High CD49d Expression Research Articles

Advancements in the study of allergen-specific Th2 cell surface markers

Allergen-specific Th2 cells refer to a subset of Th2 cells that undergo substantial expansion following allergen stimulation. They play a crucial role in allergic diseases, and an increasing amount of research has revealed a close relationship between surface molecules on allergen-specific Th2 cells and allergic diseases. In comparison to other CD4+T cells or Th2 cells, allergen-specific Th2 cells exhibit low expression of CD27 but high expression of CD154, CD69, CRTH2, CD161, ST2, hPGDs, CD49d, and COX-2. They can be used for the identification of allergen-specific Th2 cells and serve as potential targets for the prevention and treatment of specific diseases. They hold significant value in preventing the onset and exacerbation of allergic diseases.

Extended interval dosing of ocrelizumab modifies the repopulation of B cells without altering the clinical efficacy in multiple sclerosis

BackgroundRecent studies suggest that extended interval dosing of ocrelizumab, an anti-B cell therapy, does not affect its clinical effectiveness in most patients with multiple sclerosis (MS). However, it remains to be established whether certain B cell subsets are differentially repopulated after different dosing intervals and whether these subsets relate to clinical efficacy.MethodsWe performed high-dimensional single-cell characterization of the peripheral immune landscape of patients with MS after standard (SID; n = 43) or extended interval dosing (EID; n = 37) of ocrelizumab and in non-ocrelizumab-treated (control group, CG; n = 28) patients with MS, using mass cytometry by time of flight (CyTOF).ResultsThe first B cells that repopulate after both ocrelizumab dosing schemes were immature, transitional and regulatory CD1d+ CD5+ B cells. In addition, we observed a higher percentage of transitional, naïve and regulatory B cells after EID in comparison with SID, but not of memory B cells or plasmablasts. The majority of repopulated B cell subsets showed an increased migratory phenotype, characterized by higher expression of CD49d, CD11a, CD54 and CD162. Interestingly, after EID, repopulated B cells expressed increased CD20 levels compared to B cells in CG and after SID, which was associated with a delayed repopulation of B cells after a subsequent ocrelizumab infusion. Finally, the number of/changes in B cell subsets after both dosing schemes did not correlate with any relapses nor progression of the disease.ConclusionsTaken together, our data highlight that extending the dosing interval of ocrelizumab does not lead to increased repopulation of effector B cells. We show that the increase of CD20 expression on B cell subsets in EID might lead to longer depletion or less repopulation of B cells after the next infusion of ocrelizumab. Lastly, even though extending the ocrelizumab interval dosing alters B cell repopulation, it does not affect the clinical efficacy of ocrelizumab in our cohort of patients with MS.

Immune Profiling Reveals the T-Cell Effect of Ocrelizumab in Early Relapsing-Remitting Multiple Sclerosis.

Ocrelizumab (OCR), a humanized anti-CD20 monoclonal antibody, is highly efficient in patients with relapsing-remitting multiple sclerosis (RR-MS). We assessed early cellular immune profiles and their association with disease activity at treatment start and under therapy, which may provide new clues on the mechanisms of action of OCR and on the disease pathophysiology. A first group of 42 patients with an early RR-MS, never exposed to disease-modifying therapy, was included in 11 centers participating to an ancillary study of the ENSEMBLE trial (NCT03085810) to evaluate the effectiveness and safety of OCR. The phenotypic immune profile was comprehensively assessed by multiparametric spectral flow cytometry at baseline and after 24 and 48 weeks of OCR treatment on cryopreserved peripheral blood mononuclear cells and analyzed in relation to disease clinical activity. A second group of 13 untreated patients with RR-MS was included for comparative analysis of peripheral blood and CSF. The transcriptomic profile was assessed by single-cell qPCRs of 96 genes of immunologic interest. Using an unbiased analysis, we found that OCR as an effect on 4 clusters of CD4+ T cells: one corresponding to naive CD4+ T cells was increased, the other clusters corresponded to effector memory (EM) CD4+CCR6- T cells expressing homing and migration markers, 2 of them also expressing CCR5 and were decreased by the treatment. Of interest, one CD8+ T-cell cluster was decreased by OCR corresponding to EM CCR5-expressing T cells with high expression of the brain homing markers CD49d and CD11a and correlated with the time elapsed since the last relapse. These EM CD8+CCR5+ T cells were enriched in the CSF of patients with RR-MS and corresponded to activated and cytotoxic cells. Our study provides novel insights into the mode of action of anti-CD20, pointing toward the role of EM T cells, particularly a subset of CD8 T cells expressing CCR5.

