CD34 Stem Research Articles

Association of electronic cigarette use with circulating angiogenic cell levels in healthy young adults: Evidence for chronic systemic injury.

Circulating angiogenic cells (CACs) are indicative of vascular health and repair capacity; however, their relationship with chronic e-cigarette use is unclear. This study aims to assess the association between e-cigarette use and CAC levels. We analyzed CAC levels in 324 healthy participants aged 21-45 years from the cross-sectional Cardiovascular Injury due to Tobacco Use study in four groups: never tobacco users (n = 65), sole e-cigarette users (n = 19), sole combustible cigarette users (n = 212), and dual users (n = 28). A total of 15 CAC subpopulations with four cell surface markers were measured using flow cytometry: CD146 (endothelial), CD34 (stem), CD45 (leukocyte), and AC133 (early progenitor/stem). Generalized linear models with gamma distribution and log-link were generated to assess association between CACs and smoking status. Benjamini-Hochberg were used to adjust p-values for multiple comparisons. The cohort was 47% female, 51% Black/African American, with a mean (± SD) age of 31 ± 7 years. Sole cigarette use was significantly associated with higher levels of two endothelial marker CACs (Q ⩽ 0.05). Dual users had higher levels of four endothelial marker CACs and one early progenitor/stem marker CAC (Q ⩽ 0.05). Sole e-cigarette users had higher levels of one endothelial and one leukocyte marker CAC (Q ⩽ 0.05). Dual use of e-cigarettes and combustible cigarettes was associated with higher levels of endothelial origin CACs, indicative of vascular injury. Sole use of e-cigarettes was associated with higher endothelial and inflammatory CACs, suggesting ongoing systemic injury. Distinct patterns of changes in CAC subpopulations suggest that CACs may be informative biomarkers of changes in vascular health due to tobacco product use.

Trends in Collection and Utilization of Autologous Hematopoietic Stem Cells (APBHC) Intended for Transplant in Patients with Multiple Myeloma

Background Autologous stem cell transplant (ASCT) remains a mainstay of therapy in Multiple Myeloma (MM) management. At the time of stem cell collection, it is recommended to collect stem cells for two transplants. However, the use of stem cells for a tandem and salvage auto transplant has decreased over time. Here, we evaluate the trends in stem cell collection and utilization over the period of 2014 to mid 2020 at a large urban medical center. Methods We identified 155 patients with MM in the EPIC database at Montefiore Medical Center who had their stem cells collected with the purpose of autologous stem cell transplant from January 2014 to July 2020. We extracted data on patient characteristics (age, sex, race, and ethnicity), treatment factors (time from diagnosis to stem cell mobilization and induction regiments), as well as MM disease characteristics (heavy and light chain subtypes) and compared them to stem cell utilization, the number of ASCTs received, to identify trends. Results Patient demographics and information on disease characteristics are shown in Table 1. From January 2014 to July 2020, 155 patients underwent at least one apheresis session. The median number of apheresis sessions was 1 (Range 1-4). The median number of CD34 stem cells collected was 7.51 x10^6 CD34 cells/kg (2.82-33.41). The median time from MM diagnosis to apheresis was 225 days (range 82-1713). Chemo-mobilization was done in 12 patients (7.7%). Plerixafor was used in 74 patients (47.7%). Excluding those requiring chemo-mobilization, it took median of 5 days from the initiation of mobilization to collection (range 4-9). Of the 155 patients, 150 have undergone a transplant. To this date, 69 (44.5%) had a tandem transplant and only 5 patients (3.2%) had a salvage auto transplant. 104 patients (67.1%) had stem cells left. The median number of infused CD34 cells was 3.84 x10^6 CD34 cells/ kg (2.4-16.4) for the first transplant and 3.7 x 10^6 CD34 cells/ kg (2.3-9.77) for the second transplant. Table 1 shows the number of patients undergoing tandem transplant over the years. The median overall survival (OS) for the entire cohort was 34.4 months (156-2909 days). 131 patients (84.5%) are alive at the time of last follow-up. Conclusion Our study highlights the stem cell collection and utilization trends for Multiple Myeloma at a tertiary care hospital that is responsible for care of minority ethnic groups. Our data shows significant utilization of tandem transplant, however, the use is decreasing over the years. More than half of our patients still have cryopreserved stem cells left and will possibly not be used for a second transplant. It may be time to revise our apheresis goal for myeloma patients since a significant portion of patients may not end up going to a second autologous stem cell transplant. However, the utilization of stem cells in other settings is also evolving. Although tandem and salvage transplant have fallen out of favor, it is possible that with the advent of newer therapies such as CAR-T cells and bispecific T-cell engagers, these stem cells may be used in alternative setting post treatment for prolonged cytopenia. Our next steps would be to highlight the outcomes of patients undergoing tandem transplant at our center as compared to those undergoing one autologous stem cell transplant as well as report on the trends of stem cell use post novel-therapies such CAR-T cell therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MM-531 Case Control Study of Outcomes of Autologous Stem Cell Transplantation in Jehovah's Witness Multiple Myeloma Patients

