patients who developed post-transplant relapse. Materials and methods: Leukemic blasts from patients in relapse were FACS-isolated from bone marrow aspirate or peripheral blood samples. Recipient T cells or saliva were isolated from pre-transplant peripheral blood as a germline control, and donor monocyte or CD34-positive cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) at 100X coverage, paired with RNAseq at 40M reads per sample. Somatic mutations were called between recipient blasts and germline controls with mutect and mpileup, followed by annotation with SnpEff. Somatic mutations with variant allele frequency >20% were subjected to pVAC-seq for prediction of neoantigen likely presented on recipient MHC (IC50 < 500nM). 73 SNPs loci associated with publicly known minor histocompatibility antigens (miHAs) were also genotyped to find the miHA disparities between donors and recipients. Serial peripheral blood mononuclear cell samples (PBMCs) were obtained before relapse if available, at the time of relapse, and after treatment for relapse. For HLA-A*02:01-restricted neoantigens, PBMCs were stained with HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65 (control). Purified CD3 cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: Eight patients with relapsed acute leukemia after allo-SCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their transplant donors (4 matched siblings, 1 matched unrelated, 3 haploidentical) were screened for neoantigens. We analyzed samples from 7 subjects with available WES/RNAseq and detected an average of 273 somatic mutations per patient (range 108- 609) on average. Targeted next-generation sequencing was used to detect 2 somatic mutations in one subject. Among these mutations, potential neoantigen candidates were identified in seven out of eight subjects (87.5%), with a median number of 9 candidate neoantigens per subject. In contrast, only 2 subjects (25%) were found with donor:receipient miHAs at tested loci. CD8 T cells derived from UPN8, a responder to DLI, showed dominant CMVpp65-specific T cells at the time of relapse and persistent low frequencyTP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse by HLA-A*02:01 tetramer staining (Figure A). HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these T cell clones during GVHD (Figure B). In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells reactive to neoantigens were detectable by IFN-γ/CD107a production specific to mutant peptides ( SDE2.pK123X and TP53.pR248W respectively). Discussion: Our in-silico analysis suggested the possibility of somatic mutations in acute leukemia to serve as putative neoantigens in alloSCT. We were able to detect neoantigen-specific T cells by tetramer staining and cytokine production assays from a subject who achieved a durable remission after DLI. Although the sample size is very small, we demonstrated the feasibility of studying neoantigen-specific T cells in post-transplant specimens. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with post-relapse outcomes, especially for response to DLI.

Baseline Immune State and T Cell Clonal Kinetics Are Associated with Response to CAR-T Therapy in Large B-Cell Lymphoma

