Eltrombopag for Treating Thrombocytopenia after Allogeneic Stem Cell Transplantation.

Thrombocytopenia after allogeneic hematopoietic stem cell transplantation (allo-SCT) can pose significant problems in management of patients. Eltrombopag is a small-molecule thrombopoietin receptor agonist that has been approved for use in immune thrombocytopenic purpura and aplastic anemia; but its use after allo-SCT is limited. Between 2014 and 2017, we treated 13 patients with eltrombopag for poor platelet engraftment without evidence of relapse at the time of initiation, including 6 patients with primary platelet engraftment failure and 7 with secondary platelet engraftment failure. Eltrombopag was started at an initial dose of 25 or 50 mg per day, and dose adjustments were made in accordance with the manufacturer's recommendation. The cumulative incidence of platelet recovery to ≥50,000/µL without the need for transfusion for at least 7 days was defined as response. The overall response rate was 62% (n = 8). Of the 6 patients with primary isolated platelet failure, 3 (50%) responded, and of the 7 patients with secondary platelet failure, 5 (71%) responded. The median time to response was 33 days (range, 11 to 68 days). In addition, no significant differences in platelet recovery were noted in patients with adequate and decreased bone marrow megakaryocytic reserve (60% and 67%, respectively). Although eltrombopag was well tolerated, and no patient discontinued treatment because of adverse events, only 3 patients were alive at the end of the observation period, with relapse and graft-versus-host disease accounting for majority of the deaths. This suggested that despite the relatively good overall response rate to eltrombopag, inadequate platelet engraftment is a harbinger of poor outcome in allo-SCT.

Periostin and Human Teeth.

Periostin is a secreted matricellular protein that primarily interacts with type I collagen and fibronectin extracellular matrix proteins, and is widely distributed in tissues rich in collagen-rich connective tissues, including the periodontal ligament. Its expression in these tissues is especially regulated by mechanical load. While the expression and regulation of periostin in the teeth of murine models and cell lines is well known, its presence in human teeth is poorly documented. Here we update and summarize the available data on the distribution of periostin in the human periodontal ligament, gingiva and dental pulp.

Racial and ethnic associations with comprehensive cancer center access and clinical trial enrollment for acute leukemia.

BACKGROUND: Clinical trial participation at Comprehensive Cancer Centers (CCC) is inequitable for minoritized racial and ethnic groups with acute leukemia. CCCs care for a high proportion of adults with acute leukemia. It is unclear if participation inequities are due to CCC access, post-access enrollment, or both. METHODS: We conducted a retrospective cohort study of adults with acute leukemia (2010-2019) residing within Massachusetts, the designated catchment area of the Dana-Farber/Harvard Cancer Center (DF/HCC). Individuals were categorized as non-Hispanic Asian (NHA), Black (NHB), White (NHW), Hispanic White (HW), or Other. Decomposition analyses assessed covariate contributions to disparities in (1) access to DF/HCC care and (2) post-access enrollment. RESULTS: Of 3698 individuals with acute leukemia, 85.9% were NHW, 4.5% HW, 4.3% NHB, 3.7% NHA, and 1.3% Other. Access was lower for HW (age- and sex-adjusted OR = 0.64, 95% CI = 0.45 to 0.90) and reduced post-access enrollment for HW (aOR = 0.54, 95% CI =0.34 to 0.86) and NHB (aOR = 0.60, 95% CI = 0.39 to 0.92) compared to NHW. Payor and socioeconomic status (SES) accounted for 25.2% and 21.2% of the +1.1% absolute difference in HW access. Marital status and SES accounted for 8.0% and 7.0% of the -8.8% absolute disparity in HW enrollment; 76.4% of the disparity was unexplained. SES and marital status accounted for 8.2% and 7.1% of the -9.1% absolute disparity in NHB enrollment; 73.0% of the disparity was unexplained. CONCLUSIONS: A substantial proportion of racial and ethnic inequities in acute leukemia trial enrollment at CCCs are from post-access enrollment, the majority of which was not explained by sociodemographic factors.

