Mononuclear Cell Collection Research Articles

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...

Apheresis machines variably overestimate mononuclear cell collection volume.

Calculation of the actual number of CD34+ cells in the collection product is based on the volume of the collected product and its concentration of CD34+ cells measured in the lab. The number of CD34+ cells infused correlates closely with the pace of hematopoietic reconstitution following autologous or allogeneic stem cell transplantation. We studied peripheral blood stem cell collections in a single apheresis center with two Spectra Optia devices, using mononuclear cell collection or continuous mononuclear cell collection procedures. The collection volume displayed by the apheresis device was compared with the volume determined by a weight-based method. Fifty-two consecutive CD34 collections in 35 different donors (range 1-4 daily procedures per donor) were analyzed. The machines reported larger collection volumes (P < .001). The mean collection volume reported by the machine was 274.37 mL (range 162-396). The mean manually measured collection volume was 261.82 mL (range 155-371.40). Mean overestimation by machine was 12.53 mL (range -0.95 to 31.24; 95% confidence interval 10.94-14.11) or 4.88% (range -0.26 to 10.28). Median overestimation of the absolute number of CD34 was 10.29 × 106 (range -2.83 to 141.84 × 106 ). Both Spectra Optia machines overestimated the collection volume after peripheral blood stem cell collection. Although the mean variation falls within the expected range, in some cases, this overestimation may be clinically relevant if no other method of measurement is used.

Chimeric Antigen Receptor (CAR) T cells on demand: developing potent CAR T cells in less than 24 hr for adoptive immunotherapy

Background & Aim The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic “drugs” for cancer therapy. CAR T therapies generally relies upon viral transduction of T cell-receptor (TCR) activated T cell followed by ex vivo expansion of patient-derived T cells over days to weeks prior to infusion. In addition to the required time and labor, we previously reported that TCR/CD3 activation and ex vivo expansion leads to progressive differentiation of the CAR T cells with associated loss of CAR T cell potency. Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that elimination of the CD3/CD28 activation step would yield a T cell product with high functional potency. Methods, Results & Conclusion Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We further demonstrate that some of the restriction factors that limit the infection of quiescent T cells can be overcome by changes to the medium formulation. Most importantly, we show that non-activated T cells exhibit potent antitumor function with deep and durable control of leukemia achieved in vivo with greater than 1-log fewer CD19-specific CAR T cells compared with a 9-day process currently used by tisagenlecleucel (Fig. 1). These findings confirmed that as few as an estimated 12,000-32,000 non-activated CAR T cells produced within 24 hours from mononuclear cell collection could eradicate leukemia. These results demonstrate the potential for vastly reducing the time, materials and labor required to generate CAR T cells, which could be especially beneficial in patients with rapidly progressive disease and in resource-poor health care environments.

Safety, clinical efficancy, and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: Experience from a private cell collection facility

Safety, clinical efficancy, and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: Experience from a private cell collection facility

A new protocol for improving efficiency of autologous peripheral blood stem cell collection in patients with high white blood cell counts.

Collection efficiency (CE) of peripheral blood stem cell (PBSC) collections is negatively affected by increasing white blood cell (WBC) counts of the patient. This study compared a new optimized mononuclear cell (MNC) collection protocol (OPP) to the standard MNC collection protocol recommended by the manufacturer (STP) for PBSC collection in patients with WBC counts >35 000/μL. Single-center, retrospective, and observational study of 81 autologous PBSC collections on Fenwal Amicus cell separators in 70 adult patients. Median peripheral WBC count (×103 /μL; 44.2 in OPP group vs 46.5 in STP group) and median CD34+ count (105/μL in OPP group vs 40/μL in STP group) at the beginning of PBSC collection did not differ significantly. Median CE2 (45% vs 31%; P < .001) as well as CD34+ yield of the apheresis product both with regards to median absolute CD34+ content (×106 ; 793 vs 188; P = .001) as well as median CD34+ content (×106 )/kg body weight (8.93 vs 2.51; P = .002) were significantly higher for the OPP. Overall, 18/21 (86%) patients with the OPP obtained their target CD34+ amount with a single apheresis session, compared to 25/50 (50%) with the STP (P = .005). PBSC collections using OPP lasted significantly longer (median 377 minutes vs 260 minutes; P < .001) than with the STP. The OPP significantly improves CE2 for PBSC collections on Fenwal Amicus cell separators in patients with pre-apheresis WBC counts >35 000/μL and significantly reduces the necessity for multiple apheresis sessions. The OPP is therefore suited to reduce both patient burden and cost in autologous PBSC collection.

