Bone Marrow Recovery Research Articles

Abstract 4868: BI 907828: A novel, potent MDM2 inhibitor that is suitable for high-dose intermittent schedules

Abstract MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly, thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties including a reliable, dose-linear PK across species with good bioavailability after oral dosing that allows various dose schedules. Remarkably, a single oral dose of 2 mg/kg led to tumor regressions in a SJSA-1 xenograft model in all treated mice, as did treatment on a daily low dose schedule. Data on PK, PD and efficacy relationships in this xenograft model will be presented including dose-dependent induction of TP53 target genes and markers of apoptosis. Moreover, BI 907828 significantly prolonged the survival by more than 50 days with a daily oral dose of 2.5 mg/kg in a difficult to treat disseminated MOLM-13 AML model, as noted by profiling of a clinical-stage MDM2 inhibitor. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows excellent efficacy with daily low dose but also intermittent high dose schedules in preclinical models of cancer. Citation Format: Dorothea Rudolph, Andreas Gollner, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Jürgen Moll, Kraut Norbert. BI 907828: A novel, potent MDM2 inhibitor that is suitable for high-dose intermittent schedules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4868.

Abstract 4865: BI 907828: A highly potent MDM2 inhibitor with low human dose estimation, designed for high-dose intermittent schedules in the clinic

Abstract MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present pharmacokinetic data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties. Due to its high permeability, good physiological solubility and low systemic clearance, the compound shows a high bioavailability in mice, rats, dogs and minipigs. BI 907828 is not absorption-limited, even at multiples of the efficacious dose, and shows a reliable, dose-linear PK with low variability in AUC and Cmax across species. Based on a good PK-PD correlation with efficacy in preclinical models and a robust human PK prediction based on in vivo PK data across species, a low human efficacious dose is predicted. The PK properties of BI 907828 enable a variety of different clinical dosing schedules with the potential to improve the therapeutic index with respect to on-target safety. In summary, BI 907828 is a novel, potent, orally available MDM2 inhibitor with excellent PK properties and low estimated human dose suitable for high-dose intermittent dose schedules in the clinic. Citation Format: Joerg Rinnenthal, Dorothea Rudolph, Sophia Blake, Andreas Gollner, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jürgen Moll, Norbert Kraut, Darryl McConnell, Jens Quant. BI 907828: A highly potent MDM2 inhibitor with low human dose estimation, designed for high-dose intermittent schedules in the clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4865.

Mapping Bone Marrow Response in the Vertebral Column by Positron Emission Tomography Following Radiotherapy and Erlotinib Therapy of Lung Cancer

To map functional bone marrow (BM) by 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) in the vertebral column of lung cancer patients prior to, during, and after treatment. Moreover, to identify radiation- and erlotinib-induced changes in the BM. Twenty-six patients with advanced non-small cell lung cancer, receiving radiotherapy (RT) alone or concomitantly with erlotinib, were examined by [18F]FDG PET before, during, and after treatment. A total of 61 [18F]FDG PET scans were analyzed. Vertebral column BM [18F]FDG standardized uptake value normalized to the liver (SUVBMLR) was used as uptake measure. Wilcoxon signed-rank test was used to assess changes in BM uptake of [18F]FDG between sessions. Effects of erlotinib on the BM activity during and aftertreatment were assessed using Mann-Whitney U test. A homogeneous uptake of [18F]FDG was observed within the vertebral column prior to treatment. Mean SUVBMLR (± S.E.M) in the body of thoracic vertebrae receiving a total RT dose of 10Gy or higher was 0.64 ± 0.01, 0.56 ± 0.01, and 0.59 ± 0.01 at pre-, mid-, and post-therapy, respectively. A significant reduction in the mean SUVBMLR was observed from pre- to both mid- and post-therapy (p < 0.05). Mean SUVBMLR was significantly higher at post-therapy compared to mid-therapy for patients receiving erlotinib in addition to RT (p < 0.05). RT reduces BM [18F]FDG uptake in the vertebral column, especially in the high-dose region. Concomitant erlotinib may stimulate a recovery in BM [18F]FDG uptake from mid- to post-therapy. NCT02714530. Registered 10 September 2015.

Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy.