CD49d Expression Identifies a Biologically Distinct Subtype of Chronic Lymphocytic Leukemia with Inferior Progression Free Survival on BTK Inhibitor Therapy

Background: Acalabrutinib, a highly selective Bruton tyrosine kinase inhibitor (BTKi), is effective and well tolerated for patients with chronic lymphocytic leukemia (CLL). During treatment with BTKis, transient lymphocytosis is observed due to the efflux of CLL cells from lymphoid organs. CD49d, the alpha chain of the CD49d/CD29 integrin very late antigen-4 (VLA-4), is a key mediator in CLL microenvironmental interactions. High CD49d expression has been associated with inferior response to ibrutinib (Tissino, 2018). Here, we investigated the role of CD49d in response to acalabrutinib in 48 patients with CLL enrolled in a single-center, phase 2 study (NCT02337829). Methods: Patients were divided into CD49d positive (CD49d+, n = 27, 56%) and CD49d negative (CD49dneg, n = 21, 44%) groups based on the established 30% cutoff, as assessed by flow cytometry. CD49d activation was measured as VLA-4 receptor occupancy (RO), as described (Tissino, 2018). Time to progression was estimated using the Kaplan-Meier method. RNA sequencing (RNA-seq) of CD19+ selected CLL cells from CD49d+ (n = 10) and CD49dneg (n = 10) subsets was performed at baseline and 6 months on acalabrutinib. Results: After the first month on acalabrutinib, the median increase in absolute lymphocyte count (ALC) for CD49d+ CLL was 6.4% vs. 50.4% for CD49dneg (P = .02). However, the increase in ALC on day 3 of 42% and 48%, respectively, was comparable (P = .8). A subset of patients showed bimodal CD49d (bimCD49d) expression, with concomitant presence of CD49d positive and negative subpopulations. In these cases, the fraction of CD49d+ CLL cells increased significantly on day 3 (P = .002), but decreased at 6 months (P = .004), suggesting that CD49d+ cells may be better able to remain tissue bound on chronic therapy or reenter lymphoid tissues at higher rates than CD49dneg cells. While constitutive VLA-4 activation was reduced in patients on acalabrutinib, BCR and CXCR4 stimulation still increased VLA-4 RO on CLL cells even after 6 months of treatment. PI3K, in addition to BTK signaling, is implicated in inside-out activation of VLA-4. In vitro, addition of the PI3K inhibitor duvelisib to acalabrutinib-treated CLL cells completely blocked BCR- but not CXCR4- induced VLA-4 activation. The overall response rate of the study was 95.8% (Sun, 2020). Sixteen patients progressed at a median of 42.5 months, 3 with transformation. Time to progression for patients with CD49d+ and CD49dneg CLL (applying 30% cutoff) did not differ (P = .4). However, at 72 months 50% of the combined CD49d+ and bimCD49d patients (n = 37) were estimated progression-free vs 90% of patients with a homogeneous CD49dneg CLL population (n = 11, P = .02). Of 13 patients progressing with CLL, 12 (92%) were CD49d+ or bimodal CD49d and 11 (85%) had BTK and/or PLCG2 mutations. To dissect the impact of CD49d expression on CLL pathobiology and the response to acalabrutinib, we compared transcriptomes of CD49d+ and CD49dneg subsets by RNA-seq. A distinct gene expression profile separated the groups by principal component analysis. Using gene set enrichment analysis, CD49d+ CLL showed constitutively more immunoreceptor, NF-kappa B, and cytokine/JAK-STAT signaling, as well as enhanced cell survival, adhesion and migration capacity compared to CD49dneg CLL (FDR <.25). After 6 months on acalabrutinib, all 20 patients achieved a clinical response and all samples showed downregulation of B-cell receptor and NF-kappa B target genes, as expected for BTKi therapy. Interestingly, on acalabrutinib, CD49d+ samples not only maintained a higher level of activation compared to CD49dneg CLL, but differences in the degree of cytokine (IL-6, IL-10) and STAT3 signaling became more prominent. Conclusion. CD49d+ and CD49dneg CLL cells are rapidly mobilized out of lymph nodes at the start of acalabrutinib therapy, but CD49d+ cells appear able to reenter or persist in lymphoid tissues better than CD49dneg cells. Bimodal CD49dneg and CD49d+ cases share an increased risk of disease progression, with most patients having detectable BTK or PLCG2 mutations at the time of progression. The transcriptional program of CD49d+ CLL reflects upregulation of survival and activation pathways that may increase tolerance to BTKi therapy and set the stage for clonal evolution. Thus, CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance and a possible therapeutic target to improve on BTKi therapy.

Novel Mechanisms of Acalabrutinib Resistance in Patients with Chronic Lymphocytic Leukemia By Whole Genome Methylome Sequencing

Novel Mechanisms of Acalabrutinib Resistance in Patients with Chronic Lymphocytic Leukemia By Whole Genome Methylome Sequencing

In vivo quantitative assessment of therapeutic response to bortezomib therapy in disseminated animal models of multiple myeloma with [18F]FDG and [64Cu]Cu-LLP2A PET

BackgroundMultiple myeloma (MM) is a disease of cancerous plasma cells in the bone marrow. Imaging-based timely determination of therapeutic response is critical for improving outcomes in MM patients. Very late antigen-4 (VLA4, CD49d/CD29) is overexpressed in MM cells. Here, we evaluated [18F]FDG and VLA4 targeted [64Cu]Cu-LLP2A for quantitative PET imaging in disseminated MM models of variable VLA4 expression, following bortezomib therapy.MethodsIn vitro and ex vivo VLA4 expression was evaluated by flow cytometry. Human MM cells, MM.1S-CG and U266-CG (C: luciferase and G: green fluorescent protein), were injected intravenously in NOD-SCID gamma mice. Tumor progression was monitored by bioluminescence imaging (BLI). Treatment group received bortezomib (1 mg/kg, twice/week) intraperitoneally. All cohorts (treated, untreated and no tumor) were longitudinally imaged with [18F]FDG (7.4–8.0 MBq) and [64Cu]Cu-LLP2A (2–3 MBq; Molar Activity: 44.14 ± 1.40 MBq/nmol) PET, respectively.ResultsFlow cytometry confirmed high expression of CD49d in U266 cells (> 99%) and moderate expression in MM.1S cells (~ 52%). BLI showed decrease in total body flux in treated mice. In MM.1S-CG untreated versus treated mice, [64Cu]Cu-LLP2A localized with a significantly higher SUVmean in spine (0.58 versus 0.31, p < 0.01) and femur (0.72 versus 0.39, p < 0.05) at week 4 post-tumor inoculation. There was a four-fold higher uptake of [64Cu]Cu-LLP2A (SUVmean) in untreated U266-CG mice compared to treated mice at 3 weeks post-treatment. Compared to [64Cu]Cu-LLP2A, [18F]FDG PET detected treatment-related changes at later time points.Conclusion[64Cu]Cu-LLP2A is a promising tracer for timely in vivo assessment of therapeutic response in disseminated models of MM.