Jehovah's Witnesses (JW) persons do not accept blood transfusions. Autologous stem cell transplantation (ASCT) after using high-dose chemotherapy is the standard of care in treatment of multiple myeloma (MM). Patients usually require blood transfusions during ASCT. We sought to analyze safety outcomes of single or tandem ASCT in JW MM patients. We performed a case control study at the University of Arkansas for Medical Sciences for JW and non-JW MM patients who underwent single or tandem ASCT from 1990-2021. Matching (1:2) was done based on age, date of MM diagnosis, race/ethnicity, performance status, stage indicated by International Staging System (ISS), and number of ASCT. Wilcoxon Rank-Sum test was used to test continuous variables, while Fisher's exact test was used for categorical variables. A total of 13 JW MM patients received ASCT in the study period (8 single, 5 tandem) compared to 26 non-JW MM patients (16 single, 10 tandem). For JW patients who received single ASCT, median age was 66 (range:65.8-67.5 years), 50% were female, 62.5% were black. Average CD34 stem cells collected/infused were 23/5.8 million cells in JW compared to 30.1/5.1 million cells in non-JW, respectively. For JW patients who received tandem ASCT, median age was 49 (range:46-52 years), 50% were female, 62.5% were black. Average CD34 stem cells collected/infused were 34/10.8 million in JW compared to 21.3/8.8 million in non-JW, respectively. Lowest hemoglobin and platelets level in JW were 6.6 g/dl and 3000 k/uL, respectively. 100-days mortality for all patients was 0%. Multivariable analysis showed no significant difference in 30 days/100 days re-hospitalization, major and/or minor bleeding adverse events, response to treatment (very good partial response or better). Bloodless single/tandem ASCT can be done safely for selected JW MM patients with no significant difference in safety/outcomes.

The Long Non-coding RNA HOTAIR Controls the Self-renewal, Cell Senescence, and Secretion of Anti-aging Protein α-Klotho in Human Adult Renal Progenitor Cells.

The long non-coding RNAs (lncRNA) play an important role in several biological processes, including some renal diseases. Nevertheless, little is known about lncRNA that are expressed in the healthy kidneys and involved in renal cell homeostasis and development, and even less is known about lncRNA involved in the maintenance of human adult renal stem/progenitor cells (ARPCs) that have been shown to be very important for renal homeostasis and repair processes. Through a whole-genome transcriptome screening, we found that the HOTAIR lncRNA is highly expressed in renal progenitors and potentially involved in cell cycle and senescence biological processes. By CRISPR/Cas9 genome editing, we generated HOTAIR knockout ARPC lines and established a key role of this lncRNA in ARPC self-renewal properties by sustaining their proliferative capacity and limiting the apoptotic process. Intriguingly, the HOTAIR knockout led to the ARPC senescence and to a significant decrease in the CD133 stem cell marker expression which is an inverse marker of ARPC senescence and can regulate renal tubular repair after the damage. Furthermore, we found that ARPCs expressed high levels of the α-Klotho anti-aging protein and especially 2.6-fold higher levels compared to that secreted by renal proximal tubular cells (RPTECs). Finally, we showed that HOTAIR exerts its function through the epigenetic silencing of the cell cycle inhibitor p15 inducing the trimethylation of the histone H3K27. Altogether, these results shed new light on the mechanisms of regulation of these important renal cells and may support the future development of precision therapies for kidney diseases.

Glycidamide and cis-2-butene-1,4-dial (BDA) as potential carcinogens and promoters of liver cancer - An in vitro study.

Acrylamide and furan are environmental and food contaminants that are metabolized by cytochrome P450 2E1 (CYP2E1), giving rise to glycidamide and cis-2-butene-1,4-dial (BDA) metabolites, respectively. Both glycidamide and BDA are electrophilic species that react with nucleophilic groups, being able to introduce mutations in DNA and perform epigenetic remodeling. However, whereas these carcinogens are primarily metabolized in the liver, the carcinogenic potential of acrylamide and furan in this organ is still controversial, based on findings from experimental animal studies. With the ultimate goal of providing further insights into this issue, we explored in vitro, using a hepatocyte cell line and a hepatocellular carcinoma cell line, the putative effect of these metabolites as carcinogens and cancer promoters. Molecular alterations were investigated in cells that survive glycidamide and BDA toxicity. We observed that those cells express CD133 stemness marker, present a high proliferative capacity and display an adjusted expression profile of genes encoding enzymes involved in oxidative stress control, such as GCL-C, GSTP1, GSTA3 and CAT. These molecular changes seem to be underlined, at least in part, by epigenetic remodeling involving histone deacetylases (HDACs). Although more studies are needed, here we present more insights towards the carcinogenic capacity of glycidamide and BDA and also point out their effect in favoring hepatocellular carcinoma progression.

Role of large unstained cells in predicting successful stem cell collection in autologous stem cell transplantation

IntroductionSufficient stem cell collection is mandatory for Autologous stem cell transplantation (ASCT). Peripheral CD34+ stem CD34 + stem cell counting by flow cytometry is the gold standard method in both the predicting and timing of successful stem cell collection. Large unstained cells (LUC) are large peroxidase-negative cells that are displayed on certain automatic cell counters and present large lymphocytes, virocytes, blasts, abnormal cells and hematopoietic stem cells. In this study, we evaluated the role of LUC parameters in the timing and prediction of successful stem cell collection. MethodsPatients with a diagnosis of multiple myeloma, lymphoma and testis tumor who proceed to ASCT were included in this study. Preapheresis LUC parameters were analyzed with Siemens ADVIA® 2120i system., Kruskal Wallis, Mann-Whitney U, Spearman Rho and receiver–operator curve (ROC) tests were used for analyses. ResultsNinety patients were evaluated. Peripheral CD34 + cell count was positively correlated with both LUC count (p = 0014) and LUC percentage (p = 0,01). LUC percentage in peripheral blood was positively correlated with mobilized stem cell count in the yield (p = 0.003). We found a LUC count of > 0.485 × 109/L as a cut-off value for detecting > 20 × 106/L CD34 +cells in the peripheral blood with a sensitivity of 64.6% and specificity of 75%. We defined > 2.15% as a cut-off value for LUC percentage to collect > 5 × 106/kg of stem cells with a sensitivity of 64% and specificity of 63%. Additionally, total nucleated cell (TNC) count was negatively correlated with LUC percentage (p = 0.014) and positively correlated with LUC count (p = 0.001). ConclusionLUC parameters are readily available, simple and cheap tools that can be useful in both timing of CD34 count by flow cytometry in peripheral blood and in the prediction of successful mobilization. LUCs can also be an indicator of graft composition.