Severe thrombocytopenia (≤50×109 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca2+ flux, integrin a β activation and P-selectin expression when stimulated with a panelIIbof3 agonists. The patients’ platelets were also impaired in spreading on fibrinogen. Whereas the Ca2+ store content was unaffected, the patients’ platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (P<0.001) and low oxygen consumption (P<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman’s rho=−0.459, P=0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients.

Venetoclax for patients with chronic lymphocytic leukemia who progressed during or after idelalisib therapy

AbstractB-cell receptor pathway inhibitors (BCRis) have transformed treatment of chronic lymphocytic leukemia (CLL); however, the efficacy of therapies for patients whose disease is refractory to/relapses after (R/R) BCRis is unknown. Venetoclax is a selective, orally bioavailable BCL-2 inhibitor with activity in patients with CLL, including those who are heavily pretreated or have 17p deletion. This phase 2 study prospectively evaluated venetoclax in patients with R/R CLL after ibrutinib or idelalisib; here we report on patients who received idelalisib as the last BCRi before enrollment. Venetoclax was initiated at 20 mg daily, followed by intrapatient ramp-up to 400 mg daily. Primary objectives included efficacy (objective response rate [ORR]) and safety of venetoclax. The study enrolled 36 patients who previously received idelalisib (ORR, 67% [24/36]); 2 patients achieved complete remission, and 1 had complete remission with incomplete bone marrow recovery. Median progression-free survival (PFS) has not yet been reached; estimated 12-month PFS was 79%. The most common adverse events (AEs; all grades) were neutropenia (56%), diarrhea (42%), upper respiratory tract infection (39%), thrombocytopenia (36%), nausea (31%), fatigue (28%), cough (22%), rash (22%), and anemia (22%). Grade 3 or 4 AEs were primarily hematologic (neutropenia [50%], thrombocytopenia [25%], and anemia [17%]). No patients experienced tumor lysis syndrome. Venetoclax demonstrated promising clinical activity and favorable tolerability in patients with CLL whose disease progressed during or after idelalisib therapy. This trial was registered at www.clinicaltrials.gov as #NCT02141282.

The Influence of Warm Ischemic Time on the Viability of Deceased Organ Donor Derived Bone Marrow

It was recognized 60 years ago that deceased donor bone marrow (BM) could be a useful source of viable hematopoietic stem cells (HSCs) for transplantation. Validated procedures have since been published for the recovery of viable deceased donor HSCs and their safe use in immune tolerance clinical trials. It is the goal of Ossium Health to streamline the process of obtaining deceased donor derived BM on an industrial scale. It has been shown by previous studies that BM is tolerant to a wide range of cold and warm ischemic times during collection. CD34+ HSCs prefer a hypoxic environment and remain quiescent in vivo. This observation indicates that BM procured during routine organ and tissue recovery may remain viable for extended periods following cardiac arrest and, thus, will be suitable for transplantation. Ossium is evaluating the range of ischemia times to determine if organ and tissue donor bone marrow recovered using a standardized approach is suitable for transplantation.Vertebral bodies (VBs) and ilia of 34 deceased organ donors were recovered by Ossium's network of organ procurement organizations (OPOs) over warm ischemia times ranging from 1:03–22:03 (hr:min). Recovered VBs were then cooled and shipped overnight on wet ice to Ossium Health, Indianapolis, IN, for BM recovery using grinding and elution methods. The CD34+ HSC population was enumerated using flow cytometric ISHAGE protocols and a mean viability over the entire ischemia range was determined to be 79.3 ± 3.7% (mean ± SEM) as determined by 7‐Aminoactinomycin D (7‐AAD).While gross membrane viability is a useful measure, engraftment potential correlates strongly with functional viability as measured through colony forming unit (CFU) assay, and engraftment of transplanted units requires the presence of Granulocyte/Macrophage (GM) colonies specifically. To confirm that the deceased donor marrow would be suitable for engraftment, the CFU assay was performed and CFU‐GM were scored. Over the entire range of ischemic times, an average of 37.05 ±11.16 CFU‐GM (mean ± SEM) per 105 nucleated cells plated was observed.Rapid neutrophil and platelet engraftment in living donor allogeneic grafts has previously been correlated with a dose containing CFU‐GM of 5.6 per 105 TNC in for an average patient of 70kg. The results of this preliminary study indicate deceased donor marrow can easily meet this requirement. These data demonstrate the potential to recover viable and functional HSCs after extended periods of warm ischemia; thus, establishing the feasibility and utility of banking cells from deceased organ and tissue donors.Support or Funding InformationThis work was funded by a Grant from the NIH‐NIAID (1R43AI129444‐01A1, PI: Woods, EJ), and Ossium Health, Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PROGNOSIS OF ACUTE LEUKEMIA IN CHILDREN DEPENDING ON THE STAGE OF HEMATOPOIESIS AND PHASES OF CHEMOTHERAPY