Factors Influencing Mononuclear Cell Collection in Tumor Cell Biotherapy

To investigate the factors affecting counting and collection efficiency of the final product- mononuclear cells (MNCs) in the collection of mononuclear cells for tumor cell biotherapy. The collected data of 142 tumor patients and healthy donors were analyzed, including age, sex, height, weight, BMI, the total blood volume, diagnostic category, vascular access, operator, final product volume, ACD anticoagulant usage, flow rate and circulation times, pre-apheresis Hb, RBC, Plt, WBC, lymphocyte count, monocyte count, neutrophil count, circulating blood volume without anticoagulant, final product MNC and collection efficiency of MNC. CE(collection efficiency)%= final product MNC×100/(pre-apheresis MNC×circulating blood volume without anticoagulant). The factors affecting final products MNC and CE of MNC were detected by T test and multiple linear regression analysis. The CE of tumor patients was higher than that of healthy donors （24.41±1.91,vs 20.01±0.99)，(P=0.043）, and CE of MNC was different among different operators (P=0.01, H=18.59). There was a positive correlation of the final MNC with the volume of final product, ACD anticoagulant usage and pre-apheresis lymphocyte count (P= 0.00, P= 0.01, P= 0.00， r=0.811); CE of MNC negatively correlated with flow rate and pre-apheresis RBC, but positively correlated with operator's working age and ACD anticoagulant usage (P=0.01, P=0.04, P=0.03, P= 0.00, r=0.495). more higher pre-apheresis lymphocyte , more amount of the final product and ACD anticoagulant usage, and more high the final MNC. During the collecting process, more ACD anticoagulant usage and more high operator's seniority, lead to the higher MNC'S CE; while more high pre-apheresis RBC and more fast flow rate, cause the lower the CE of MNC.

Abstract A66: Intracavity infusions of autologous cellular immunotherapy in the treatment of refractory malignant pleural and ascites: A Chinese population-based cohort study

Abstract Objectives: we have explored the potential efficacy and safety of autologous adoptive T cells represented by dendritic cell (DC)-cytokine-induced killer (CIK) infused within cavity in advanced cancer patients with malignant pleural effusion and ascites. Methods: The apheresis for collection of peripheral blood mononuclear cells was performed in 37 recruited patients. DC-CIKs were expanded ex vivo until qualified for intracavity perfusions for three consecutive times. The quantitative volume of malignant effusions was recorded by radiologic imaging assay. The clinical adverse reactions were analyzed. Results: For the quantitative assay for those effusions, 1 patient has achieved complete response, 13 patients have achieved PR, 12 patients have stable diseases, and 11 patients have progressive diseases, respectively, yielding a total clinical control rate of 70.3%. After adjusting for competing risk factors, number of concurrent metastasis distribution of organs (p=0.007) and infused number of CIKs (p=0.013) remained independent factors associated with malignant effusion remission. Further exploration demonstrated that the infused CD3+/CD8+ and CD8+/CD28- T-cell populations were able to determine the clinical responses (P&amp;lt;0.05). Moreover, elevated peripheral blood CD3+/CD8+ and declined CD4+/CD25+ T-cell population were noticed in malignant effusion remission group compared with non-remission group (P &amp;lt;0.05). Fewer adverse reactions were recorded. Conclusions: We have provided the novel therapeutic approach by autologous cellular immunotherapy with confirmed clinical efficacy and safety in the stably controllable malignant effusions. The intracavity infusions were featured by the immunologic T-cell recovery and clinical remission of those effusions. Note:This abstract was not presented at the conference. Citation Format: Guoliang Qiao. Intracavity infusions of autologous cellular immunotherapy in the treatment of refractory malignant pleural and ascites: A Chinese population-based cohort study [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A66.

Phase II trial of a novel immunotherapy combination of pembrolizumab and HER2 bi-armed activated T cells (BATs) in metastatic castrate resistant prostate cancer.