Engineered adoptive cellular therapy with CD19-targeting chimeric antigen receptor T-cells (CART) has revolutionized the treatment landscape for patients with relapsed/refractory (R/R) large B cell lymphoma (LBCL); however, failure rates remain high. Thus, improved molecular understanding of patient and treatment characteristics linked to response are urgently needed to define the basis for achieving sustained remission. Single cell technologies have the power to evaluate heterogenous populations and provide unprecedented ability to track T cell clonal kinetics. Here we focused on patients with R/R LBCL treated with axicabtagene ciloleucel (axi-cel), from whom we collected serial peripheral blood mononuclear cell (PBMC) samples from Day -30 to +28 relative to CAR-T infusion, as well as bag washings from infusion products (IPs). We evaluated 308,357 single cell transcriptomes (10x Genomics) from serial PBMCs and IPs from 29 patients (16 responders [R], 13 non-responders [NR], R defined as complete [CR] or partial response [PR] at 1 year, NR defined as progressive disease [PD] by 1 year), of which 220,653 originated from 19 patients in a previously reported cohort generated from our center (Haradhvala et al., Nat Med, 2022) and 87,704 newly generated from 10 of 50 patients (29 R, 21 NR) from an extension cohort on whom flow cytometry and mass cytometry (CyTOF) were also generated. With this expanded power, we systematically evaluated baseline and immediate post-treatment samples for features associated with durable response. While we observed no quantitative differences in post-infusion circulating immune subsets in R versus NR, scRNA-seq analysis yielded two notable findings at baseline (Day -30). First, the proportion of B cells (mostly naïve B cells) was increased in R (n=12) compared to NR (n=7) (p=0.012). Protein expression by flow cytometry in the full extension cohort (of whom 20 [13 R, 7 NR] had available PBMC data at baseline) revealed a trend towards increased B cells in R (p=0.16), dominated by four exceptional outliers in whom B cells comprised 2-30% of total PBMCs, all of whom were CR. Resegregating these 20 patients to 11 CR vs 9 non-CR confirmed higher frequency of circulating B cells (median CR 0.18% vs non-CR 0.011%, p=0.0057). Patients with B cells had longer time from anti-CD20 therapy (60, 41, 22, and 10 months) whereas patients without B cells had median 1 month since last anti-CD20 therapy. Second, lymphocyte to monocyte ratio [ALC/AMC] by scRNA-seq trended higher in R (n=12) compared to NR (n=7) (median 1.36 vs 1.02, p=0.22). This was confirmed in the larger flow cytometry cohort (median 2.38 vs 0.69, p=0.0013) and supported by both increased percent of total lymphocytes in R (n=13) vs NR (n=7) (median 31% vs 9.4%, p=0.011) as well as decreased percent of monocytes in R vs NR (median 68% vs 91%, p=0.019). As validation, we detected an elevated ALC/AMC upon evaluation of our entire cohort of patients treated with commercial axi-cel at DFCI from 2019-2022 (n=157 R, 123 NR) based on complete blood count and differential on Day -30 (median 1.31 for R vs 0.97 for NR, p=0.0034). Since we detected evidence of immunologic differences at baseline between R and NR, we asked whether differences in IPs contributed to therapy outcome. By scRNA-seq, IP cells demonstrated increased proportion of CD8 T cells in R (62% vs 32%, p=0.024, Fig 1A) with a trend toward higher proportion of a T effector memory (T EM) expression profile (median 45% vs 21%, p=0.079). By scTCR-seq analysis, we observed greater clonal expansion of CD8 T EM cells in R vs NR, whereas non-clonally expanded cells tended towards a T CM profile (median ratio of clonal TEM to clonal TCM 2.61 for R vs 0.71 for NR, p=0.042, Fig 1B). Differential gene expression of clonally expanded vs nonclonally expanded CD8 T cells revealed increased expression of T cell activation genes ( GZMH, IFNG, TNF, KLRD1, B3GAT1, HLA-class II genes) among clonally expanded cells. These data implicate clonal expansion and T cell activation at infusion as key indicators of CAR-T response. Our results suggest that baseline patient immunologic state affects likelihood of response to CART through modulation of the T cell apheresis product composition and promoting a more favorable circulating immune compartment prior to therapy. These features, detectable by highly accessible clinical measures, could be leveraged for improved patient selection and enhanced CAR-T manufacturing.

Abstract 3238: Integrative biomarker analysis of regorafenib plus nivolumab (RegoNivo) in unresectable hepatocellular carcinoma (uHCC): A multicenter phase 2 RENOBATE trial

Abstract RegoNivo combination has shown promising anti-cancer activity in multiple cancer types. REBNOBATE is a single-arm multicenter phase 2 trial of first-line RegoNivo in patients (pts) with uHCC (NCT04310709). In this study, we present the final clinical outcomes of RENOBATE and integrative biomarker analysis. Pts with >19 years, ECOG PS 0 or 1, BCLC stage B or C, and no prior systemic therapy were eligible. Pts received Nivo 480 mg iv, every 4 wks, and Rego 80 mg daily po, 3 wks on/1 wk off, every 4 wks. Tumor response was assessed according to RECIST v1.1 every 8 wks. ctDNA analysis was performed using the Guardant 360 CDx platform (n=42). scRNA-seq was performed using peripheral blood mononuclear cells (PBMCs) at baseline (e.g., C1D1, n=28) and C1D15 (n=19) from early progression (EP) group (pts with PD or tumor burden increases towards PD on the first evaluation) and long-term response (LR) group (pts with PR or SD > 6 months). A total of 42 pts was enrolled. Response rates were 31.0% and median progression-free survival (PFS) was 7.4 mo (95% CI, 4.2-13.0) and 1-year OS rate was 80.5% (95% CI, 63.0-90.3%). Aberrations of Wnt/β-Catenin and PI3K/mTOR pathways identified in ctDNA were not associated with efficacy outcomes. scRNA-seq revealed that genes upregulated in immune cells on C1D15 (vs. baseline) were associated with immune activation and regulation of angiogenesis. Unsupervised clustering identified 13 immune subets; among them, classical monocyte showed most prominent changes in their proportion and gene expression profiles. After RegoNivo treatment, CD8 T cell clusters had enhanced expressions of genes related to cytoxicity, tissue homing (i.e., CXCR6, CX3CR1, and CXCR3) and proliferation (MKI67), and monophaghocyte clusters showed an enrichment of the gene signatures representing polarization towards M1-related features. Importantly, enrichment of cytotoxic features of the T cell clusters and M1-polarizing features of monophagocyte clusters were more pronounced in LR group than in EP group. From the analysis of differentially expressed genes monophagocyte clusters between the EP and LR groups, a surface molecule ‘X’ was identified as a potential biomarker involved in resistance to RegoNivo. RegoNivo showed promising efficacy outcomes in pts with uHCC. Through scRNA-seq of serial PBMCs, we identified enhanced cellular immune responses induced by RegoNivo and found a potential predictive marker for immunotherapy in pts with uHCC. Our study highlighted that Rego, a multikinase inhibitor, might synergize with anti-PD-1 in pts with uHCC. Association between differential features of T cell and monophagocyte clusters induced by this combination and distinct clinical outcomes highlights the importance of immune-modulation in HCC pts and suggests a potential to further harness these signals. Citation Format: Hyung-Don Kim, Seyoung Jung, Baek-Yeol Ryoo, Min-Hee Ryu, Beodeul Kang, Hong Jae Chon, Jung Yong Hong, Ho Yeong Lim, Jeong Seok Lee, June-Young Koh, Changhoon Yoo. Integrative biomarker analysis of regorafenib plus nivolumab (RegoNivo) in unresectable hepatocellular carcinoma (uHCC): A multicenter phase 2 RENOBATE trial [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 3238.