Patients with acute leukemia were examined before the start of chemotherapy (CT), and after the І-st and ІІ-nd phase of CT. According myelogram data the bone marrow recovery after myelosuppression occured with predominance with one of these links of hematopoiesis: erythroid, granulocyte, granulocyte-monocyte, monocyte and uniform mixed types. It is shown, that in the ІІ-nd examination phase the reduced number of cases with erythroid recovery prevalence were existed and the number of children with activation of granulocyte compound of hematopoiesis were increased compared to the І-st phase of СТ. Indices of megakaryocyte link in both periods of chemotherapy practically were no different. The correlation between the percentage of erythroid cells in myelogram and child survival were established: if the of erythroid cells in the first stage of treatment exceeded 30 %, the survival of patients with acute leukemia decreased (U test. – 39) and it can serve as the additional criterion for detection of high risk patients and correction of further chemotherapy.

Efficacy and safety of biosimilar filgrastim for autologous peripheral blood stem cell harvest and transplantation: a single-institutional retrospective analysis

Three biosimilar filgrastim products are currently available in Japan. Among these, the safety and efficacy of two imported drugs for autologous peripheral blood stem cell harvest (autoPBSCH) and autologous peripheral blood stem cell transplantation (autoPBSCT) have been studied widely; however, evidence of the safety and efficacy of domestically manufactured filgrastim is limited. Therefore, we compared the efficacy and safety of domestic biosimilar filgrastim (BF1, n=23) with those of originator filgrastim (OF, n=21) for autoPBSCH and autoPBSCT. Before autoPBSCH, the same median total dose of 3.3 mg filgrastim was administered to patients in the BF1 and OF groups. Median numbers of CD34-positive cells harvested did not significantly differ between BF1 (4.32×106/kg) and OF (4.75×106/kg) groups. After autoPBSCT, the median total doses of BF1 and OF used for neutrophil recovery were 2.7 and 3.3 mg, respectively. There were no significant inter-group differences in the time to bone marrow recovery, total transfusion units, hospitalization duration, overall survival at 1 year, or adverse events. Compared with OF, the cost of BF1 was considerably lower by 229,529 yen per transplantation case. Thus, the efficacy and safety of BF1 were comparable to those of OF, making BF1 an effective and economical alternative to OF.

SMER28 is a mTOR-independent small molecule enhancer of autophagy that protects mouse bone marrow and liver against radiotherapy.

Effective cytoprotectors that are selective for normal tissues could decrease radiotherapy and chemotherapy sequelae and facilitate the safe administration of higher radiation doses. This could improve the cure rates of radiotherapy for cancer patients. Autophagy is a cytoplasmic cellular process that is necessary for the clearance of damaged or aged proteins and organelles. It is a strong determinant of post-irradiation cell fate. In this study, we investigated the effect of the mTOR-independent small molecule enhancer of autophagy (SMER28) on mouse liver autophagy and post-irradiation recovery of mouse bone marrow and liver. SMER28 enhanced the autophagy flux and improved the survival of normal hepatocytes. This effect was specific for normal cells because SMER28 had no protective effect on hepatoma or other cancer cell line survival in vitro. In vivo subcutaneous administration of SMER28 protected mouse liver and bone marrow against radiation damage and facilitated survival of mice after lethal whole body or abdominal irradiation. These findings open a new field of research on autophagy-targeting radioprotectors with clinical applications in oncology, occupational, and space medicine.

Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Factor

Bone marrow formation requires an orchestrated interplay between osteogenesis, angiogenesis, and hematopoiesis that is thought to be mediated by endothelial cells. The nature of the endothelial cells and the molecular mechanisms underlying these events remain unclear in humans. Here, we identify a subset of endoglin-expressing endothelial cells enriched in human bone marrow during fetal ontogeny and upon regeneration after chemotherapeutic injury. Comprehensive transcriptional characterization by massive parallel RNA sequencing of these cells reveals a phenotypic and molecular similarity to murine type H endothelium and activation of angiocrine factors implicated in hematopoiesis, osteogenesis, and angiogenesis. Interleukin-33 (IL-33) was significantly overexpressed in these endothelial cells and promoted the expansion of distinct subsets of hematopoietic precursor cells, endothelial cells, as well as osteogenic differentiation. The identification and molecular characterization of these human regeneration-associated endothelial cells is thus anticipated to instruct the discovery of angiocrine factors driving bone marrow formation and recovery after injury.

Adequate Neutrophil Responses and Non-Inferior Clinical Outcomes Can Be Achieved by a Two-Day Course of Low-Dose Filgrastim: A Retrospective Single Institution Experience

BackgroundFilgrastim is used in the setting of chemotherapy-induced neutropenia to stimulate recovery of bone marrow, which allows for further chemotherapy administration without delay. The recommended dose is 5 ug/kg. The commercially available vials of the drug come in two strengths; 300 ug and 480 ug. Due to these limitations in dosage formulations, it is a frequent occurrence to administer a lower dosage to patients weighing more than 60 kg, in whom the ideal dose would have been more than 300 ug but less than 480 ug. It is also a frequent practice to administer the drug for two consecutive days as it often leads to adequate response that will render patients eligible for their next cycle administration.ObjectiveTo determine whether a course of 300 ug of filgrastim administered daily for two consecutive days was as successful at reducing chemotherapy-induced neutropenia-related complications in patients with a higher weight (>60 kg) and hence receiving suboptimal dose as compared to those with weight less than 60 kg who are receiving the recommended dose.MethodsWe identified 91 patients from our facility with chemotherapy-induced neutropenia treated with 300 ug of filgrastim daily for two consecutive days, and we separated them into low, medium, and high weight groups. Multivariate logistic regression models examined correlations between outcomes (e.g., increases in absolute neutrophil count) and predictors (e.g., weight groups).ResultsThe vast majority of encounters demonstrated rises in white blood cell (WBC) and absolute neutrophil count (ANC). Infection rates were not significantly different between low and medium weight groups (5% vs 0%; p = 0.1658), but the high weight group’s infection rate was significantly higher than the medium weight group (5% vs 33%; p = 0.001). The high weight group did have an increased rate of febrile neutropenia as compared to medium and low weight groups, but these differences were not significant. Incidences of chemotherapy delay and dose reduction were comparable across the three weight groups.LimitationsRetrospective study, small sample size, heterogeneous cancer sites and different chemotherapy regimens administered limit generalizability of findings.ConclusionPatients with weights <85 kg receiving a two-day course of 300 ug of filgrastim have similar neutropenia-related complication rates with a potential percent cost-savings of roughly 43%.

Imaging of subclinical haemopoiesis after stem-cell transplantation in patients with haematological malignancies: a prospective pilot study

Haemopoietic stem-cell transplantation (HSCT) eradicates host haemopoiesis before venous infusion of haemopoietic stem cells (HSCs). The pathway to cellular recovery has been difficult to study in human beings because of risks associated with interventions during aplasia. We investigated whether 18F-fluorothymidine (18F-FLT) imaging was safe during allogenic HSCT and allowed visualisation of early cellular proliferation and detection of patterns of cellular engraftment after HSCT. Eligible patients were aged 18-55 years, had high-risk haematological malignancies. All patients underwent myeloablation followed by HSCT. The imaging primary endpoint was detection of early subclinical engraftment after HSCT with 18F-FLT PET or CT. Imaging was done 1 day before and 5 or 9, and 28 days, and 1 year after HSCT. This study is registered with ClinicalTrials.gov, number NCT01338987. Between April 1, 2014, and Dec 31, 2015, 23 patients were enrolled and assessable for toxic effects after completing accrual. 18F-FLT was not associated with any adverse events or delayed engraftment. 18F-FLT imaging objectively identified subclinical bone-marrow recovery within 5 days of HSC infusion, which was up to 20 days before engraftment became clinically evident. Quantitatively, 18F-FLT intensity differed significantly between myeloablative infusion before HSCT and subclinical HSC recovery (p=0·00031). 18F-FLT biodistribution over time revealed a previously unknown path of cellular recovery of haemopoiesis in vivo that mirrored fetal ontogeny. 18F-FLT allowed quantification and tracking of subclinical bone-marrow repopulation in human beings and revealed new insights into the biology of HSC recovery after HSCT. National Institutes of Health, Ben's Run/Ben's Gift, Albert and Elizabeth Tucker Foundation, Mex Frates Leukemia Fund, Jones Family fund, and Oklahoma Center for Adult Stem Cell Research.

