Peripheral Blood Haploidentical Allogeneic Stem Cell Transplantation in Older Adults with Acute Myeloid Leukemia and Myelodysplastic Syndromes Demonstrates Long Term Survival, Results from Australia and New Zealand Transplant and Cellular Therapies.

There is a limited body of evidence for haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in older patients. Previous studies have used a high proportion of bone marrow-derived grafts and a variety of conditioning regimens. In Australia and New Zealand, haplo-HCST is predominantly performed using peripheral blood (PB) with universal use of post-transplantation cyclophosphamide (PTCy). To characterize the outcomes of older recipients undergoing haplo-HSCT for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Data were collected through the Australasian Bone Marrow Transplant Recipient Registry (ABMTRR) for patients aged 65 or older receiving a PB haplo-HSCT for AML/MDS between January 2010 and July 2020. A total of 44 patients were included in the analysis. The median follow-up time was 377 days. The median age was 68 (range 65-74) with a median Karnofsky performance status of 90. Thirty patients (68.2%) had AML, whereas 14 (31.8%) had MDS. The median donor age was 40. The most common conditioning regimen was nonmyeloablative fludarabine, cyclophosphamide, and total body irradiation (75%); the remainder of the patients received either melphalan- or busulfan-based regimens, and the majority were reduced intensity, with only 2 patients undergoing myeloablative conditioning. All patients received post-transplantation cyclophosphamide and mycophenolate mofetil, with the majority also receiving tacrolimus (90.5%) and the remainder receiving cyclosporine (9.5%). No patients received anti-thymocyte globulin. Neutrophil engraftment was achieved in 97.6% of patients at a median of 18 days, whereas platelet engraftment was achieved in 92.7% of patients at a median of 28 days. The cumulative incidences of cytomegalovirus (CMV) reactivation and CMV disease were 52.5% and 5.1% at 1 year. The incidence of grade 2-4 acute Graft Versus Host Disease (GVHD) was 18.2%. The incidence of chronic GVHD at 2 years was 40.7%, with extensive chronic GVHD occurring in 17.7% of patients. The incidences of relapse and non-relapse mortality (NRM) at 2 years were 8.8% and 20.7% respectively. The leading causes of death were infection (64.7%) followed by relapse (14.2%). The 2-year overall survival was 74%. Relapse free survival and GVHD free, relapse free survival at 2 years was 70% and 48%. Haplo-HSCT using a peripheral blood graft and PTCy GVHD prophylaxis demonstrates long-term disease control with acceptable rates of NRM for older patients with AML/MDS.

Novel modes of MPL activation in triple-negative myeloproliferative neoplasms.

The identification of a somatic mutation associated with myeloid malignancy is of diagnostic importance in myeloproliferative neoplasms (MPNs). Individuals with no mutation detected in common screening tests for variants in JAK2, CALR, and MPL are described as 'triple-negative' and pose a diagnostic challenge if there is no other evidence of a clonal disorder. To identify potential drivers that might explain the clinical phenotype, we used an extended sequencing panel to characterise a cohort of 44 previously diagnosed triple-negative MPN patients for canonical mutations in JAK2, MPL and CALR at low variant allele frequency (found in 4/44 patients), less common variants in the JAK-STAT signalling pathway (12 patients), or other variants in recurrently mutated genes from myeloid malignancies (18 patients), including hotspot variants of potential clinical relevance in eight patients. In one patient with thrombocytosis we identified biallelic germline MPL variants. Neither MPL variant was activating in cell proliferation assays, and one of the variants was not expressed on the cell surface, yet co-expression of both variants led to thrombopoietin hypersensitivity. Our results highlight the clinical value of extended sequencing including germline variant analysis and illustrate the need for detailed functional assays to determine whether rare variants in JAK2 or MPL are pathogenic.

The mutational burden of therapy-related myeloid neoplasms is similar to primary myelodysplastic syndrome but has a distinctive distribution.