Abstract 1280: Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) are becoming the standard of care for ovarian cancer. The greatest benefit has been observed in patients who have mutations in BRCA1/2 or related genes in the homologous recombination (HR) DNA repair pathway. Nonetheless, responses to PARPi were not durable, indicating that strategies to prevent recurrence and overcome resistance, such as combinations with immune checkpoint inhibitors (ICIs), are of high priority. However, most studies on PARPi have focused on the tumor itself and synthetic lethality. The immunological effect, particularly the effect on adaptive immune response, has been overlooked. PARP is expressed in almost all cells, and it regulates the transcription of various genes through poly(ADP-ribosyl)ation. Therefore, we hypothesized that PARPi affect the composition and functions of T cells in the maintenance therapy setting, and consequently reconstitute the anti-tumor immune response in ovarian cancer. Here, we collected serial peripheral blood mononuclear cells (PBMCs) from PARPi-treated patients and analyzed their dynamic immunologic changes using muticolor flow cytometry. The PBMCs were collected during PARPi maintenance therapy, and the time points included pre-treatment as well as 1 month and 3 months after the initiation of treatment. Olaparib or niraparib was used as maintenance therapy. First, the percentages of CD4, CD8, and FoxP3+regulatory T cells (Treg cells) were compared at each time point and by PARPi type. Interestingly, the proportion of Treg cells increased at 1 and 3 months after initiation, especially in the niraparib group. However, there were no significant changes in the proportion of CD4 and CD8 T cells. Second, we analyzed immune checkpoints and properties in each T cell population. We found that the PD-1 expression on Treg cells decreased after 1 and 3 months. Although the percentage of Treg cells increased, proliferative Ki-67+Treg cells decreased after 1 and 3 months, especially in the niraparib group. Third, intracellular staining assay was performed to measure functional changes of T cells during PARPi treatment. IFN-γ and/or TNF-α producing CD8 and CD4 T cells decreased after 1 month compared to pre-treatment. Finally, proliferative Ki-67+CD8 T cells and PD-1+CD8 T cells decreased after 72 hours of in vitro olaparib treatment compared to the control group. In conclusion, long-term PAPRi treatment may affect Treg cells and CD8 T cells. However, strategies to overcome negative effects of PARPi and their detailed mechanisms need to be further investigated. Additional data in this regard will be presented at the meeting. Citation Format: Junsik Park, Miran Lee, Yup Kim, Yoo-Na Kim, Yong-Jae Lee, Jung-Yun Lee. Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1280.

Impaired Lymphocyte Responses in Pediatric Sepsis Vary by Pathogen Type and are Associated with Features of Immunometabolic Dysregulation.