Outcomes of Immunocompetent Children Presenting with Fever and Neutropenia

BackgroundNeutropenia may alarm clinicians and prompt extensive evaluation in children with fever, even in immunocompetent patients. ObjectiveOur aim was to determine outcomes in previously healthy febrile children presenting to the emergency department with severe neutropenia. MethodsWe reviewed data from infants and children aged 3–36 months with fever and severe neutropenia, defined as a peripheral neutrophil count <500 × 103/μL, at our institution between January 1, 2012 and December 31, 2015. We compared our results to those from a similar study of children with a peripheral neutrophil count of 500–1000 × 103/μL. ResultsSevere neutropenia was recorded in 52 patients; severe bacterial infection (SBI) was found in 1 (1.9%), but none had a positive blood culture. Incidence of SBI was not different from that found in a study of similar patients with moderate neutropenia. ConclusionsImmunocompetent patients with fever and severe neutropenia do not carry a higher risk for SBI compared to patients with fever and moderate neutropenia. Such patients could potentially be followed closely with serial blood counts to ensure bone marrow recovery, without the use of antibiotics.

Safety of discharge for children with cancer and febrile neutropenia off antibiotics using absolute neutrophil count threshold values as a surrogate marker for adequate bone marrow recovery.

Febrile neutropenia (F&N) is common among pediatric oncology patients. However, there is a lack of clarity regarding parameters whereby such patients have demonstrated adequate bone marrow recovery for hospital discharge and empiric antibiotic discontinuation. A retrospective review was performed for 350 episodes of F&N occurring at a single institution between 2007 and 2012 in pediatric oncology patients who were afebrile for 24hr and had no bacterial source identified. Seven-day postdischarge outcomes were assessed and compared based on absolute neutrophil count (ANC) at discharge in order to identify an optimal threshold. Overall, 7-day readmission rates were low (17/350, 4.6%), with patients discharged with post-nadir ANC of 100-199/μl (2/51, 3.9%), 200-499/μl (5/125, 4.0%), and ≥500/μl (8/160, 5.0%), all having similar rates. Patients with a discharge ANC<100/μl (2/14, 14.3%) had a higher readmission rate. A new bloodstream infection was identified upon readmission in one patient in each discharge ANC range except for ANC of 100-199/μl, in which none occurred. In a subset of 217 episodes where the ANC fell below 200/μl during the admission and subsequently rose above 100/μl, 94 episodes resulted in 126 additional hospital days while subjects awaited further count recovery. One death occurred in a patient whose ANC at discharge was 290/μl. This patient had received additional chemotherapy after count recovery and prior to discharge, and was readmitted with Clostridium tertium bacteremia. These results suggest that a post-nadir ANC>100/μl is a safe threshold value for empiric antibiotic discontinuation and discharge home.

Eruption of lymphocyte recovery with atypical lymphocytes mimicking a primary cutaneous T-cell lymphoma: a series of 12 patients

Eruption of lymphocyte recovery (ELR) may occur during bone marrow aplasia after chemotherapies. We reviewed the clinical and pathologic features of 12 patients (male-female ratio, 7:5; median age, 61 years) with an atypical ELR histologically mimicking a primary cutaneous T-cell lymphoma such as Sézary syndrome or CD30+ T-cell lymphoproliferative disorder. All the patients displayed an erythematous maculopapular eruption on the trunk and the limbs associated with fever. All but one had received a polychemotherapy for an acute myeloid leukemia (n=10) or a urothelial carcinoma (n=1) before the occurrence of the skin eruption. One had an autoimmune lymphoproliferative syndrome causing chronic agranulocytosis requiring granulocyte colony-stimulating factor injection. In all patients, the skin eruption was associated with a slight increase of white blood cell count followed by bone marrow recovery within the next weeks. All skin biopsies showed a dermal perivascular lymphocytic infiltrate containing atypical medium- to large-sized CD3+, CD4+ and CD8+, CD25+, ICOS+, PD1- lymphocytes with a strong CD30 expression in most instances (n=10), suggesting the recruitment of strongly activated T cells in the skin. In 6 patients, a diagnosis of CD30+ lymphoproliferative disorder or Sézary syndrome was proposed or suspected histopathologically, and only the clinical context allowed the diagnosis of ELR with a peculiar presentation with atypical lymphocytes. We describe a series of patients with an unusual form of ELR characterized by the presence of atypical activated T cells in the skin. On a practical ground, pathologists should be aware of this distinctive and misleading presentation.