A novel nitidine chloride nanoparticle overcomes the stemness of CD133+EPCAM+ Huh7 hepatocellular carcinoma cells for liver cancer therapy

BackgroundStemness of CD133+EPCAM+ hepatocellular carcinoma cells ensures cancer resistance to apoptosis,which is a challenge to current liver cancer treatments. In this study, we evaluated the tumorcidal activity of a novel nanoparticle of nitidine chloride (TPGS-FA/NC, TPGS-FA: folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate, NC: nitidine chloride), against human hepatocellular carcinoma (HCC) cell line Huh7 growth in vitro and in vivo.MethodsHuh7 cells were treated with TPGS-FA/NC. Cell proliferation was assessed using MTT and colony assays. The expression of cell markers and signaling proteins was detected using western blot analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Huh7 cells. TPGS-FA/NC (7.5, 15, 30, 60, 120 μg/mL) dose-dependently inhibited the proliferation of HCC cells, which associated with a reduction in AQP3 and STAT3 expression. Importantly,TPGS-FA/NC (10, 20, and 40 μg/mL) significantly reduced the EpCAM+/CD133+cell numbers, suppressed the sphere formation. The in vivo antitumor efficacy of TPGS-FA/NC was proved in Huh7 cell xenograft model in BALB/c nude mice, which were administered TPGS-FA/NC(4 mg· kg − 1· d − 1, ig) for 2 weeks.ResultsTPGS-FA/NC dose-dependently suppressed the AQP3/STAT3/CD133 axis in Huh7 cells. In Huh7 xenograft bearing nude mice, TPGS-FA/NC administration markedly inhibited Huh7 xenograft tumor growth .ConclusionsTPGS-FA/NC inhibit HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the clinical therapy of hepatocellular carcinoma.

ScPCOR-seq enables co-profiling of chromatin occupancy and RNAs in single cells

Cell-to-cell variation in gene expression is a widespread phenomenon, which may play important roles in cellular differentiation, function, and disease development1–9. Chromatin is implicated in contributing to the cellular heterogeneity in gene expression10–16. Fully understanding the mechanisms of cellular heterogeneity requires simultaneous measurement of RNA and occupancy of histone modifications and transcription factors on chromatin due to their critical roles in transcriptional regulation17,18. We generally term the occupancy of histone modifications and transcription factors as Chromatin occupancy. Here, we report a technique, termed scPCOR-seq (single-cell Profiling of Chromatin Occupancy and RNAs Sequencing), for simultaneously profiling genome-wide chromatin protein binding or histone modification marks and RNA expression in the same cell. We demonstrated that scPCOR-seq can profile either H3K4me3 or RNAPII and RNAs in a mixture of human H1, GM12878 and 293 T cells at a single-cell resolution and either H3K4me3, RNAPII, or RNA profile can correctly separate the cells. Application of scPCOR-seq to the in vitro differentiation of the erythrocyte precursor CD36 cells from human CD34 stem or progenitor cells revealed that H3K4me3 and RNA exhibit distinct properties in clustering cells during differentiation. Overall, our work provides a promising approach to understand the relationships among different omics layers.

P1343: TRANSPLANT OUTCOMES OF LYMPHOMA PATIENTS UNDERGOING STEM CELL MOBILIZATION WITH PLERIXAFOR

Background: Autologous stem cell transplantation is a standard treatment modality widely used in relapsed refractory Hodgkin and Non-Hodgkin Lymphoma. In order to perform an autologous stem cell transplantation, it is necessary to collect sufficient stem cells. The accepted minimal number of stem cells that will provide the reconstruction of hematopoiesis after high-dose chemotherapy is >2x106/kg CD34(+), and the optimal number is >5x106/kg CD34(+) stem cells. Aims: In this study, we aimed to compare the engraftment kinetics and long-term survival data after autologous stem cell transplantation in lymphoma patients who underwent stem cell mobilization with plerixafor. Methods: Between 2006 and 2020, 138 lymphoma patients who underwent autologous stem cell transplantation in our clinic were retrospectively analyzed. The last follow-up and survival data of the patients were obtained in January 2022. First-line peripheral stem cell mobilization with chemotherapy+G-CSF (5-10 μg/kg/day) was planned for all patients. Patients whom were mobilized at least 2x106/kg CD34(+) stem cells with this regimen were defined as “good mobilizers”. Patients who could not be mobilized with chemotherapy + G-CSF in the first line were mobilized with Plerixafor + G-CSF who could collect at least 2x106/kg CD34(+) stem cells with this collection regimen was defined as “poor mobilizers”. Results: While 110 of the patients (79.8%) were considered as “good mobilizers”, 28 patients (20.2%) took place in the “poor mobilizer” group. The mean age was 37.6 years in the good mobilizer group, while the mean age was 48.3 years in the poor mobilizer group.The good mobilizers group were significantly younger than poor mobilizer group (p<0.001). There was no significant difference between the two groups in terms of gender, histological diagnosis, stage, presence of B symptoms, bone marrow involvement, Bulky disease, prior chmo lines, disease status at the time of transplant, and ECOG performance score. The amount of collected and infused stem cells in the poor mobilizers group was significantly lower than in the good mobilzers arm (4.54x106/kg vs 3.6 x106/kg, p<0.001). While no significant difference was observed in the median neutrophil engrafment day(11 vs 10 day, p=0.06), median platellet engrafment day was significantly longer in the poor mobilizers group (12 vs 13 day, p=0.011). After a median 38 month follow up time; The 5-year progression-free survival rate was 49.7% in the good mobilizers arm and 48.6% in the poor mobilizers arm, with a median PFS of 73 months in the good mobilizers arm and 59 months in the poor mobilizers arm, and no significant difference was found between the two groups in terms of PFS. (p=0.912).The 5-year Overall Survival was 62.1% in the good mobilizers arm and 58.5% in the poor mobilizers arm with a median OS was not reached in the good mobilizers arm, the median OS was 83 months in the poor mobilziers group. There was no significant difference in overall survival between the two groups (p=0.748) Image:Summary/Conclusion: In our study, no difference was found between poor mobilizers and good mobilizers in terms of progression-free survival and overall survival. However, the amount of mobilized stem cells were lesser and the median platelet engraftment day was longer in the poor mobilizers arm. We conclude that plerixafor administration is safe and efficient for mobilization in lymphoma patients who were poor mobilizers.

