Marker-positive Cells Research Articles

A pair of cell preservation solutions for therapy with human adipose tissue-derived mesenchymal stromal cells.

IntroductionStem cells for therapy are often suspended in a preservation solution, such as normal saline or lactated Ringer's solution, for a short time before intravenous infusion. However, these solutions are not necessarily ideal for maintaining cell viability and preventing the sedimentation of cells during storage and infusion. In this study, we attempted to optimize the compositions of preservation solutions, which could affect the efficacy and safety of stem cell therapy.MethodsWe determined the characteristics of a preservation solution that would optimize cell viability and the percentage of cells in the supernatant using human adipose-derived mesenchymal stromal cells (hADSCs). We compared solutions that differed by electrolytes (e.g., normal saline and Ringer's solution) and the concentrations of dextran 40 and trehalose. The effects of the solutions on hADSCs were evaluated by assessing cell surface markers, colony-forming capacity, differentiation potential, and cell concentrations in the infusion line.ResultsOptimized preservation solutions consisted of lactated Ringer's solution with 3% trehalose without or with 5% dextran 40 (LR-3T and LR-3T-5D, respectively). The cell viabilities after 24 h of storage at 5 °C in LR-3T and LR-3T-5D were 94.9% ± 2.4% and 97.6% ± 2.4%, respectively. The percentage of cells in the supernatant after 1 h of storage at room temperature in LR-3T-5D was 83.5% ± 7.6%. These solutions preserved the percentage of cell surface marker-positive cells, the colony-forming capacity, and the adipogenic and osteogenic differentiation ability in hADSCs for at least 24 h after preservation at 5 °C and 25 °C.DiscussionWe determined the optimal composition of preservation solutions for hADSCs and confirmed the effects of these solutions on cell viability and the stability of cell characteristics in vitro. Our results suggest that LR-3T and LR-3T-5D can help maintain the quality of stem cells for therapy during preservation and infusion. However, further in vivo research is needed on the efficacy and safety of the solutions in different therapeutic cell lines before clinical use.

Indoleamine 2,3-dioxgenase-transfected mesenchymal stem cells suppress heart allograft rejection by increasing the production and activity of dendritic cells and regulatory T cells

Expression of indoleamine 2,3-dioxygenase (IDO) in mesenchymal stem cells (MSC) is thought to contribute to MSC-mediated immunosuppression. A lentiviral-based transgenic system was used to generate bone marrow stem cells (BMSC)...

Sonic Hedgehog, a Novel Endogenous Damage Signal, Activates Multiple Beneficial Functions of Human Endometrial Stem Cells.

Local endometrial stem cells play an important role in regulating endometrial thickness, which is an essential factor for successful embryo implantation and pregnancy outcomes. Importantly, defects in endometrial stem cell function can be responsible for thin endometrium and subsequent recurrent pregnancy losses. Therefore, many researchers have directed their efforts toward finding a novel stimulatory factor that can enhance the regenerative capacity of endometrial stem cells. Sonic hedgehog (SHH) is a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. In addition to this canonical function, we identified for the first time that SHH is actively secreted as a stem cell-activating factor in response to tissue injury and subsequently stimulates tissue regeneration by promoting various beneficial functions of endometrial stem cells. Our results also showed that SHH exerts stimulatory effects on endometrial stem cells via the FAK/ERK1/2 and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. More importantly, we also observed that endometrial stem cells stimulated with SHH showed markedly enhanced differentiation and migratory capacities and subsequent invivo therapeutic effects in an endometrial ablation animal model.

Multicolor Immunophenotyping of Candidate Leukemic Stem Cell Markers in CD34+CD38- Chronic Myeloid Leukemia Stem Cells

Introduction: Detection of leukemic stem cells (LSCs) may represent a new potential prognostic parameter in chronic myeloid leukemia (CML) and a tool for minimal residual disease monitoring in combination with standard qPCR method. To date CD26 is studied as a most specific marker for CML LSC detection. Several other candidate LSC markers have been reported, such as IL1-RAP, CD25 and CD93, however a side-by-side testing of their specificity is lacking. Recently, we have identified CD69 molecule to be overexpressed in CD34+CD38-CD26+ cells, which makes this antigen another candidate marker for LSC. Aim: To compare the surface expression of LSC markers CD69, CD26, CD25, CD56, IL1-RAP, CD56, CD93 in bulk CD34+CD38- population at diagnosis using a multicolor phenotypization assay Methods: In total, 44 patients were analyzed at diagnosis of chronic phase CML before administration of any treatment. Fresh (n=38) or cryopreserved (n=6) leukocytes obtained by erythrolysis were stained with CD45, CD34, CD38, CD25, CD26, CD56, CD69, CD93, IL1-RAP antibodies and 7-AAD for selection of live cells. Analysis was performed on FACSAria Fusion (BD Biosciences). Acquisition of live mononuclear cells ranged from 2×104 to 2.5×106. Results: Expression of candidate LSC markers CD26, CD25, CD56, IL1-RAP, CD56, CD93 was analyzed in BM of 35 patients who carried at least 30 CD34+CD38- cells. Median percentage of marker positive cells was 34% for IL1-RAP, 31% for CD25, 23% for CD26, 16% for CD56 and 2% for CD93, from the parent CD34+CD38- population. Next we analyzed the overlap and combination for the three best markers - IL1-RAP, CD25 and CD26. The 3-combination (defined as IL1-RAP or CD25 or CD26 expression) identified 40% of CD34+CD38- positive cells, which was more than any of the markers alone. Expression of the three markers showed good overlap and ruled out mutually exclusive expression of the markers. This was demonstrated by median difference of 0.4% of CD34+CD38- cells (range: 0-18%) and a correlation coefficient r2=0.9914, when comparing the 3-combination and the best performing marker in each patient. In contrast, in 12/35 (34%) of patients, one of the three markers failed to identify at least half of the cells positive for another marker. In 21/35 patients, we also analyzed the expression of CD69 in the CD34+CD38- compartment. The CD69 showed similar performance as the 3-combination of CD26, CD25, and IL1-RAP, 59% vs 54% positive cells, respectively. We observed excellent overlap between CD69 and 3-combination expression in individual patients with median difference of 0% of CD34+CD38- cells (range 0-15%) and correlation coefficient r2=0.9890. Furthermore, we compared marker positivity in BM vs PB in 19 paired samples. Both, sample types showed similar frequency of CD34+CD38- cells (5×10-3 in BM, and 2×10-3 for PB), but PB carried higher percentage of LSCs identified by the 3-combination - median 76 vs 52% cells. Conclusions: We show an overlap in surface expression of three previously reported CML LSC markers - IL1-RAP, CD25 and CD26. Nevertheless, a combination of these markers can detect more positive cells than any of the markers alone. Moreover, we demonstrate that CD69 identifies the same cells within the CD34+CD38- compartment as the combination of three above mentioned markers, which makes CD69 the best candidate for routine CML LSC quantification. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