Material Viscoelastic Properties Modulate the Mesenchymal Stem Cell Secretome for Applications in Hematopoietic Recovery.

Human mesenchymal stem cells (MSCs) exhibit morphological and phenotypic changes that correlate with mechanical cues presented by the substratum material to which those cells adhere. Such mechanosensitivity has been explored in vitro to promote differentiation of MSCs along tissue cell lineages for direct tissue repair. However, MSCs are increasingly understood to facilitate indirect tissue repair in vivo through paracrine signaling via secreted biomolecules. Here we leveraged cell-material interactions in vitro to induce human bone marrow-derived MSCs to preferentially secrete factors that are beneficial to hematopoietic cell proliferation. Specifically, we varied the viscoelastic properties of cell-culture-compatible polydimethylsiloxane (PDMS) substrata to demonstrate modulated MSC expression of biomolecules, including osteopontin, a secreted phosphoprotein implicated in tissue repair and regeneration. We observed an approximately 3-fold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli <1 kPa) and highest ratio of loss modulus to storage modulus (tan(δ) > 1). A specific subpopulation of these cells, shown previously to express increased osteopontin in vitro and to promote bone marrow recovery in vivo, also exhibited up to a 5-fold increase in osteopontin expression when grown on compliant PDMS relative to heterogeneous MSCs on polystyrene. Importantly, this mechanically modulated increase in protein expression preceded detectable changes in the terminal differentiation capacity of MSCs. In coculture with human CD34+ hematopoietic stem and progenitor cells (HSPCs) that repopulate the blood cell lineages, these mechanically modulated MSCs promoted in vitro proliferation of HSPCs without altering the multipotency for either myeloid or lymphoid lineages. Cytokine and protein expression by human MSCs can thus be manipulated directly by mechanical cues conferred by the material substrata prior to and instead of tissue lineage differentiation. This approach enables enhanced in vitro production of both mesenchymal and hematopoietic stem and progenitor cells that aid regenerative clinical applications.

Absolute lymphocyte counts at end of induction correlate with distinct immune cell compartments in pediatric B cell precursor acute lymphoblastic leukemia.

Several retrospective studies in children with B cell precursor (BCP) acute lymphoblastic leukemia (ALL) provided clinical evidence that higher absolute lymphocyte counts (ALC) early into treatment significantly correlated with improved relapse-free and overall survival. It still remains unknown, however, whether the predictive role of higher ALCs reflects general bone marrow recovery or a more specific attribute of immune function. To investigate this question, we implemented a prospective observational cohort study in 20 children with BCP ALL on day 29 (D29) of induction chemotherapy and immunophenotyped their lymphoid (T, B and natural killer cells) and myeloid (neutrophils, monocytes, dendritic cells) compartments. In a first evaluation of a cohort treated with Children's Oncology Group-based induction chemotherapy, the immune cell compartments were differentially depleted at D29. Neither gender, risk status, minimal residual disease, nor bone marrowrecovery markers correlated with D29 ALC. In contrast, both CD3+ T cell and dendritic cell compartments, which did not correlate with age, significantly correlated with D29 ALC (p<0.0001). In addition, subset complexity of cellular immune compartments was preserved at D29. This study reveals that D29 ALC significantly correlates with distinct immune cell compartments but not with bone marrowrecovery markers, suggesting that higher D29 ALCs may contribute to leukemia control by inducing specific host immune activity.