Enhanced AC133-specific CAR T cell therapy induces durable remissions in mice with metastatic small cell lung cancer

Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133+ cancer stem cells and PD-L1+CD73+ myeloid cells were detectable in primary human SCLC tissues, suggesting that patients may benefit from the triple-immunotherapy. We conclude that the combination of AC133-specific CAR T cells, anti-PD-1-antibody and CD73-inhibitor specifically eliminates chemo-resistant tumor stem cells, overcomes SCLC-mediated T cell inhibition, and might induce long-term complete remission in an otherwise incurable disease.

Colorectal cancer stem cell-derived exosomal long intergenic noncoding RNA 01315 (LINC01315) promotes proliferation, migration, and stemness of colorectal cancer cells

ABSTRACT The effect of long intergenic noncoding RNA 01315 (LINC01315) on colorectal cancer has widely been proved. Nevertheless, how LINC01315 functions in the stemness of colorectal cancer and whether LINC01315 exists in colorectal cancer stem-like cell-derived exosomes remain dim, which are thus investigated in this research. CD133+/CD44+ colorectal cancer stem cells were sorted and verified through flow cytometry. Exosomes derived from CD133+/CD44+ colorectal cancer stem cells were collected. The viability, proliferation, stemness and migration of CD133+/CD44+, CD133−/CD44−, and colorectal cancer cells after transfection or the co-culture with exosomes were detected by MTT, colony formation, spheroid, and wound healing assays, respectively. Expressions of LINC01315, BCL-2, Bax, cleaved caspase-3, MMP-9, E-cadherin, and vimentin in cells or exosomes were analyzed using western blot or qRT-PCR. Genes interacted with LINC01315 in colorectal cancer were predicted by bioinformatics analysis. The results showed that LINC01315 was high-expressed in CD133+/CD44+ colorectal cancer stem cells and exosomes. Compared with colorectal cancer cells, the viability, proliferation, stemness, and migration of CD133+/CD44+ cancer cells were stronger, while these of CD133−/CD44− cancer cells were weaker. Besides, LINC01315 silencing decreased the viability, proliferation, stemness, and migration of CD133+/CD44+ cancer cells, while sh-LINC01315 inhibited the promotive effects of CD133+/CD44+ cancer cell-derived exosomes on the viability, proliferation, stemness, and migration of colorectal cancer cells. LINC01315 was also found to be correlated with DPEP1, KRT23, ASCL2, AXIN2, and DUSP4 in colorectal cancer. In conclusion, colorectal cancer stem cell-derived exosomal LINC01315 promotes the proliferation, migration, and stemness of colorectal cancer cells.

Fluorescent-Activated Cell Sorting (Flow Cytometry).

Flow cytometry is a widely used diagnostic tool in many laboratories, which generates information that is essential for the diagnosis and classification of different hematolymphoid neoplasms (Reichard KK, KS, Flow cytometry in the assessment of hematologic disorders. In: Orazi A, Foucar K, Knowles DM et al (eds) Neoplastic hematopathology. Lippincott, Williams and Wilkins, Baltimore, MD, pp 119-145, 2013). Flow cytometry allows us to identify individual cells within heterogeneous populations. It is also useful for the quantification of cells, such as CD4 counts in HIV patients and CD34 stem cells on bone marrow and peripheral blood specimens. Lastly, it can also be used to describe the pattern of antigen expression on cells known as immunophenotyped (Craig FE, Foon KA, Blood 111(8):3941-3967, 2008).