Profiling the Immune Tumor Microenvironment of Newly Diagnosed Diffuse Large B Cell Lymphoma Identifies Three Major Immune Infiltration Patterns

Background: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease of malignant B cells most often classified by tumor gene expression and/or mutations. DLBCL is also characterized by a tumor microenvironmental influence that has not been well described. Immuno-oncology-targeting agents such as immune checkpoint inhibitors have limited clinical activity in DLBCL, highlighting the need for a better understanding of the DLBCL tumor microenvironment for rational drug development, combinations, and disease stratification. Here, we systematically characterize the immune composition of more than 100 DLBCL tumors using 2 imaging technologies and provide insight into the complexity of DLBCL disease biology. Methods: A total of 110 cases of newly diagnosed DLBCL were analyzed by multiplex immunohistochemistry (IHC; n=70) and multiplexed ion beam imaging (MIBI) (n=40), each with 10 common markers (CD20, CD3, CD8, Foxp3, CD56, CD163, CD11c, CD56, PD-1, PD-L1) and a few platform-specific markers. Both IHC and MIBI images were digitalized to generate marker-positive cell counts (including single, double, or triple positivity), cell density (cell count/mm2), and population fraction (% of total nucleated cells). Marker density was analyzed for the major components of tumor-infiltrating cell types and correlation between any pair of markers. In addition, RNAseq data and fluorescence in situ hybridization (FISH) data on MYC, BCL-2, and BCL-6 translocation were generated. Results: In the IHC cohort, T cells (CD3+), dendritic cells (DCs; by CD11c+), and macrophages (CD163+)were the major immune components, with median population fractions of 22%, 16%, and 2.7%, respectively. Natural killer cells (CD56+CD20−) were a minor component at a median of 0.1%. A significant negative correlation was observed between tumor cells (CD20+) and CD4+ (Spearman ρ = −0.47; P = 1.3 × 10−04), and CD8+ T cells (Spearman ρ = −0.42; P = 1.4 × 10−03) cells, and an unexpectedly negative correlation between DCs (CD11c+) and macrophages (CD163+, r = −0.63; P = 9.8 × 10−06) was found. Similar to follicular lymphoma, 2 PD-1+ T-cell populations were identified: PD-1bright and PD-1dim. The PD-1bright cells co-stained with CXCR5, indicating T follicular helper cells (Tfh). The PD-1dim were expressed on exhausted effector cells that co-stained with Tim3 or Lag3. The median population fraction of Tim3+ or Lag3+ among T cells was 25%, whereas that of PD-1dim among T cells was 0.2%, indicating that PD-1 was not a useful marker for exhausted T cells. PD-L1 was predominantly found on DCs and macrophages, and the median population fraction of PD-L1+ among tumor cells was only 6.2%. By unsupervised hierarchical clustering on marker density, 3 major immune-infiltration patterns (P1, P2, and P3) were identified. The first 2 segments (P1 and P2) were 20% and 25% of the total cases, respectively. Both were characterized by high T-cell, macrophage, and DC infiltration. P1 was enriched for PD-1+ T cells, whereas P2 was void of any PD-1+ cells. The third segment (P3) that comprised 55% of the cases was predominantly tumor cells with low T-cell, macrophage, and DC infiltration. PD-L1+ cells were primarily found in segments P1 and P2 but rare in segment P3. Additional analysis on the associations between the 3 immune-infiltration patterns and prognostic DLBCL molecular features such as cell-of-origin, double-hit gene signature, and double-hit FISH will also be presented. Conclusions: These data show the complexity of DLBCL disease biology and show classification of DLBCL at the immune-infiltration level as 3 distinct patterns. The overall low expression of PD-1+ T cells and the restricted pattern of PD-L1+ tumor cells provide a possible explanation for the lack of clinical activity of PD-1/PD-L1 blockade in DLBCL. We also observed other exhausted T cells expressing Lag3 and Tim3, suggesting alternative therapeutic opportunities in stratified populations. These data also highlight the opportunity to develop rational immuno-oncology-targeted agents based on the immune infiltration pattern of DLBCL and selection of patients who may respond more favorably to particular agents. Disclosures Huang: Celgene Corporation: Employment, Equity Ownership. Nakayama:Celgene Corporation: Employment, Equity Ownership. Stokes:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Ren:Celgene Corporation: Employment, Equity Ownership. Marella:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Newhall:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

