Positive Immunomagnetic Selection Research Articles

Abstract PO1-16-05: Matched multi-analyte and multi-omic liquid biopsy of cell-free DNA, circulating tumor cells and extracellular vesicles in HER2-positive metastatic breast cancer patients

Abstract Background: Liquid biopsy, specifically cell-free DNA (cfDNA) mutation analysis, has recently entered clinical practice for treatment decision-making in hormone receptor (HR)-positive (+)/HER2-negative (-) metastatic breast cancer (mBC) patients. However, we showed that the analysis of multiple liquid biopsy analytes in the course of treatment in this BC subgroup has additive value. Since no multi-analyte studies are currently available for the subgroup of HER2+ mBC patients, we here studied multiple analytes [cfDNA, circulating tumor cells (CTCs) and extracellular vesicles (EVs)] and utilized our multi-omics perspective (CpG island methylation, mutations and mRNA expression) to get insight into their utility in therapy management of HER2+ mBC. Methods: 2x 9 ml blood was drawn from 21 HER2+ (HR+ n=16 and HR- n=5) mBC patients at the time of disease progression and at two consecutive staging time points. CTCs and their mRNA were isolated from 2x5 ml blood by positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select/Detect). Plasma of CTC-depleted blood was used for cfDNA isolation, while mRNA from EVs was isolated from 4 ml pre-filtered plasma by affinity-based binding to a spin column (exoRNeasy) using the remaining blood. The mRNA purified from CTCs and EVs by Oligo-dT beads was reverse transcribed, pre-amplified and analyzed by a multi-marker (18 genes) qPCR panel. qPCR data was normalized to CD45 (for CTC analysis) and data of 20 healthy female donor controls to identify BC specific overexpression signals with a specificity of >90% for all targets. Variants in the cfDNA were analyzed with a customized QIAseq Targeted DNA Panel with unique molecular indices (UMIs) and high coverage (mean 22.000x), while CpG island methylation in the cfDNA was analyzed with a customized QIAseq Targeted Methyl Panel with UMIs covering 9786 CpG sites (mean 900x). Consumables: QIAGEN, Germany. Results: In general, substantial differences occurred between the CTC and EV mRNA profiles. Transcripts involved in the PI3K pathway (mTOR, AKT2, PIK3CA) as well as ERBB2 signals were significantly more prevalent in CTCs compared to EVs and mTOR, ERCC1, SRC, AKT2 und PIK3CA CTC overexpression signals were found in >50% of these patients. In contrast, PARP1, AURKA, SRC und ERCC1 signals were significantly more prominent in the EVs and detected in >50% of the patients. ERCC1 signals were present in EVs with highest prevalence whereas ERBB2 signals were only present in CTCs. ERBB3+ CTCs were only detected in patients showing disease progression. Currently, cfDNA mutations as well as cfDNA CpG island methylation are under evaluation and the complete multi-analyte and multi-omic data set as well as their relation to clinical outcome will have been finalized to be presented at the meeting. Conclusion: We successfully established a workflow for parallel isolation of multiple liquid biopsy analytes from a minimized blood volume in HER2+ mBC. Preliminary mRNA profile results for CTCs and EVs show the complementary nature of these two liquid biopsy analytes. Besides the multi-analyte nature of this workflow, the pending results for genomic and epigenomic information will show which one of the analytes, alone or in combination, will help to optimize therapy management in HER2+ mBC patients. Citation Format: Sabine Kasimir-Bauer, Markus Storbeck, Ioanna Andreou, Siegfried hauch, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Matched multi-analyte and multi-omic liquid biopsy of cell-free DNA, circulating tumor cells and extracellular vesicles in HER2-positive metastatic breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-05.

Abstract 1491: The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host

Abstract Background: Diabetes is associated with increased risk of cancer development and progression. Recent studies have shown that neutrophils in diabetic patients are prone to forming neutrophil extracellular traps (NETs) which have been reported to capture circulating cancer cells, leading to the promotion of liver and lung metastasis. This study aims to clarify the causative relationship between NETs and the peritoneal metastasis (PM) in diabetic condition. Methods: Outcome of the diabetic patients who received curative gastrectomy for gastric cancer from 2010 to 2021 in our institute was comparatively investigated with those without diabetes. Neutrophils were isolated from the bone marrow from C57BL/JHamSlc-ob/ob mouse (type 2 diabetes model) and wild type mouse using Ly6G-based positive immunomagnetic selection. The neutrophils were stimulated with 100nM of PMA for 4 hours and NETs formation was evaluated by the addition of Sytox Green under fluorescent microscope. A mouse gastric cancer cell, YTN16P (5 × 105 cells) was intraperitoneally (IP) injected, the number of metastatic nodules on mesentery was comparatively evaluated two weeks later. Results: Disease-free survival (p=0.004, HR=1.65) and overall survival (p=0.035, HR=1.46) of 203 diabetic patients were significantly worse those of 975 non-diabetic patients. Neutrophils isolated from ob/ob mice produced markedly increased NETs than that from wild-type mice. NETs produced by neutrophils obtained from the peritoneal cavity of ob/ob mice after IP injection of a 9% casein solution effectively captured many YTN16P in vitro. The concentration of double strand DNA (dsDNA) in plasma in ob/ob mice compared with their counterparts (676ng/mL vs 590ng/mL, p=0.036, n=3). The number of PM in ob/ob mice was significantly more than that in wild-type controls (39.1 vs 15.5, p=0.027, n=10). Conclusion: Enhanced NETs formation in diabetic conditions may be associated with an increased risk of PM in gastric cancer. Citation Format: Rei Takahashi, Hideyuki Ohzawa, Yuki Kaneko, Misaki Matsumiya, Yurie Futoh, Kohei Tamura, Kazuya Takahashi, Yuki Kimura, Akira Saito, Hideyo Miyato, Naohiro Sata, Joji Kitayama. The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1491.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

Abstract P5-02-25: Transcriptional profiling of CTCs in metastatic breast cancer patients in the course of CDK4/6 inhibition