Interferon Regulatory Factor 4 (IRF4) Is a Master Regulator in the Pathogenesis of Chronic Lymphocytic Leukemia Patients Harboring Trisomy 12

BACKGROUND:Chronic lymphocytic leukemia (CLL) represents the most common leukemia in the Western countries. Although its natural history is not fully elucidated, it may be determined by a combination of genetic factors and by the complex relationship that leukemic cells entertain with the surrounding microenvironment. Interferon regulatory factor 4 (IRF4, also known as MUM1) is a transcriptional regulator that exerts critical functions in different cell types of the immune system. In B cells IRF4 plays a role in the function and development, by controlling the transition from cycling pre-B to small pre-B cells and by regulating receptor editing, germinal center (GC) reaction and plasma cell differentiation. Deregulation of the biological programs regulated by IRF4 has been related to the pathogenesis of different B-cell malignancies. Tumor cells in CLL patients express reduced levels of IRF4 protein compared to normal B cells and a causal relationship between low levels of IRF4 and the development of CLL has been demonstrated by two elegant in vivo mouse studies.AIM OF THE WORK:The goal of our study was to characterize the expression of IRF4 in CLL patients and to identify its biological functions in order to devise a possible involvement of IRF4 in the pathogenesis of CLL.METHODS:IRF4 expression was monitored by real time PCR in purified CLL cells. Modulation of IRF4 expression in vitro was performed by using a Myc-DDK-tagged IRF4 vector or by siRNA strategy.RESULTS:Among a large cohort of CLL samples (n=250), we identified a peculiar lower expression of IRF4 in patients harboring trisomy 12 (Tri12) compared with other cytogenetic aberrations. We focused our attention on the biological features of Tri12 CLL cells as adhesion, migration and viability dissecting the underlying molecular aspects.First, we tested the ability of Tri12 versus noTri12 CLL cells to adhere to VCAM1 coated plate. As expected, Tri12 CLL cells showed a higher ability to adhere to VCAM1 compared to no Tri12 due to high CD49d expression. We correlated the low expression of IRF4 in Tri12 CLL cells with an increased ability of leukemic cells to adhere to VCAM1 and endothelial cells. Forcing the expression of IRF4 by using a IRF4 vector is able to revert the adhesive features of CLL cells decreasing the surface expression of CD49d. We compared the capacity of Tri12 versus no Tri12 CLL cells to migrate towards CXCL12 in chemotaxis assay. Tri12 CLL cells tended to migrate towards CXCL12 at higher level than no Tri12 CLL cells. The induction of IRF4 in Tri12 CLL cells affected the expression of CXCR4 determining an impaired migration either in response or not to CXCL12. This result is in line with a reduced activation of signaling pathway induced by the reconstitution of IRF4 expression upon CXCL12 stimulation. Investigating the spontaneous apoptotic response of Tri12 CLL cells and no Tri12 CLL cells, we found a prominent resistance to spontaneous death in the first compared to the second subset. Since IRF4 is a regulator of Notch2 during CLL development, we compared the expression of Notch2 and its targets in Tri12 and no Tri12 CLL cells. At the molecular level, Tri12 CLL cells are characterized by an increased expression of Notch2 that is linked to a more pronounced expression of Mcl1 and Hes1 compared to no Tri12 CLL cells. Again, forcing the expression of IRF4 in Tri12 CLL cells, there was a dramatic drop in CLL cells viability that correlates with a reduced expression of Notch2, Mcl1 and CD23. Conversely, reducing the expression of IRF4 by using a siRNA strategy restores Tri12 CLL cells viability by inducing Notch2, Mcl1 and CD23 expression. Finally, we interfered with Notch2 expression using a siRNA or a pharmacological strategy. Silencing of Notch2 in Tri12 CLL cells affected CLL cells viability through the downregulation of CD23 and Mcl1 expression. On this line, using the pharmacological compound gliotoxin, known to interfere with Notch signaling pathway, we found a strong induction of CLL cells apoptosis due to the complete Notch2 degradation.CONCLUSIONS:Collectively, these results indicate that IRF4 plays a relevant role in determining the pathobiology of CLL harboring trisomy 12. The forced restoration of IRF4 expression is followed by marked decrease in adhesion and homing and induction of intrinsic apoptosis in Tri12 CLL cells, suggesting that targeting IRF4 expression may be effective for Tri12 CLL. DisclosuresNo relevant conflicts of interest to declare.