97 Background: Immune checkpoint inhibitors (ICI) have demonstrated only modest efficacy in metastatic castrate resistant prostate cancer (mCRPC). Anti-HER2 and Anti-CD3 bispecific antibody armed activated T cells (HER2 BATs) demonstrated efficacy in prostate cancer cell lines, irrespective of HER2 expression status. Safety and preliminary responses in refractory mCRPC were noted in a phase I trial with HER2 BATs. Preclinical synergy was noted in prostate cancer with ICI and BATs. We conducted a phase II study with pembrolizumab and HER2 BATs that would potentially transform immune cells to exhibit cytotoxicity against prostate cancer. Methods: mCRPC patients with 0-1 performance status (PS) and normal liver, kidney and marrow function, pre or post docetaxel chemotherapy were eligible. Primary endpoint was 6 month progression free survival. Patients underwent apheresis for mononuclear cell collection which was shipped to the University of Virginia for manufacturing of HER2 BATs, which were infused twice weekly for 4 weeks. Pembrolizumab was administered every 21 days for a maximum duration of 6 months starting 1 week prior to HER2 BATs infusion. Progression was determined by clinical evaluation, PSA, and radiologic assessment at 3 and 6 months and every 6 months thereafter. Results: Twelve patients have been enrolled on the study of which one withdrew consent. Median age was 69 years (Range 57 to 76 years). Five of 11 patients had prior docetaxel chemotherapy. Eight of 11 patients had bone and lymph node metastases, 1 had lymph node metastases only, and 2 had peritoneal metastasis. Median PSA was 111ng/ml [Range 5.2 - 378 ng/ml]. Five and 6 patients had PS of 0 and 1 respectively. The regimen was well tolerated with no unexpected toxicities. To date 3 of 6 patients have completed study treatment and remain progression free at 6 months, 1 death occurred due to an unrelated cause, and 2 patients progressed at 12 weeks of therapy. Two patients demonstrated an 88% and 36% PSA decline. Immune correlates will be presented. Conclusions: The combination regimen of pembrolizumab and BATs appears to be worthy of future exploration in a larger trial in mCRPC. Clinical trial information: NCT03406858.

Peripheral Blood Cell Sorting Strategies for Transcriptomic Analysis in Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by sustained peripheral blood (PB) monocytosis and an inherent risk for leukemic transformation. Clonal origins of the disease can be detected in hematopoietic progenitor cells (CD34+/CD38-), while the complete spectrum of mutational evolution can be seen in circulating monocytes (CD14+). Cell sorting strategies have been employed to select cells in CMML, and while there are adequate monocyte numbers in the PB, there are very few circulating progenitor cells. In addition, attrition related to the selection process significantly depletes primary cells available for biological experiments and multiomics studies such as RNA-seq, ChIP-seq, ATAC-seq, and DIP-seq. While single-cell methods may be able to overcome this challenge, bulk sequencing methods remain a robust and cost-effective approach. We hypothesized that, secondary to the stem cell origin of this disease and significant myeloproliferation, PB mononuclear cells (MNC) would provide comparable results with regards to transcriptomic analysis, in comparison to cell selection procedures. Methods: Peripheral blood obtained from 15 molecularly annotated patients with WHO-defined CMML was ACK-lysed and subjected to a Ficoll procedure for collection of MNC. MNC were left unsorted (n=5) or further selected for CD34+/CD38- (n=5) and CD14+ (n=5) using a fully automated RoboSep-S (StemCell Technologies) protocol. All samples were then subjected to bulk whole transcriptome shotgun sequencing (using Illumina TruSeq and an Illumina HiSeq 4000). After data quality control, counts of detectable transcripts were log2-normalized and Pearson's product-moment correlation coefficients were calculated to evaluate the correlation between the two cell-sorting strategies and unsorted cells in terms of detectable transcripts. To visualize sample differences log2-normalized transcripts counts were centered and scaled per gene for a select number of genes relevant to myeloid biology as well as a number of housekeeping genes. Results: Fifteen patients with WHO-defined CMML, median age 69 years (55-73 years), 66% male, were included. Next generation sequencing for somatic mutations was performed on PB MNC obtained at CMML diagnosis (Figure 1, top heatmap). Considering the small sample size, mutations were evenly distributed among groups with the exception of ASXL1 (higher frequency in CD14+ and CD34+/CD38- cells), ZRSR2 (higher frequency in unsorted cells), and TET2 (lower frequency in CD14+ cells). The three groups were also well matched with regards to other CMML-related variables such as WHO and FAB morphological subtypes, cytogenetic abnormalities, and risk stratification by the Mayo Molecular Model. Transcriptomic analysis revealed a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD34+/CD38- cells (ρ = 0.96, p &lt; 0.001, top scatterplot). Likewise, there was a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD14+ cells (ρ = 0.91, p &lt; 0.001, bottom scatterplot). The latter correlation was marginally lower, which was explained by increased global gene expression in 3 of the 5 CD14+ samples (bottom heatmap). Increased gene expression in these 3 samples involved key myeloid genes and housekeeping genes known to have stable expression across human tissues alike. In comparison to PB MNC, both cell sorting strategies resulted in significant depletion of primary cells required for other experiments, and for procedures such as ChIP-seq, DIP-seq and ATAC-seq (CD34+/CD38- had greater depletion than CD14+). Additional experiments to assess this strategy for the above mentioned epigenetic studies are currently being planned. Conclusions: Accounting for sample differences, different cell sorting strategies (unsorted, CD34+/CD38- selection, and CD14+ selection) yielded similar results when performing bulk transcriptomic assessments on PB MNC from patients with CMML. For the purpose of gene expression profiling there was no clear advantage with CD34+/CD38- or CD14+ selection. These results support the use of unsorted cells for bulk transcriptomic analysis in CMML. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