Lymphatic‐to‐blood vessel transition in adult microvascular networks: A discovery made possible by a top‐down approach to biomimetic model development

Emerging areas of vascular biology focus on lymphatic/blood vessel mispatterning and the regulation of endothelial cell identity. However, a fundamental question remains unanswered: Can lymphatic vessels become blood vessels in adult tissues? Leveraging a novel tissue culture model, the objective of this study was to track lymphatic endothelial cell fate over the time course of adult microvascular network remodeling. Cultured adult Wistar rat mesenteric tissues were labeled with BSI-lectin and time-lapse images were captured over five days of serum-stimulated remodeling. Additionally, rat mesenteric tissues on day 0 and day 3 and 5 post-culture were labeled for PECAM+LYVE-1 or PECAM+podoplanin. Cultured networks were characterized by increases in blood capillary sprouting, lymphatic sprouting, and the number of lymphatic/blood vessel connections. Comparison of images from the same network regions identified incorporation of lymphatic vessels into blood vessels. Mosaic lymphatic/blood vessels contained lymphatic marker positive and negative endothelial cells. Our results reveal the ability for lymphatic vessels to transition into blood vessels in adult microvascular networks and discover a new paradigm for investigating lymphatic/blood endothelial cell dynamics during microvascular remodeling.

The Effects of TGF-β Signaling on Cancer Cells and Cancer Stem Cells in the Bone Microenvironment.

Background: Transforming growth factor-β (TGF-β) plays a key role in bone metastasis formation; we hypothesized the possible involvement of TGF-β in the induction of cancer stem cells (CSCs) in the bone microenvironment (micro-E), which may be responsible for chemo-resistance. Methods: Mouse mammary tumor cells were implanted under the dorsal skin flap over the calvaria and into a subcutaneous (subQ) lesions in female mice, generating tumors in the bone and subQ micro-Es. After implantation of the tumor cells, mice were treated with a TGF-β R1 kinase inhibitor (R1-Ki). Results: Treatment with R1-Ki decreased tumor volume and cell proliferation in the bone micro-E, but not in the subQ micro-E. R1-Ki treatment did not affect the induction of necrosis or apoptosis in either bone or subQ micro-E. The number of cells positive for the CSC markers, SOX2, and CD166 in the bone micro-E, were significantly higher than those in the subQ micro-E. R1-Ki treatment significantly decreased the number of CSC marker positive cells in the bone micro-E but not in the subQ micro-E. TGF-β activation of the MAPK/ERK and AKT pathways was the underlying mechanism of cell proliferation in the bone micro-E. BMP signaling did not play a role in cell proliferation in either micro-E. Conclusion: Our results indicated that the bone micro-E is a key niche for CSC generation, and TGF-β signaling has important roles in generating CSCs and tumor cell proliferation in the bone micro-E. Therefore, it is critically important to evaluate responses to chemotherapeutic agents on both cancer stem cells and proliferating tumor cells in different tumor microenvironments in vivo.

Three Dimensional Culture of Potential Epithelial Progenitor Cells in Human Lacrimal Gland.

PurposeWe investigate human lacrimal gland tissue to determine the presence of progenitor cells in this adult human tissue.MethodsSix human lacrimal gland tissues from donors were collected and stored immediately in the culture medium at 4°C until the next procedure. One part of the lacrimal gland tissue was prepared for immunofluorescence staining and the other part was prepared for primary cell culture. Immunofluorescence analysis was conducted to evaluate cultured lacrimal epithelial phenotype and progenitor cell markers for five passages. Real-time polymerase chain reaction (PCR) was performed to assess proliferation markers in the different passages. Three-dimensional culture and PCR were conducted to determine the differentiation potential of cultured human lacrimal gland cells.ResultsHuman lacrimal gland tissue expressed a number of epithelial progenitor cell markers. Precursor cell markers C-Kit, K15, Nestin, and P63 were observed in lacrimal gland tissues. Lacrimal gland epithelial cells were cultured successfully and passaged to P5. The cultured lacrimal gland epithelial cells were positive for pan-cytokeratin (PCK), AQP5, Rab3D, ABCB5, C-kit, K15, Ki67, and P63. Human lacrimal gland cells could form spheroids in vitro and then grow into mini-gland-like structures. PCR results showed proliferation and differentiation capability of those cultured cells.ConclusionsHuman lacrimal gland tissues contain precursor marker-positive cells and marker expression also was detected in ex vivo cultured cells, which showed differentiation capability.Translational RelevanceFuture studies of differentiation in human lacrimal gland tissue may aid in developing stem cell-based therapies for dry eye disease.

Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice.

Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are unclear. In this study, we investigated the effects of low-level, chronic permethrin exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to the acceptable daily intake level of permethrin (0.3ppm) in drinking water during the prenatal and postnatal periods. We then examined the effects on the central nervous system in adult male offspring. In the permethrin group, we detected behavior that displayed incomplete adaptation to a novel environment, as well as an impairment in learning and memory. In addition, immunohistochemical analysis revealed an increase in doublecortin- (an immature neuron marker) positive cells in the hippocampal dentate gyrus in the permethrin exposure group compared with the control group. Additionally, in the permethrin exposure group there was a decrease in astrocyte number in the hilus of the dentate gyrus, and remaining astrocytes were often irregularly shaped. These results suggest that exposure to permethrin at low levels in early life affects the formation of the neural circuit base and behavior after maturation. Therefore, in the central nervous system of male mice, low-level, chronic permethrin exposure during the prenatal and postnatal periods has effects that were not expected based on the known effects of permethrin exposure in mature animals.

Distribution of Neuroendocrine Marker-Positive Cells in Colorectal Cancer Tissue and Normal Mucosal Tissue: Consideration of Histogenesis of Neuroendocrine Cancer

Background: Colorectal neuroendocrine carcinoma (NEC) is a rare disease, and mixed cases with colorectal adenocarcinoma also exist. The histogenesis of this disease remains unclear. We studied the numbers of neuroendocrine marker-positive cells in adenocarcinoma tissue and in normal ­mucosal tissue to investigate the relation between adenocarcinoma and NEC and to discuss the histogenesis of NEC. Methods: We studied a total of 354 curatively resected cases of stage II or III colon cancer and 36 cases of rectal cancer treated at the Tokai University Hospital between 2007 and 2012. Adenocarcinoma tissue and normal mucosal tissue were immunohistochemically stained with chromogranin A, synaptophysin, and CD56. Cases in which neuroendocrine marker-positive cells were found in cancer tissue were defined as positive. In normal mucosa, the numbers of positive cells per 15 high-power fields (HPF) were counted. Results: Among the 390 cases, 181 cases had right sided colon cancer, 173 cases had left sided colon cancer, and 36 cases had rectal cancer. The rates of positive staining for chromogranin A, synaptophysin, and CD56 were significantly higher in the right sided colon than in the left sided colon, consistent with the preferred sites of NEC as reported previously. Cells positive for chromogranin A and synaptophysin in normal mucosa were significantly more common in the rectum and the left sided colon than in the right sided colon. No site-specific differences were found for CD56. Conclusions: Neuroendocrine marker-positive cells in colorectal cancer tissue are more common in the right sided colon, whereas neuroendocrine marker-positive cells in normal mucosa are more common in the rectum. These results suggest that NEC may arise from preceding adenocarcinomas.

Abstract CT128: Palbociclib in combination with fulvestrant or tamoxifen as treatment for hormone receptor positive metastatic breast cancer with prior chemotherapy for advanced disease (TBCRC 035): A Phase II study with pharmacodynamic markers

Abstract Background: Addition of the cyclin dependent kinase 4/6 inhibitor (CDK4/6i) palbociclib to endocrine therapy significantly improves progression free survival (PFS) in patients with HR+ MBC. The primary toxicity is neutropenia (ntp). TBCRC035 explored rates of neutropenia in patients with prior chemotherapy for MBC with 2 dose levels of palbociclib, and correlated changes in retinoblastoma protein phosphorylation (pRB) and Ki67 expression in proliferating keratinocytes and tumor, as well as mutations in cell-free DNA (cfDNA) with response. Methods: TBCRC035 is a 1:1 randomized multicenter Phase II study evaluating palbociclib at either 125 or 100 mg in combination with physician choice fulvestrant or tamoxifen. Eligible patients (pts) with HR+ MBC had received 1-3 lines of chemotherapy for MBC, any number of hormone therapies, and were naïve to CDK4/6i. The primary endpoint was grade 3/4 ntp; secondary endpoints included PFS, clinical benefit rate (CBR), safety/tolerability, inhibition of RB phosphorylation (pRB) and change in Ki67 expression in skin and tumor (FFPE sections of skin and tumor biopsies) at day 14-21 of treatment compared to baseline, and correlation of response with mutations in cell free DNA (cfDNA). FFPE sections of skin punch and tumor biopsies were stained using antibodies to Ki67, total RB, and phospho-RB-S780 using BOND polymer red detection. Stained slides were scanned into the Aperio image analysis platform; the percentage of marker positive cells was determined. Whole blood was collected at baseline & processed for plasma; cfDNA was extracted. Using a combination of digital PCR and ultradeep next generation sequencing, cfDNA was analyzed for ESR1 and PIK3CA mutations. Results: 70 pts were enrolled; 35 were randomized to 100 vs 125 mg of palbociclib respectively. 12 pts (100 mg), and 19 pts (125 mg) had >1 episode of grade 3/4 ntp (p=0.091). Pts on 100 mg had fewer total episodes of grade 3/4 ntp (p=0.036). Dose reductions were more frequent in patients starting with 125 mg compared to those starting with 100 mg (12 vs6). CBR and PFS were similar (100 vs 125 mg; CBR: 67 vs 74%; PFS: 6.5 vs 10 mo,p=0.18). In skin and tumor, the % of Ki67-positive nuclei was significantly lower in post-treatment biopsies (p<0.0001 for both) and was similarly reduced for pRb in skin, (tumor data pending); there was no significant difference in % change in pRB and Ki67 by palbociclib dose, or by CBR or PFS. Higher baseline tumor Ki67 was associated with worse PFS (p=0.006), regardless of dose. Presence of PIK3CA mutations in cfDNA correlated with worse PFS (p=0.008), but ESR1 mutations did not. Conclusion: In pts with prior chemotherapy for HR+ MBC, treatment with 100 mg of palbociclib significantly reduced episodes of ntp and dose reductions; efficacy was comparable to patients treated with 125mg. Reductions in expression of Ki67 in tumor and keratinocytes and pRB in keratinocytes were comparable between dose levels indicating robust inhibition of CDK4/6 at both doses. The presence of PIK3CA mutations correlated with reduced PFS. These data suggest that dose reduction of palbociclib to mitigate toxicity should not compromise efficacy and provide additional prognostic information for use in treatment selection. Citation Format: Hope S. Rugo, Erica Mayer, Anna Maria Storniolo, Claudine Isaacs, Ingrid Mayer, Vered Stearns, Rita Nanda, Julie Nangia, Michelle Melisko, Chiara Wabl, Alona Muzikansky, Bose Kochupurakkal, Ben H. Park, Antonio Wolff, Geoffrey Shapiro. Palbociclib in combination with fulvestrant or tamoxifen as treatment for hormone receptor positive metastatic breast cancer with prior chemotherapy for advanced disease (TBCRC 035): A Phase II study with pharmacodynamic markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT128.