Overcoming Barriers to Referral for Chimeric Antigen Receptor T Cell Therapy in Patients with Relapsed/Refractory Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin lymphoma. Although outcomes to frontline therapy are encouraging, patients who are refractory to or in relapse after first-line therapy experience inferior outcomes. A significant proportion of patients treated with additional lines of cytotoxic chemotherapy ultimately succumb to their disease, as established in the SCHOLAR-1 study. Chimeric antigen receptor (CAR) T cell therapy is a novel approach to cancer management that reprograms a patient's own T cells to better target and eliminate cancer cells. It was initially approved by the US Food and Drug Administration for patients with relapsed/refractory (r/r) DLBCL as a third line of treatment. Based on recently published randomized data, CAR-T therapy (axicabtagene ciloleucel and lisocabtagene maraleucel) also has been approved as a second line of treatment for patients who are primary refractory or relapse within 12 months of first-line therapy. Despite the proven efficacy in treating r/r DLBCL with CD19-directed CAR-T therapy, several barriers may prevent eligible patients from receiving treatment. Barriers to CAR-T therapy include cost of therapy, patient hesitancy, required travel to academic treatment centers, nonreferrals, poor understanding of CAR-T therapy, lack of caregiver support, knowledge of available resources, and timely patient selection by referring oncologists. In this review, we provide an overview of the FDA-approved CD19-directed CAR-T cell therapies (tisagenlecleucel, axicabtagene ciloleucel, and lisocabtagene maraleucel) from pivotal clinical trials and supporting real-world evidence from retrospective studies. In both clinical trials and real-world settings, CAR-T therapy has been shown to be safe and efficacious for treating patients with r/r DLBCL: however, several barriers prevent eligible patients from accessing these therapies. Barriers to referrals for CAR-T therapy are described, along with recommendations to improve collaboration between community oncologists and physicians from CAR-T therapy treatment centers and subsequent long-term care of patients in community treatment centers.

Manifestations and evolution of Wilson disease in pediatric patients carrying ATP7B mutation L708P.

OBJECTIVES: The aim of the study was to characterize a group of 11 pediatric patients, ages 3 to 13 years, affected by Wilson disease (WD) in the island of Gran Canaria, Spain. PATIENTS AND METHODS: Genetic, biochemical, and pathological features, together with their response to treatment and clinical evolution, have been analyzed for this group of patients. RESULTS: Genetically, the group was rather homogeneous, with an extremely high prevalence of the L708P mutation (4 homozygotes and 5 heterozygotes). Despite being initially screened because of asymptomatic hypertransaminemia, all of the patients presented with some degree of liver damage that was never accompanied by any neurological manifestation. Hepatic damage was most severe in a compound heterozygote with a novel mutation, G1266W, affecting a motif in the ATP7B polypeptide that is greatly conserved in similar proteins among metazoans. Ceruloplasmin and copper serum levels, together with the determination of hepatic copper content, were found to be of great diagnostic value, whereas urine copper measurements were found to be much less conclusive. All of the patients responded well to treatment with D-penicillamine with no documented adverse reactions. CONCLUSIONS: The patients in Gran Canaria constitute, overall, one of the largest groups of patients with WD with a high incidence of a single mutation, allowing us to define the early clinical symptoms and the evolution of the disease in patients carrying the ATP7B L708P mutant allele, and the study of WD in a genetically homogeneous background.

Complement-dependent inflammation and injury in a murine model of brain dead donor hearts.

RATIONALE: Donor brain death (BD) is an unavoidable occurrence in heart transplantation and results in profound physiological derangements that render the heart more susceptible to ischemia/reperfusion injury in the recipient and likely has negative long-term consequences to allograft survival. OBJECTIVE: We developed a novel mouse model of BD and investigated the role of complement in BD-induced myocardial inflammation and injury. METHODS AND RESULTS: BD was induced by inflation of a balloon catheter in the cranial cavity. BD in wild-type mice resulted in a significant increase in serum concentrations of the complement activation product complement component (C)3a, and immunohistochemical analysis of heart sections demonstrated C3 deposition on the vascular endothelium and surrounding myocytes. Following induction of BD in complement (C3)-deficient mice, cardiac troponin levels, and histological evidence of injury were significantly reduced compared to wild-type mice. C3 deficiency was also associated with reduced myocardial leukocyte infiltration and reduced or absent expression of P-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor-alpha, and interleukin-1beta. CONCLUSIONS: These data indicate an important role for complement in BD-induced inflammation and injury and suggest that a complement inhibitory strategy applied to the donor (in addition to the recipient) may provide graft protection.

Impact of Next-Generation Sequencing Cell-free Pathogen DNA Test on Antimicrobial Management in Adults with Hematological Malignancies and Transplant Recipients with Suspected Infections.