How to Collect the Minimum-Targeted CD3+ Cells for CAR-T Therapy- the Bern Approach

Introduction: An autologous CD3+ cell collection is the first step required for a CAR-T therapy. Up to now, in the commercial setting, one of the available models predicting the time or processed patient blood volume needed in order to gain the minimum-targeted absolute count of CD3+ cells relies on a formula provided by Novartis (Reference Model - RM), which assumes these parameters to be inversely proportional. We here aimed to develop a refined and more precise model, which enables to predict the needed processed blood volume in patients with low pre-apheresis peripheral CD3+ counts for gaining the targeted CD3+ cell count of e.g. 1.0x10⁹, using the patient's pre-apheresis peripheral CD3+ cell values, the collected absolute CD3+ cell counts, and the effectively processed blood volumes (Bern model - BM). Methods: We analyzed 16 lymphocyte apheresis procedures in adult patients (ALL: n=1; B-cell lymphomas: n=15) with a median age of 70.5 years (r 25 - 81 years) performed at the University Hospital Bern, Switzerland, for the production of Kymriah® (Novartis) in the period from December 2018 till July 2019. All cell collections were performed on the Spectra Optia (Terumo BCT©) device. Continuous Mononuclear Cell Collection (CMNC) procedure was used. We measured the peripheral CD3+ cell counts of the patients' blood and the collected products by means of multi-parameter flow cytometry (BD FACSCanto II). The inversely proportional function (RM) did not fit well our data, which was the trigger to look for an alternative function. Best fit of the series of data points and establishing of the function calculation has been performed by using Excel 2019 statistics, using power function modeling. Results: The median peripheral CD3+ cell level at start of apheresis was 1.29x10⁹/ml (r 0.11x10⁹/ml - 2.66x10⁹/ml). Low peripheral CD3+ counts (defined as &lt; 0.7x10⁹/ml) were found in 5 out of 16 patients, whereas 11 patients showed normal peripheral CD3+ counts. Interestingly, in our hands lymphocyte collections at low pre-apheresis CD3+ counts was successful reaching the target &gt;1.0x10⁹ CD3+ cells defined by the manufacturer. The median collection duration was 176 minutes (r 114 - 286 minutes) and the median ratio of processed blood volume to WBV (patient whole blood volume) was 3.1 (r 1.50 - 6.70). There was a significant negative correlation between CD3+ cell levels and blood volume processed/WBV (Spearman rank correlation, r: -0.92, p=0.0285). The median collected CD3+ cell number in the lymphocyte harvests was 6.43 x109 (r 1.88x109 - 11.90x109), fulfilling the required minimum of CD3+ cells in all 16 patients. In Figure 1, each data point labeled by 1-9 or A- G represents an individual patient. The function of RM y=2.5x-1 (plotted in gray) is the intuitive, inverse proportional model provided by Novartis to calculate the minimum targeted cell count for production of Kymriah®. The function of BM y=2.167x-0,759 (plotted in blue) is our empiric model best fitting to our patients' data. Both models provide numerically similar results in case of normal pre-apheresis CD3+ cell counts, but differ significantly in the area of low pre-apheresis CD3+ values. To illustrate all similarities and differences between BM and RM in a single plot, one can present this relation as follows: BM = 2.167(pCD3+)-0,759 is equal to 2.5(pCD3+) -1(RM) * 0.8671(pCD3+)0,241 where pCD3+ is the pre-apheresis CD3+ cell count expressed in 109 cells/ ml. Using this approach we obtain the following final formula: BM = RM * BF where BF (Bern Factor) is defined here as 0.8671 * (pCD3+)0.241. Figure 2 presents a graphic illustration of the formula for the BF. The function in Figure 2 is linking the RM and BM. For low CD3+ values, the BF is lower than for the normal CD3+ values. Conclusion: In the area of lower pre-apheresis CD3+ values, lymphocyte apheresis showed to be more efficient than expected by the manufacturer's model and the collection could be performed faster than assumed. According to our data the function y=2.167x-0,759 is more precise for calculating the needed processed blood volume for gaining the minimum required CD3+ cell count of 1.0x10⁹. Further investigations are needed to evaluate the applicability of the above Bern Model to predict processed blood volumes in patients with low peripheral CD3+ counts and to explain the phenomenon why the collection seems less efficient for patients with normal peripheral CD3+ cells. Disclosures Jalowiec: Pfizer: Other: Travel grant; Amgen: Other: Travel grant; Novartis: Other: Travel grant. Baerlocher:Novartis: Research Funding. Zeerleder:Alexion: Speakers Bureau; Jazz Pharma: Speakers Bureau; Sanofi/Genzyme: Speakers Bureau.