Abstract 11398: Association of Electronic Nicotine Delivery Systems Use with Circulating Angiogenic Cell Levels

Background: Circulating Angiogenic Cells (CAC) are known to aid in endothelial repair and are a promising marker of vascular function. Electronic Nicotine Delivery Systems (ENDS) have been hypothesized to impact vascular health, however the association between ENDS and CACs is unknown. Hypothesis: We hypothesize that ENDS use, sole or dual, will be significantly associated with CAC levels. Methods: We analyzed CAC levels of 314 human participants enrolled in the Cardiovascular Injury due to Tobacco Use study, which is a multi-center cross-sectional study of healthy participants 21-45 years age, without known cardiovascular disease or cardiovascular disease risk factors. Four study groups were determined a priori - non-smokers (n=62), sole cigarette users (n=207), sole ENDS users (n=17), and dual users (cigarette and ENDS) (n=28). A total of 15 CACs were measured via a standardized flowcytometry protocol using four cell surface markers - CD146 (endothelial), CD34 (stem cell), CD45 (leukocyte), and ACC133 (early progenitor/stem cell). Regression analysis was conducted using a General Linear Model with gamma distribution and log-link. Model was adjusted for age, sex, race, ethnicity, and enrollment site. Results: Among sole ENDS users, two CACs were significantly higher and four CACs were significantly lower, as compared to referent non-smokers (all P&lt;0.05) (Figure 1-A). Similarly, among dual users, seven CACs were significantly higher and one CAC was significantly lower, as compared to non-smokers (all P&lt;0.05) (Figure 1-B). Conclusion: CACs are sensitive to ENDS use, including dual use. Although CACs with endothelial surface markers were most commonly associated with ENDS/dual use, CACs with stem and early progenitor surface markers were also associated with ENDS/dual use. CACs are promising biomarkers of vascular function and further research is needed to quantify the effect of individual CAC on vascular function.

Trends in Utilization of Stored Cryopreserved Autologous Peripheral Hematopoietic Cells (APBHC) Intended for a Second (or beyond) Autologous Hematopoietic Cell Transplantation (AHCT) in Patients with Multiple Myeloma (MM): A Single Center Experience

▪Background: AHCT is a standard of care treatment for eligible patients (pts) with MM. Most consensus guidelines recommend that at the time of first AHCT, enough APBHC should be collected to perform two AHCT. However, recent studies have shown that with each decade after 1990, the use of cryopreserved APBHC for a salvage AHCT has declined and is now in the range of 4.6-15% in the decade between 2010-2018. The collection and storage of APBHC for two AHCT incurs additional costs and a prior single center cost analysis revealed that having had collected for just one AHCT in a cohort of 726 MM pts would have saved 3.8 million USD between 1993 and 2011. 1 Most centers that perform AHCT for MM pts continue to collect and store PBAHC for 2 AHCT. Herein we evaluate utilization trends and costs associated with cryopreserved APBHC in patients with MM.Methods: We retrospectively evaluated the clinicopathologic data from 440 patients with MM who underwent APBHC mobilization and collection at Mayo Clinic Florida between 2010 and 2019. The number of apheresis/collection sessions and APBHC product collection yields were determined for patients who were mobilized by any method. We estimated the costs involved in PBSC collection (apheresis) and cryopreservation storage based on our institution-specific charges as of May 2021. The cost of 1 session of APBHC collection/apheresis was $4,680 and the cost of 1 year of APBHC cryopreservation was $4,790 per patient. Statistical analysis was performed using JMP Pro 15 (SAS). Chi-square and Fischer exact tests were used to carry out univariate analysis for categorical variables and Wilcoxon rank sum/Kruskal Wallis for continuous variables.Results: Patient demographics are shown in Table 1. 440 pts underwent at least one collection session and 347 underwent at least 1 AHCT. The median number of mobilization sessions was 1 (95% CI 1.0-1.03), the median number of collection/apheresis sessions was 2.0 (95% CI 2.39-2.59), 84.3% (n=371) required ≥1 collection session. The median number of CD34 stem cells collected was 7.6 x10 6 (95% CI 7.76-8.19 x10 6), and the median number of infused CD34 cells was 4 x10 6(95% CI 4.13-4.37x10 6). The median cost of total collection sessions (defined as # of apheresis/collection sessions x $4,680) was $9,360 per pt (95% CI $11,181-$12,100). The median cost of cryopreservation (defined as # of years of storage x $4,790) was $19,160 per pt (95% CI $9,682-$22,252). The median number of years of storage was 4 (95% CI 4.11-4.65). 77(17.5%) pts have had APBHC in storage for <2 years, 218 (49.5%) have had APBHC in storage for 2-5 years, and 145 (33%) had have APBHC in storage for >5 years. The median time from MM diagnosis to AHCT was 9.0 months (range 13.8-18.5). 82% of patients collected enough APBHC (≥6x10 6, at least 3x10 6 per AHCT)for 2 AHCT. 8% of patients had all APBHC infused during their first AHCT, 83.5% had half of their APBHC infused during their first AHCT, and 8.1% had a different amount of APBHC infused during their first AHCT thus 91.6% of patients who collected APBHC had cells in storage. The median OS for the entire cohort after AHCT was 94.7 months (95% CI 88.6-100). Out of 347 patients who had stem cells in cryopreservation, 5 (1.4%) underwent a salvage AHCT and 3 (0.9%) underwent a tandem AHCT. 61% percent of patients had ≥1 excess collection sessions for APBHC that ultimately went unused. The median cost of excess collection sessions was $4,680 per pt (range, $4,680-$32,760) and the median total cost for excess collection sessions plus costs for storage was $23,840 per pt (range, $4,680-$85,450). The sum of costs of excess collection sessions was $2,077,920 and the sum of costs of cryopreservation was $5,812,665.Conclusion: The results of this single center analysis show that only 1.4% of patients underwent a salvage AHCT between 2010 and 2019 while 91.6% had APBHC left in storage which confirms the increase in storage of cryopreserved APBHC and declining trend in the use of stored APBHC for salvage AHCT in pts with MM. This is likely due to the advent of next generation novel therapies such as monoclonal antibodies, proteasome inhibitors and immunomodulatory agents. Excess collections, and cryopreservation of unused APBHC incur a cost of nearly $8 million. Institutional policies regarding universal APBHC collection and long-term storage should be reevaluated.1Phipps C, et al. Bone Marrow Transplantation 2015:50;663-667. [Display omitted] DisclosuresMurthy: CRISPR Therapeutics: Research Funding. Ailawadhi: Sanofi: Consultancy; Medimmune: Research Funding; Genentech: Consultancy; Ascentage: Research Funding; Pharmacyclics: Consultancy, Research Funding; Xencor: Research Funding; Janssen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy; Beigene: Consultancy; GSK: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Takeda: Consultancy; Cellectar: Research Funding; Karyopharm: Consultancy.

