A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

Racial Disparities in Access to Alternative Donor Allografts Persist in the Era of "Donors for All"

Background: Understanding disparities in allograft access is a prerequisite to interpret outcomes. Moreover, while alternative donors extend access, the extent to which there are racial disparities in availability of optimal donors is not established. Methods: We evaluated access to alternative donor allografts (all other than HLA-identical sibling donors) in adults 19-65 years according to recipient ancestry over time between 1/2016-4/2021. During this period an 8/8 HLA allele-matched unrelated donor (URD) had priority followed by double unit cord blood (dCB) (usually preferred if < 60 years) or haploidentical donors with mismatched URDs being considered most recently. We examined access to any acceptable donor, as well as an optimal donor, by recipient ancestry. To determine trends over time, we compared early (1/2016-1/2018, 25 months), middle (2/2018-2/2020, 25 months), & pandemic (3/2020-4/2021, 14 months) time periods. Results: 592 adults (median 53.5 years, range 19-65) received alternative donor allografts. 374 (63%) had European & 218 (37%) non-European origins (66 African, 56 Asian, 55 White Hispanic, 41 other). Overall, 340 (56%) patients received 8/8 URD, 139 (23%) dCB, 69 (11%) haploidentical, & 44 (7%) 5-7/8 URD grafts with 14 (2%) patients having no graft. Europeans (263/374, 70%) mostly received 8/8 URD donors, whereas only one-third of non-Europeans (77/218, 35%) did (p < 0.01). Moreover, non-European patients were more likely than Europeans to receive HLA-disparate donors of all types: 36% of non-Europeans received dCB vs 16% of Europeans, 18% vs 8% for haploidentical donors, 10% vs 6% for 5-7/8 URD grafts. African ancestry patients (n = 66) were the least likely to receive 8/8 URDs (13/66, 20%) with 27/66 (41%) of them receiving dCB, 16/66 (24%) haploidentical, & 10/66 (15%) 5-7/8 URD grafts. When analyzing by period, the relative proportion of patients receiving allografts from 8/8 URDs, dCB, & haploidentical donors remained unchanged over time (Figure 1). However, while 14 patients (13 non-Europeans including 11 of African ancestry) had no graft, the utilization of 5-7/8 URDs (4% of alternative donor allografts 1/2016-1/2018, 8% 2/2018-2/2020, 14% 3/2020-4/2021) has decreased the "no graft" incidence to 1% of patients most recently (Figure 1). We then analyzed access to an "optimal donor" defined as an 8/8 URD < 35 years (Shaw et al., BBMT 2018), a dCB graft with each unit with a CD34+ dose > 1.5 x10^5/kg & > 4/8 HLA-match (Politikos et al., BBMT 2020), or a haploidentical donor < 40 years without recipient high titer donor-specific antibodies (McCurdy et al., Seminars in Hematology 2016 & others). Mismatched URDs were excluded based on lack of literature guiding an "optimal" definition. Of 8/8 URDs/ dCB/ haploidentical transplant recipients, 424/548 (77%) received an optimal donor with 269/340 (79%) URD, 94/139 (68%) dCB, & 61/69 (88%) haploidentical grafts being optimal. Transplanted non-Europeans were less likely to receive an optimal 8/8 URD / dCB / haploidentical donor than transplanted Europeans (67% vs 84%, p < 0.01) with White Hispanic & African patients having the lowest chances at 56% & 61%, respectively. Analysis of the 3 periods showed the likelihood that non-European patients received an optimal 8/8 URD / dCB / haploidentical donor is not improving: optimal allografts in 63% of non-Europeans vs 78% of Europeans 1/2016-1/2018, 68% vs 88% 2/2018-2/2020 & 68% vs 92% 3/2020-4/2021. Notably, the greatest disparity was seen at the pandemic's onset (3/2020-9/2020, Figure 2). Conclusion: Our data suggests access to 8/8 URDs for non-Europeans is not improving but utilization of all potential alternatives (dCB, haploidentical, 5-7/8 URD) is increasingly providing "donors for all". However, when incorporating the concept of an "optimal" 8/8 URD/ dCB/ haploidentical donor, there is a significant disparity in access to optimal donors for non-Europeans, with Africans & White Hispanics the least likely to receive an optimal graft. This disparity is also not improving, and worsened at the pandemic's onset. Optimization of dCB, haploidentical, & mismatched URD transplants, & recognition of optimal donor definitions for each, is critical to further improve allograft outcomes. Future studies must also investigate the extent to which futile 8/8 URD pursuits adversely impact non-European patient transplant outcomes. Figure 1 Figure 1. Disclosures Politikos: Merck: Research Funding; ExcellThera, Inc: Other: Member of DSMB - Uncompensated. Giralt: AMGEN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Gyurkocza: Actinium Pharmaceutical Inc.: Research Funding. Perales: Omeros: Honoraria; Novartis: Honoraria, Other; NexImmune: Honoraria; Nektar Therapeutics: Honoraria, Other; MorphoSys: Honoraria; Miltenyi Biotec: Honoraria, Other; Merck: Honoraria; Medigene: Honoraria; Kite/Gilead: Honoraria, Other; Karyopharm: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Cidara: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Sellas Life Sciences: Honoraria; Servier: Honoraria; Takeda: Honoraria. Ponce: Ceramedix: Consultancy, Honoraria; Takeda Pharmaceuticals: Research Funding; CareDx: Consultancy, Honoraria; Kadmon pharmaceuticals: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Research Funding; Generon Pharmaceuticals: Consultancy.