Automated mononuclear cell collection: a feasibility study employing a new software for extracorporeal photopheresis

Very recently, Fresenius Kabi, improved the software (autoMNC lymphocytes, SW 04.03.08) for mononuclear cells (MNCs) collection with the aim to ameliorate the quality of harvest, employing the automated autoMNC lymphocytes software SW 04.03.09. Herein, we report the results of an observational study evaluating the feasibility of MNCs collection in patients undergoing extracorporeal photopheresis (ECP) at our centre, using the new COM.TEC software 04.03.08c for MNC collection, afterwards integrated in the software 04.03.09, available on the market since November 2018. Thirty adult patients (21 males and 9 females) with GvHD, Chronic Lung Allograft Dysfunction or renal rejection, were consecutively enrolled to undergo 1 ECP procedure by the offline technique, according to our internal protocol, processing 1·5 blood volumes. Feasibility of collection was defined as: Hct in collection bag ≤5%, MNCs purity (percentage of MNCs/bag) ≥80%, MNCs collection efficiency (CE2) ≥60%, patient's platelet depletion ≤50%. Thirty ECP procedures were evaluated. Feasibility (defined by the four parameters previously described) of MNCs collection was observed in 1 out of the 30 harvests analysed. Median Hct in the product was 3·45% (IQR: 2·6-5·0), and median MNCs purity was 97·2% (IQR 89·1-98·6). Median CE2 for MNCs was 21·4% (IQR: 11·9-41·2), and median patient's platelet depletion was 36·2% (IQR 21·9-51·4). The autoMNC lymphocytes software SW 04.03.08c for MNCs collection in ECP setting demonstrated to collect a good quality product in terms of purity and RBC contamination even if the collection efficiency and platelet contamination must be improved.

A Study on the Collection of Peripheral Blood Mononuclear Cells From Healthy Volunteers for the Development of Allogeneic Cellular Immunotherapy Products

A Study on the Collection of Peripheral Blood Mononuclear Cells From Healthy Volunteers for the Development of Allogeneic Cellular Immunotherapy Products

Phase II trial of AKT inhibitor MK-2206 in patients with advanced breast cancer who have tumors with PIK3CA or AKT mutations, and/or PTEN loss/PTEN mutation