Immunologic Autograft Engineering: 13 Years Follow-up

Our group published a double blind, randomized phase III study (Porrata et al, BBMT 2016; 22: 1017-1023) showing that the infusion of autograft absolute lymphocyte count (A-ALC) ≥ 0.5 x 10 9 cells/kg is a survival prognostic factor for lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). However, a limitation of the study was the short-term follow-up duration of two years post-APBHSCT. To assess if the A-ALC still provide survival benefit we looked at the 121 lymphoma patients enrolled in the phase III from December 10, 2007 until October 12, 2010 survival outcomes with a longer follow-up duration of up to 13 years post-APBHSCT. Since the accrual for the study, 52 patients had died of recurrence lymphoma, 4 patients of therapy-related acute myelogenous leukemia, 3 patients of unknown causes, 1 patient of acute respiratory distress syndrome, 1 patient of anaplastic astrocytoma, and 1 patient of septic shock. The current median follow-up is 86 months (range: 2.1-158.1 months) for the entire cohort and 127.8 months (range: 5.9-158.1 months) for patients still alive. Patients infused with an A-ALC ≥ 0.5 x 10 9 cells/kg continue to experience superior overall survival (OS) (HR = 0.392, 95%CI 0.224-0.687, P < 0.001) and progression-free survival (PFS) (HR = 0.413, 95%CI 0.253-0.677, P < 0.0004). The 10-year OS rates for the A-ALC ≥ 0.5 x10 9 cells/kg patients was 70% (95% confidence interval [CI], 55%-81%) and for the A-ALC < 0.5 x10 9 cells/kg patients was 36% (95% CI, 25%-49%) (Figure 1A). The 10-year PFS rates for the A-ALC ≥ 0.5 x10 9 cells/kg group was 57% (95% CI, 43%-70%) and for the A-ALC < 0.5 x10 9 cells/kg group was 28% (95% CI, 19%-40%) (Figure 1B). Autograft lymphocyte subset analysis including autograft-CD3 (A-CD3), autograft CD4 (A-CD4), autograft CD8 (A-CD8), and autograft natural killer cells (A-NK), showed that the infusion of A-NK was a predictor for OS (HR = 0.518, 95%CI 0.292-0.919, P < 0.02) and PFS (HR = 0.393, 95%CI 0.230-0.670, P < 0.0006). The 10-year OS rates for the A-NK ≥ 0.09 x10 9 cells/kg patients was 67% (95% confidence interval [CI], 50%-80%) and for the A-NK < 0.09 x10 9 cells/kg patients was 42% (95% CI, 31%-54%) (Figure 1C). The 10-year PFS rates for the A-NK ≥ 0.09 x10 9 cells/kg group was 59% (95% CI, 43%-73%) and for the A-NK < 0.09 x10 9 cells/kg group was 32% (95% CI, 23%-44%) (Figure 1D). Both the A-ALC and A-NK were independent predictor for OS [ A-ALC: HR = 0.367, 95%CI 0.195-0.690, P < 0.001) and A-NK: HR = 0.411, 95%CI 0.275-0.892, P < 0.01] and for PFS [ A-ALC: HR = 0.477, 95%CI 0.271-0.840, P < 0.01) and A-NK: HR = 0.490, 95%CI 0.266-0.903, P < 0.02]. With a longer follow-up of 13 years, this study shows that the infusion of A-ALC continues to provide better clinical outcomes to lymphoma patients undergoing APBHSCT. These study findings support our standard practice changed of not only collecting CD34 stem cell for hematologic engraftment, but also A-ALC for lymphoma patients undergoing APBHSCT to improve clinical outcomes. Further, autograft immune effector studies are warranted such as A-NK to develop a more immunocompetent targeted autologous graft verus tumor effect. [Display omitted] DisclosuresAnsell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Paludo: Karyopharm: Research Funding.

Preferential interaction of platelets with prostate cancer cells with stem cell markers