Natalizumab differentially affects plasmablasts and B cells in multiple sclerosis

BackgroundNatalizumab treatment increases the frequencies of B cells in blood but reduces IgG in blood and CSF. Plasmablasts are important in the production of IgG, and the development of plasmablasts is CD49d dependent. ObjectiveWe hypothesized that natalizumab treatment affects the development of plasmablasts. MethodsWe retrospectively analyzed frequencies and absolute counts of B cell subsets by flow cytometry from a longitudinal cohort of 9 progressive multiple sclerosis (MS) patients treated with natalizumab for 60 weeks, and a cross-sectional relapsing-remitting MS (RRMS) cohort with 17 untreated and 37 treated with natalizumab (17 stable and 20 unstable patients with relapse activity). Additionally, CD49d expression on B cell subsets was examined in 10 healthy controls, and blood and cerebrospinal fluid (CSF) frequencies of B cell subsets were quantified in untreated and natalizumab treated RRMS patients. ResultsIn progressive MS, levels of IgG decreased in plasma (p<0.001) from baseline to 60 weeks follow-up. In the progressive MS and RRMS cohorts we observed that natalizumab treatment significantly increased the frequency of B cells (p=0.004; p<0.0001) and several B cell subsets, most pronounced for memory B cell subsets (p=0.0001; p<0.0001), while there was a decrease in plasmablast frequency (p=0.008; p=0.008). In both progressive MS and RRMS the absolute cell counts of B cells increased (p=0.004; p<0.001), which was explained by a significant increase in all subsets, except for plasmablasts. Furthermore, we found decreased memory B cell counts in unstable compared to stable natalizumab-treated patients (p=0.02). The expression of CD49d was higher on plasmablasts compared to other B cell subsets (p<0.0001). In CSF, plasmablasts could not be detected in patients treated with natalizumab, in contrast to an increased frequency in untreated RRMS patients. ConclusionWe confirm previous studies showing that natalizumab increases circulating number of B cells, particularly memory cells, concomitant with a decrease in plasma IgG concentrations. Moreover, we demonstrate in two separate cohorts that natalizumab treatment markedly decreases frequencies of plasmablasts while the absolute number is stable. Additionally, plasmablasts have high expression of CD49d, and plasmablasts could not be detected in the CSF of natalizumab-treated patients. Finally, memory B cells were found to be reduced in unstable natalizumab-treated patients, which could possibly indicate increased recruitment to the CNS.

Risk factors for hypogammaglobulinemia in chronic lymphocytic leukemia patients treated with anti-CD20 monoclonal antibody-based therapies

Hypogammaglobinemia is a well-recognized complication in chronic lymphocytic leukemia (CLL); however, the risk factors that lead to this complication after treatment with anti-CD20 monoclonal antibodies are not well understood. Using the Mayo Clinic CLL database, a retrospective chart review was conducted on patients enrolled between January 1995 and September 2017 who had pre- and post-treatment immunoglobulin values. The relationship between immunoglobulin values prior to treatment as well as post-treatment with clinical variables and overall survival (OS) was evaluated. Hypogammaglobinemia was defined as serum immunoglobulin G (IgG) levels <500 mg/dL and evaluated at both baseline and following treatment. Follow-up hypogammaglobinemia was treated as a time-dependent variable in Cox models. A total of 179 patients met the eligibility criteria, 24 (13%) of whom had hypogammaglobinemia pre-treatment. Patients with hypogammaglobulinemia prior to treatment were more likely to have higher expression of CD49d (p value = 0.007). There was no difference in infection risk, other comorbidities, or OS between the pre- and post-treatment hypogammaglobinemia. In the subset of 155 subjects without hypogammaglobinemia at baseline, 22 developed hypogammaglobinemia. The median time to hypogammaglobinemia for these subjects was 36.6 months. Hypogammaglobinemia is commonly observed in CLL patients, at baseline as well as with the additional use of anti-CD20 therapies. The mean time to onset of hypogammaglobinemia after anti-CD20 therapies was 36 months. Time to hypogammaglobinemia does not significantly differ based on any of the CLL clinical factors studied. The presence of hypogammaglobinemia did not appear to impact survival or time to infection.

High Surface Expression of CD49d (VLA-4) and CD79b Correlates with Acalabrutinib Resistance in Patients with Chronic Lymphocytic Leukemia (CLL)

High Surface Expression of CD49d (VLA-4) and CD79b Correlates with Acalabrutinib Resistance in Patients with Chronic Lymphocytic Leukemia (CLL)

The Role of Imaging in Predicting Time to First Treatment and Overall Survival in Individuals with CLL-like High Count Monoclonal B-Cell Lymphocytosis