BackgroundThe PI3K/AKT pathway is activated through PIK3CA or AKT1 mutations and PTEN loss in breast cancer. We conducted a phase II trial with an allosteric AKT inhibitor MK-2206 in patients with advanced breast cancer who had tumors with PIK3CA/AKT1 mutations and/or PTEN loss/mutation.MethodsThe primary endpoint was objective response rate (ORR). Secondary endpoints were 6-month progression-free survival (6 m PFS), predictive and pharmacodynamic markers, safety, and tolerability. Patients had pre-treatment and on-treatment biopsies as well as collection of peripheral blood mononuclear cells (PBMC) and platelet-rich plasma (PRP). Next-generation sequencing, immunohistochemistry, and reverse phase protein arrays (RPPA) were performed.ResultsTwenty-seven patients received MK-2206. Eighteen patients were enrolled into the PIK3CA/AKT1 mutation arm (cohort A): 13 had PIK3CA mutations, four had AKT1 mutations, and one had a PIK3CA mutation as well as PTEN loss. ORR and 6 m PFS were both 5.6% (1/18), with one patient with HR+ breast cancer and a PIK3CA E542K mutation experiencing a partial response (on treatment for 36 weeks). Nine patients were enrolled on the PTEN loss/mutation arm (cohort B). ORR was 0% and 6 m PFS was 11% (1/9), observed in a patient with triple-negative breast cancer and PTEN loss. The study was stopped early due to futility. The most common adverse events were fatigue (48%) and rash (44%). On pre-treatment biopsy, PIK3CA and AKT1 mutation status was concordant with archival tissue testing. However, two patients with PTEN loss based on archival testing had PTEN expression on the pre-treatment biopsy. MK-2206 treatment was associated with a significant decline in pAKT S473 and pAKT T308 and PI3K activation score in PBMC and PRPs, but not in tumor biopsies. By IHC, there was no significant decrease in median pAKT S473 or Ki-67 staining, but a drop was observed in both responders.ConclusionsMK-2206 monotherapy had limited clinical activity in advanced breast cancer patients selected for PIK3CA/AKT1 or PTEN mutations or PTEN loss. This may, in part, be due to inadequate target inhibition at tolerable doses in heavily pre-treated patients with pathway activation, as well as tumor heterogeneity and evolution in markers such as PTEN conferring challenges in patient selection.Trial registrationClinicalTrials.gov, NCT01277757. Registered 13 January 2011.

The effects of prebiotics on growth performance and in vitro immune biomarkers in weaned pigs.

The objective of the experiment was to investigate the effects of prebiotics in nursery pigs on growth performance and immune biomarkers. Sixty-four weaned pigs (31 ± 1 d; BW 8 ± 0.1 kg) of mixed gender were housed (4 pigs/pen) in an environmentally controlled nursery with ad libitum access to feed and water over a 35-d study. Pigs were randomly assigned to one of four treatments: control (53% corn, 32% SBM, 7% fishmeal, 8% others), control + 2.5% GroBiotic-S (GS), control + 0.05% chicory (CL), or control + 0.5% chicory (CH). Feeders and pigs were weighed weekly. On day 21, blood samples were obtained from three pigs/treatment for collection of peripheral blood mononuclear cells (PBMC). Isolated PBMC were cultured and subsequently challenged with lipopolysaccharide (LPS; 20 ng/mL). Cell culture supernatants were collected for quantification of the pro- and anti-inflammatory cytokines, interleukin (IL)-8 and IL-10, respectively. Dietary treatment had no effect on BW. At days 28 to 35, pigs fed GS (790 ± 15 g), CL (704 ± 15 g), or CH (692 ± 15 g) had greater (P < 0.05) ADG compared with control (643 ± 15 g) pigs. In addition, overall (days 0–35), pigs fed GS (823 ± 18 g), CL (783 ± 18 g), or CH (782 ± 18 g) had greater (P < 0.05) ADFI compared with control, and ADFI for GS-fed pigs was greater (P < 0.05) than either CL or CH. There was no difference in G:F among treatments. In vitro LPS challenge increased (P < 0.05) IL-8 secretion from PBMC isolated from CL (23,731 ± 3,221 pg/mL) pigs compared with control (10,061 ± 3,221 pg/mL) and CH (12,411 ± 3,221 pg/mL) pigs. Secretion of IL-10 from PBMC isolated from CL (63 ± 9 pg/mL) pigs was greater (P < 0.05) compared with control (22 ± 9 pg/mL) pigs and tended (P < 0.1) to be greater compared with CH (34 ± 9 pg/mL) pigs. Results indicate that inclusion of prebiotics in nursery pig diets has positive effects on growth performance and may have immunomodulatory effects (in vitro) on cells isolated from prebiotic-fed pigs.

Performance prediction algorithm for autologous PBSC collection in adults and pediatric patients using large volume leukapheresis.

The number of CD34+ cells collected in apheresis procedures depends mainly on the collection efficiency of the device and the blood volume processed. Large volume leukapheresis (LVL) can improve CD34+ cell yield and has previously been investigated using the COBE Spectra device (Terumo BCT, USA). This was a retrospective analysis of LVL performance in patients undergoing continuous mononuclear cell collection (CMNC) using the new Spectra Optia apheresis system (Terumo BCT, USA) at the University Hospital Center, Zagreb, from March 2016 to September 2016. CD34+ cell yield predictability, determined using a customized algorithm, was also assessed. In total, 67 procedures performed in 46 adults and 14 performed in 11 children were included in the analysis. In adults, 30 (65.2%) patients successfully reached their target preapheresis CD34+ cell count on day 1, with a median (interquartile range [IQR]) CD34+ collected cell dose of 4.8 × 106 /kg (2.3-10.6 × 106 /kg). In the pediatric group, 81.8% successfully collected the target CD34+ cell dose on the first day, with a median (IQR) CD34+ collected cell dose of 11.1 × 106 /kg (3.2-16.3 × 106 /kg). The customized algorithm showed a strong and significant linear correlation with actual CD34+ cell dose (P < 0.0001). The results of this study support the use of LVL and the customized prediction algorithm in apheresis procedures. The ability to tailor the procedure to meet the needs of the individual patient may help to minimize the blood volume processed, shorten the duration, reduce the volume of infused anticoagulants, and improve patient comfort.