Sepsis is the leading cause of death in hospitalized children worldwide. Despite its hypothesized immune-mediated mechanism, targeted immunotherapy for sepsis is not available for clinical use. To determine the association between longitudinal cytometric, proteomic, bioenergetic, and metabolomic markers of immunometabolic dysregulation and pathogen type in pediatric sepsis. Serial peripheral blood mononuclear cell (PBMC) samples were obtained from 14 sepsis patients (34 total samples) and 7 control patients for this observational study. Flow cytometry was used to define immunophenotype, including T cell subset frequency and activation state, and assess intracellular cytokine production. Global immune dysfunction was assessed by tumor necrosis factor-α (TNF-α) production capacity and monocyte human leukocyte antigen DR (HLA-DR) expression. Mitochondrial function was assessed by bulk respirometry. Plasma cytokine levels were determined via Luminex assay. Metabolites were measured by liquid chromatography-mass spectrometry. Results were compared by timepoint and pathogen type. Sepsis patients were older (15.9 years vs. 10.4 years, P = 0.02) and had higher illness severity by PRISM-III (12.0 vs. 2.0, P < 0.001) compared to controls; demographics were otherwise similar, though control patients were predominately male. Compared to controls, sepsis patients at timepoint 1 demonstrated lower monocyte HLA-DR expression (75% vs. 92%, P = 0.02), loss of peripheral of non-naïve CD4+ T cells (62.4% vs. 77.6%, P = 0.04), and reduced PBMC mitochondrial spare residual capacity (SRC; 4.0 pmol/s/106 cells vs. 8.4 pmol/s/106 cells, P = 0.01). At sepsis onset, immunoparalysis (defined as TNF-α production capacity < 200 pg/mL) was present in 39% of sepsis patients and not identified among controls. Metabolomic findings in sepsis patients were most pronounced at sepsis onset and included elevated uridine and 2-dehydrogluconate and depleted citrulline. Loss of peripheral non-naïve CD4+ T cells was associated with immune dysfunction and reduced cytokine production despite increased T cell activation. CD4+ T cell differentiation and corresponding pro- and anti-inflammatory cytokines varied by pathogen. Pediatric sepsis patients exhibit a complex, dynamic physiologic state characterized by impaired T cell function and immunometabolic dysregulation which varies by pathogen type.

Translational randomized phase II trial of cabozantinib in combination with nivolumab in advanced, recurrent, or metastatic endometrial cancer

BackgroundCombining immunotherapy and antiangiogenic agents is a promising treatment strategy in endometrial cancer. To date, no biomarkers for response have been identified and data on post-immunotherapy progression are lacking. We...

Prognostic Value of Minimal Residual Disease (MRD) Among Patients with Classical Hodgkin Lymphoma Undergoing Autologous Stem Cell Transplantation

Prognostic Value of Minimal Residual Disease (MRD) Among Patients with Classical Hodgkin Lymphoma Undergoing Autologous Stem Cell Transplantation