Abstract Background: De novo resistance defined as progression within six months and acquired resistance are one of the major problems in the subset of metastatic (M), hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) patients without visceral crisis receiving CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (TX). Here, we aim to identify predictive and monitoring markers of CDK4/6i resistance by conducting transcriptional profiling of circulating tumor cells (CTCs) that represent a real-time snapshot of the heterogeneity. Methods: Blood of (A) 60 HR+/HER2- MBC patients drawn at baseline of Palbociclib plus endocrine TX (TX as first line n=31, second or more lines n=29), (B) 19 HR+/HER2- MBC patients drawn before the initiation of endocrine monoTX (control) and matched blood samples of these patients after six months under TX (n=72) and at the time of progression (n=42) were analyzed. To enlarge the global CDK4/6i cohort at baseline, blood of (C) 32 patients before the initiation of Ribociclib plus endocrine TX was also drawn. Patients with progression within six months were defined as non-responders. Isolation of CTCs was conducted using positive immunomagnetic selection (AdnaTest EMT2/StemCell Select) and preamplified cDNA was analyzed by a multimarker qPCR panel utilizing QuantiNova LNA Probe assays targeting 25 genes involved in the DNA damage -, MAPK -, STAT -, Hippo – pathway or cell cycle, chemokine sensing, multidrug resistance and cell adhesion. qPCR data was normalized to CD45 and data of 20 healthy female donor controls to identify BC CTC specific overexpression signals with a specificity of >90% for all targets. Consumables: QIAGEN, Germany. Statistical analysis included log-rank testing and univariate Cox regression. Results: For first line CDK4/6i treated patients at baseline, CETN2 and E2F1 signals correlated significantly with worse progression-free survival (PFS) while CETN2 signals also related significantly to non-response. Furthermore, CETN2 and PCNA signals were significantly associated with worse overall survival (OS). Analyzing the Palbociclib cohort after six months of TX, PCNA signals correlated significantly with a decreased PFS while EpCAM signals showed a significant association with OS. In addition, CETN2, CXCR4, EpCAM, MLH3, WWTR1 signals after six months were shown to correlate significantly with a decreased OS and PFS and MAPK1 signals were only found in the non-responders. While non-response was related to appearing (from baseline to six months under TX) ABCC2, JUN and MAPK1 signals, disappearing ABCC2 signals were only found in the responders. Dynamics of ABCC2, CXCR4, EpCAM, JUN, MAPK1, MLH3, STAT1 and WWTR1 signals from baseline to six months under TX correlated significantly with OS and CXCR4 signal dynamics significantly with a worse PFS. At the time of progression, the presence of E2F1, JUN, MAPK1 and STAT1 signals correlated significantly with a decreased OS and in comparison to baseline analysis, the prevalence of ABCC2, EpCAM, E2F1, CETN2 and CXCR4 signals increased. Conclusion: CTC overexpression signals at baseline of targets involved in the cell cycle (CETN2 and E2F1) might be predictive markers for de novo resistance to CDK4/6i as first line TX, while ABCC2 (multidrug resistance), EpCAM (cell adhesion), E2F1, CETN2 and CXCR4 (chemokine sensing) signals could indicate acquired resistance to Palbociclib. In case of disease progression, E2F1, JUN (cell cycle) and targets of the MAPK- and STAT- pathway could be relevant targets for therapeutic strategies beyond Palbociclib TX. Monitoring and prognostic value was shown for single and repeated measurement of signals under TX in genes involved in resistance, cell cycle progression, DNA damage response (MLH3, PCNA), chemokine sensing, cell adhesion and the MAPK-, STAT- and Hippo (WWTR1) pathway. Citation Format: Sabine Kasimir-Bauer, Charlotte Gruber, Stefanos Moukas, Mitra Tewes, Hans-Christian Kolberg, Rainer Kimmig, Corinna Keup. Transcriptional profiling of CTCs in metastatic breast cancer patients in the course of CDK4/6 inhibition [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-25.

Hepatocellular carcinoma risk-stratification based on ASGR1 in circulating epithelial cells for cancer interception.

Purpose: Lack of diagnostic and prognostic biomarkers in hepatocellular carcinoma impedes stratifying patients based on their risk of developing cancer. The aim of this study was to evaluate phenotypic and genetic heterogeneity of circulating epithelial cells (CECs) based on asialoglycoprotein receptor 1 (ASGR1) and miR-122-5p expression as potential diagnostic and prognostic tools in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Methods: Peripheral blood samples were extracted from LC and HCC patients at different disease stages. CECs were isolated using positive immunomagnetic selection. Genetic and phenotypic characterization was validated by double immunocytochemistry for cytokeratin (CK) and ASGR1 or by in situ hybridization with miR-122-5p and CECs were visualized by confocal microscopy. Results: The presence of CECs increased HCC risk by 2.58-fold, however, this was only significant for patients with previous LC (p = 0.028) and not for those without prior LC (p = 0.23). Furthermore, the number of CECs lacking ASGR1 expression correlated significantly with HCC incidence and absence of miR-122-5p expression (p = 0.014; r = 0.23). Finally, overall survival was significantly greater for patients at earlier cancer stages (p = 0.018), but this difference was only maintained in the group with the presence of CECs (p = 0.021) whereas progression-free survival was influenced by the absence of ASGR1 expression. Conclusion: Identification and characterization of CECs by ASGR1 and/or miR-122-5p expression may be used as a risk-stratification tool in LC patients, as it was shown to be an independent prognostic and risk-stratification marker in LC and early disease stage HCC patients.

P-090 Genotypic sperm sorting: A less invasive Assisted Reproductive Technology (ART) to prevent genetic disorders in newborns

Abstract Study question Is the prevention of genetic disorders in newborns possible by genotypically sorting sperm cells with specially characterized proteins on the sperm surface membrane? Summary answer For Cystic Fibrosis and X-linked Disorders prevention model, we utilized certain monoclonal antibodies to sort sperm cells by targeting proteins expressed on their surface membrane. What is known already Our study focused on Cystic Fibrosis (CF) and X-linked Diseases (XLD) which are genetic disorders inherited by offspring from parents who are healthy carriers of the autosomal recessive or allosomal genes. More than 10million Caucasians are healthy carriers of a mutant cystic-fibrosis gene (predominantly F508del) while 4% of all newborns are at risk of being born with an X-linked disease. The current clinically approved mitigation plan for preventing such genetic disorders in newborns – is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M). PGT-M involves an invasive microsurgical procedure that requires the removal of cells from 3-5day old embryos. Study design, size, duration For our study design, semen samples purchased from FairFax Cryobank from an anonymous CF-carrier and a non-carrier were utilized. The samples were assessed for the presence of H-Y male antigen and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, which are both selectively expressed on the sperm surface – to delineate genotypic sperm sort models for XLD and CF respectively. Participants/materials, setting, methods Methods utilized in the genotypic sperm sorting protocols included the use of phycoerythrin (PE) binding Tetrameric Antibody Complexes (TAC) coupled with magnetic beads. Immuno-magnetic sorting was conducted with a conventional sort method in tubes and with specially fabricated microfluidic sperm sorting (MSS) chips. Sort yield and purities of positively selected sperm populations were assessed by flow cytometry, immunofluorescence, fluorescent in situ hybridization (FISH) and by quantitative-PCR (qPCR – Cq & Tm). Main results and the role of chance Our study was able to elucidate a potential less invasive clinical “ART” application or method of genotypically sorting sperm cells with H-Y/CFTR monoclonal-antibody-functionalized microfluidic chips or conventional immuno-magnetic sorting protocols in sort tubes. Preliminary assessments showed that flow cytometry may be utilized to confirm sort purities of positively selected sperm populations (with HY or CFTR markers) from immuno-magnetic sort tubes – based on their morphological and cellular characteristics. For a genotypic sperm sort in the pathogenic CF-carrier sample, positively and negatively selected CF-sperm fractions (being CFTR-Wt and F508del-Mut sperm) from immuno-magnetic sort tubes could not be adequately distinguished by qPCR-Cq – because of their intrinsic and slightly insignificant genetic variabilities. However, the microfluidic sperm sorting chip fabricated for this study enabled a differential and positive immuno-magnetic selection of CFTR-Wt sperm with a comparably higher but statistically insignificant sort yield, relative to the population of the F508del-Mut sperm negatively selected – when assessed by qPCR-Cq (p value = 0.092; significant at < 0.05). Immunofluorescence and FiSH evaluations of genotypically sorted sperm cells in both XLD and CF models also showed pictorial comparisons with the observed genetic assessment outcomes. Limitations, reasons for caution One of the limitations of this study is the immuno-magnetic sort efficiency. The sorting efficiency may however be improved if freshly collected normospermic semen samples were utilized rather than a frozen-thawed sample which may have been subjected to the stress of cryopreservation and potential cellular damage with reduced motility. Wider implications of the findings This novel less invasive method of Assisted Reproductive Technology may also usher in a new “ART” called Intra-cytoplasmic Genotypically-selected Sperm Injection (IGSI). The IGSI method would potentially enable the selection of non-diseased sperm cells – for the sole purpose of helping to prevent genetic disorders in newborns. Trial registration number Not Applicable