Background: The 2008 International Working Group for CLL (IWCLL) criteria define MBL as a clonal B-cell disorder with a peripheral blood B-cell count of &lt;5 x 109/L, in the absence of cytopenias(s), lymphadenopathy and/or organomegaly on physical examination, and no other concomitant lymphoproliferative disorder. In the presence of palpable lymphadenopathy and/or organomegaly, and a B-cell count of &lt; 5 x 109/L, a diagnosis of small lymphocytic lymphoma (SLL) is established. A study by Gentile et al (Am J Hematol. 2013) reported that 29 out of 62 individuals with clinically identified MBL (42%) had lymphadenopathy identified on CT imaging. Notably, however, after a median follow-up of 35 months, the rate of progression among cases of MBL with lymphadenopathy identified on CT imaging was only 6.9% (2/29 patients), which was no different than in cases of MBL without lymphadenopathy on CT imaging (rate of progression, 9%; 3/33 patients). Our study sought to 1) determine if lymphadenopathy and/or organomegaly detected by imaging studies performed for unrelated reasons in individuals with MBL can identify a subset with shorter time to first CLL therapy (TFT) and overall survival (OS); and 2) compare outcomes of individuals with MBL who have incidentally detected lymphadenopathy/splenomegaly and SLL patients. Methods: We used the Mayo Clinic CLL Database to identify individuals with high-count MBL (absolute B-cell count: 0.5 - 4.9 x 109/L) and divided them into the following three cohorts: a) Cohort A: no imaging studies available at MBL diagnosis; b) Cohort B: imaging studies done within one year of MBL with no evidence of lymphadenopathy and/or organomegaly; and c) Cohort C: imaging studies done within one year of MBL with lymphadenopathy and/or organomegaly, attributable to MBL. All individuals with MBL were first seen at Mayo Clinic after 1/1/2002; those with a concomitant lymphoproliferative disorder within 3 months of diagnosis were excluded. Patients with SLL seen during the same time interval were identified as a comparison cohort. We compared the baseline characteristics (Kruskal Wallis for continuous and Chi-square for categorical variables) and TFT and OS across the three MBL cohorts, and patients with SLL. Cumulative incidence of TFT was adjusted for the competing risk of death. Age- and sex-adjusted OS was analyzed using inverse weights methods. Results: 657 individuals with high-count MBL with a median follow-up of 6.7 years were identified: 415 (63%) individuals were in Cohort A, 142 (22%) in Cohort B, and 100 in (15%) Cohort C. Compared to Cohort B, individuals in Cohort C were more likely to have unmutated IGHV, high expression of CD38, and higher beta-2 microglobulin level (Table 1). SLL patients were younger, had higher rate of unmutated IGHV, and had high expression of CD49d compared to MBL individuals in Cohort C (Table 1). There was suggestive evidence of a difference in TFT between Cohorts B and C (estimated 10-year treatment rate 28.3% vs. 41.6%; p=0.16; HR 1.62 95% CI 0.84-3.15). TFT was significantly shorter in SLL patients compared to those with Cohort C (estimated 10-year treatment rate 71% vs. 41.6%, respectively, P&lt;0.001; Figure 1A). There was a trend toward shorter OS in Cohort C compared to Cohort B (estimated 10-year OS rate 53.7% vs. 64.2%, respectively, P=0.09; Figure 1B). In contrast, there was no difference in OS among MBL individuals in Cohort C compared to those with SLL (estimated 10-year OS rate 56.9% vs. 62.6%, P=0.72). Summary/Conclusion: Our findings demonstrate that individuals with MBL who have incidentally identified lymphadenopathy/splenomegaly at diagnosis are more likely to have an unfavorable risk profile, shorter TFT and OS compared to patients who did not have lymphadenopathy/splenomegaly at MBL diagnosis. Additionally, these individuals have a longer TFT but similar OS compared to SLL patients seen during the same time interval. If externally validated, these findings have significant implications for individuals with newly diagnosed MBL. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Kenderian:Novartis: Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Parikh:AstraZeneca: Honoraria, Research Funding; Janssen: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding.

T cell trafficking within the peritoneal tumor environment and ovarian cancer progression.

5573 Background: Ovarian cancer is associated with high mortality due to detection at late stages with widespread peritoneal metastases at diagnosis in a majority of patients. Ovarian cancer recurrences are primarily found in the peritoneal cavity, and peritoneal disease is the primary cause of morbidity and mortality in this disease. This pattern of tumor engraftment and recurrence indicates that the peritoneal tumor environment is distinct from other niches. Our data indicate that selective peritoneal dissemination is immunologically mediated, based on functional differences in peritoneal and systemic T cells during ovarian cancer progression. This is consistent with data in other solid tumors demonstrating that tumor immunity is driven by regional lymphocyte populations. However, less is known about the mechanisms that establish a permissive immune environment in the peritoneal cavity. We hypothesize that pathways regulating T cell recruitment and retention in the peritoneal cavity (PC) are co-opted by ovarian cancer cells to enable intraperitoneal cancer dissemination and recurrence. Methods: We have developed a novel model that uses direct in vivo labeling of peritoneal cells in an established immune-competent high grade serous murine cancer model. This functional approach enables us to identify T cell subsets retained in the PC with tumor engraftment and progression. Results: We identified high expression of CD49d (a4 integrin) as the most prevalent cell surface marker on T cells retained in the peritoneal cavity, consistent with prior published data in healthy mice and people. We demonstrated a functional role for CD49dhi in T cell retention by showing preferential binding to VCAM. A role for tumor cells mediating this interaction was observed based on enhanced binding affinity in vitro with tumor monolayers. The importance of this mechanism is supported by high VCAM expression in multiple murine and human ovarian cancer cell lines, and T cell localization to VCAM rich areas within ovarian cancer tumors in vivo. Conclusions: CD49d not only defines a lymphocyte subset with a significant role in tumor immunity but presents itself as an important potential therapeutic target to modulate T cell trafficking.

Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia.

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF (KITLG, KIT, PTPN11, GNB1, KRAS and NRAS) were mutated in 12 patients (2.6%), and genes downstream of BRAF (MAPK2K1, MAPK2K2, and MAPK1) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P=0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited.

