The subject was a 62-year-old man who presented to the hospital on June 2013 with a one-month history of hematuria. TURBT revealed multiple lesions measuring 0.6 × 0.5 × 0.5 cm, 0.7 × 0.5 × 0.5 cm, and 1.0 × 0.5 × 0.5 cm. Pathological analysis demonstrated high-grade NMIBC, T1N0M0 .The patient received the institutional standard 2-weekly pirarubicin and 6-weekly thiotepa intravesical instillations, and 3 monthly hydroxycamptothecin instillations concurrently with gemcitabine plus cisplatin intravenous chemotherapy for 5 cycles, each at 21-day intervals. Comparing the PET/CT from August 8, 2020 with that performed on June 8, 2013, we noted metabolic activity of the bladder consistent with early progression of disease. The subject declined radical cystectomy and subsequently enrolled into this study after providing written informed consent.
Study design and procedures
This was an N-of-1 study, performed in accordance with the Declaration of Helsinki after receiving approval from the Institutional Review Board of Beijing Shijitan Hospital. The DC-CIK cell product was prepared as described in our previous studies [10, 14]. For the induction of DC-CIKs, peripheral blood mononuclear cells were mobilized by G-CSF. Apheresis was performed using the COBE Spectra cell separator (COBE BCT, Lakewood, CO, USA) until CD34+ cells reached ≥ 4.5 × 106/kg. A portion of the apheresis product (25–50 ml) was co-cultured with IL-4, TNF-α and GM-CSF in vitro to generate autologous DCs. PBMCs were activated in vitro with IL-2, INF-gamma and anti-CD3 antibody to generate the CIK cells. After meeting lot release criteria, the cultured cells were infused intravenously over 20 minutes.
Beginning in the first month post-TURBT, the ex vivo expanded DC-CIK cells were administered intravenously (median infusion of 1.95 × 109 CIK cells, consisting of ~ 75% CD8+ T cells) for a total of 18 infusions over a 63 month period. Blood samples were collected for TCR repertoire and T cell phenotypic analysis and ctDNA mutational analysis during this time period. Peripheral blood CD8+/CD28− T cell levels were used as predictors for determining the timing of adoptive T cell infusion. We chose 24.2% of CD8+/CD28− as the threshold to determine the timing of DC-CIK infusions.
ctDNA mutational analysis
Next generation sequencing was performed on peripheral blood ctDNA by a commercial vendor (Geneplus-Beijing Institute, Beijing). Targeted sequencing was performed in 60 plasma ctDNA and 30 germ line DNA samples. The target region is about 1.1 Mb, which includes coding exons and selected introns of 1021 genes selected from four sources: 1) known oncogenes and tumor suppressor genes; 2) genes that are targets of agents approved by the FDA or have been assessed in clinical trials; 3) genes implicated in major cancer-related signaling pathways; and, 4) genes identified in the findings of the TCGA network which covers 12 cancer types. Sequencing libraries were prepared from ctDNA using KAPA DNA Library Preparation Kits (Kapa Biosystems, Inc.), and gDNA sequencing libraries were prepared using the protocols recommended by the Illumina TruSeq DNA Library Preparation Kit. For samples close to the minimum input requirement, additional pre-capture PCR cycles were performed to generate sufficient PCR product for hybridization. Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Integrated DNA Technology, Coralville, USA) covering the target region sequence. DNA sequencing was carried out with the HiSeq3000 Sequencing System (Illumina, San Diego, CA).
Somatic SNVs and InDels were detected using the Mutect 2.0 algorithm (https://software.broadinstitute.org/gatk/gatkdocs/current/org_broadinstitute_gatk_tools_walkers_cancer_m2_MuTect2.php) Somatic copy number alterations and structure variations were analyzed using local algorithms.
T cell receptor (TCR) sequencing of peripheral blood T cells
DNA was extracted from peripheral blood T cells using a Qiagen DNA FFPE kit, DNA blood kit, or DNA blood mini kit (Qiagen). TCR Vβ CDR3 sequencing was performed using the deep (Peripheral blood mononuclear cell, PBMC) resolution Immunoseq platforms. Bio-informatic and bio-statistical analyses of productive clones were performed to assess the dynamics of T cells. The Shannon diversity index , TCR clonality and Evenness  of T cells were used to evaluate the diversity of TCR CDR3 V-beta sequences.
Phenotypic analysis of peripheral blood immune cells
Whole blood (100 µl) was incubated in the dark with primary antibody at 4 °C for 15 min. Anti-CD3-FITC/anti-CD56-RPE (Dako), anti-CD3-FITC (fluorescein isothiocyanate), anti-CD4-RPE, anti-CD8-RPE, anti-CD45RO, and anti-CD4-FITC/anti-CD25-PE (BD Bioscience) were used. After hemolysis for 10 minutes, samples were centrifuged for 10 min at 1,500 rpm at room temperature, and then washed twice in PBS and subjected to three-color flow cytometry to determine cell phenotypes using an FC500 (Beckman-Coulter), and CXP analysis software (Beckman-Coulter). Lymphocytes were identified on scattergrams as the low forward scatter/low side scatter population. Analysis was performed on ≥ 5,000 gated events.
At the time of each cycle of cell therapy, standard physical exam, pelvic CT and cystoscopy were performed to assess the status of the tumor.
Continuous variables were expressed as mean ± SD (standard deviation) and compared using a two-tailed unpaired Student’s t test. Categorical variables were compared using χ2 or Fisher analysis. All statistical evaluations were carried out using SPSS software (Statistical Package for the Social Science, version 15.0, SPSS Inc, Chicago, IL) and GraphPad Prism 5 (Version 5.01, GraphPad Software, Inc., USA). A value of p < 0.05 was considered to be statistically significant in all the analyses.