In situ cell cycle analysis in giant cell tumor of bone reveals patients with elevated risk of reduced progression-free survival.

Giant cell tumor of bone (GCTB) is a frequently recurring locally aggressive osteolytic lesion, where pathological osteoclastogenesis and bone destruction are driven by neoplastic stromal cells. Here, we studied if cell cycle fractions within the mononuclear cell compartment of GCTB can predict its progression-free survival (PFS). 154 cases (100 primaries and 54 recurrent) from 139 patients of 40 progression events, was studied using tissue microarrays. Ploidy and in situ cell cycle progression related proteins including Ki67 and those linked with replication licensing (mcm2), G1-phase (cyclin D1, Cdk4), and S-G2-M-phase (cyclin A; Cdk2) fractions; cell cycle control (p21waf1) and repression (geminin), were tested. The Prentice-Williams-Peterson (PWP) gap-time models with the Akaike information criterion (AIC) were used for PFS analysis. Cluster analysis showed good correlation between functionally related marker positive cell fractions indicating no major cell cycle arrested cell populations in GCTB. Increasing hazard of progression was statistically associated with the elevated post-G1/S-phase cell fractions. Univariate analysis revealed significant negative association of poly-/aneuploidy (p < 0.0001), and elevated cyclin A (p < 0.001), geminin (p = 0.015), mcm2 (p = 0.016), cyclin D1 (p = 0.022) and Ki67 (B56: p = 0.0543; and Mib1: p = 0.0564 -strong trend) positive cell fractions with PFS. The highest-ranked multivariate interaction model (AIC = 269.5) also included ploidy (HR 5.68, 95%CI: 2.62-12.31, p < 0.0001), mcm2 (p = 0.609), cyclin D1 (HR 1.89, 95%CI: 0.88-4.09, p = 0.105) and cyclin A (p < 0.0001). The first and second best prognostic models without interaction (AIC = 271.6) and the sensitivity analysis (AIC = 265.7) further confirmed the prognostic relevance of combining these markers. Ploidy and elevated replication licensing (mcm2), G1-phase (cyclin D1) and post-G1 phase (cyclin A) marker positive cell fractions, indicating enhanced cell cycle progression, can assist in identifying GCTB patients with increased risk for a reduced PFS.

PF266 COMBINING FLOW CYTOMETRY‐BASED LEUKEMIC CELL ENRICHMENT AND MUTATIONAL ANALYSIS FOR DETECTION OF RESIDUAL DISEASE IN ACUTE MYELOID LEUKEMIA: A PILOT STUDY