PB1954: COMPARING STR PROFILES OF DNA ISOLATED FROM DIVERSE TUMOR SITES IN MULTIPLE MYELOMA

Background: Multiple myeloma (MM) course is often complicated by extramedullary lesions - plasmacytomas, which pathogenesis is still not completely understood. Genome instability underlies dissemination in various neoplasias, including MM. An important manifestation of genetic instability is the loss of heterozygosity, i.e., the loss of the second allele in the regions of the genome featuring inherited polymorphisms. STR profiling is a method that allows simultaneously assessing the degree of DNA degradation and giving an integral assessment of the stability of the tumor genome. To assess the heterogeneity of MM, not only the tumor substrate in the bone marrow and plasmacytoma but also in circulating cell-free DNA (cfDNA) present in the peripheral blood plasma could be effectively analyzed. Aims: To compare STR profiles of DNA isolated from various anatomical locations (plasma, bone marrow, plasmacytoma) in MM patients. Methods: The prospective study included 21 patients with newly diagnosed MM (11 women, 10 men) aged 39 to 82 years (median 53 years). MM was diagnosed according to the IMWG-2014 criteria. In 15 patients, MM was complicated by plasmacytoma, in 6 of them a tumor biopsy was performed and tumor DNA was isolated. In all patients, plasma circulating cfDNA and control DNA were isolated from buccal epithelium using a commercial kit (Qiagen, Germany) according to the manufacturer’s protocol. In 11 patients, positive immunomagnetic selection of CD138+ bone marrow cells was performed using a monoclonal antibody to CD138 (Miltenyi Biotec, Germany). STR-profiling using COrDIS Plus panel (Gordiz LLC, Russia) of isolated DNA samples (21 plasma circulating cfDNA samples, 11 CD138+ DNA samples of bone marrow cells, 6 plasmacytoma DNA samples) was performed. STR profiles were analyzed using GeneMapper v.4 software (Applied Biosystems, USA). Results: In 10 out of 11 patients, analysis of STR profiles in CD138+ bone marrow cells revealed loss of heterozygosity (LOH) variants with different allelic load, suggesting either a deletion/copy number neutral LOH, or a duplication of one of the two alleles affecting from one to six STR loci for every patient. In all patients for whom plasmacytoma DNA was isolated (n=6), various variants of LOH were also identified (from 1 to 6 loci) (Table 1). Of the 21 plasma circulating cfDNA samples, LOH variants were detected in ten cases (48%) (from 1 to 4 loci), in the other 10 cases, no tumor DNA was detected, the samples matched the control DNA from the buccal epithelium; only in one case, the result turned out to be non-informative due to a significant degree of degradation of the cfDNA. STR profiles of DNA isolated from different tumor sites of the same patient in most cases were identical. In one case aberrant STR loci in plasma circulating cfDNA and bone marrow CD138+ cells were different (patient No. 20). In this patient, MM was complicated by plasmacytomas of various anatomical locations (Th7, 8th rib). One can speculate that the discrepancy between plasma circulating cfDNA and bone marrow CD138+ STR profiles indicates the presence of various MM clones in different tumor loci. Image:Summary/Conclusion: Evaluation of plasma circulating cfDNA might be an effective approach for molecular diagnosis in MM. In 48% of MM patients, it is possible to detect tumor cfDNA in the plasma at the onset of the disease. Comparison of DNA STR profiles from different lesions might reveal the diversity of tumor clones in MM.

Abstract P2-02-04: In early breast cancer, the ratios of neutrophils, monocyctes and platelets to lymphocytes significantly correlate with the presence of subsets of circulating tumor cells in blood and disseminated tumor cells in the bone marrow

Abstract Background: Circulating tumor cells (CTCs) are the precursors of metastasis and while travelling through the peripheral blood, they crosstalk with different types of blood cells before they finally reach distant organs to settle down as disseminated tumor cells (DTCs). Proinflammatory markers include the neutrophil lymphocyte ratio (NLR), the platelet lymphocyte ratio (PLR) as well as the monocyte lymphocyte ratio (MLR) and the presence of tumor cells as well as specific proinflammatory markers are two independent predictors of worse outcome in breast cancer (BC). However, little is known about the correlation between NLR, PLR, MLR and subsets of CTCs as well as DTCs in early, non-metastatic BC. Here we evaluated the correlation of NLR, PLR, MLR and the presence of epithelial CTCs (eCTCs), CTCs in epithelial mesenchymal transition (EMT-CTCs) as well as DTCs to better identify patients at risk, to monitor treatment reponse and probably adjust therapeutic options. Methods: The counts of peripheral neutrophils, lymphocytes, monocytes and platelets to determine NLR, PLR and MLR as well as clinicopathological data during diagnosis were retrospectively recorded for 171 patients (pts) diagnosed with BC from July 2006 to December 2012, before the start of therapy. NLR, PLR and MLR were calculated from peripheral blood cell counts and their optimal cutoff levels were determined by the 75% percentile resulting in the following values: NLR: 3.13, MLR: 0.39 and PLR: 222.3, respectively. 170/171 pts were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibodyA45-B/B3 (Clodronate intake was recommended in case of DTC-positivity). CTCs were determined in 155/171 pts applying positive immunomagnetic selection using the AdnaTest BreastCancerSelect. The recovered cDNA was tested for the presence of eCTCs and EMT-CTCs using the AdnaTests BreastCancerDetect and EMTDetect. Pts were considered CTC-positive if at least one of the two tests were positive. Statistical analysis included descriptive reporting, U-test for group differences, Rho correlations for dependencies and Fisher's chi-square test for distributional differences. Rho correlations were used to assess the relationship between NLR, PLR, MLR and tumor cells as well as progression-free survival (PFS) and overall survival (OS). Results: DTCs were detected in 34% of the pts and at least one CTC-subtype was found in 28% of the pts. Whereas the presence of DTCs was not associated with PFS or OS, the presence of CTCs significantly correlated with a shorter PFS (p=0.046) and OS (p=0.018). However, neither eCTCs nor EMT-CTSs alone were of prognostic significance. Enhanced lymphocyte (p=0.025) and monocyte counts (p=0.039) as well as a low PLR (p=0.032) significantly correlated with a reduced PFS in pts still alive whereas an enhanced MLR showed a high correlation with a shorter PFS (p=0.007) and OS (p=0.021) in deceased pts. Whereas MLR significantly correlated with the presence of EMT-CTCs (p=0.045), the presence of eCTCs significantly associated with an elevated NLR (p=0.003) and enhanced lymphocyte (p=0.007) as well as monocyte counts (p=0.012). No significant correlations were found for NLR, PLR and MLR with DTCs, however, DTC-positive pts, harboring a lower PLR, had a significant shorter OS (p=0.043). Conclusion: Here we show that proinflammatory markers in blood are closely related to the presence of different CTC subsets in early BC while no direct correlation was found for tumor cell spread to the bone. These findings might improve the prognostication of these pts and probably help to monitor response to therapy and adjust treatment options. Citation Format: Sabine Kasimir-Bauer, Ebru Karaaslan, Olaf Hars, Ann-Kathrin Bittner, Oliver Hoffmann, Rainer Kimmig. In early breast cancer, the ratios of neutrophils, monocyctes and platelets to lymphocytes significantly correlate with the presence of subsets of circulating tumor cells in blood and disseminated tumor cells in the bone marrow [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-04.