Identification of Human B-1 Helper T Cells With a Th1-Like Memory Phenotype and High Integrin CD49d Expression.

Human B-1 cells have been proposed to be CD20+CD27+CD43+CD1c− B cells found in the umbilical cord and adult peripheral blood, but their regulatory mechanisms have not been well elucidated. Previously, we reported that mouse CD49dhigh CD4+ T cells could enhance the secretion of natural antibodies by B-1 cells. In this study, we aimed to investigate the presence and helper functions of the human equivalents of murine CD49dhigh CD4+ T cells. Here, we showed that human CD49dhigh CD4+ T cells found in the peritoneal cavity (PEC), spleen, and peripheral blood can enhance the production of IgM antibodies by B-1 cells. As revealed in mouse, CD49dhigh CD4+ T cells were more abundant in the PEC and showed a higher tendency to form conjugates with B cells than CD49dlow CD4+ T cells. Moreover, CD49dhigh CD4+ T cells showed a Th1-like memory phenotype, characterized by high expression of CD44 and CXCR3; low expression of CD62L and CCR7; rapid production of IFN-γ, tumor necrosis factor-α, and IL-2 upon stimulation with phorbol myristate acetate and ionomycin; and rapid proliferation upon stimulation with anti-CD3 and anti-CD28 antibodies. These cells also expressed high levels of PD-1, ICOS, and CD5, suggesting that they are undergoing chronic stimulation. Remarkably, CD49dhigh CD4+ T cells specifically helped B-1 cells, but not follicular memory B cells (CD27+ CD43−CD1c−) or marginal zone B cells (CD27+CD43−CD1c+), produce IgM and IgG antibodies. In parallel, the titer of human anti-blood group A IgM was positively correlated with the frequency of CD49dhigh CD4+ T cells. In conclusion, we identified human CD49dhigh CD4+ T cells with a Th1-like memory phenotype that secrete Th1 proinflammatory cytokines and help B-1 cells secrete antibodies, thereby aiding in primary defense. We suggest that these CD49dhigh CD4+ T cells are a unique type of B-cell helper T cells distinct from follicular helper T cells.

Adipose-derived and bone marrow mesenchymal stem cells: a donor-matched comparison

BackgroundAdipose-derived stem cells (ASCs) have been introduced as an alternative to bone marrow mesenchymal stem cells (BMSCs) for cell-based therapy. However, different studies comparing ASCs and BMSCs have shown conflicting results. In fact, harvesting ASCs and BMSCs from different individuals might influence the results, making comparison difficult. Therefore, this study aimed to characterize donor-matched ASCs and BMSCs in order to investigate proliferation, differentiation potential and possible effects of donor variation on these mesenchymal stem cells (MSCs).MethodsHuman bone marrow and adipose tissue samples were obtained from nine donors aged 8–14. ASCs and BMSCs were isolated and characterized based on expression of surface markers using flow cytometry. The proliferation up to 21 days was investigated. Multi-lineage differentiation was induced using osteogenic, chondrogenic and adipogenic differentiation media. Alkaline phosphatase (ALP) activity was monitored and collagen type I formation was evaluated by immunofluorescence staining. In vitro multi-potency was studied using tissue-specific stains and lineage-specific gene expression. In addition, the osteogenic lineage was evaluated at protein level.ResultsIsolated ASCs and BMSCs from all donors demonstrated morphologic and immunophenotypic characteristics of MSCs, with expression of MSCs markers and negative expression of hematopoietic markers. Unlike BMSCs, ASCs showed high expression of CD49d and low expression of Stro-1. In general, ASCs showed significantly higher proliferation and adipogenic capacity with more lipid vesicle formation and expression of the adipogenesis-related genes than BMSCs. In contrast, BMSCs showed significantly higher osteogenic and chondrogenic capacity compared to ASCs. BMSCs had earlier and higher ALP activity, calcium deposition, and expression of the osteogenesis- and chondrogenesis-related genes and the osteogenesis-related protein osteopontin. Proliferation and differentiation capacity of ASCs and BMSCs varied significantly among the donors.ConclusionsASCs and BMSCs showed tissue-specific differentiation abilities, but with significant variation between donors. The similarities and differences in the properties of ASCs and BMSCs should be taken into consideration when planning stem cell-based therapy.

Relevance of CD49d and CD38 expressions as predictors of disease progression in chronic lymphocytic leukemia

Background Chronic lymphocytic leukemia is a heterogeneous disease characterized by a highly variable clinical course. The majority of cases do not require therapy and show identical survival rate with their chronic lymphocytic leukemia-free counterparts of the same age. However, other patients may suffer from an aggressive course of the disease with early need for therapy and shorter overall survival. Objective The aim of this study was to investigate the expression pattern and prognostic value of the adhesion molecules, CD49d and CD38, in chronic lymphocytic leukemia (CLL) patients. Moreover, we attempted to investigate their correlation with trisomy 12, which could help in the clarification of the distinctive clinical features of the trisomy 12+ CLL subset. Patients and methods Seventy newly diagnosed chronic lymphocytic leukemia patients and 50 age-matched and sex-matched controls were included in this study. Patients were subjected to full history taking, clinical examination and abdominal ultrasonography. Laboratory investigations included complete blood count, cytogenetic analysis for the presence of trisomy 12, and flow cytometric assessment. Results A significant positive correlation was detected between CD49d expression and CD38 expression. CD49d showed positive correlation with poor prognostic parameters that included organomegaly, high count of white blood cells, lymphocyte count and trisomy 12. CD38 showed a significant positive correlation with high white blood cell count, lymphocyte count, and splenomegaly. In addition, trisomy 12+ chronic lymphocytic leukemia patients showed significant higher lymphocyte count and splenomegaly. Conclusion Our data confirm the suggested role of CD38 and CD49d as predictors for poor clinical outcome of CLL patients. Moreover, trisomy 12+ CLL cells exhibit a high CD49d expression that may account for the unique clinical characteristics of this group.