Background:Persistent minimal residual disease (MRD) is an independent prognostic marker in acute myeloid leukemia (AML) that has become increasingly important for risk stratification and treatment planning. MRD can be evaluated by multi‐parameter flow cytometry (MFC) and molecular methods including PCR‐based methods detecting leukemia‐specific fusion transcripts and targeted deep sequencing of genes recurrently mutated in AML. However, while MFC is highly operator‐dependent, PCR‐based methods are only available for a minority of AML patients. In addition, targeted deep sequencing approaches are hampered to distinguish between mutations responsible for preleukemic clonal hematopoiesis and true leukemia‐specific mutations.Aims:In this study we aimed for developing a highly sensitive and broadly applicable method for MRD detection in AML by combining MFC‐based leukemic cell enrichment followed by mutational analysis.Methods:To identify markers suitable for leukemic cell enrichment, AML samples from 150 newly diagnosed patients were analyzed for 24 surface markers using MFC with a strictly standardized protocol. In 25 cases, samples were also available at relapse. To assess the normal hematopoietic stem and progenitor cell (HSPC) compartments normal bone marrow (BM) samples were obtained from 12 lymphoma patients without any evidence of disease in the BM. For validation of our method, BM samples were prospectively collected from a total of 42 patients in complete remission (CR) after induction chemotherapy. Mutational analysis of sorted leukemic cell enriched samples was done using targeted deep sequencing of 39 recurrently mutated genes using an Ion Torrent sequencing platform. In some NPM1‐mutated samples analysis was performed using a mutation‐specific digital droplet PCR assay.Results:A combination of antibodies against CLL‐1, TIM‐3, CD123 and CD117 was identified to perform best for leukemic cell enrichment by enabling staining of &gt;90% of AML cells in 137 of 150 diagnostic AML samples (91.3%) and in 25 of 25 (100%) relapse samples. In dilution experiments using NPM1‐mutated samples and normal BM, leukemic cell enrichment by these markers followed by mutational analysis showed a sensitivity of 10–5 for residual disease detection. In contrast, NPM1‐mutations were not detected in cells negative for the marker combination in dilutions higher than 1:100. Using this marker combination for cell sorting allowed a 30‐ to 250‐fold cell enrichment in prospectively collected BM samples of 42 patients in complete remission. In 39 samples DNA quality of sorted cells was sufficient for sequencing. Twenty‐one samples tested MRD positive whereas 18 were negative. With a median follow‐up of 372 days 71% of MRD positive (15/21) and 28% (5/18) of MRD negative patients relapsed (p = 0.007). Accordingly, median relapse free survival was significantly longer in MRD negative patients (497 vs. 242 days, p = 0.0035). Of note, in 8 out of 10 MRD positive remission samples, in which we also analyzed marker negative cells, mutations were exclusively found in marker positive cells emphasizing the importance of MFC‐based cell enrichment prior to sequencing.Summary/Conclusion:MFC‐based leukemic cell enrichment using antibodies against CLL‐1, TIM‐3, CD123 and CD117 followed by mutational analysis is feasible for MRD detection with high sensitivity and informative on relapse risk in AML patients. Standardization of this method as well as its comparability to other approaches of MRD detection need to be evaluated in a multi‐center clinical trial.

Computer-assisted image analysis of the tumor microenvironment on an oral tongue squamous cell carcinoma tissue microarray

Oral tongue squamous cell carcinoma (OTSCC) displays variable levels of immune cells within the tumor microenvironment. The quantity and localization of tumor infiltrating lymphocytes (TILs), specific functional TIL subsets (e.g., CD8+), and biomarker-expressing cells (e.g., PD-L1+) may have prognostic and predictive value. The purpose of this study was to evaluate the robustness and utility of computer-assisted image analysis tools to quantify and localize immunohistochemistry-based biomarkers within the tumor microenvironment on a tissue microarray (TMA). We stained a 91-patient OTSCC TMA with antibodies targeting CD3, CD4, CD8, FOXP3, IDO, and PD-L1. Cell populations were segmented into epithelial (tumor) or stromal compartments according to a mask derived from a pan-cytokeratin stain. Definiens Tissue Studio was used to enumerate marker-positive cells or to quantify the staining intensity. Automated methods were validated against manual tissue segmentation, cell count, and stain intensity quantification. Univariate associations of cell count and stain intensity with smoking status, stage, overall survival (OS), and disease-free survival (DFS) were determined. Our results revealed that the accuracy of automated tissue segmentation was dependent on the distance of the tissue section from the cytokeratin mask and the proportion of the tissue containing tumor vs. stroma. Automated and manual cell counts and stain intensities were highly correlated (Pearson coefficient range: 0.46–0.90; p < 0.001). Within this OTSCC cohort, smokers had significantly stronger PD-L1 stain intensity and higher numbers of CD3+, CD4+ and FOXP3+ TILs. In the subset of patients who had received adjuvant radiotherapy, a higher number of CD8+ TILs was associated with inferior OS and DFS. Taken together, this proof-of-principle study demonstrates the robustness and utility of computer-assisted image analysis for high-throughput assessment of multiple IHC markers on TMAs, with potential implications for studies on prognostic and predictive biomarkers.

Anemia Severity in β-Thalassemia Correlates with Elevated Levels of microRNA-125b in Activated Phagocytic Monocytes

β-Thalassemia (β-thal), is an inherited blood disorder caused by reduced or absent synthesis of β-globin chains leading to imbalance of globin chain synthesis. The clearance of β-thalassemic abnormal red blood cells (RBCs) that result from excessive unbound α-globin is mainly achieved by activated monocytes. The phagocytic activity of β-thal monocytes significantly increases when co-cultured with normal and β-thal RBC individuals compare to that of normal monocytes co-cultured with normal RBCs. The present study indicates that microRNA (miR) plays a role in monocyte activation. In this study, we identified the higher miR-125b expression in CD14 marker-positive monocytic cells of β-thal patients. Moreover, miR-125b expression levels positively correlate with the phagocytic activity of monocytes. Remarkably, miR-125b expression levels are negatively correlated with RBC count, hemoglobin (Hb) and hematocrit [or packed cell volume (PCV)], which are the indices for the severity of anemia. From these findings, our future studies will be to prove the hypothesis that miR-125b expression in activated monocytes may be a genetic modifier related to the severity of anemia in β-thal patients.