The Effect of Pasteurization on the Antioxidant Properties of Human Milk: A Literature Review

High rates of oxidative stress are common in preterm born infants and have short- and long-term consequences. The antioxidant properties of human milk limits the consequences of excessive oxidative damage. However, as the mother’s own milk it is not always available, donor milk may be provided as the best alternative. Donor milk needs to be pasteurized before use to ensure safety. Although pasteurization is necessary for safety reasons, it may affect the activity and concentration of several biological factors, including antioxidants. This literature review describes the effect of different pasteurization methods on antioxidant properties of human milk and aims to provide evidence to guide donor milk banks in choosing the best pasteurization method from an antioxidant perspective. The current literature suggests that Holder pasteurization reduces the antioxidant properties of human milk. Alternative pasteurization methods seem promising as less reduction is observed in several studies.

Clinical Outcomes of Unrelated Umbilical Cord Blood Graft vs. Haploidentical Donor Transplantation: Critical Issues for an Adequate Comparison

Unrelated umbilical cord blood (UCB) and haploidentical grafts have been used for allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation in patients without a related or non-related human leukocyte antigen (HLA)-matched donor. The less stringent HLA-matching requirement in both sources raises an important possibility for patients in need of urgent transplantation to treat any hematological disease. Selection of the best alternative donor is a difficult task that will depend on donor criteria, center experience, patient disease conditions, and risk, among others. Most comparisons available in scientific publications between both graft sources are obtained from retrospective analysis in wide time windows and a heterogeneous number of patients, types of disease, disease stages, previous treatments, graft source, conditioning regimen, graft vs. host disease (GVHD) approach, and evaluable endpoints. There is also an evident impact of the economic traits since low-income countries must consider less expensive treatments to satisfy the needs of the patients in the most effective possible path. Therefore, haploidentical transplantation could be an appealing option, even though it has not been completely established if any chronic treatment derived from the procedure could become a higher cost. In Colombia, there is a huge experience in UCB transplantation especially in units of pediatric transplantation where benign indications are more common than in adults. Due to the availability of a public UCB bank and HLA high-resolution typing in Colombia, there is a wider inventory of cord blood donors. Unfortunately, we do not have an unrelated bone marrow donor registry, so UCB is an important source along with haploidentical transplantation to consider in decision-making. This minireview focuses on comparing the main issues associated with the use of both HSCP sources and provides tools for physicians who face the difficult decision between these alternative donor sources.

Metagenomic Next Generation Sequencing in the Detection of Pathogens in Cerebrospinal Fluid of Patients After Alternative Donor Transplantation: A Feasibility Analysis

Central nervous system (CNS) complications can occur in 9%–15% of patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical manifestations of the CNS complications are non-specific, with most of them being disturbances of consciousness, convulsions, headaches, fever, and epilepsy, making it difficult to infer the cause of the complications based on clinical manifestations. We retrospectively analyzed the sensitivity and feasibility of metagenomic next generation sequencing (mNGS) in the diagnosis of CNS infections after allo-HSCT. Lumbar punctures were performed on 20 patients with CNS symptoms after receiving alternative donor HSCT(AD-HSCT) at the Affiliated Cancer Hospital of Zhengzhou University from February 2019 to December 2020, and their cerebrospinal fluid (CSF) was collected. The mNGS technique was used to detect pathogens in the CSF. Routine CSF testing, biochemical analyses, G experiments, GM experiments, ink staining, acid-fast staining, and bacterial cultures were carried out, and quantitative PCR (qPCR) tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKPyV), and human alphaherpesvirus (HHV). A total of 29 tests were performed with 21 of them being positive. Of the five negative patients, three were diagnosed with a posterior reversible encephalopathy syndrome, one as having transplantation-associated thrombotic microangiopathy, and one with transient seizure caused by hypertension. Fifteen patients tested positive, of which four had single infections and eleven had mixed infections. Five cases of fungal infections, six cases of bacterial infections, and 13 cases of viral infections were detected. Among the 13 cases of viral infections, ten cases were CMV(HHV-5); three were BKPyV; two were Torque teno virus (TTV); Two were HHV-1,two were EBV(HHV4), and one each of HpyV5 and HHV-6B. Thirteen patients tested positive for virus while the qPCR detection method of 6 identical specimens were below the minimum detection limit(<1×103 U/ml). The mNGS technique is highly sensitive, and it can be used to diagnose CNS infections after allo-HSCT.

Ruxolitinib early administration reduces acute GVHD after alternative donor hematopoietic stem cell transplantation in acute leukemia

This study aimed to observe the safety and clinical efficacy of early application of ruxolitinib to prevent acute graft-versus-host disease (aGVHD) after alternative donor transplantation in acute leukemia. There were 57 patients undergoing allo-HSCT at the Affiliated Cancer Hospital of Zhengzhou University from July 2017 to October 2019. They were divided into control(16 patients) and ruxolitinib (41 patients) groups. For aGVHD prophylaxis, the control group received post-transplantation cyclophosphamide, antithymocyte globulin-Fresenius, cyclosporine A, and mycophenolate mofetil, while in the ruxolitinib group, ruxolitinib 5 mg/d in adults or 0.07–0.1 mg/(kg d) in children was administered from the day of neutrophil engraftment to 100 days post-transplantation based on control group. We found 55 patients had successful reconstitution of hematopoiesis; No significant difference was found in cGVHD, hemorrhagic cystitis, pulmonary infection, intestinal infection, Epstein-Barr virus infection, cytomegalovirus infection, relapse, death, and nonrelapse mortality. The incidences of aGVHD (50 vs. 22%, P = 0.046) and grade II–IV aGVHD (42.9 vs. 12.2%, P = 0.013) were significantly higher in the control group than in the ruxolitinib group. No significant differences were observed in overall survival (P = 0.514), disease-free survival (P = 0.691), and cumulative platelet transfusion within 100 days post-transplantation between two groups. This suggests early application of ruxolitinib can reduce the incidence and severity of aGVHD and patients are well tolerated.