In vivo imaging to monitor differentiation and therapeutic effects of transplanted mesenchymal stem cells in myocardial infarction

Here, we used a noninvasive multimodality imaging approach to monitor differentiation of transplanted bone marrow mesenchymal stem cells (BMSCs) and recovery of cardiac function in an in vivo model of myocardial infarction (MI). We established a rat MI model by coronary artery ligation. Ninety rats were randomly assigned into four groups: sham-operated, MI model, and α-MHC-HSV1-tk-transfected or un-transfected BMSCs-treated MI model. We used 18F-Fluro-deoxyglucose (18F-FDG) positron emission tomography (PET) to monitor recovery of cardiac function, and 18F-FHBG PET/CT imaging to monitor transplanted BMSCs differentiation 24 h after 18F-FDG imaging. The uptake of 18F-FDG at 3, 16, 30 and 45 days after BMSCs injection was 0.39 ± 0.03, 0.57 ± 0.05, 0.59 ± 0.04, and 0.71 ± 0.05% ID/g, respectively. Uptake of 18F-FHBG increased significantly in large areas in the BMSCs-treated group over time. Ex vivo experiments indicated that expression of the cardiomyocyte markers GATA-4 and cardiac troponin I markedly increased in the BMSCs-treated group. Additionally, immunohistochemistry revealed that HSV-tk-labelled BMSCs-derived cells were positive for cardiac troponin I. Multimodal imaging systems combining an α-MHC-HSV1-tk/18F-FHBG reporter gene and 18F-FDG metabolism imaging could be used to track differentiation of transplanted BMSCs and recovery of cardiac function in MI.

Investigating the Abscopal Effects of Radioablation on Shielded Bone Marrow in Rodent Models Using Multimodality Imaging.

The abscopal effect is the response to radiation at sites that are distant from the irradiated site of an organism, and it is thought to play a role in bone marrow (BM) recovery by initiating responses in the unirradiated bone marrow. Understanding the mechanism of this effect has applications in treating BM failure (BMF) and BM transplantation (BMT), and improving survival of nuclear disaster victims. Here, we investigated the use of multimodality imaging as a translational tool to longitudinally assess bone marrow recovery. We used positron emission tomography/computed tomography (PET/CT), magnetic resonance imaging (MRI) and optical imaging to quantify bone marrow activity, vascular response and marrow repopulation in fully and partially irradiated rodent models. We further measured the effects of radiation on serum cytokine levels, hematopoietic cell counts and histology. PET/CT imaging revealed a radiation-induced increase in proliferation in the shielded bone marrow (SBM) compared to exposed bone marrow (EBM) and sham controls. T2-weighted MRI showed radiation-induced hemorrhaging in the EBM and unirradiated SBM. In the EBM and SBM groups, we found alterations in serum cytokine and hormone levels and in hematopoietic cell population proportions, and histological evidence of osteoblast activation at the bone marrow interface. Importantly, we generated a BMT mouse model using fluorescent-labeled bone marrow donor cells and performed fluorescent imaging to reveal the migration of bone marrow cells from shielded to radioablated sites. Our study validates the use of multimodality imaging to monitor bone marrow recovery and provides evidence for the abscopal response in promoting bone marrow recovery after irradiation.

Bone marrow T cells populations are differently affected by sepsis than their splenic counterparts in the murine sepsis cecal ligation and puncture (CLP) model

Abstract Sepsis is known to induce lymphopenia which correlates with secondary infections and mortality. In sepsis survivors the lymphocyte count restores with time, although the T cell responses are impaired. So far, the mechanisms of T cells recovery are not fully understood and the murine studies were focused on the peripheral lymphatic organs in which the spleen was most common. Our study was designed to elucidate the recovery of bone marrow (BM) T cells which contain population of memory cells, after the CLP murine sepsis. In our model, mild CLP produced peripheral lymphopenia which was lowest at 48h after CLP but after 7 days circulating T cells count was rebounding. 7 days after CLP the BM frequencies of naïve and central memory (CD44+CD62L+) CD4 cells were unchanged while they were reduced in the spleen. Percentage of effector memory (CD44+CD62L−) CD4 and CD8 cells increased in the BM and at the same time they dramatically decreased in the spleen. On the contrary, the effector (CD44−CD62L+) CD4 and CD8 cells expanded at that time point in the spleen while they did not change in the BM. At D7 after CLP, the rates of BrdU+ proliferating CD4+ and CD8+ cells were markedly higher in the BM than spleen. Among the BM CD4+ cells most of the proliferating cells were the effector memory cells, while the effector CD4+ had highest proliferation rate in the spleen. Similarly, CD8 effector memory cells in the BM and CD8 effector cells were most extensively proliferating in the BM and the spleen, respectively. Taken together, our data point to the BM as the place of effector memory T cell proliferation during post-sepsis recovery.