Abstract P5-13-33: Longitudinal transcriptional profiling of CTCs in metastatic breast cancer patients receiving the CDK4/6 inhibitor Palbociclib to predict therapy response

Abstract Background: CDK4/6 inhibitors represent a new treatment option for metastatic (M), hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) patients. However, predictive markers have not been defined to decide who will benefit from CDK4/6 inhibitors. Circulating tumor cells (CTCs) represent the tumoral heterogeneity in real time and are available for molecular characterization via minimal-invasive sequential blood sampling. Here, we aim to identify biomarkers of resistance to Palbociclib by conducting transcriptional profiling of CTCs before therapy initiation (baseline) and after six months under treatment. Methods: Blood of 50 HR+/HER2-MBC patients drawn at baseline of Palbociclib plus endocrine treatment and blood of 20 HR+/HER2-MBC patients drawn before initiation of endocrine monotherapy, as well as blood samples of these 70 patients after six months under treatment will be analyzed. Patients with progressive disease within the first six months of treatment were defined as non-responders. Isolation of CTCs was conducted using positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select). cDNA was analyzed by a new multimarker qPCR panel utilizing QuantiNova LNA Probe assays targeting 25 genes. qPCR data was normalized to CD45 to substract the effect of contaminating leukocytes. Additionally, data was normalized to 20 healthy female donor controls to identify BC specific overexpression signals with a specificity of >90% for all targets. Consumables: QIAGEN, Germany. Results: We successfully established the multimarker qPCR according to the MIQE guidelines. Up to now, the binary mRNA overexpression signals of 25 targets in CTCs isolated at baseline were analyzed in 40 of the total 70 planned MBC patients. In detail, 24 of those 40 patients were treated with Palbociclib plus endocrine therapy, including seven responders and 15 non-resonders, while the 16 other patients were treated with endocrine therapy alone (seven non-responders and nine responders). In the entire cohort, STAT1 signals were significantly related to a decreased overall survival (p=0.0034) and NFKB1 signals correlated significantly with an increased progression free survival (PFS; p=0.027). In the Palbociclib group, CDK2, WWTR1 and YAP1 overexpression signals were more common in non-responders compared with responders. MLH1 and NFKB1 signals were more common in responders than in non-responders. In this cohort, MLH3 overexpression at baseline was significantly correlated with prolonged PFS (p=0.047) and the patient with the longest PFS (35 months) was the only patient with detectable ERBB4, JUN, CETN2 and TEAD2 signals. In the control group treated with endocrine therapy alone, signal prevalence of CXCR4 and STAT1 were increased in the non-responders versus responders and ABCC2, ESR1, FAT4 and FGFR1 signals were more frequently detected in the responders. Conclusion: Transcriptional profiling of CTCs represents a real-time snapshot of the disease complexity. Characterization of the CTCs isolated from blood drawn at baseline is promising for the identification of urgently needed predictive markers (here MLH3 overexpression), while characterization of CTCs isolated under treatment can elucidate the transcriptional dynamics and might be used as monitoring marker by differentiation of non-responders and responders. We will finalize CTC analysis of the entire cohort and will continue blood sampling of additional Palbociclib treated patients, also in the follow-up of the disease, to validate CTC MLH3 overexpression as predictive biomarker for CDK4/6 inhibitor treatment and moreover, to find further overexpression signals in CTCs relevant for MBC therapy management. Citation Format: Sabine Kasimir-Bauer, Charlotte Gruber, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Longitudinal transcriptional profiling of CTCs in metastatic breast cancer patients receiving the CDK4/6 inhibitor Palbociclib to predict therapy response [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-33.

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting

PurposeThe incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. MethodsSprague-Dawley rats (3–4 weeks, 120–150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. ResultsThe purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12–16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. ConclusionAEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Circulating Tumor Cells Enumeration from the Portal Vein for Risk Stratification in Early Pancreatic Cancer Patients.

Simple SummaryEffective biomarkers are needed to enable personalized medicine for pancreatic cancer patients. This study analyzes the prognostic value, in early pancreatic cancer, of circulating tumor cells and clusters from the central venous catheter and portal blood. Circulating tumor cells were isolated using an immunomagnetic selection and were detected by microscopy using immunocytochemistry staining. In conclusion, the circulating tumor cell number in portal blood identifies a death risk in patients with early pancreatic cancer.Background. Effective biomarkers are needed to enable personalized medicine for pancreatic cancer patients. This study analyzes the prognostic value, in early pancreatic cancer, of single circulating tumor cell (CTC) and CTC clusters from the central venous catheter (CVC) and portal blood (PV). Methods. In total, 7 mL of PV and CVC blood from 35 patients with early pancreatic cancer were analyzed. CTC were isolated using a positive immunomagnetic selection. The detection and identification of CTC were performed by immunocytochemistry (ICC) and were analyzed by Epi-fluorescence and confocal microscopy. Results. CTC and the clusters were detected both in PV and CVC. In both samples, the CTC number per cluster was higher in patients with grade three or poorly differentiated tumors (G3) than in patients with well (G1) or moderately (G2) differentiated. Patients with fewer than 185 CTC in PV exhibited a longer OS than patients with more than 185 CTC (24.5 vs. 10.0 months; p = 0.018). Similarly, patients with fewer than 15 clusters in PV showed a longer OS than patients with more than 15 clusters (19 vs. 10 months; p = 0.004). These significant correlations were not observed in CVC analyses. Conclusions. CTC presence in PV could be an important prognostic factor to predict poor prognosis in early pancreatic cancer. In addition, the number of clustered-CTC correlate to a tumor negative differentiation degree and, therefore, could be used as a diagnostic biomarker for pancreatic cancer.