Infections in adult patients with hematological malignancies (HM) and stem cell transplant (SCT) recipients are a significant cause of morbidity and mortality. A timely diagnosis of infections can have a major impact on outcomes. Tools that help rule out infectious causes of fever can decrease antibiotic use, toxicities, hospitalization costs, and potentially decrease antibiotic resistance in the long term. We retrospectively evaluated the ability of cell-free DNA next-generation sequencing (NGS) testing in the timely identification of pathogenic microorganisms and its impact on the antimicrobial management of immunocompromised patients with hematologic malignancies. In the period between 2018 to 2020, 95 samples were reviewed, of which 31 adult patients (32 tests) had hematologic malignancies or were recipients of SCT. The NGS tests were performed in the following patients: (a) patients with prolonged fever and negative conventional tests, (b) persistent fever despite positive conventional test and appropriate antimicrobials, and (c) fever-free patients with imaging suspicious for infection. The median time from fever to NGS sampling was 5 days (range, 1-28). The median time to NGS results was 2 days (range, 1-6). The NGS resulted in an escalation of antibiotics in 28% of cases (9/32) and de-escalation of antibiotics in 31% of cases (10/32). Overall, NGS testing changed management in nearly 59% (19/32) of patients. The sensitivity and specificity of NGS to detect clinically significant infection was 80% and 58%, respectively. The test identified uncommon and difficult to diagnose organisms such as Nocardia, Legionella, Toxoplasma and Pneumocystis jirovecii resulting in rapid antimicrobial interventions. In conclusion, in patients with HM or SCT recipients, microbial cell-free DNA sequencing allowed rapid and actionable treatment. This strategy can target appropriate antibiotic use, avoid overtreatment, and potentially decrease the hospital length-of-stay.

Enhancement of antibody-dependent mechanisms of tumor cell lysis by a targeted activator of complement.

Complement inhibitors expressed on tumor cells provide a hindrance to the therapeutic efficacy of some monoclonal antibodies (mAb). We investigated a novel strategy to overwhelm complement inhibitor activity and amplify complement activation on tumor cells. The C3-binding domain of human complement receptor 2 (CR2; CD21) was linked to the complement-activating Fc region of human IgG1 (CR2-Fc), and the ability of the construct to target and amplify complement deposition on tumor cells was investigated. CR2 binds C3 activation fragments, and CR2-Fc targeted tumor cells by binding to C3 initially deposited by a tumor-specific antibody. Complement deposition on Du145 cells (human prostate cancer cell line) and anti-MUC1 mAb-mediated complement-dependent lysis of Du145 cells were significantly enhanced by CR2-Fc. Anti-MUC1 antibody-dependent cell-mediated cytotoxicity of Du145 by human peripheral blood mononuclear cells was also significantly enhanced by CR2-Fc in both the presence and the absence of complement. Radiolabeled CR2-Fc targeted to s.c. Du145 tumors in nude mice treated with anti-MUC1 mAb, validating the targeting strategy in vivo. A metastatic model was used to investigate the effect of CR2-Fc in a therapeutic paradigm. Administration of CR2-Fc together with mAb therapy significantly improved long-term survival of nude mice challenged with an i.v. injection of EL4 cells. The data show that CR2-Fc enhances the therapeutic efficacy of antibody therapy, and the construct may provide particular benefits under conditions of limiting antibody concentration or low tumor antigen density.

The Basics of Artificial Antigen Presenting Cells in T Cell-Based Cancer Immunotherapies.

Adoptive T cell transfer (ACT) can mediate objective responses in patients with advanced malignancies. There have been major advances in this field, including the optimization of the ex vivo generation of tumor-reactive lymphocytes to ample numbers for effective ACT therapy via the use of natural and artificial antigen presenting cells (APCs). Herein we review the basic properties of APCs and how they have been manufactured through the years to augment vaccine and T cell-based cancer therapies. We then discuss how these novel APCs impact the function and memory properties of T cells. Finally, we propose new ways to synthesize aAPCs to augment the therapeutic effectiveness of antitumor T cells for ACT therapy.

ß-catenin and PI3Kδ inhibition expands precursor Th17 cells with heightened stemness and antitumor activity.

ICOS costimulation generates Th17 cells with durable memory responses to tumor. Herein, we found that ICOS induces PI3K/p110δ/Akt and Wnt/ß-catenin pathways in Th17 cells. Coinhibiting PI3Kδ and ß-catenin altered the biological fate of Th17 cells. Th17 cells inhibited of both pathways expressed less RORγt, which, in turn, reduced their ability to secrete IL-17. Unexpectedly, these cells were more effective (than uninhibited cells) at regressing tumor when infused into mice, leading to long-term curative responses. PI3Kδ inhibition expanded precursor Th17 cells with a central memory phenotype that expressed nominal regulatory properties (low FoxP3), while ß-catenin inhibition enhanced Th17 multifunctionality in vivo. Remarkably, upon TCR restimulation, RORγt and IL-17 rebounded in Th17 cells treated with PI3Kδ and ß-catenin inhibitors. Moreover, these cells regained ß-catenin, Tcf7, and Akt expression, licensing them to secrete heightened IL-2, persist, and eradicate solid tumors without help from endogenous NK and CD8 T cells. This finding shines a light on ways to repurpose FDA-approved drugs to augment T cell-based cancer immunotherapies.

