Imaging of Nitric Oxide in Nitrergic Neuromuscular Neurotransmission in the Gut

Background: Numerous functional studies have shown that nitrergic neurotransmission plays a central role in peristalsis and sphincter relaxation throughout the gut and impaired nitrergic neurotransmission has been implicated in clinical disorders of all parts of the gut. However, the role of nitric oxide (NO) as a neurotransmitter continues to be controversial because: 1) the cellular site of production during neurotransmission is not well established; 2) NO may interacts with other inhibitory neurotransmitter candidates, making it difficult to understand its precise role. Methodology/Principal Findings: Imaging NO can help resolve many of the controversies regarding the role of NO in nitrergic neurotransmission. Imaging of NO and its cellular site of production is now possible. NO forms quantifiable fluorescent compound with diaminofluorescein (DAF) and allows imaging of NO with good specificity and sensitivity in living cells. In this report we describe visualization and regulation of NO and calcium ($Ca^{2+}$) in the myenteric nerve varicosities during neurotransmission using multiphoton microscopy. Our results in mice gastric muscle strips provide visual proof that NO is produced de novo in the nitrergic nerve varicosities upon nonadrenergic noncholinergic (NANC) nerve stimulation. These studies show that NO is a neurotransmitter rather than a mediator. Changes in NO production in response to various pharmacological treatments correlated well with changes in slow inhibitory junction potential of smooth muscles. Conclusions/Significance: Dual imaging and electrophysiologic studies provide visual proof that during nitrergic neurotransmission NO is produced in the nerve terminals. Such studies may help define whether NO production or its signaling pathway is responsible for impaired nitrergic neurotransmission in pathological states

FRA2A is a CGG repeat expansion associated with silencing of AFF3

Folate-sensitive fragile sites (FSFS) are a rare cytogenetically visible subset of dynamic mutations. Of the eight molecularly characterized FSFS, four are associated with intellectual disability (ID). Cytogenetic expression results from CGG tri-nucleotide-repeat expansion mutation associated with local CpG hypermethylation and transcriptional silencing. The best studied is the FRAXA site in the FMR1 gene, where large expansions cause fragile X syndrome, the most common inherited ID syndrome. Here we studied three families with FRA2A expression at 2q11 associated with a wide spectrum of neurodevelopmental phenotypes. We identified a polymorphic CGG repeat in a conserved, brain-active alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene: Expansion of the AFF2 CGG repeat causes FRAXE ID. We found that FRA2A-expressing individuals have mosaic expansions of the AFF3 CGG repeat in the range of several hundred repeat units. Moreover, bisulfite sequencing and pyrosequencing both suggest AFF3 promoter hypermethylation. cSNP-analysis demonstrates monoallelic expression of the AFF3 gene in FRA2A carriers thus predicting that FRA2A expression results in functional haploinsufficiency for AFF3 at least in a subset of tissues. By whole-mount in situ hybridization the mouse AFF3 ortholog shows strong regional expression in the developing brain, somites and limb buds in 9.5-12.5dpc mouse embryos. Our data suggest that there may be an association between FRA2A and a delay in the acquisition of motor and language skills in the families studied here. However, additional cases are required to firmly establish a causal relationship

Allogeneic hematopoietic cell transplantation provides effective salvage despite refractory disease or failed prior autologous transplant in angioimmunoblastic T-cell lymphoma: a CIBMTR analysis

Abstract Background There is a paucity of data on the role of allogeneic hematopoietic cell transplantation (allo-HCT) in patients with angioimmunoblastic T-cell lymphoma (AITL). Using the CIBMTR registry, we report here the outcomes of AITL patients undergoing an allo-HCT. Methods We evaluated 249 adult AITL patients who received their first allo-HCT during 2000–2016. Results The median patient age was 56 years (range = 21–77). Majority of the patients were Caucasians (86%), with a male predominance (60%). Graft-versus-host disease (GVHD) prophylaxis was predominantly calcineurin inhibitor-based approaches while the most common graft source was peripheral blood (97%). Median follow-up of survivors was 49 months (range = 4–170 months). The cumulative incidence of grade 2–4 and grade 3–4 acute GVHD at day 180 were 36% (95% CI = 30–42) and 12 (95% CI = 8–17), respectively. The cumulative incidence of chronic GVHD at 1 year was 49% (95%CI 43–56). The 1-year non-relapse mortality (NRM) was 19% (95% CI = 14–24), while the 4-year relapse/progression, progression-free survival (PFS), and overall survival (OS) were 21% (95% CI = 16–27), 49% (95% CI = 42–56), and 56% (95% CI = 49–63), respectively. On multivariate analysis, chemoresistant status at the time of allo-HCT was associated with a significantly higher risk for therapy failure (inverse of PFS) (RR = 1.73 95% CI = 1.08–2.77), while KPS < 90% was associated with a significantly higher risk of mortality (inverse of OS) (RR = 3.46 95% CI = 1.75–6.87). Conclusion Our analysis shows that allo-HCT provides durable disease control even in AITL patients who failed a prior auto-HCT and in those subjects with refractory disease at the time of allografting.https://deepblue.lib.umich.edu/bitstream/2027.42/146782/1/13045_2018_Article_696.pd