SCRI-CAR19x22v2 T Cell Product Demonstrates Bispecific Activity in B-ALL

Introduction: CAR T cells in B-ALL have recently focused on the dual targeting of CD19 and CD22 to enhance long term remissions and prevent antigen negative recurrence that is frequently encountered with single antigen targeting. However, a barrier to this approach has been the retention of dual specificity killing and ongoing persistence. PLAT-05 is a multisite phase 1 trial (NCT03330691) that was undertaken to evaluate the safety and feasibility of SCRI-CAR19x22v1, a dual transduced patient-derived product with lentiviral vectors encoding for either a CD19- or CD22-specific CAR, both with 4-1BB co-stimulation. Early results of the first 27 subjects infused demonstrated feasibility and a favorable safety profile with encouraging CR rates. Products were fractionated evenly between CD19 CAR, CD19+CD22 CAR and CD22 CAR. However, engraftment was predominated by the single CD19 CAR population, leading to unsuccessful eradication of CD19-CD22+ leukemia. This finding led to re-engineering the CD22 CAR construct for enhanced CD22 targeting, and re-initiation of dose finding with the new product, SCRI-CAR19v2.Methods: After enrollment, subjects undergo apheresis followed by a combined CD4/CD8 positive immunomagnetic selection and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two lentiviral vectors that encode for either a CD19- or CD22-specific CAR. After flu/cy lymphodepletion, CAR T cells are infused at one of three dose levels: 0.5, 1 or 3 X 10 6 CAR T cells/kg. Toxicity is graded according to CTCAEv5 except for CRS and ICANS which are graded per ASTCT criteria. Leukemic response and CAR T cell persistence are evaluated by flow cytometry.Results: 14 subjects enrolled onto PLAT-05 for the SCRI-CAR19x22v2 dose escalation and products were successfully manufactured in all subjects with an average of 8.9 days in culture (range 7-12 days). In contrast to v1 products, the CAR composition of v2 products was skewed in favor of CD22 CAR expression, with median expression of each population as follows: 42% CD22 only, 33% CD19 and CD22, 3.2% CD19 only. Twelve subjects were infused (0.5x10 6/kg n=3, 1x10 6/kg n=3, 3x10 6/kg n=6), 11 of whom had prior exposure to CD19 or CD22 targeted therapies with diverse expression of CD19 and CD22 on the leukemic blasts. No dose limiting toxicities occurred in the 11 fully evaluable subjects (1 subject is pending) and the recommended phase 2 dose was determined as 3x10 6 CAR + cells/kg. CRS was present in 45% of subjects, all grade 1. Neurotoxicity occurred in 45% of subjects, all grade 1 except a single self-limited grade 3 ICANS event (due to a single time point CAPD score). 91% of infused subjects obtained a CR, of which 100% were MRD negative. The non-responder had persistent disease that was CD19-CD22-. The in vivo engraftment of CAR T cells peaked most frequently between day +7 and +14 and was predominated by the CD22 CAR T cells, with some minimal contribution of dual and CD19 CAR T cells. Of the 4 subjects who had previously received an FMC63 based CD19 CAR, expansion was due to solely to the CD22 CARs in all 4 subjects, with apparent rejection of the T cells expressing CD19 CAR.Conclusions: We demonstrate enhanced activity of SCRI-CAR19x22v2 compared to v1, now with dual activity against both CD19 and CD22 demonstrated by elimination of ALL with single antigen expression. We maintained encouraging CR rates with a favorable toxicity profile. Interestingly, the product is predominated by CD22 CAR and CD19/CD22 CAR populations, while in vivo engraftment is predominated by single CD22 CAR expressing T cells. Subjects exposed to prior CD19 murine based CAR rejected the CD19 CAR but engrafted the CD22 CAR with demonstratable activity, a potential advantage of a dual transduced product. The impact of lower CD19 CAR engraftment on durable remissions is unknown. While limited expansion of the CD19 CAR population could be protective against exhaustion, the uneven engraftment of the CAR populations may ultimately lead to single antigen targeting. Optimization of transduction may be required for a more balanced product to maintain dual targeting and give further insight into the behavior of dual-expressing CAR T cells. An expansion cohort is currently underway to further characterize engraftment kinetics and in vivo performance to best inform future development of this product. [Display omitted] DisclosuresPulsipher: Jasper Therapeutics: Honoraria; Adaptive: Research Funding; Equillium: Membership on an entity's Board of Directors or advisory committees. Li: Novartis Canada: Membership on an entity's Board of Directors or advisory committees. Jensen: Bluebird Bio: Research Funding; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Patents & Royalties. Gardner: Novartis: Consultancy; BMS: Patents & Royalties. OffLabel Disclosure:investigational use of SCRI-CAR19x22 will be discussed

Procédé de préparation, contrôles de qualité et spécifications des immunosélections CD34+ : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC)

CD34+ immunomagnetic positive selection allows for CD34+ hematopoietic progenitors separation from CD3+ lymphocytes subsets, usually from an apheresis product collected from a previously mobilized donor. This T-cell depleted stem cell graft is primarily intended for rare cases (around 2% of allotransplanted patients in France) of severe, persistent, symptomatic bi- or tri-cytopenia post-allotransplantation, in order to allow for hematologic reconstitution without increasing the risk of GvHD occurrence. Although semi-manual and complex, the process is of sufficient robustness to consistently generate a cellular product with distinctive features and specifications, based on iterative in-process quality controls, that are discussed within these guidelines.

Abstract 5366: Metastatic breast cancer subgroup specific mRNA profiles of circulating tumor cells and extracellular vesicles

Abstract Background: We recently demonstrated great differences in the RNA profiles of matched circulating tumor cells (CTCs) and extracellular vesicles (EVs) in HER2 negative (HER2-) hormone receptor positive (HR+) metastatic breast cancer (MBC) patients. To elucidate the clinical utility of CTC and EV RNA profiling, we here a) tested the RNA profiles of another MBC subgroup, namely the HER2 positive (HER2+) patients, and b) compared the profiles in the two cohorts. Patients and methods: Blood was collected from 25 HER2+ (HR+ and HR-) and 35 HER2- (only HR+) MBC patients at the time of disease progression and at two consecutive staging time points. CTCs were isolated from 5 ml blood by positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select). EVs were isolated from 4 ml pre-filtered plasma by affinity-based binding to a spin column (exoRNeasy). The mRNA was purified by Oligo-dT beads and reverse transcribed (AdnaTest EMT2/StemCell Detect). Pre-amplified cDNA was analyzed by multimarker qPCR assays (AdnaTest TNBC Panel prototype) for 18 genes (including AKT2, ALK, AR, AURKA, BRCA1, KIT, MET, EGFR, ERCC1, ERBB2, ERBB3, KRT5, mTOR, NOTCH1, PARP1, PIK3CA, SRC, GAPDH). Consumables: QIAGEN, Germany. Results: In the HER2+ cohort, transcripts involved in the PI3K pathway were significantly more prevalent in CTCs compared to EVs and were found in more than 50% of these patients. In contrast, NOTCH1 and PARP1 were significantly more prominent in EVs as compared to their matched CTCs and EV PARP1 and EV AURKA overexpression signals were detected in more than 50% of patients. ERCC1 and SRC signals were present in CTCs as well as EVs in the majority of patients. ERCC1 signals in CTCs and EVs showed a significantly higher frequency in the HER2+ compared to the HER2-HR+ cohort. However, ERCC1 signals in CTCs of the HER2-HR+ cohort correlated with shorter survival time after first diagnosis of metastasis (p=0.011). In general, the overexpression signal prevalence was similar in both cohorts and both blood analytes. A small overlap of identical signals in CTCs and EVs of 15%/5% in the HER2+/HER2-HR+ cohort was observed. Interestingly, ERBB2 signals were only found in CTCs, not EVs, and in both cohorts with a similar prevalence (36%/40%). ERBB3 overexpression signals in CTCs significantly correlated with non-response only in the HER2-HR+ cohort (p=0.021). However, in HER2+ patients ERBB3 signals were only present at the time of progressive disease, but not at the time point of stable disease. Conclusions: RNA profiling indicated great differences in CTCs and EVs in both subgroups. Moreover, data emphasize the immense diversity between HER2+ versus HER2-HR+ MBC patients, revealing the need for blood analyte specific and subgroup specific multimarker panel for therapy management based on liquid biopsies in the future. Citation Format: Corinna Keup, Siegfried Hauch, Mitra Tewes, Hans-Christian Kolberg, Oliver Hoffmann, Rainer Kimmig, Sabine Kasimir-Bauer. Metastatic breast cancer subgroup specific mRNA profiles of circulating tumor cells and extracellular vesicles [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 5366.