A standardized methodical approach to characterize the influence of key parameters on the in vitro efficacy of extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) is an autologous immunomodulatory cell therapy that consists of the ex vivo collection of mononuclear cells (MNCs), which are irradiated with UVA in the presence of the photosensitizing agent 8-methoxypsoralen (8-MOP) to induce cell apoptosis. This photoactivated cell preparation is then reinfused into the patient. While the clinical benefits of ECP are well-demonstrated, no study has yet characterized the influence of variations in the composition of the cell preparation on the efficacy of ECP in vitro. Here, we describe a standardized methodology for the in vitro assessment of ECP that uses the human lymphoma T-cell line and mimics the clinical procedure. By quantifying cell apoptosis, inhibition of cell proliferation, and 8-MOP consumption, we used this approach to characterize the specific influence of key variables on the cellular response to ECP. We found that (i) increases in hematocrit and plasma concentrations attenuated the cellular response to ECP; (ii) plasma concentration was the only variable tested that influenced 8-MOP consumption; and (iii) the loss of efficacy due to variations in the concentration of certain blood components could be counteracted by modulating the UVA dose. This methodology may enable evaluation of other leukapheresis preparation protocols and better determination of the optimal working parameters for ECP.

Evaluation of peripheral blood mononuclear cell collection by leukapheresis.

Adoptive immunotherapy using engineered lymphocytes has shown promising results in treating cancers even in patients who have failed other treatments. As the first essential step, the number of peripheral mononuclear cell (MNC) collection procedures is rapidly increasing. In this retrospective study, we reviewed the collection results to determine factors that affect MNC collection. We reviewed 184 collections that were performed on 169 adult allogenic donors and patients with acute lymphoid leukemia, chronic lymphoid leukemia, lymphoma, multiple myeloma, or solid-organ tumors. All the leukapheresis procedures were performed after a complete cell count with differential was obtained. Total blood volume (TBV) was defined as processed blood volume divided by patient blood volume. There was a significant association between the precollection MNC count (pre-MNC) and the MNC yields normalized by TBV (r = 0.926; p < 0.001) and a regression formula was created to predict MNC yields. Multiple regression analyses showed that pre-MNC, TBV, and precollection hemoglobin were strongly associated with MNC yield (R 2 = 0.866; F (3180) = 388.472; p < 0.001), and pre-MNC had the greatest influence on MNC yield (β = 0.960; p < 0.001) followed by TBV (β = 0.302; p < 0.001), and Hgb (β = 0.136; p < 0.001). Our results suggest that the optimal time for MNC collection can be determined based on pre-MNC and that processing volume should be determined based on collection goal and pre-MNC to optimize and personalize the harvesting procedure.

A dual strategy to optimize hematopoietic progenitor cell collections: validation of a simple prediction algorithm and use of collect flow rates guided by mononuclear cell count.

Previous prediction algorithms to achieve target CD34+ goals have not been widely adopted, with many centers still using a set volume to process for hematopoietic progenitor cell collections. This may be because previous algorithms are challenging to implement. Additionally, no study has yet examined the utility of adjusting the collect flow rate (CFR) based on the donor's preprocedure total mononuclear cell (MNC) count, which correlates with CD34+ yield. In this retrospective analysis of mobilized allogeneic donors collected using MNC (COBE Spectra, Terumo BCT) or continuous mononuclear cell collection (CMNC) (Spectra Optia, Terumo BCT) procedures, we validated a one-step prediction algorithm to achieve the target CD34+ product dose (Appendix S1, available as supporting information in the online version of this paper). The COBE Spectra MNC Collect Flow Tool (Appendix S2, available as supporting information in the online version of this paper) was used to select the collect flow rate for both MNC and CMNC procedures. Procedural collection efficiency (CE) was compared to that of historical procedures utilizing fixed CFRs (1.0-1.5 mL/min). Ninety-three percent of collections achieved the target CD34+ goal using our algorithm-calculated process volumes. The remaining 7% of cases had CEs lower than the algorithm CE (0.40), and thus were below goal. Second, an MNC-based CFR improved MNC and CD34+ CEs in patients with higher MNC counts compared to our historical controls. We validated that this simple, single-step prediction algorithm achieves the target CD34+ goal in most HPC collections. Secondly, we showed that an MNC-based CFR for hematopoietic progenitor cell collections improves CE at higher MNCs; this may be preferable to a WBC-based CFR because of variability of MNC counts at a given WBC count.