Pilot Study of Pembrolizumab for Immunoprevention in Smoldering Multiple Myeloma

Background: The current management of patients with smoldering multiple myeloma (SMM) is watchful waiting. However, a fraction of these patients are at significant risk of progression to symptomatic multiple myeloma (MM). In a recent study, expression of PD-L1 on tumor cells correlated with increased risk of progression from SMM to MM (Dhodapkar et al Blood. 2015; 126(22): 2475-2478) suggesting a role for immune checkpoint blockade for immunoprevention of MM. Pembrolizumab is a humanized monoclonal antibody against programmed death receptor-1 (PD-1) and blocks the interaction between PD-1 and PD-L1. Here, we report the results of a pilot study evaluating the safety and efficacy of pembrolizumab in intermediate- and high-risk SMM patients (I-HR-SMM). Methods: Patients with I-HR-SMM by PETHEMA, Mayo or SWOG criteria were eligible. Permbrolizumab was administered at a dose of 200 mg IV every 21 days for up to 8 cycles. Patients were eligible to continue treatment for up to 24 cycles if they achieved ≥ minor response after 8 cycles. The primary objective was the overall response rate (ORR) after 8 cycles. The target ORR was 25%. The trial was conducted by the Simon's Minimax design and the ORR was estimated accordingly (Simon, 1989). Twelve patients were planned for the first stage. If at least one patient had ≥ partial response, 4 more patients could enroll for a total of 16 patients. Serial peripheral blood mononuclear cells and bone marrow samples were collected for comprehensive immunophenotyping using multiparametric flow cytometry, and molecular profiling with gene expression profiling (GEP) and exome sequencing. Results: Twelve patients with I-HR-SMM were dosed on the first stage between 08/2016 and 06/2017 (Table 1a/b). Median age was 67.5 years (range 49-74). Median number of cycles received was 8 (range 3-8) and all 12 were evaluable for safety and efficacy. Median follow up was 8.54 months (4.7 to 9.6 months). One patient achieved a stringent complete remission (sCR) (8%), ten patients had stable disease (83%), and one patient had progressive disease (8%). The patient with sCR had HR-SMM by GEP70 and FISH (deletion 17p and amplification of CKS1B) and 50% plasma cells infiltration in the bone marrow at study entry. The sCR with negative flow cytometry on bone marrow was noted in this patient after only 3 cycles of therapy and response is ongoing 8 months after the end of treatment. Five patients discontinued therapy due to related adverse events: grade 2 elevation in LFT (n=2), grade 3 myalgia (n=1), and grade 3 acute kidney injury (AKI) due to tubulo-interstitial nephritis (n=2) [all resolved with discontinuation of treatment or prednisone administration]. There were no grade 4/5 adverse events. Enrollment on second stage is ongoing. Imunophenotyping of paired baseline and end of treatment bone marrow samples showed that the patient who achieved sCR had high levels of PD-1 expression on CD4+ and CD8+ T cells at baseline and showed evidence of CD8+ T-cell activation after therapy with greater upregulation of CD69, CTLA-4, PD-1, TIM-3, 4-1BB, and ICOS relative to non-responders. In contrast, expression of other immune checkpoint molecules such as TIM-3, LAG-3, and CD244 were greater on T cells in non-responders at baseline relative to the responder suggesting greater exhaustion of T cells in non-responders. Molecular profiling including GEP, exome sequencing, and neoantigens analysis is ongoing and will be presented at the meeting. Conclusions: This pilot study of pembrolizumab monotherapy showed that it is tolerable and can induce sCR in patients with SMM. More importantly, it suggested that immunoprevention to prevent progression of SMM to MM is possible. Additional biomarker studies are needed to identify patients that are most likely to respond to this approach. Our correlative studies also provide insights into potential future combination approaches to further improve efficacy of immunoprevention strategies in SMM. [Display omitted] DisclosuresManasanch:takeda: Consultancy; celgene: Consultancy; sanofi: Research Funding; quest diagnostics: Research Funding; merck: Research Funding; adaptive biotechnologies: Consultancy. Lee:Eutropics Pharmaceuticals: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees; Pimera Inc: Consultancy; Celgene: Consultancy; Daiichi Sankyo: Research Funding; Takeda: Consultancy. Patel:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Consultancy; Celgene: Consultancy. Thomas:Celgene: Research Funding; Bristol Myers Squibb: Research Funding. Neelapu:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karus: Research Funding; Cellectis Inc.: Research Funding; Poseida Therapeutics, Inc: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Orlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees.

High Complete Response Rates with Pembrolizumab in Combination with Rituximab in Patients with Relapsed Follicular Lymphoma: Results of an Open-Label, Phase II Study

High Complete Response Rates with Pembrolizumab in Combination with Rituximab in Patients with Relapsed Follicular Lymphoma: Results of an Open-Label, Phase II Study

Abstract 5512: Identification of circulating myeloid cells induced in advanced biliary cancer patients responding to anti-PD1 through combined single cell RNA sequencing and protein expression analysis

Abstract Background: Patients with advanced biliary cancer (ABC) have low rates of response to immune checkpoint inhibition (CPI) monotherapy. We conducted a phase II clinical trial studying combination immunotherapy with GM-CSF and the anti-PD-1 antibody, pembrolizumab (NCT02703714), with preliminary objective response rate of 19%, higher than the reported response rate of 5.8% for pembrolizumab monotherapy in ABC. Mechanisms of response to CPI in ABC are unknown, and to our knowledge, there have not been studies of the immune response in this patient population at the single cell level. Methods: Cryopreserved serial peripheral blood mononuclear cells (PBMC) samples from n=27 ABC patients treated with pembrolizumab plus GM-CSF were assessed with single cell RNA sequencing combined with CITE-Seq, the staining of cells with &amp;gt;90 oligo-tagged antibodies. This allows for the simultaneous identification of protein and RNA expression on an individual cell basis. We used high-throughput multiplexing in which PBMC from different patients could be deconvolved from other mixed samples using individual specific SNPs (“demuxlet”). &amp;gt;100 PBMC samples, including pre-treatment and 1 week, 3 weeks, and 6 weeks post-treatment, were studied. Nine of 27 patients were characterized as responders, while 18 were non-responders. We also performed CITE-Seq on PBMC from age/gender-matched healthy controls (n=9). Results: Unbiased clustering revealed all major immune populations were present in PBMC from both ABC and controls, though the immune compositions differed, most strikingly within the myeloid compartment. A distinct population of CXCL5-expressing “cancer-associated myeloid cells”, distinct from classical and non-classical monocytes and dendritic cells, was found only within cancer patients. We also analyzed the effects of treatment on immune sub-populations within responders and non-responders and found that following anti-PD-1 monotherapy, there was an increased frequency and absolute number of non-classical monocytes in responders. Conversely, the addition of GM-CSF to anti-PD-1 therapy decreased the frequency of circulating non-classical monocytes, though there was a robust induction of pro-inflammatory genes following treatment with GM-CSF in responders. Regardless of responder status, GM-CSF also dramatically induced monocyte-derived dendritic cells within 1 week of administration. Conclusions: The compositions of the myeloid compartment differed between ABC patients and healthy individuals, with a circulating cancer-associated myeloid population identified in ABC patients. Circulating non-classical monocytes increased following anti-PD-1 monotherapy in all patients, but declined after combination with GM-CSF in responders, suggesting that anti-PD-1-induced myeloid populations may be associated with response to anti-PD1. Pre-existing and induced myeloid cell states may represent a mechanism of response to anti-PD-1 and GM-CSF. Citation Format: Bridget P. Keenan, Elizabeth E. McCarthy, Arielle Ilano, David S. Lee, Yang Sun, Brenna Sheldon, Alexander Cheung, Chun Jimmie Ye, Robin Kate Kelley, Lawrence Fong. Identification of circulating myeloid cells induced in advanced biliary cancer patients responding to anti-PD1 through combined single cell RNA sequencing and protein expression analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5512.