NOTCH1 mutations are associated with high CD49d expression in chronic lymphocytic leukemia: link between the NOTCH1 and the NF-κB pathways

In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10-15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (P<0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.

CD49d associates with nodal presentation and subsequent development of lymphadenopathy in patients with chronic lymphocytic leukaemia.

CD49d is a surface integrin that is expressed on chronic lymphocytic leukaemia (CLL) cells, and strongly correlates with more aggressive disease. Given its association with cell-cell adhesion and leucocyte trafficking, we hypothesized that patients with high CD49d expression would experience a clinical course dominated by lymphadenopathy. CD49d expression was measured by flow cytometry and considered positive if expressed by ≥30% of CLL cells. The study included 797 newly diagnosed CLL/small lymphocytic leukaemia patients; 279 (35%) were CD49d positive. CD49d-positive patients were more likely to present with lymphadenopathy (P<0·001); a finding that persisted after adjusting for fluorescence insitu hybridisation (FISH) and IGHV mutation status [odds ratio (OR) 2·51; 95% confidence interval (CI) 1·64-3·83; P<0·001]. Among CLL Rai 0 patients, CD49d positivity was associated with shorter time to development of lymphadenopathy (3·2years vs not reached, P<0·01). This association was maintained after adjusting for either FISH [hazard ratio (HR) 2·18; 95% CI 1·25-3·81; P=0·006) or IGHV status (HR 2·02; 95% CI 1·11-3·69; P=0·02) individually, but was attenuated when adjusting by both (HR 1·72; 95% CI 0·88-3·38; P=0·11).These data demonstrate that CD49d-positive CLL patients experience a disease course dominated by lymphadenopathy. These findings could have implications for therapy selection and disease monitoring.

How to Improve the Definition of Chronic Lymphocytic Leukemia Outcome Using a Simple Flow Cytometric Score Based on CD49d and Homing Marker Expression

The identification of new molecular markers in Chronic Lymphocytic Leukemia (CLL) allowed to better define prognosis and clinical outcome. The actual staging systems could estimate the prognosis, but not the rapidity of disease evolution. Neither the identification of new molecular markers did allow to foresee the evolution and clinical response, because discordant findings were mostly reported. The aim of the present study was (1) to confirm the independent prognostic role of CD49d as a single marker in CLL patients, (2) to investigate the relationship between CD49d and other well-established CLL-membrane predictor markers (CD5, CD11c, CD20 and CD38) or clinical staging systems and (3) to evaluate the role of an immunophenotypic score based on the flow-cytometric detection of CD5, CD11c, CD20, CD38 and CD49d in the work up of CLL staging.Heparinized whole blood was collected from 68 CLL patients for immunophenotyping using the following antibodies: anti kappa, anti-lambda, CD5, CD11c, CD19, CD20, CD23, CD38, CD45, CD49d. A scoring system was elaborated combining 5 membrane markers: CD5, CD11c, CD20, CD38 and CD49d. Antigens were divided in two groups, favorable (CD5 and CD20) and unfavorable (CD11c, CD38 and CD49d) prognostic markers, and the cut-off of positivity was chosen according to the literature (30% for CD5, CD11c, CD20 and CD38, and 45% for CD49d). A value of "0" or "1" ("2" only for CD49d positivity) was assigned according to antigen expression. Finally, we defined a favorable phenotype when the sum of all the cytometric features was equal or less than 2, conversely the unfavorable phenotype was defined for a sum equal or greater than 3 (between 3 and 6).Flow cytometric analysis showed high CD49d expression in CD19+ cells in 47% of patients (n=32), and high CD38 expression in 44% of subjects (n=30), simultaneously expressed in 28% of patients (n=19). The 19% (n=13) of all CLL patients were CD5-, and interestingly the 85% of them showed higher expression of CD49d. Linear correlation was found between CD49d and CD38 (r2=0.08772, p=0.0142), and between CD49d and CD20 expression (r2=0.2490, p<0.0001). For CD5, the opposite tendency was registered (r2=0.1944, p=0.0002). Strong negative correlation between higher CD49d expression and total lymphocyte count was found (Pearson r = -0.3577, r2=0.1279, p=0.0068), but not for hemoglobin level and platelet count. The statistical power of each parameter of the score was also calculated by Chi-square test and all markers displayed a statistically significant weight (p<0.0001). After assessed the prognostic power of each marker, we applied the score to staged patients. Forty patients (59%) had a Favorable Phenotype and 28 of them (70%) an early stage disease; in this group of patients, only 7.5% (n=3) showed CD49d high expression. The overall response rate (ORR) was of 58%. The other 28 patients (41%) showed an Unfavorable Phenotype and 21 of them (75%) had an early stage disease. In this arm, all subjects carried>45% of CD49d positive cells. Four patients with Unfavorable Phenotype received chemotherapy with an ORR of 25%. Furthermore, a small population (n=16) of our CLL cohort was also studied for genetic abnormalities using FISH technique. According to FISH analysis, 25% of studied patients were classifies as very low-risk and, interestingly, no one of them showed an Unfavorable Phenotype (only one patient carried CD49d as unique negative marker). In our cohort, 50% of patients were low-risk with no genetic abnormalities or +12, but 63% of them showed an Unfavorable Phenotype with high CD49d and CD38 expression in 100% and 60% of cases, respectively.Our data confirm the independent negative prognostic role of CD49d and suggest a stronger prognostic power compared to CD38 in the definition of CLL outcome, because of its complex activity as homing marker, signaling receptor and anti-apoptotic molecule. Thus, the prognostic significance of CD49d may be enhanced when considered in comparison with other established markers, as CD11c and CD38. In conclusion, our results propose the use of the CD49d marker in combination with other B-cell membrane antigens as an additional tool for routine diagnosis and risk-stratification of CLL patients. Identification of high-risk phenotype with a simple scoring method could improve the treatment of these patients, who could take advantage of the most recent molecular targeting therapies. DisclosuresNo relevant conflicts of interest to declare.