Abstract PD2-12: Palbociclib in combination with fulvestrant or tamoxifen as treatment for hormone receptor positive (HR+) metastatic breast cancer (MBC) with prior chemotherapy for advanced disease (TBCRC 035) A phase II study with pharmacodynamics markers

Abstract Background Addition of the cyclin dependent kinase 4/6 inhibitor (CDK4/6i) palbociclib to endocrine therapy in the first and later line settings significantly improves progression free survival (PFS) in patients with HR+ MBC. The primary toxicity is neutropenia without an increase in febrile neutropenia. TBCRC035 explored rates of neutropenia in patients who had received prior chemotherapy for MBC with 2 dose levels of palbociclib, and correlated changes in retinoblastoma protein phosphorylation (pRB) and Ki67 expression in proliferating keratinocytes and tumor with response. Methods TBCRC035 is a 1:1 randomized multicenter phase II study evaluating palbociclib at either 125 or 100 mg in combination with physician choice fulvestrant or tamoxifen. Eligible patients (pts) with HR+ MBC had received &amp;gt;1 but &amp;lt;3 lines of chemotherapy for MBC, any number of prior hormone therapies, and were naïve to CDK4/6i. The primary endpoint was grade 3/4 neutropenia; secondary endpoints included response, safety/tolerability, inhibition of pRB and change in Ki67 in skin and tumor at day 14-21 of treatment compared to baseline. FFPE sections of skin punch and tumor biopsies obtained before and on treatment were stained using antibodies to Ki67, total RB, and phospho-RB-S780 using BOND polymer red detection. Stained slides were scanned into the Aperio image analysis platform; the percentage of marker positive cells and H-score was determined. Results 70 pts were enrolled (fully accrued); 35 randomized to 100 vs 125 mg of palbociclib respectively; data for the last 3 pts on the 125 mg arm is pending. Grade 3/4 neutropenia was more common in the 125 mg vs the 100 mg arm (56 vs 34%); dose adjustments for adverse events (AEs) occurred in 47 vs 43%, 4 vs 0 pts discontinued treatment due to AEs. Grade 3 febrile neutropenia was rare (1 patient each arm). Median duration of treatment was 5.2 vs 7.2 months. Response data and correlation with changes in pRB and Ki67 expression in skin and tumor by treatment arm will be reported. Conclusion In pts with prior chemotherapy for HR+ MBC, treatment with 100 mg of palbociclib in patients is associated with a lower rate of &amp;gt; grade 3 neutropenia compared to 125 mg. Correlation of response by dose with pRB and Ki67 has the potential to inform palbociclib dosing and reduce toxicity for pts with HR+ MBC. Citation Format: Rugo HS, Mayer EL, Storniolo AM, Isaacs C, Mayer I, Stearns V, Nanda R, Nangia J, Wabl C, Deluca A, Kochupurakkal B, Wolff AC, Shapiro GI. Palbociclib in combination with fulvestrant or tamoxifen as treatment for hormone receptor positive (HR+) metastatic breast cancer (MBC) with prior chemotherapy for advanced disease (TBCRC 035) A phase II study with pharmacodynamics markers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-12.

Distribution of neuroendocrine marker-positive cells in colorectal cancer tissue and adjacent mucosa.

617 Background: Neuroendocrine carcinoma (NEC) is a rare disease and has been reported to most frequently arise in the right side of the colon. In the 2010 WHO classification, mixed adenoneuroendocrine carcinoma (MANEC) was defined as a neoplasm consisting of NEC and adenocarcinoma components. To clarify the histogenesis of NEC, we attempted to detect neuroendocrine marker-positive cells in cancer tissue and in the adjacent mucosa in patients with adenocarcinoma. Methods: The study group comprised 390 patients with Stage II or III colorectal adenocarcinoma between 2007 and 2012. Immunostaining was performed with anti chromogranin A, synaptophysin, and CD56 antibodies. Cases with positively stained cells in cancer tissue were defined as positive. In the adjacent mucosa, at least 5 cm from the tumor, the numbers of positive cells per 15 HPF were measured. Results: Tumor location was right side in 181 patients, left side in 173, and the rectum in 36 patients. Positive rates of Chromogranin A in cancer tissues were 23.7% in the right colon, 13.2% in the left colon, and 19.4% in the rectum. Those of synaptopysin were 35.3%, 21.9%, and 30.6%, respectively. Those of CD56 were 22.6%, 8.0%, and 16.7%, respectively. Positive rates of these three markers in right colon were significantly higher than those in left colon and rectum. (p = 0.0115, p = 0.0054, p = 0.0062). In the adjacent mucosa, the mean numbers of positive cells for chromogranin A were 62.2 ± 20.5 in the right colon, 131.9 ± 44.7 in the left colon, and 243.7 ± 60.2 in the rectum (p &lt; 0.001). Those for synaptophysin were 47.7 ± 23.5, 95.3 ± 35.1, and 156.9 ± 56.8, respectively. (p &lt; 0.001). There were no significant differences in the number of positive cells for CD56 among the sites (p = 0.295). Conclusions: In cancer tissue, the rate of positive staining for neuroendocrine marker-positive cells was higher in the right side of the colon, whereas in normal mucosa the rates of positive staining for these cells were higher in the sigmoid colon and the rectum. These results suggest that neuroendocrine marker-positive cells are an acquired characteristic of cancer tissue.

NQO1 is Required for β-Lapachone-Mediated Downregulation of Breast-Cancer Stem-Cell Activity.