Therapy-related myeloid neoplasms (T-MN) are poorly characterized secondary hematological malignancies following chemotherapy/radiotherapy exposure. We compared the clinical and mutational characteristics of T-MN (n = 129) and primary myelodysplastic syndrome (P-MDS, n = 108) patients. Although the somatic mutation frequency was similar between T-MN and P-MDS patients (93% in both groups), the pattern was distinct. TP53 mutations were more frequent in T-MN (29.5 vs. 7%), while spliceosomal complex mutations were more common in P-MDS (56.5 vs. 25.6%). In contrast to P-MDS, the ring sideroblasts (RS) phenotype was not associated with better survival in T-MN, most probably due to genetic association with TP53 mutations. SF3B1 was mutated in 96% of P-MDS with ≥15% RS, but in only 32% T-MN. TP53 mutations were detected in 92% T-MN with ≥15% RS and SF3B1 wild-type cases. Interestingly, T-MN and P-MDS patients with "Very low" or "Low" Revised International Prognostic Scoring System (IPSS-R) showed similar biological and clinical characteristics. In a Cox regression analysis, TP53 mutation was a poor prognostic factor in T-MN, independent of IPSS-R cytogenetics, disease-modifying therapy, and NRAS mutation. Our data have direct implications for T-MN management and provide evidence that, in addition to conventional disease parameters, mutational analysis should be incorporated in T-MN risk stratification.

Red cell alloimmunization is associated with development of autoantibodies and increased red cell transfusion requirements in myelodysplastic syndrome.

Up to 90% of patients with a myelodysplastic syndrome require red blood cell transfusion; nevertheless, comprehensive data on red cell alloimmunization in such patients are limited. This study evaluates the incidence and clinical impact of red cell alloimmunization in a large cohort of patients with myelodysplastic syndrome registered in the statewide South Australian-MDS registry. The median age of the 817 patients studied was 73 years, and 66% were male. The cumulative incidence of alloimmunization was 11%. Disease-modifying therapy was associated with a lower risk of alloimmunization while alloimmunization was significantly higher in patients with a revised International Prognostic Scoring System classification of Very Low, Low or Intermediate risk compared to those with a High or Very High risk (P=0.03). Alloantibodies were most commonly directed against antigens in the Rh (54%) and Kell (24%) systems. Multiple alloantibodies were present in 49% of alloimmunized patients. Although 73% of alloimmunized patients developed alloantibodies during the period in which they received their first 20 red cell units, the total number of units transfused was significantly higher in alloimmunized patients than in non-alloimmunized patients (90±100 versus 30±52; P<0.0001). In individual patients, red cell transfusion intensity increased significantly following alloimmunization (2.8±1.3 versus 4.1±2.0; P<0.0001). A significantly higher proportion of alloimmunized patients than non-alloimmunized patients had detectable autoantibodies (65% versus 18%; P<0.0001) and the majority of autoantibodies were detected within a short period of alloimmunization. In conclusion, this study characterizes alloimmunization in a large cohort of patients with myelodysplastic syndrome and demonstrates a signficant increase in red cell transfusion requirements following alloimmunization, most probably due to development of additional alloantibodies and autoantibodies, resulting in subclinical/clinical hemolysis. Strategies to mitigate alloimmunization risk are critical for optimizing red cell transfusion support.

Galactomannan and PCR versus culture and histology for directing use of antifungal treatment for invasive aspergillosis in high-risk haematology patients: a randomised controlled trial.

BACKGROUND: Empirical treatment with antifungal drugs is often used in haematology patients at high risk of invasive aspergillosis. We compared a standard diagnostic strategy (culture and histology) with a rapid biomarker-based diagnostic strategy (aspergillus galactomannan and PCR) for directing the use of antifungal treatment in this group of patients. METHODS: In this open-label, parallel-group, randomised controlled trial, eligible patients were adults undergoing allogeneic stem-cell transplantation or chemotherapy for acute leukaemia, with no history of invasive fungal disease. Enrolled patients were randomly assigned (1:1) by a computer-generated schedule to follow either a standard diagnostic strategy (based on culture and histology) or a biomarker-based diagnostic strategy (aspergillus galactomannan and PCR) to direct treatment with antifungal drugs. Patients, were followed up for 26 weeks or until death. Masking of the use of different diagnostic tests was not possible for patients, treating physicians, or investigators. The primary endpoint was empirical treatment with antifungal drugs in the 26 weeks after enrolment (for the biomarker-based diagnostic strategy, a single postive galactomannan or PCR result was deemed insufficient to confirm invasive aspergillosis, so treatment in this context was classified as empirical). This outcome was assessed by an independent data review committee from which the study allocations were masked. Analyses were by intention to treat and included all enrolled patients. This study is registered with ClinicalTrial.gov, number NCT00163722. FINDINGS: 240 eligible patients were recruited from six Australian centres between Sept 30, 2005, and Nov 19, 2009. 122 were assigned the standard diagnostic strategy and 118 the biomarker-based diagnostic strategy. 39 patients (32%) in the standard diagnosis group and 18 (15%) in the biomarker diagnosis group received empirical antifungal treatment (difference 17%, 95% CI 4-26; p=0·002). The numbers of patients who had hepatotoxic and nephrotoxic effects did not differ significantly between the standard diagnosis and biomarker diagnosis groups (hepatotoxic effects: 21 [17%] vs 12 [10%], p=0·11; nephrotoxic effects: 52 [43%] vs 60 [51%], p=0·20). INTERPRETATION: Use of aspergillus galactomannan and PCR to direct treatment reduced use of empirical antifungal treatment. This approach is an effective strategy for the management of invasive aspergillosis in high-risk haematology patients. FUNDING: Australian National Health and Medical Research Council, Cancer Council New South Wales, Pfizer, Merck, Gilead Sciences.