Ex Vivo Evaluation of VLA-4 Expression in Primary Human Multiple Myeloma Bone Marrow Samples and In Vivo Mobilization of Murine Multiple Myeloma Cells with Small Molecule VLA-4 Inhibitors

Background: The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) affects disease progression and provides resistance to therapeutic agents. Very-late-antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) is a noncovalent, heterodimeric transmembrane receptor that is strongly implicated in the pathogenesis of MM via altering cell trafficking, proliferation and drug resistance. LLP2A is a high-affinity peptidomimetic ligand for activated VLA-4. We recently reported (Soodgupta et al. J. Nucl. Med 2016) the sensitive and specific molecular imaging of activated VLA-4 in mouse MM tumors using 64Cu-LLP2A and LLP2A-Cy5. Here we extended these studies by further characterizing VLA-4 expression in primary human MM samples and malignant plasma cells in mouse models of MM.Methods: We evaluated VLA-4 expression in 5 human MM cell lines (U266, OPM2, H929, RPMI-8226 and MM1.S), one mouse MM cell line (5TGM1) and seventeen primary human MM bone marrow samples by flow cytometry using LLP2A-Cy5, soluble VCAM-1/Fc recombinant protein and CD49d (α4) and CD29 (β1) antibodies. The relative mean fluorescence intensity (RMFI) of LLP2A-Cy5 binding was calculated by dividing the MFI of LLP2A-Cy5 binding in the absence of BIO5192 (small molecule VLA-4 inhibitor) by the MFI of LLP2A-Cy5 binding in the presence of excess BIO5192. The 5TGM1/KaLwRij immunocompetent mouse model of MM was used for in vivo study.Results: The expression of activated VLA-4 on MM cell lines as measured by LLP2A-Cy5+ mean fluorescent intensity (MFI) varied 10-fold as follows (LLP2A-Cy5 MFI in parentheses): 5TGM1 (23.7) > U266 (16.1) > OPM2 (4.6) > H929 (3.4) > RPMI-8226 (3.2) > MM1.S (2.1). We observed similar variable expression of LLP2A-Cy5 binding to primary human CD138+CD38+ MM plasma cells (PCs), with 76.47% (13/17) of MM patients exhibiting greater than 20% LLP2A-Cy5+ PCs. expressing VLA-4 on CD138+CD38+ cells. Overall, the mean percentage of positive cells and LLP2A-Cy5 relative MFI (RMFI) on malignant CD138+ PCs from these 13 patients were 78.2% (43.8-98.3%) and 4.3 (1.7-10.8), respectively. Other hematopoietic cells within the BM samples expressed less VLA-4 in descending order as follows; monocytes (58.2%, RMFI 3.0), T-lymphocytes (34.4%, RMFI 2.1) and B-lymphocytes (21.6%, RMFI 1.6). These levels of VLA-4 expression on normal cell subsets within MM patients were comparable to normal blood donors. In general, there was good correlation between LLP2A-Cy5 binding and expression of CD49d and CD29 on CD138+ PCs in MM patients. To our surprise, the four MM patients with <20% LLP2A-Cy5 binding demonstrated high expression of CD49d (92.1%) but very low percentages of CD29 positive cells (17.3%). Using BIO5192 (VLA-4 inhibitor), we found that the LLP2A-Cy5 reagent allowed more accurate detection of activated VLA-4 than the soluble VCAM-1 binding assay as determined by the magnitude of inhibition of binding in the presence of inhibitor. We next evaluated targeting VLA-4 molecule in murine MM model. Preliminary mouse mobilization studies demonstrated that VLA-4 inhibitors effectively and rapidly mobilized murine 5TGM1 MM cells from the bone marrow to the blood (2.49-fold increase in circulating GFP+CD138+ cells) within 1 hour of injection.Summary:This study is the first demonstration that activated VLA4 can be detected on primary human MM cells using LLP2A. These data support the continued development of LLP2A as a molecular diagnostic imaging reagent for MM and as a potential therapeutic target of VLA-4 in MM. Ongoing studies are testing whether small molecule VLA-4 inhibitors can sensitize MM cells to cytotoxic therapy in vivo. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.