An open-label, phase II multicohort study of an oral hypomethylating agent CC-486 and durvalumab in advanced solid tumors

PurposeTo evaluate whether administration of the oral DNA hypomethylating agent CC-486 enhances the poor response rate of immunologically ‘cold’ solid tumors to immune checkpoint inhibitor durvalumab.Experimental designPD-L1/PD-1 inhibitor naïve patients...

Abstract CT190: A Phase II basket study of hypomethylating agent oral cc-486 and durvalumab in advanced solid tumors (METADUR)

Abstract Background Aberrant DNA methylation contributes to cancer initiation and progression. In preclinical models of solid tumors, chronic low dose administration of DNA hypomethylating agents can induce T-cell mediated immune activation responses by stimulating expression of endogenous retroviral elements, culminating in an IFN-mediated response. The addition of physiological levels of vitamin C may potentiate viral mimicry and increase anti-tumor immune priming. Immunologically ‘cold’ tumors were selected to evaluate whether these strategies can enhance their otherwise poor responses to immune checkpoint blockade. Methods PD-L1/PD-1 inhibitor naïve patients (pts) with advanced microsatellite stable colorectal cancer (MSS CRC); platinum resistant ovarian cancer (OC); estrogen receptor positive, HER2 negative breast cancer (ER+HER2- BC) were enrolled in this single institution, multi-cohort, investigator-initiated trial. The initial regimen (regimen A) consisted of oral CC-486 300mg QD Days 1-14 (cycles 1-3 only) in combination with durvalumab 1500mg IV Day 15, in 28 day-cycles. A protocol amendment (regimen B) after 19 patients changed CC-486 to 100 mg QD Days 1-21 (cycles 1-3), added oral vitamin C 500mg QD continuously and kept durvalumab 1500mg IV Day 15, in 28-day cycles. Adverse events (AEs) assessed by CTCAEv4.03 grade (G); tumor response by RECIST 1.1 every 2 cycles; paired tumor biopsies at baseline and cycle 2 days 10-14; and serial peripheral blood mononuclear cells (PBMCs) for immune-profiling (IP) and epigenetic analysis. PD-L1 by IHC was assessed using SP263 assay, positivity defined by TC&amp;gt;25%. Results A total of 28 pts with MSS CRC (n=15), OC (n=4), ER+HER2- BC (n=9) were enrolled, 19 pts treated on regimen A, 9 on regimen B. Median age was 56 (range 36-78), ECOG 0:1=7:21, 100% had ≥3 prior lines of therapy, all tumors were PD-L1 negative. Best response was SD (3/28 pts received 3, 3 and 4 cycles respectively) with DCR 7.1%. Median follow-up of 4.7 months, mPFS was 1.9 months (95% CI 1.5-2.3) and mOS was 5 months (95% CI 4.5-10). Three patients (all regimen A) experienced DLTs (2 G4 neutropenia and 1 G3 anemia). Fifteen patients (54%) experienced G1/2 fatigue, 46% experienced G1/2 nausea, vomiting or diarrhea. Toxicity was comparable with addition of vitamin C. Initial analysis of PBMCs by flow cytometry in 3 of 4 OC pts demonstrated an increase in PD-1 and Ki67 expression in CD8 T cells only following administration of durvalumab. EPIC methylation array on 4 pts’ paired tumors and LINE 1 assay on 19 pts’ serial PBMCs (regimen A) demonstrated minimal change in global methylation. Conclusion No meaningful clinical responses to CC-486 plus durvalumab were observed. Tumor tissue and PBMCs both demonstrate minimal global DNA demethylation, with or without physiological dose vitamin C. Incremental immune activation beyond PD-L1 blockade was not demonstrated. Clinical trial information: NCT02811497 Citation Format: Kirsty Taylor, Helen Loo Yau, Ben X. Wang, Philippe L. Bedard, Albiruni R. Razak, Aaron R. Hansen, Anna Spreafico, Dave Cescon, Marcus O. Butler, Amit M. Oza, Stephanie Lheureux, Neda Stjepanovic, Lisa Wang, Brendan Van As, Sarah Boross-Harmer, Trevor Pugh, Lillian L. Siu, Daniel D. De Carvalho. A Phase II basket study of hypomethylating agent oral cc-486 and durvalumab in advanced solid tumors (METADUR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT190.