Cancer stem cells (CSCs) exhibit self-renewal activity and give rise to other cell types in tumors. Due to the infinite proliferative potential of CSCs, drugs targeting these cells are necessary to completely inhibit cancer development. The β-lapachone (bL) compound is widely used to treat cancer development; however, its effect on cancer stem cells remain elusive. Thus, we investigated the effect of bL on mammosphere formation using breast-cancer stem-cell (BCSC) marker-positive cells, MDA-MB-231. MDA-MB-231 cells, which are negative for reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H):quinone oxidoreductase (NQO1) expression, were constructed to stably express NQO1 (NQO1 stable cells). The effect of bL on these cells was evaluated by wound healing and Transwell cell-culture chambers, ALDEFLUOR assay, and mammosphere formation assay. Here, we show that bL inhibited the proliferative ability of mammospheres derived from BCSC marker-positive cells, MDA-MB-231, in an NQO1-dependent manner. The bL treatment efficiently downregulated the expression level of BCSC markers cluster of differentiation 44 (CD44), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), and discs large (DLG)-associated protein 5 (DLGAP5) that was recently identified as a stem-cell proliferation marker in both cultured cells and mammosphered cells. Moreover, bL efficiently downregulated cell proliferation and migration activities. These results strongly suggest that bL could be a therapeutic agent for targeting breast-cancer stem-cells with proper NQO1 expression.

Loss of ZBTB7A Facilitates RUNX1/RUNX1T1-Dependent Clonal Expansion and Sensitizes for Metabolic Inhibition

ZBTB7A is a transcription factor involved in the regulation of metabolism and hematopoietic linage fate decisions. Recently, we found ZBTB7A mutated in 23% of Acute Myeloid Leukemia (AML) patients with t(8;21) translocation (Hartmann et al., 2016, Nat Commun). However, the oncogenic collaboration between ZBTB7A alterations and the RUNX1/RUNX1T1 fusion in AML t(8;21) remains poorly understood.To study ZBTB7A mutations in the context of RUNX1/RUNX1T1-dependent transformation, we used human CD34+ cells co-transduced with a truncated form of RUNX1/RUNX1T1 and ZBTB7A wild-type (WT) or its mutants (R402C and A175fs). We then followed the evolution of fluorescence marker positive cells over a period of 60 days. While expression of RUNX1/RUNX1T1 alone caused clonal expansion, co-expression of ZBTB7A WT impaired the outgrowth of CD34+ cells (Figure 1a). In contrast, the anti-proliferative effect of ZBTB7A was lost for both of its mutants tested resulting in a rescue of the clonal expansion (Figure 1b).To investigate the effect of ZBTB7A mutations on tumor metabolism, we used CRISPR/Cas9 to knockout (KO) ZBTB7A in the myeloid K562 cell line. As ZBTB7A is a known negative regulator of glycolysis, we treated KO and control cells with the glycolysis inhibitor 2-deoxy-d-glucouse (2-DG). KO cells were more sensitive to 2-DG compared to control cells (mean IC50: 3.06 vs 6.82 mM; p-value=0.087) (Figure 1c). These results are in line with the observed upregulation of glycolytic genes in ZBTB7A-mutant AML t(8;21) and suggest that these patients may benefit from the treatment with metabolic inhibitors. To learn more about deregulation of ZBTB7A target genes we are currently performing RNA-Seq analysis of WT vs KO K562.Moreover, we used the K562 KO model to investigate the impact of loss of ZBTB7A on myeloid differentiation. The baseline expression of the erythroid marker CD235a was reduced in KO cells. Ectopic expression of ZBTB7A WT in the KO cells restored the CD235a levels to a control level, while expression of mutants or vector showed no effect. These findings are in agreement with previous reports of ZBTB7A involvement in erythroid differentiation.To study the effect of ZBTB7A mutations on granulopoiesis, we established HL60 cells stably expressing WT or mutant ZBTB7A. We then differentiated the cells into granulocytes through all-trans retinoic acid (ATRA) treatment. Expression of WT, but not the mutants, resulted in a 4-fold increase of the granulocytic marker CD11b. Additionally, we induced monocytic differentiation through Phorbol 12-myristate 13-acetate (PMA) treatment. The mutant expressing cells showed similar levels of the monocytic marker CD14 as control cells. WT overexpressing cells had a 50% decrease in the number of monocytes. We then used CRISPR/Cas9 to establish ZBTB7A KO HL60, which exhibited a 5.5-fold increase in CD14 compared to control cells (Figure 1d). This data supports a previously unknown negative regulatory role of ZBTB7A in monocytic differentiation.With regards to potential therapeutic applications, we tested the PMA sensitivity of HL60 and found a significantly lower IC50 in absence of ZBTB7A (mean: 124.5 vs 269.8 pM; p-value=0.001). Hence, loss of ZBTB7A may facilitate the pharmacological differentiation of leukemia cells.In conclusion, ZBTB7A mutations in AML t(8;21) display a loss-of-function phenotype. Inactivating mutations of ZBTB7A allow for hCD34+ RUNX1/RUNX1T1-mediated expansion and deregulated tumor metabolism. Finally, loss of ZBTB7A expression perturbs myeloid development and thus may complement the block of differentiation induced by RUNX1/RUNX1T1. These findings open up avenues to novel therapies for ZBTB7A mutated patients including metabolic inhibition and pharmacological differentiation. [Display omitted] DisclosuresHiddemann:F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

559P - Distribution of endocrine tumor marker-positive cells in adenocarcinoma tissue between right-sided and left-sided colon cancer

559P - Distribution of endocrine tumor marker-positive cells in adenocarcinoma tissue between right-sided and left-sided colon cancer