BackgroundProstate cancer (PCa) may be initiated by CD133+/CD44+ expressing stem cell-like cells (PCSC), which are also thought to drive metastasis. Platelets also contribute to metastasis via tumor cell-induced platelet aggregation (TCIPA), which in part enhances cancer cell invasion. Moreover, activated platelets secrete stromal derived growth factor-1α (SDF-1α) that can mobilize CSCs via the CXCR4 receptor. However, the potential reciprocal interactions between CSCs and platelets have not been investigated. ObjectiveTo characterize the mechanisms behind PCSC-platelet interaction. MethodsFluorescence Activated Cell Sorting was utilized to separate DU145 and PC3 PCa cells into CD133+/CD44+, CD133+/CD44-, CD44+/CD133-, and CD133-/CD44- subpopulations and to measure their CXCR4 surface expression. PCa subpopulation TCIPA experiments were performed using aggregometry and immunoblot was used to measure prothrombin. Platelet SDF-1α secretion was measured by ELISA. Modified-Boyden chamber assays were used to assess the role of SDF-1α:CXCR4 pathway in platelet-PCSC interactions. ResultsDU145 and PC3 expressing both CD133 and CD44 stem cell markers accounted for only small fractions of total cells (DU145: CD133+/CD44+ 3.44 ± 1.45% vs. CD133+/CD44- 1.56 ± 0.45% vs. CD44+/CD133- 68.19 ± 6.25% vs. CD133-/CD44- 20.36 ± 4.51%). However, CD133+ subpopulations induced the greatest amount of aggregation compared to CD44+/CD133- and double-negative DU145, and this aggregation potency of CD133+ PCa cells corresponded with high levels of prothrombin expression. Additionally, CD133+ subpopulations expressed significantly higher level of CXCR4 compared to CD133-/CD44- and CD44+/CD133-. Disruption of SDF-1α:CXCR4 pathway reduced platelet-induced PCSC invasion. ConclusionsCD133+/CD44+ and CD133+/CD44- PCSCs have highest platelet aggregation potency, which could be attributed to their increased prothrombin expression. Reciprocally, platelet-derived SDF-1α stimulates PCSC invasion.

CD44 Promotes Lung Cancer Cell Metastasis through ERK-ZEB1 Signaling.

Simple SummaryLung cancer treatment has always been challenging as its metastasis rate is higher than other cancers. Cancer stemness promotes cancer metastasis, and it is important to understand the mechanism behind the cancer-stemness-mediated metastasis. CD44 is a well-known cancer stem cell marker and plays a role in tumor metastasis in different cancer types. In this study, we investigated the role of CD44 in lung cancer metastasis by using clinical studies as well as in vitro and in vivo studies. We found that CD44 promotes the migration and invasion abilities of lung cancer cells through ERK–ZEB1 signaling. This study provided evidence that CD44 may be a possible therapeutic target to decrease lung cancer metastasis.Lung cancer is a malignancy with high mortality worldwide, and metastasis occurs at a high frequency even when cancer spread is not detectable at primary operation. Cancer stemness plays an important role in malignant cancer behavior, treatment resistance, and cancer metastasis. Therefore, understanding the molecular pathogenesis behind cancer-stemness-mediated metastasis and developing effective approaches to prevent metastasis are key issues for improving cancer treatment. In this study, we investigated the role of CD44 stemness marker in lung cancer using in vitro and clinical studies. Immunohistochemical staining of lung cancer tissue specimens revealed that primary tumors with higher CD44 expression showed increased metastasis to regional lymph nodes. Flow cytometry analysis suggested that CD44 positive cells were enriched in the metastatic lymph nodes compared to the primary tumors. CD44 overexpression significantly increased migration and invasion abilities of lung cancer cells through CD44-induced ERK phosphorylation, ZEB1 upregulation, and Claudin-1 downregulation. Furthermore, ERK inhibition suppressed the migration and invasion abilities of CD44-overexpressing lung cancer cells. In summary, our in vitro and clinical results indicate that CD44 may be a potential prognostic and therapeutic marker for lung cancer patients.

Oncogene-regulated release of extracellular vesicles.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

Abstract 1913: Pre-existing HSV-1 Immunity enhances anti-cancer efficacy of a novel immune stimulating oncolytic virus - VG161

Abstract It is not clear if pre-existing anti-virus immunity or multicycle treatment of oncolytic virus (OV) can affect the treatment outcome. This study is to investigate the impact of pre-existing anti Herpes Simplex Virus type-1 (HSV-1) immunity or multicycle OV treatment on the efficacy of VG161, a novel HSV-1 OV armed with IL12, IL15 with its receptor α unit (IL15RA), and a PD-L1 peptide blocker (PDL1B). VG161 has entered phase I trial in Australia and China. Seventy Balb/c mice were divided into 2 groups. One group (35 mice) was subcutaneously immunized by mVG161 (a version of VG161 expresses mouse IL12, human IL15 and PDL1B) on day 1 (5x105 PFU/mouse) followed by a boost on day 7. The other group (35 mice) was non-immunized. On day 7 post-boost, the blood was collected from tail to determine anti-HSV antibody. At the same time, all 70 mice were subcutaneously implanted with CT26 mouse colon cancer cells. Once the tumors reached 100 mm3, 20 mice (10 from each group) were treated with intra-tumoral (IT) injection of either mVG161 at a dose of 5×106 PFU/mouse or Vehicle, once a day for 5 days. Hepatic cancer (HC) PDX tissues were dissected into pieces of 27 mm3 and subcutaneously implanted into humanized mice established by transplantation of 1×104 human CD34 stem cells (HLA matched with PDX). When the tumor reached 100-150 mm3, animals were divided into 4 groups (10 mice/group): vehicle control, positive control (oxaliplatin 5 mg/kg and gemcitabine 100 mg/kg), and 2 groups treated with VG161 at high dose (7.75×106 PFU) or low dose (3.10×105 PFU). The OV treatments were given for 3 cycles with IT injection of VG161 once a day for day 1-5 of each cycle. Tumor, spleen, and peripheral blood mononuclear cells were collected for further analysis. All HSV pre-immunized mice but not naïve mice were HSV-1 antibody positive. By day 19 post-treatment, mVG161 significantly inhibited CT26 tumor growth in both HSV-1 pre-immunized and non-immunized groups, while all control mice were euthanized due to tumor size reached 2000 mm3. Starting from day 27, tumors grew back in non-immunized group but not in pre-immunized group, resulting in a significant difference of tumor size between the 2 groups on day 31 to 39 (p&amp;lt;0.01). As compared to the negative controls, HC PDX tumors in both treatment groups were significantly smaller with percentage of T/C less than 40% on day 72. The median time for 3-fold increase of tumor size is 41.5 days (95%CI: 32-58) in the vehicle control, 74 days (95%CI: 51-undefined) in high dose group, and not reached in low dose group (p&amp;lt;0.01). Repeated treatment (2-3 cycles) increased the anti-cancer efficacy of VG161. The anti-cancer efficacy of VG161 can be enhanced by pre-immunization of HSV-1 or multicycle administration when the virus is given by IT, suggesting potential clinical benefits of HSV-1 based OV therapy in HSV sero-positive patients and multicycle administration of VG161 for long term maintenance treatment. Citation Format: Jun Ding, Jason Sun, Yana Murad, Erica Lee, Ismael Samudio, William Jia, Ronghua Zhao. Pre-existing HSV-1 Immunity enhances anti-cancer efficacy of a novel immune stimulating oncolytic virus - VG161 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1913.