Association between the choice of the conditioning regimen and outcomes of allogeneic hematopoietic cell transplantation for myelofibrosis

Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only curative treatment for myelofibrosis. However, the optimal conditioning regimen either with reduced intensity conditioning (RIC) or myeloablative conditioning (MAC) is not well known. Using the Center for International Blood and Marrow Transplant Research database, we identified adults aged ≥18 years with myelofibrosis undergoing allo-HCT between 2008-2019 and analyzed the outcomes separately in the RIC and MAC cohorts based on the conditioning regimens used. Among 872 eligible patients, 493 underwent allo-HCT using RIC (Fludarabine/busulfan=166, Fludarabine/melphalan=327) and 379 using MAC (Fludarabine/busulfan=247, Busulfan/cyclophosphamide=132). In multivariable analysis with RIC, Fludarabine/melphalan was associated with inferior overall survival (HR 1.80, 95% CI 1.15-2.81, p=0.009), higher early non-relapse mortality (HR 1.81, 95% CI 1.12-2.91, p=0.01) and higher acute graft versus host disease (GVHD) (grade II-IV- HR 1.45, 95% CI 1.03-2.03, p=0.03; grade III-IV HR 2.21, 95%CI 1.28-3.83, p=0.004) compared to Fludarabine/busulfan. In the MAC setting, Busulfan/cyclophosphamide was associated with a higher acute GVHD (grade II-IV HR 2.33, 95% CI 1.67-3.25, p\u3c0.001; grade III-IV HR 2.31, 95% CI 1.52-3.52, p\u3c0.001) and inferior GVHD-free relapse-free survival (GRFS) (HR 1.94, 95% CI 1.49-2.53, p\u3c0.001) as compared to Fludarabine/busulfan. Hence, our study suggests that Fludarabine/busulfan is associated with better outcomes in RIC (better overall survival, lower early non-relapse mortality, lower acute GVHD) and MAC (lower acute GVHD and better GRFS) in myelofibrosis

Outcomes of Medicare-age eligible NHL patients receiving RIC allogeneic transplantation: a CIBMTR analysis

The application of allogeneic hematopoietic cell transplantation (allo-HCT) in non-Hodgkin lymphoma (NHL) patients ≥65 years in the United States is limited by lack of Medicare coverage for this indication. Using the Center for International Blood and Marrow Transplant Research (CIBMTR) database, we report allo-HCT outcomes of NHL patients aged ≥65 years (older cohort; n = 446) compared with a cohort of younger NHL patients aged 55-64 years (n = 1183). We identified 1629 NHL patients undergoing a first reduced-intensity conditioning (RIC) or nonmyeloablative conditioning allo-HCT from 2008 to 2015 in the United States. Cord blood or haploidentical transplants were excluded. The median age was 68 years (range 65-77) for the older cohort vs 60 years (range 55-64) in the younger cohort. The 4-year adjusted probabilities of nonrelapse mortality (NRM), relapse/progression (R/P), progression-free survival (PFS), and overall survival (OS) of the younger and older groups were 24% vs 30% (P = .03), 41% vs 42% (P = .82), 37% vs 31% (P = .03), and 51% vs 46% (P = .07), respectively. Using multivariate analysis, compared with the younger group, the older cohort was associated with increased NRM, but there was no difference between the 2 cohorts in terms of R/P, PFS, or OS. The most common cause of death was disease relapse in both groups. In NHL patients eligible for allo-HCT, there was no difference in OS between the 2 cohorts. Age alone should not determine allo-HCT eligibility in NHL, and Medicare should expand allo-HCT coverage to older adults

Haploidentical vs. sibling, unrelated, or cord blood hematopoietic cell transplantation for acute lymphoblastic leukemia