Abstract P4-01-11: Circulating tumor cells expressing the prostate specific membrane antigen PSMA indicate worse outcome in triple-negative breast cancer patients at primary diagnosis

Abstract Background: Triple negative breast cancer (TNBC) is known for its aggressive behavior and poor prognosis since treatment options are limited. Specific biomarkers are urgently needed to treat patients (pts) accordingly. In this regard, circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options. We here comprehensively analyzed mRNA profiles of CTCs from TNBC pts before and after neoadjuvant chemotherapy including the expression of prostate related genes [androgen receptor (AR), its splice variant ARV7, the prostate specific antigen (PSA) and the prostate specific membrane antigen (PSMA)] to further elucidate these markers as targets for personalized treatment. Methods: 2 × 5 ml blood of 34 TNBC pts before and 22 pts after therapy were analyzed for CTCs applying positive immunomagnetic selection using the AdnaTest EMT-2/Stem Cell Select (QIAGEN, Germany). Subsequently, cDNA was pre-amplified for specific genes using TaqMan PreAmp Master Mix according to in house designed assays. A 19 gene qPCR panel was performed including AKT2, ALK, AR, AURKA, BRCA1, EGFR, ERCC1, ERBB2, ERBB3, KIT, KRT5, MET, MTOR, NOTCH1, PARP1, PIK3CA, SRC, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taking the false positive rate in healthy donors into account, and defined as Ct (cutoff) - Ct (sample) - [Ct (CD45cutoff) - Ct (CD45sample)]. Using the AdnaPanel Prostate Cancer (QIAGEN, Germany), cDNA was further analyzed for the expression profile of AR, ARV7, PSA and PSMA, respectively. Results: Before therapy, at least one of the prostate related genes was detected in 50% of the pts, resulting in the expression of AR in 35%, ARV7 in 24%, PSMA in 18%, and PSA in 15% of cases, respectively. Notably, in 75% of AR-positive cases, ARV7 was also expressed on CTCs. After therapy, prostate related genes were only detected in two pts, expressing AR, ARV7 and PSA (n=1) and AR (n=1), respectively. Before therapy, the presence of PSMA-positive CTCs alone (p=0.026) or in combination with ARwildtype/ARV7-positive CTCs (p=0.014) significantly correlated with early relapse. Interestingly, this expression pattern was highly related to the expression of genes belonging to the ERBB family (EGFR, ERBB2, ERBB3; p=0.034). Either ERRB family or PSMA expression on CTCs in TNBC was detected in about 70% of the cases analyzed with 90% relapses predicted. Conclusion: Although AR and its splice variant were detected on CTCs in one third of the pts before therapy, these markers do not seem to be relevant with regard to be considered as targets for additional treatment options. In contrast, although expressed in a minority of pts, PSMA-positive CTCs at primary diagnosis indicated worse outcome and should be included in CTC-multimarker panel analysis to identify pts at higher risk for relapse. This study is ongoing and the analysis of more pts will finally show whether these results can be confirmed. Citation Format: Sabine Kasimir-Bauer, Corinna Keup, Oliver Hoffmann, Siegfried Hauch, Rainer Kimmig, Ann-Kathrin Bittner. Circulating tumor cells expressing the prostate specific membrane antigen PSMA indicate worse outcome in triple-negative breast cancer patients at primary diagnosis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-11.

Analysis of Deficiency of Adenosine Deaminase 2 Pathogenesis Based on Single Cell RNA Sequencing of Monocytes

Deficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive disease caused by loss-of-function mutations in the ADA2 gene. DADA2 typically presents in childhood and is characterized by vasculopathy, stroke, inflammation, and immunodeficiency as well as hematologic manifestations, such as bone marrow failure and lymphoproliferation. The ADA2 protein is predominantly expressed in stimulated monocytes, dendritic cells and macrophages. ADA2 increases in the setting of inflammation and/or infection conditions. ADA2 has been reported to have a critical role in maintaining the balance between M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. Macrophages of DADA2 patients are polarized towards M1 subset., DADA2 pathogenesis is not well characterized. To elucidate molecular mechanisms in DADA2 deficiency, we analyzed a gene expression profile of CD14+ monocytes derived from peripheral blood using single cell RNA sequencing (scRNA-seq). Blood was collected from DADA2 patients and age- and sex-matched healthy donors; all patients were studied in a registered research protocol (clinicaltrials.gov NCT00071045). Samples were obtained from 14 DADA2 patients and 6 healthy donors; median age of the DADA2 patients was 23 years old (range, 5 - 57 years). Among the 14 patients, 7 had hematological phenotypes: 5 lymphopenia, 3 neutropenia, 3 thrombocytopenia, and 2 with hypocellular bone marrow histology. Low serum immunoglobulins and cutaneous findings were frequent. Nine of the 14 patients had been treated with TNF inhibitors (etanercept and adalimumab). Mutations were distributed throughout the ADA2 gene; although two siblings had the same mutation, even they showed poor genotype-phenotype correlation. Monocytes were isolated by immunomagnetic positive selection with the EasySep™ positive CD14 selection kit Ⅱ, then subjected to scRNA-seq using Single Cell 3' Reagent Kits v2 (10X Genomics). Libraries for scRNA-seq were sequenced on the HiSeq-3000 instrument. Based on scRNA-seq data, we could classify monocytes into three populations by conventional flow cytometric criteria using cell surface protein expression imputed from scRNA-seq: CD14++CD16- classical, CD14++CD16+ intermediate, and CD14+CD16++ nonclassical monocytes (Figure A). CD16 expression was higher in DADA2 patients than in healthy donors (Figure B). A proportion of nonclassical monocytes among total monocytes were significantly higher in DADA2 patients compared to healthy donors (Figure C). On comparison of gene expression of each monocyte subtypes in DADA2 patients with that of healthy donors, there were 215, 237, and 267 differentially expressed upregulated genes in classical, intermediate, and nonclassical monocytes, respectively (at a threshold avg_logFC > 0.2). Approximately 35% of upregulated genes were overlapped among the three monocyte subtypes of DADA2 patients, including immune response genes such as IFITM1, IFITM2, IFITM3, and C3AR1 (Figure D). Common gene pathways were associated with immune function, such as interferon alpha/beta signaling and interferon gamma signaling. Specific genes to classical and intermediate monocytes were less than 10% of all the upregulated genes. Distinctively, the NF-κB pathway was upregulated in nonclassical monocytes, this might contribute to the pathogenesis of DADA2 as inflammatory disease. Overall, each monocyte subtype of DADA2 patients showed upregulation of immune response gene sets compared to controls. DADA2 patients have increased numbers of nonclassical monocytes which may contribute the immune dysregulation and increased inflammation observed in the disease. Figure Disclosures No relevant conflicts of interest to declare.