HIV DNA levels and decay in a cohort of 111 long-term virally suppressed patients.

We examined specimens from 111 HIV-infected participants virally suppressed on ART for a minimum of 5 years who had donated serial peripheral blood mononuclear cell (PBMC) specimens to the University of Washington/Fred Hutch Center for AIDS Research (CFAR) Specimen Repository. We determined the HIV proviral copy number per million PBMCs, corrected for CD4 cell count, in 477 specimens collected after a minimum of 5 years of follow-up and up to 15.5 years of clinical viral suppression. Generalized estimating equation regression was used to examine the association between the reservoir size and time, age at study entry, antiretroviral regimen, and risk factors for HIV acquisition. We found that the inter-participant baseline HIV DNA level varied widely between 0.01 and 4.8 pol-copies per microgram genomic DNA and per CD4 cell number/micoliter; the HIV DNA level declined with time (half-life was estimated at 12 years, 95% confidence interval of 6.2-240 years); the HIV DNA level was lower for those who achieved viral suppression at a younger age; and the HIV DNA level was not affected by the specific antiretroviral regimen used to achieve and maintain suppression.

Low-Dose Interleukin-2 Therapy Activates Circulating T Follicular Regulatory Cells and Suppresses Circulating T Follicular Helper Cells in Patients with Chronic Gvhd

Introduction: T follicular helper (TFH) and T follicular regulatory (TFR) cells play important roles in the regulation of B-cell immunity. While TFH promote B cell functions in the germinal center (GC), TFR function as negative regulators of the GC response. Previous studies in murine models established that TFH and GC B cells are required for the development of chronic graft-versus-host disease (cGVHD). We previously reported that circulating TFH (cTFH) were more functionally activated in patients with active cGVHD compared with patients with no cGVHD. Low-dose IL-2 therapy has been shown to selectively expand CD4Treg and improve cGVHD symptoms. In the current study, we examined the effects of IL-2 therapy on cTFH and circulating TFR (cTFR) in patients with steroid resistant cGVHD.Methods: Single cell mass cytomtery (CyTOF) was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors and 17 adult patients with active cGVHD receiving daily low-dose IL-2 therapy (Koreth et al. Blood 2016). A panel of 35 metal-tagged monoclonal antibodies was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct lymphocyte subsets and 13 intracellular markers to measure functional status and activation of specific signaling pathways. Before staining for surface and intracellular antigens, serial samples from individual patients were barcoded to ensure uniformity of analysis. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data.Results: In PBMC from healthy donors, expression of CD25 (IL-2Rα), CD95, CTLA-4, BLIMP-1 and GITR was higher in cTFR compared with cTFH. To examine the response to IL-2 in vitro, PBMC from healthy donors were stimulated with IL-2 for 15 minutes (Figure 1A). At low IL-2 concentrations (1 to 10 IU/mL), phospho-STAT5 (p-STAT5) was selectively activated in cTFR compared with cTFH. At high IL-2 concentrations (100 to 1,000 IU/mL), p-STAT5 was activated in both cTFR and cTFH. To examine the response to IL-2 in vivo, we used mass cytometry to examine serial PBMC samples from cGVHD patients receiving daily low dose IL-2 therapy (1x106 IU/M2/day). Selective expansion of cTFR was noted after 1 week of treatment and cTFR expansion remained stable for the 12 week duration of therapy. Expanded cTFR increased expression of p-STAT5, FoxP3, BCL6, HLA-DR (Figure 1B) and CD25, CD95, CTLA-4, ICOS, Ki67 and Helios 1 week after starting IL-2. cTFR:cTFH ratio increased rapidly after starting low dose IL-2 and paralleled the increased Treg:Tcon ratio (Figure 1C). Activated TFH and TFR can be identified by expression of ICOS and PD-1. The expansion of ICOS+PD-1+ cTFR was evident after 1 week of IL-2 and remained elevated at the end of therapy. In contrast, ICOS+PD-1+ cTFH increased 1 week after starting IL-2 therapy but subsequently decreased and fell below baseline 6 and 12 weeks after starting IL-2 (Figure 1D). Activated ICOS+PD-1+ cTFR expressed higher levels of p-STAT5, BCL-6, FoxP3, HLA-DR and CD25 during low dose IL-2 therapy. In contrast, these functional markers were not increased in ICOS+PD-1+ cTFH during IL-2 therapy (Figure 1B).Conclusion: Single cell mass cytometry analysis revealed that daily low dose IL-2 therapy induces selective activation and increased expression of functional proteins in ICOS+PD-1+ cTFR. In contrast, activated ICOS+PD-1+ cTFH were suppressed during IL-2 therapy. The selective activation of cTFR and suppression of cTFH provide a mechanism whereby low dose IL-2 therapy can promote B cell tolerance as well as T cell tolerance in patients with cGVHD. [Display omitted] DisclosuresForcade:Neovii: Other: Travel grant. Koreth:Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Tensha: Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Affimed: Research Funding; Sigma Tau: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding. Cutler:Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