An innovative microarray strategy identities informative molecular markers for the detection of micrometastatic breast cancer.

There is increasing evidence that molecular detection of micrometastatic breast cancer in the axillary lymph nodes (ALN) of breast cancer patients can improve staging. Molecular analyses of samples obtained from the Minimally Invasive Molecular Staging of Breast Cancer Trial (n = 489 patients) indicate that whereas the majority of molecular markers are informative for the detection of metastatic breast cancer (significant disease burden), only a few are sensitive for the detection of micrometastatic disease (limited disease burden). Frequency distribution and linear regression analyses reveal that relative levels of gene expression are highly correlated with apparent sensitivity for the detection of micrometastic breast cancer (P < 0.05). These data provides statistical validation of the concept that the most informative markers for detection of micrometastatic disease are those that are most highly expressed in metastatic disease. To test this hypothesis, we developed an innovative microarray strategy. RNA from a metastatic breast cancer ALN was diluted into RNA from a normal lymph node and analyzed using Affymetrix microarrays. Expression analysis indicated that only two genes [mammaglobin (mam) and trefoil factor 1 (TFF1)] were significantly overexpressed at a dilution of 1:50. Real-time reverse transcription-PCR analysis of pathology-negative ALN (n = 72) confirm that of all the markers tested, mam and TFF1 have the highest apparent sensitivity for detection of micrometastatic breast cancer. We conclude that a dilutional microarray approach is a simple and reliable method for the identification of informative molecular markers for the detection of micrometastatic cancer.

Temporal patterns of gene expression in murine cutaneous burn wound healing.

The global changes in gene expression in injured murine skin were characterized following a second-degree scald burn. Dorsal skin was harvested from uninjured and from burned mice at 2 h and at 3 and 14 days following immersion in 65 degrees C water for 45 s. Gene expression was surveyed using an Affymetrix U74Av2 GeneChip, and patterns of gene expression were analyzed using hierarchical clustering and supervised analysis. Burn injury produced significant alterations in the expression of a number of genes, with the greatest changes seen 3 and 14 days after the scald burn. Using a supervised analysis with a false discovery rate of 1% or 5%, differences in the expression of 192 or 1,116 genes, respectively, discriminated among the unburned skin and the three time points after the burn injury. Gene expression was primarily a transient and time-dependent upregulation. The expression of only 24 of the 192 discriminating genes was downregulated after the burn injury. No gene exhibited a sustained increase in expression over the entire 14 days following the burn injury. Gene ontologies revealed an integrated upregulation of inflammatory and protease genes at acute time intervals, and a diminution of cytoskeletal and muscle contractile genes at 3 or 14 days after the injury. Following a second-degree scald burn, global patterns of gene expression in the burn wound change dramatically over several weeks in a time-dependent manner, and these changes can be categorized based on the biological relevance of the genes.

Microarray analysis of gene expression patterns during healing of rat corneas after excimer laser photorefractive keratectomy.