PF539 MIR‐223 IS SUPPRESSED IN EXOSOMES OF PATIENTS WITH HIGH‐RISK MYELODYSPLASTIC SYNDROMES AND MAY BE A MARKER OF RESIDUAL GRANULOCYTIC‐MONOCYTIC DIFFERENTIATION

Background:Quantification of exosomal miRNAs in body fluids represents a prominent source of biomarkers for a variety of conditions. Several miRNAs have been described as potential predictors of severity and progression in neoplastic diseases, pointing to a class of less invasive and readily accessible prognostic tools. Among them, mir‐223 has been studied with contradictory findings: mir‐223 has been shown to play an important role in quimiorresistence in solid tumors (Cheng Q, et al., Mol Med Rep. 2017;15:597–604); however, suppression of mir‐223 in Acute Myeloid Leukemia (AML) is associated with lack of differentiation and adverse cytogenetic profile (Akhter A, et al., Appl Immunohistochem Mol Morphol, 2015;23:733–9).Aims:The aim of this study was to evaluate the expression of mir‐223 in exosomes extracted from serum of patients with myelodysplastic syndromes (MDS), AML and healthy blood donors and to explore the role of mir‐223 in the differentiation of human hematopoietic stem cells.Methods:Samples of venous blood were obtained from 14 patients with AML, 28 patients with MDS (prior to any treatment), and 19 healthy controls. Exosomes were isolated using the ExoQuick® precipitation solution; their concentration and size distribution were characterized by Nanoparticle Tracking Analysis (NanoSight, UK). Exosomal RNA was prepared using miRNeasy Micro Kit and submitted to reverse transcription with miScript II RT kit (QIAGEN, USA) and expression levels of mir‐223 and mir‐192 (endogenous control) were assessed by RT‐PCR using specific primers. CD34+ progenitors were purified from human umbilical cord blood using positive immunomagnetic selection and expanded in culture medium supplemented with G‐CSF, IL‐3 and SCF. The expression levels of mir‐223 were analyzed in cultured cells on days 0, 7, and 14; expression of CD34, CD14 and CD15 was verified by flow cytometry and morphologic changes were observed by examining Wright‐Giemsa‐stained cytospin smears.Results:The concentration of exosomes in serum samples ranged from 5.6 x 1011 to 3.3 x 1013 particles per μl of liquid suspension, with significantly lower concentrations in the patient samples in relation to healthy individuals (p = 0.004). A statistically significant decrease in the expression of mir‐223 was observed in exosomes from individuals with high‐risk MDS compared to healthy subjects (p = 0.002) and to patients with low‐risk MDS (p = 0.012). Interestingly, in the total group of patients, there was a statistically significant positive correlation between the expression of mir‐223 in the exosomes and countings of: total leukocytes (Spearman's r s = 0.37, p = 0.003), neutrophils (r s = 0.392, p = 0.002), monocytes (r s = 0.484, p < .001), lymphocytes (r s = 0.442, p < .001) and platelets (r s = 0.387, p = 0.002) in peripheral blood. During granulocytic‐monocytic differentiation of normal human CD34+ cells, we observed a 4‐fold increase in mir‐223 expression in cultured cells after 7 days, reaching almost 6‐fold increase after 14 days of culture, with granulocytic‐monocytic differentiation demonstrated by cytology and immunophenotyping with CD14 and CD15 markers.Summary/Conclusion:The results here obtained showed that levels of mir‐223 in peripheral blood exosomes are suppressed in patients with high‐risk MDS and correlate with cell counts in both MDS and AML. As mir‐223 levels progressively increase during normal granulocytic‐monocytic differentiation, we speculate that mir‐223 may represent a marker of residual differentiation in MDS and AML.

Automated Manufacturing of CD20.19 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cells at an Academic Center for a Phase I Clinical Trial in Relapsed, Refractory NHL

Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, but limitations exist with off-site commercial CAR-T production: (1) manufacturing can take several weeks and requires shipping patient cells; (2) off-site manufacturing limits treatment options for patients with rapidly progressive disease; (3) high cost of the products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy GMP-compliant device to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR). Using reagents from Miltenyi Biotec, production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory. Manufacturing time was 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) collected from patients by apheresis were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. The T cells were cultured in 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. Next day, lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3z and 4-1BB stimulatory domains was added to the cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on day 14. Eligible patients either received fresh CAR-T cells, or cells that had been cryopreserved and administered on a later date. CAR-T cell products have been successfully generated for all 8 patients enrolled thus far on the Phase 1 clinical trial with no production failures, and all products passed release criteria for infusion. Three patients received cryopreserved product and 5 patients received fresh product. Average T cell recovery from the apheresis cell products was 66.0% (56.0-76.0%), and the enriched T cells were 94.3% CD3+ (87.8-97.4%) (Table 1). Protein L staining of the final cell products indicated 19.6% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFNg upon antigen stimulation. An average yield of 5.27e+8 CAR T cells was obtained at harvest, exceeding the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with greater percentages of CD4 T cells. The majority of T cells (avg. 83.2%) had an effector-memory phenotype. In summary, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. A major clinical advantage of CAR-T cells generated on-site is flexibility in treatment, with either immediate (i.e., fresh) or later (cryopreserved/thaw) infusion, dependent on patient need.