Abstract 549: Clustering analysis of next-generation sequencing T cell repertoire data in sipuleucel-T treated prostate cancer patients

Abstract Immunotherapy has demonstrated significant clinical benefit in different cancers. T cells are a crucial component of the adaptive immune system and mediate anti-tumoral immunity. Antigen-specific recognition by T cells is via T cell receptor (TCR) which is the product of somatic V(D)J gene recombination with the addition/subtraction of nontemplated bases at recombination junctions. Next generation TCR sequencing effectively profiles the TCR repertoire. Currently TCR analyses quantify diversity across single clones, however, due to the low overlap of clones across samples, such analyses are limited to a single sample. Here we extend our previous analysis pipeline to track and examine TCR repertoire across time by focusing on V and J gene segments, overcoming the limitation of contemporary analytical approaches and thus obtain statistical inference across subjects directly. The published data of TCR profiling of serial peripheral blood mononuclear cells (PBMC) from three healthy subjects and five prostate cancer patients enrolled in an open-label, Phase II clinical trial of neoadjuvant sipuleucel-T (sip-T) were used as the basis of this analyses. After consolidating the clonal abundance for each combination of V and J gene segments, we calculated Pielou’s evenness, which demonstrates that sip-T treated subjects displayed greater clonal expansion compared to healthy subjects (P&amp;lt;0.001). The Circos table viewer was used to circularly visualize the distribution of the combination of V and J gene segments. Sip-T treated subjects were successfully distinguished from healthy subjects with 12 V and J gene combinations explaining the majority of variance of the patients by applying principle components analysis (PCA). Furthermore, we developed a customized clustering workflow to cluster the combination of V and J gene segments based on the change in abundance over time, with gap statistics employed to estimate the optimal number of clusters and k-means algorithm used for partitioning. We found that for all the prostate cancer patients assessed, TCRBV06/TCRBJ01, TCRBV05/TCRBJ02 and TCRBV06/TCRBJ02, which were in the same cluster with the highest frequencies, expanded after first treatment and were maintained at high frequencies at later time points. The results demonstrate that in the setting of immune modulation after sip-T treatment, the V(D)J recombination is not entirely random, and that this non-randomness can be used to distinguish T cell repertoires from sip-T treated subjects versus healthy subjects. The use of additional information of V and J gene segments enables to investigate the profiling of TCR repertoire from a different angle and add another layer of understanding of TCR repertoire. The application of PCA and the customized clustering complete our initial workflow for TCR sequencing data. Citation Format: Li Zhang, Sounak Chakraborty, Jason Cham, David Oh, Nadeem Sheikh, Lawrence Fong. Clustering analysis of next-generation sequencing T cell repertoire data in sipuleucel-T treated prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 549. doi:10.1158/1538-7445.AM2017-549