PURPOSE: To characterize changes over time in the genomic expression profile of rat corneas after excimer laser photorefractive keratectomy (PRK), in an effort to better understand the cellular response to injury and the dynamic changes that occur in gene expression patterns as a wound heals. METHODS: The corneal gene expression profile of 1176 genes at 3 and 7 days after PRK was determined and compared with untreated corneal gene expression patterns by interrogating commercially available cDNA arrays with labeled target cDNA prepared from pooled total RNA harvested from the respective treatment group of adult male rats. The gene expression patterns were inferred based on the hybridization intensities of the probes on the cDNA arrays. The hybridization signals were globally normalized and filtered. The data were analyzed by using hierarchical and k-means clustering algorithms before and after normalization of variances. RESULTS: Of the 1176 cDNA elements on the array, 588 consistently produced similar results in replicate experiments and comprised the data set analyzed in this work. In total, 73 genes were identified, with expression levels that differed by at least threefold at either 3 or 7 days after PRK. At 3 days after PRK, 70 genes were identified with expression levels that differed by more than threefold, compared with the expression level in untreated animals. The expression of 42 genes increased by threefold or more, whereas expression of 28 genes decreased by threefold or more. By day 7 after PRK, the number of genes displaying more than a threefold difference in expression pattern was reduced to 27 genes, 20 of which showed elevated levels, whereas 7 exhibited decreased levels. Hierarchical clustering of the 588 studied genes produced 10 clusters with correlation coefficients of 0.9 or greater. To determine whether any of the clusters were overrepresented by genes with related functions, the cumulative hypergeometric probability was calculated by obtaining the observed number of functionally related genes within each of the 10 clusters. Seven of the clusters were statistically overrepresented by one or more categories of functionally related genes, such as cell cycle regulators, transcription factors, and metabolic pathway genes. Clustering analysis of 56 genes generally considered to influence corneal wound healing produced 10 gene clusters with correlation coefficients of at least 0.9. Expression of 23 of these 56 genes increased at day 3, then decreased at day 7 to levels similar to those on day 0. These included several growth factors (VEGF, FGF, IGF-I), proteases (PAI-1, PAI-2A) and protease inhibitors (TIMP-2 and TIMP-3). Expression of nine genes increased on both days 3 and 7 compared with expression on day 0 (e.g., TGFB1, TGFBIIR, M6P/IGFR-2), and no genes decreased on both days 3 and 7, compared with day 0. CONCLUSIONS: Microarray analysis of 1176 identified 588 genes with reproducible patterns of expression in rat corneas on days 3 and 7 after PRK and 73 genes with a threefold change in expression compared with untreated corneas. Hierarchical clustering of these 588 genes identified 10 clusters of genes with very similar patterns of expression. Clustering of genes with similar patterns of expression implies a common regulatory pathway for the genes within a cluster, and identifies potential new targets for regulating corneal wound healing.

Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy.

PURPOSE: Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS: Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS: All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS: These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.

Alterations in gene expression of complement components in chronic rhinosinusitis.

BACKGROUND: The complement cascade forms part of the initial innate response to pathogens in the airway. Complement activation is important in the maintenance of host homeostasis, but excessive and uncontrolled activation may lead to inflammation and disease. The role of the complement pathway in the innate response in chronic rhinosinusitis (CRS) is poorly characterized Methods: Sinus mucosa biopsy specimens from the anterior ethmoid or uncinate process of patients with allergic fungal rhinosinusitis (AFRS), CRS without NPs (CRS-NPs), and controls were harvested and gene and protein expression of C3, factor B (fB), C5, and C7 complement proteins were analyzed using quantitative polymerase chain reaction and immunohistochemical techniques. RESULTS: fB, C3, and C5 gene expression were increased in both AFRS and CRS-NPs compared with controls (p < 0.05). Transcriptional activity for the terminal pathway protein C7 was not significantly increased when compared with controls, with C7 levels actually reduced in AFRS patients when compared with controls. Immunohistochemistry studies showed the presence of C3 and fB on the mucosal surface and in submucosa of both AFRS and CRS-NPs, but not normal controls. Terminal pathway protein C9 was not found in our specimens. CONCLUSION: Both AFRS and CRS-NPs display up-regulation of the complement pathway, in particular, the alternative pathway (fB) and common pathways (C3 and C5). Enhanced innate responses as shown by alterations in complement components may play a pivotal role in the inflammatory response noted in CRS and provide potential therapeutic targets in the future.

Modulation of protective T cell immunity by complement inhibitor expression on tumor cells.

Complement-inhibitory proteins expressed on cancer cells can provide protection from antitumor antibodies and may potentially modulate the induction of an immune response to tumor-associated antigens. In the current study, we investigated the consequences of complement inhibitor down-regulation on the effector and inductive phases of an immune response. Stable small interfering RNA-mediated down-regulation of the complement inhibitor Crry on MB49 murine bladder cancer cells increased their susceptibility to monoclonal antibody and complement in vitro. In a syngeneic model of metastatic cancer, the down-regulation of Crry on i.v.-injected MB49 cells was associated with a significant decrease in tumor burden and an increase in the survival of challenged mice. However, monoclonal antibody therapy had no additional benefit. There was an antitumor IgG response, but the response was not effected by Crry down-regulation on inoculated tumor cells. Down-regulation of Crry on MB49 cells resulted in an enhanced antitumor T-cell response in challenged mice (measured by lymphocyte IFN-gamma secretion), and CD8+ T cell depletion of mice prior to injection of MB49 cells completely abrogated the effect of Crry down-regulation on tumor burden and survival. Deficiency of C3 also abrogated the effect of Crry down-regulation on the survival of MB49-challenged mice, indicating a complement-dependent mechanism. These data indicate that complement inhibitors expressed on a tumor cell can suppress a T cell response and that enhancing complement activation on a tumor cell surface can promote protective T cell immunity.