Point-of-Care Manufacturing of CD20.19 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cells in a Standard Academic Cell Processing Facility for a Phase I Clinical Trial in Relapsed, Refractory NHL

Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, which has led to FDA approvals for two CAR-T products. However, limitations exist with commercial CAR-T centralized production: (1) off-site manufacturing can take several weeks and requires shipping from and to the treating facility; (2) off-site manufacturing limits treatment options for progressing patients; (3) high cost of the commercial products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy device, a GMP-compliant closed system, to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR).CAR-T manufacturing was performed exclusively using the CliniMACS Prodigy device and reagents obtained from Miltenyi Biotec. Production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory, an ISO7 air handling environment. Manufacturing was set at 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) were collected by apheresis, with a collection goal of 4 blood volumes to eliminate risk of a low CD3 yield in heavily pre-treated patients. Next, MNC were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. To start the culture process, enriched T cells were suspended in TexMACS medium supplemented with 3% human AB serum and 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. The following day (day 1), lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3ζ and 4-1BB stimulatory domains was added to the stimulated cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on Day 14. Protein L staining was used to detect expression of CD20.19 CAR on the T cells. On Day 14, eligible patients received fresh CAR-T cells, while for others the product was cryopreserved and administered on a later date.To date, the MCW Cell Therapy Laboratory has successfully generated CAR-T cell products for all 6 patients enrolled thus far on the Phase 1 clinical trial with no production failures (Table 1). Three patients received cryopreserved product and 3 patients received fresh product. The enriched T cells were 94.3% CD3+ (87.8-97.4%), and average T cell recovery from the apheresis cell products was 65.2% (54.2-80.0%). Protein L staining indicated 20.8% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFN-gamma in response to the same target cells. An average yield of 5.9e+8 (4.3-7.9e+8) CAR T cells was obtained at harvest, which exceeded the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with higher expression on CD4 T cells; average CAR-T CD4:CD8 ratio on the final products was 2.8. The majority of T cells (average of 81.5%) had an effector-memory phenotype. In-process testing performed on Day 8 of manufacturing demonstrated sufficient numbers of CAR-T cells needed for patient infusions were already present, and that the CAR-T cells only expanded an additional 1.9 to 3.5-fold between Days 8 and 14.In conclusion, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. Time to production was efficient (14 days), and patient-derived CAR-T cell products were reproducibly generated in a standard cell processing laboratory within an academic medical center. A major clinical advantage of CAR-T cells generated on-site is the flexibility in treatment. Patients can receive cells either immediately (i.e., fresh) or the cells can be cryopreserved for later infusion if the patient is not able to receive fresh cells. Based on our results, we intend to decrease the cell processing time to 10 days. [Display omitted] DisclosuresZhu:Lentigen Technology Inc., A Miltenyi Biotec Company: Research Funding. Shah:Juno Pharmaceuticals: Honoraria; Oncosec: Equity Ownership; Geron: Equity Ownership; Exelexis: Equity Ownership; Miltenyi: Other: Travel funding, Research Funding; Lentigen Technology: Research Funding. Schneider:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Keever-Taylor:Medical College of Wisconsin: Research Funding. Dropulic:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Hari:Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Kite Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding. Johnson:Miltenyi: Research Funding.

Improved Expansion and Function of CAR T Cell Products from Cultures Initiated at Defined CD4:CD8 Ratios

▪BACKGROUND: Current manufacturing paradigms for chimeric antigen receptor (CAR) modified T cells require ex vivo T cell activation, genetic modification, and expansion in cytokine-containing cell cultures. Most CAR T cell studies infuse cell products generated from unselected cells, in which the CD4:CD8 ratio is determined by what is collected during leukapheresis. The proportion of each subset can vary greatly in these products, reflecting the high heterogeneity of the CD4:CD8 ratio present in patients' (pts) blood at the time of treatment. Preclinical data demonstrate superior in vivo anti-tumor efficacy of cell products consisting of equal numbers of CD4+ and CD8+ T cells, and a recent clinical trial, in which CD19-targeted CAR T cell products were infused at a 1:1 ratio of CD4:CD8 T cells, showed clear dose-response and dose-toxicity relationships. However, CAR T cell manufacturing using parallel CD4+ and CD8+ CAR T cell cultures adds significant cost and complexity compared with a single-stream culture. Additionally, we have found that CD8+ T cell cultures from heavily treated pts often exhibit suboptimal expansion. We therefore evaluated whether CAR T cell products with approximately equal ratios of CD4+ to CD8+ cells could be generated by mixing CD4+ and CD8+ cells at a defined ratio at culture initiation, and whether the presence of ex vivo CD4+ help can improve CD8+ cell expansion.METHODS: CD4+ and CD8+ T cells were isolated from apheresis peripheral blood mononuclear cell products of lymphoma pts (n=15) treated in one of three CD20-targeted CAR T cell trials or healthy donors (n=5) using positive (CD4) and negative (CD8) immunomagnetic bead selection. Cell cultures (1x106 cells each) were established by activating CD4+ and CD8+ cells with anti-CD3/CD28-coated paramagnetic beads, mixing them at various ratios in 24-well plates, and transducing 24 hours later with a lentivirus encoding the 1.5.3-NQ-28-BB-z CD20 CAR. Beads were removed at day 4, and cells were expanded in IL-2 containing medium. At day 7, cells were counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, and then restimulated 1:1 with irradiated CD20+ lymphoblastoid cells to boost growth. On day 14 cells were again counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, as well as immunophenotype. In some cases CAR+CD4+ and CAR+CD8+ cells were flow-sorted from either CD4-only, CD8-only, and mixed cultures, stained with CFSE, and restimulated with irradiated CD20+ Raji tumor cells. Proliferation and cytokine secretion were measured by flow cytometry and Luminex, respectively. In vivo T cell activity was assessed using an NSG mouse model in which T cells were infused 7 days after i.v. injection of Raji-ffLuc cells. Suboptimal doses of T cells were used to distinguish differences between conditions, since higher doses cure most mice. Mice received either: (1) 70:30 mixed CD4:CD8 CAR+ cultures (n=8), (2) 1:1 ratio of CD4:CD8 CAR+ cells grown separately (n=8), (3) Mixed CD4:CD8 empty vector cells (n=5), or (4) no treatment (n=5).RESULTS: An initial CD4:CD8 ratio of 70:30 yielded a median CD4/CD8 ratio of 1.1 for pts (Figure 1A) and 1.3 for donors at day 7, and a ratio of 0.6 and 1.0 for pts and donors, respectively, at day 14. Mixed cultures resulted in CD8+ cell expansion that was significantly higher than in separate cultures. At day 7, mean CD8+ cell expansion was 12.1-fold vs. 4.6-fold for 70:30 mixed vs. separate cultures for donors, and 2.9-fold vs. 0.7-fold for pts (Figure 1B). At day 14 CD8+ mean cell expansion was 105-fold (70:30 mixed) vs. 25-fold (separate) for donors and 40-fold vs. 1.9-fold for pts. CD8+ cells grown in mixed cultures also exhibited higher expression of memory markers (CD62L and CCR7), lower levels of exhaustion markers (Lag-3 and PD-1), and better proliferation compared with CD8 cells grown in separate cultures. In the mice we found that tumor growth inhibition was superior in the 70:30 mixed culture group than in mice receiving a 1:1 ratio of CD4:CD8 cells grown separately (Figure 1C).CONCLUSIONS: A single-stream CAR T cell culture initiated at a defined CD4:CD8 ratio of 70:30 yielded on average approximately equal numbers of CD4+ and CD8+ cells in the final cell product. Mixed CD4+ and CD8+ cultures generated CD8+ T cells with a less differentiated phenotype, and superior expansion, proliferative capacity, and in vivo activity compared with CD4+ and CD8+ cells manufactured in parallel. [Display omitted] DisclosuresGreen:Juno Therapeutics: Patents & Royalties, Research Funding. Till:Mustang Bio: Patents & Royalties, Research Funding.