H3K27M/I Mutations Promote Context-Dependent Transformation in Acute Myeloid Leukemia with RUNX1 Alterations

Neomorphic missense mutations in histone H3 genes (most notably K27M and K36M) are frequent in solid cancers such as diffuse intrinsic pontine gliomas (DIPG), supratentorial glioblastoma multiforme (GBM) or chondroblastomas. Despite high incidences of these mutations and their profound impact on global histone H3 lysine methylation, their relevance has not been studied in hematologic cancers.Here, we report the first identification of histone H3 mutations in AML. We find that two of 415 AML patients in the Leucegene cohort and one of 200 patients in the TCGA cohort carry either K27M (n=2) or K27I (n=1) mutations in one of twelve canonical H3 genes. No K36M or other lysine-to-methionine mutations on any of the H3 genes, and no K27M mutation on any of the replication-independent H3.3 variant genes were detected in the same cohorts. H3K27 mutations invariably occurred in the context of either truncating RUNX1 mutations or t(8;21)(q22;q22), suggesting an association with altered RUNX1 function (p < 0.003; Fisher exact test). Characterization of two of the identified H3K27 mutant patient specimens revealed that both samples contained major sub-clones with markedly reduced H3K27me2/3 levels. The H3K27 mutants but not co-occurring mutant ASXL2 or USP7 alleles were only detectable in the H3K27me2/3-low sub-clones, demonstrating that the H3K27 mutations had the most profound impact on H3K27me2/3 in these patients. Moreover, although a heterozygous TET2S1203R mutation was equally present in both H3K27me2/3-high and low sub-fractions, a homozygous RUNX1L98Sfs*22 mutation was restricted to the H3K27me2/3-low sub-clone, further strengthening the possibility that impaired RUNX1 function and reduced H3K27me2/3 associate with each other.We tested the possibility that H3K27 mutants may specifically collaborate with the product of the t(8;21) translocation (AML1-ETO-9a ; AE9a) in transduced mouse hematopoietic stem and progenitor cells (HSPCs). Selectivity of this collaboration was assessed using the combination of the H3K27 mutant gene with the MLL-AF9 fusion product. Whereas expression of H3 WT resulted in a mild competitive proliferation disadvantage in both AE9a and MLL-AF9 cells, expression of H3.3K27I/M conferred a proliferative advantage in AE9a cells in three of four H3K27I -infected and in all H3K27M- infected cultures. This was not observed in MLL-AF9 cells, where H3K27I/M expressing cells were progressively outcompeted in the same culture period. H3K27I/M -infected AE9a cells exhibited significant reductions in H3K27me2/3 and elevated levels of H3K27 acetylation (H3K27ac) compared to H3WT -infected control cells despite undiminished Ezh1, Ezh2 and Suz12 protein levels. In the context of AE9a driven AML in vivo, expression of H3K27M caused a striking disease acceleration leading to all mice reaching end stage disease with a median of 88 days, in contrast to recipients of AE9a ; empty vector or AE9a ; H3WT cells, of which none or only two of nine transplanted mice succumbed to AML within 330 days, respectively (p<0.0001, Log-rank test). Seven of nine AE9a ; H3.3K27I transplanted mice developed AML with a median latency of 208 days (p=0.0004, Log-rank test, compared to controls). Importantly, combined expression of H3K27I or H3K27M with MLL-AF9 did not change disease latencies compared to control groups, indicating selectivity.We next tested the influence of the H3K27M mutation on the differentiation and proliferation of human cord blood CD34+ HSPCs ex vivo . Relative to controls, H3.3K27M expression led to an 8-fold net expansion of a phenotypically primitive CD34+/CD45RA-/CD90+ cell population, which surprisingly lacked expression of the CD133 stem cell marker. Interestingly, granulocytic/granulo-monocytic colony-forming cells (CFU-G/GM) but not multipotent colony-forming cells (granulo/erythroid/monocytic/megakaryocytic; CFU-GEMM) were expanded in 14d cultures initiated with H3K27M transduced CD34+ CB cells when compared to controls. Xenografts of transduced CD34+ CB cells indicated a marginal loss in in vivo reconstitution activity by K27M-transduced cells, mostly at 6 and 12-week time-points.Taken together, our results represent the first description of neomorphic H3K27M/I mutations in AML. We demonstrate that these mutations have a profound impact on H3K27me2/3 and provide evidence that they selectively collaborate with impaired RUNX1 function. DisclosuresNo relevant conflicts of interest to declare.