The role of haploidentical hematopoietic cell transplantation (HCT) using posttransplant cyclophosphamide (PTCy) for acute lymphoblastic leukemia (ALL) is being defined. We performed a retrospective, multivariable analysis comparing outcomes of HCT approaches by donor for adults with ALL in remission. The primary objective was to compare overall survival (OS) among haploidentical HCTs using PTCy and HLA-matched sibling donor (MSD), 8/8 HLAmatched unrelated donor (MUD), 7 /8 HLA-MUD, or umbilical cord blood (UCB) HCT. Comparing haploidentical HCT to MSD HCT, we found that OS, leukemia-free survival (LFS), nonrelapse mortality (NRM), relapse, and acute graft-versus-host disease (aGVHD) were not different but chronic GVHD (cGVHD) was higher in MSD HCT. Compared with MUD HCT, OS, LFS, and relapse were not different, but MUD HCT had increased NRM (hazard ratio [HR], 1.42; P = .02), grade 3 to 4 aGVHD (HR, 1.59; P = .005), and cGVHD. Compared with 7/8 UD HCT, LFS and relapse were not different, but 7/8 UD HCT had worse OS (HR, 1.38; P = .01) and increased NRM (HR, 2.13; P <_ .001), grade 3 to 4 aGVHD (HR, 1.86; P = .003), and cGVHD (HR, 1.72; P <_ .001). Compared with UCB HCT, late OS, late LFS, relapse, and cGVHD were not different but UCB HCT had worse early OS (<_18 months; HR, 1.93; P < .001), worse early LFS (HR, 1.40; P = .007) and increased incidences of NRM (HR, 2.08; P < .001) and grade 3 to 4 aGVHD (HR, 1.97; P < .001). Haploidentical HCT using PTCy showed no difference in survival but less GVHD compared with traditional MSD and MUD HCT and is the preferred alternative donor HCT option for adults with ALL in complete remission

Impact of Second Primary Malignancy Post–Autologous Transplantation on Outcomes of Multiple Myeloma: A CIBMTR Analysis

The overall survival (OS) has improved significantly in multiple myeloma (MM) over the last decade with the use of proteasome inhibitor and immunomodulatory drug-based combinations, followed by high-dose melphalan and autologous hematopoietic stem cell transplantation (auto-HSCT) and subsequent maintenance therapies in eligible newly diagnosed patients. However, clinical trials using auto-HSCT followed by lenalidomide maintenance have shown an increased risk of second primary malignancies (SPM), including second hematological malignancies (SHM). We evaluated the impact of SPM and SHM on progression-free survival (PFS) and OS in patients with MM after auto-HSCT using CIBMTR registry data. Adult patients with MM who underwent first auto-HSCT in the United States with melphalan conditioning regimen from 2011 to 2018 and received maintenance therapy were included (n = 3948). At a median follow-up of 37 months, 175 (4%) patients developed SPM, including 112 (64%) solid, 36 (20%) myeloid, 24 (14%) SHM, not otherwise specified, and 3 (2%) lymphoid malignancies. Multivariate analysis demonstrated that SPM and SHM were associated with an inferior PFS (hazard ratio [HR] 2.62, P \u3c .001 and HR 5.01, P \u3c .001, respectively) and OS (HR 3.85, P \u3c .001 and HR 8.13, P \u3c .001, respectively). In patients who developed SPM and SHM, MM remained the most frequent primary cause of death (42% vs 30% and 53% vs 18%, respectively). We conclude the development of SPM and SHM leads to a poor survival in patients with MM and is an important survivorship challenge. Given the median survival for MM continues to improve, continued vigilance is needed to assess the risks of SPM and SHM with maintenance therapy post-auto-HSCT

The Mutational Landscape in Chronic Myelomonocytic Leukemia and Its Impact on Allogeneic Hematopoietic Cell Transplantation Outcomes: A Center for Blood and Marrow Transplantation Research (CIBMTR) Analysis

Somatic mutations are recognized as an important prognostic factor in chronic myelomonocytic leukemia (CMML). However, limited data are available regarding their impact on outcomes after allogeneic hematopoietic cell transplantation (HCT). In this registry analysis conducted in collaboration with the Center for International Blood and Marrow Transplantation Registry database/sample repository, we identified 313 adult patients with CMML (median age: 64 years, range, 28- 77) who underwent allogeneic HCT during 2001-2017 and had an available biospecimen in the form of a peripheral blood sample obtained prior to the start of conditioning. In multivariate analysis, a CMML-specific prognostic scoring system (CPSS) score of intermediate-2 (HR=1.46, P=0.049) or high (HR=3.22, P=0.0004) correlated significantly with overall survival. When the molecularly informed CPSS-Mol prognostic model was applied, a high CPSS-Mol score (HR=2 P=0.0079) correlated significantly with overall survival. The most common somatic mutations were in ASXL1 (62%), TET2 (35%), KRAS/NRAS (33% combined), and SRSF2 (31%). DNMT3A and TP53 mutations were associated with decreased overall survival (HR=1.70 [95% CI: 1.11-2.60], P=0.0147 and HR=2.72 [95% CI: 1.37-5.39], P=0.0042, respectively) while DNMT3A, JAK2, and TP53 mutations were associated with decreased disease-free survival (HR=1.66 [95% CI: 1.11-2.49], P=0.0138, HR=1.79 [95% CI: 1.06-3.03], P=0.0293, and HR=2.94 [95% CI: 1.50-5.79], P=0.0018, respectively). The only mutation associated with increased relapse was TP53 (HR=2.94, P=0.0201). Nonetheless, the impact of TP53 mutations specifically should be interpreted cautiously given their rarity in CMML. We calculated the goodness of fit measured by Harrell\u27s C-index for both the CPSS and CPSS-Mol, which were very similar. In summary, via registry data we have determined the mutational landscape in patients with CMML who underwent allogeneic HCT, and demonstrated an association between CPSS-Mol and transplant outcomes although without major improvement in the risk prediction beyond that provided by the CPSS