A paired trial comparing mononuclear cell collection in two machines for further inactivation through an inline or offline extracorporeal photopheresis procedure.

Extracorporeal photopheresis (ECP) is an effective treatment. However, protocols differ widely, and some questions, such as the number of cells to be collected or the number of ECP treatment days per treatment cycle, are still unsolved. The aim of this study was to compare a multistep (offline) (Spectra Optia and Macogenic G2) against an integrated (inline) ECP system (Therakos Cellex system) with respect to mononuclear cell (MNC) collection. The number and quality parameters of the MNC products collected were evaluated together with some machine parameters, such as collection time. Comparisons were made through paired sample analysis with the t test. Fourteen patients underwent 15 double-paired procedures using both ECP protocols. The average MNC collected in the multistep procedure was 77.4 × 108 , four times more than in the integrated procedure (18.5 × 108 ). MNC purity (84.4% vs. 63.8%) and enrichment (27.9 vs. 5.9) in the product collected were also higher in the multistep procedure. The whole ECP time was higher in the multistep than in the integrated procedure (272 vs. 106 min), but the calculated time to collect 25 × 108 MNCs in the multistep was shorter compared with the one-step procedure (77.8 vs. 172 min). All these differences between the two protocols were statistically significant. These two ECP protocols are different with respect to MNC collection and length of procedure. Some unresolved questions, such as the better MNC dose to inactivate or the number of consecutive days that ECP should be performed for optimal clinical efficacy, require further review.

Abstract 5584: Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients

Abstract Introduction The number of circulating tumor cells (CTC) isolated by the FDA approved CellSearch (CS) system in 7.5 mL of blood of metastatic and non-metastatic non-small cell lung cancer (NSCLC) patients is low or absent. Processing a larger blood volume could yield a greater amount of CTC from these patients. An approach to probe larger volumes is using diagnostic leukapheresis (DLA) where the blood can be split into its components after which selective harvesting of mononuclear cells (MNC) including CTC can take place. Methods Patients with histologically proven NSCLC who either have been recently diagnosed or are eligible for new therapy can participate in this ongoing study. Patients had DLA for MNC collection derived from one total body blood volume. Before and after DLA, whole blood was drawn in a CellSave blood collection tube and analyzed by CS. The obtained DLA product was divided for analysis. 2x108 cells (on average 2mL of DLA product, representing ~100mL of blood) was diluted in 7.5 mL buffer, and analyzed by CS. 9 mL of the DLA product (on average 1x109 cells) was fixed and depleted of leukocytes using the RosetteSep CD45 depletion kit (Stemcell Technologies), allowing processing of a larger volume, followed by CS analysis. Results of molecular characterization of the detected CTC is pending. Results So far, a total of 8 DLA's were performed in 7 stage 4 NSCLC patients. Sample collection took place before (t=0) and/or after (t=1) treatment. Patients underwent DLA in about 110 minutes, at this time an average of 4860 mL of blood was processed. No complications occurred, no post-procedure complaints were recorded. Similar DLA products with an average of 95 mL were obtained. CTC counts for these patient samples are shown in the table below. CTC counts blood and DLAPatientt=#CTC pre blood#CTC post blood#CTC DLA (~2mL, 200x106 cells)#CTC depleted DLA (~9mL, 900x106 cells)101n/a114113020203514602031000040n/a04495000166000029020114 Conclusion DLA is a novel method to collect CTC from the blood. DLA can result in the detection of CTC where the use of 7.5mL of blood is not sufficient. Additionally, DLA shows an increase of CTC yield in patients with CTC detected in 7.5 mL of blood. Citation Format: Kiki C. Andree, Menno Tamminga, Anouk Mentink, T Jeroen Hilterman, Harry J. Groen, Leon W. Terstappen. Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5584.