Risk Factors for Graft-versus-Host Disease in Haploidentical Hematopoietic Cell Transplantation Using Post-Transplant Cyclophosphamide

Post-transplant cyclophosphamide (PTCy) has significantly increased the successful use of haploidentical donors with a relatively low incidence of graft-versus-host disease (GVHD). Given its increasing use, we sought to determine risk factors for GVHD after haploidentical hematopoietic cell transplantation (haplo-HCT) using PTCy. Data from the Center for International Blood and Marrow Transplant Research on adult patients with acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, or chronic myeloid leukemia who underwent PTCy-based haplo-HCT (2013 to 2016) were analyzed and categorized into 4 groups based on myeloablative (MA) or reduced-intensity conditioning (RIC) and bone marrow (BM) or peripheral blood (PB) graft source. In total, 646 patients were identified (MA-BM = 79, MA-PB = 183, RIC-BM = 192, RIC-PB = 192). The incidence of grade 2 to 4 acute GVHD at 6 months was highest in MA-PB (44%), followed by RIC-PB (36%), MA-BM (36%), and RIC-BM (30%) (P = .002). The incidence of chronic GVHD at 1 year was 40%, 34%, 24%, and 20%, respectively (P < .001). In multivariable analysis, there was no impact of stem cell source or conditioning regimen on grade 2 to 4 acute GVHD; however, older donor age (30 to 49 versus <29 years) was significantly associated with higher rates of grade 2 to 4 acute GVHD (hazard ratio [HR], 1.53; 95% confidence interval [CI], 1.11 to 2.12; P = .01). In contrast, PB compared to BM as a stem cell source was a significant risk factor for the development of chronic GVHD (HR, 1.70; 95% CI, 1.11 to 2.62; P = .01) in the RIC setting. There were no differences in relapse or overall survival between groups. Donor age and graft source are risk factors for acute and chronic GVHD, respectively, after PTCy-based haplo-HCT. Our results indicate that in RIC haplo-HCT, the risk of chronic GVHD is higher with PB stem cells, without any difference in relapse or overall survival

Allogeneic Hematopoietic Cell Transplantation for Blastic Plasmacytoid Dendritic Cell Neoplasm: A CIBMTR Analysis

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy with a poor prognosis and considered incurable with conventional chemotherapy. Small observational studies reported allogeneic hematopoietic cell transplantation (allo-HCT) offers durable remissions in patients with BPDCN. We report an analysis of patients with BPDCN who received an allo-HCT, using data reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). We identified 164 patients with BPDCN from 78 centers who underwent allo-HCT between 2007 and 2018. The 5-year overall survival (OS), disease-free survival (DFS), relapse, and nonrelapse mortality (NRM) rates were 51.2% (95% confidence interval [CI], 42.5-59.8), 44.4% (95% CI, 36.2-52.8), 32.2% (95% CI, 24.7-40.3), and 23.3% (95% CI, 16.9-30.4), respectively. Disease relapse was the most common cause of death. On multivariate analyses, age of ≥60 years was predictive for inferior OS (hazard ratio [HR], 2.16; 95% CI, 1.35-3.46; P = .001), and higher NRM (HR, 2.19; 95% CI, 1.13-4.22; P = .02). Remission status at time of allo-HCT (CR2/primary induction failure/relapse vs CR1) was predictive of inferior OS (HR, 1.87; 95% CI, 1.14-3.06; P = .01) and DFS (HR, 1.75; 95% CI, 1.11-2.76; P = .02). Use of myeloablative conditioning with total body irradiation (MAC-TBI) was predictive of improved DFS and reduced relapse risk. Allo-HCT is effective in providing durable remissions and long-term survival in BPDCN. Younger age and allo-HCT in CR1 predicted for improved survival, whereas MAC-TBI predicted for less relapse and improved DFS. Novel strategies incorporating allo-HCT are needed to further improve outcomes