A no-prophylaxis platelet-transfusion strategy for hematologic cancers.

BACKGROUND: The effectiveness of platelet transfusions to prevent bleeding in patients with hematologic cancers remains unclear. This trial assessed whether a policy of not giving prophylactic platelet transfusions was as effective and safe as a policy of providing prophylaxis. METHODS: We conducted this randomized, open-label, noninferiority trial at 14 centers in the United Kingdom and Australia. Patients were randomly assigned to receive, or not to receive, prophylactic platelet transfusions when morning platelet counts were less than 10×10(9) per liter. Eligible patients were persons 16 years of age or older who were receiving chemotherapy or undergoing stem-cell transplantation and who had or were expected to have thrombocytopenia. The primary end point was bleeding of World Health Organization (WHO) grade 2, 3, or 4 up to 30 days after randomization. RESULTS: A total of 600 patients (301 in the no-prophylaxis group and 299 in the prophylaxis group) underwent randomization between 2006 and 2011. Bleeding of WHO grade 2, 3, or 4 occurred in 151 of 300 patients (50%) in the no-prophylaxis group, as compared with 128 of 298 (43%) in the prophylaxis group (adjusted difference in proportions, 8.4 percentage points; 90% confidence interval, 1.7 to 15.2; P=0.06 for noninferiority). Patients in the no-prophylaxis group had more days with bleeding and a shorter time to the first bleeding episode than did patients in the prophylaxis group. Platelet use was markedly reduced in the no-prophylaxis group. A prespecified subgroup analysis identified similar rates of bleeding in the two study groups among patients undergoing autologous stem-cell transplantation. CONCLUSIONS: The results of our study support the need for the continued use of prophylaxis with platelet transfusion and show the benefit of such prophylaxis for reducing bleeding, as compared with no prophylaxis. A significant number of patients had bleeding despite prophylaxis. (Funded by the National Health Service Blood and Transplant Research and Development Committee and the Australian Red Cross Blood Service; TOPPS Controlled-Trials.com number, ISRCTN08758735.).

Polymorphism in human cytomegalovirus UL40 impacts on recognition of human leukocyte antigen-E (HLA-E) by natural killer cells.

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.

Loss-of-function germline GATA2 mutations in patients with MDS/AML or MonoMAC syndrome and primary lymphedema reveal a key role for GATA2 in the lymphatic vasculature.

Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), "MonoMAC" syndrome, and DCML deficiency. Here, we describe a previously unreported MDS family carrying a missense GATA2 mutation (p.Thr354Met), one patient with MDS/AML carrying a frameshift GATA2 mutation (p.Leu332Thrfs*53), another with MDS harboring a GATA2 splice site mutation, and 3 patients exhibiting MDS or MDS/AML who have large deletions encompassing the GATA2 locus. Intriguingly, 2 MDS/AML or "MonoMAC" syndrome patients with GATA2 deletions and one with a frameshift mutation also have primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present at high levels in lymphatic vessel valves and that GATA2 controls the expression of genes important for programming lymphatic valve development. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and suggest that complete haploinsufficiency or loss of function of GATA2, rather than missense mutations, is the key predisposing factor for lymphedema onset. Moreover, we reveal a crucial role for GATA2 in lymphatic vascular development.

Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia.

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

Human gastrointestinal neoplasia-associated myofibroblasts can develop from bone marrow-derived cells following allogeneic stem cell transplantation.

This study characterized the contribution of bone marrow-derived cells to human neoplasia and the perineoplastic stroma. The Australasian Bone Marrow Transplant Recipient Registry was used to identify solid organ neoplasia that developed in female recipients of male allogeneic stem cell transplants. Eighteen suitable cases were identified including several skin cancers, two gastric cancers, and one rectal adenoma. Light microscopy, fluorescence and chromogenic in situ hybridization, and immunohistochemistry were performed to determine the nature and origin of the neoplastic and stromal cells. In contrast to recent reports, donor-derived neoplastic cells were not detected. Bone marrow-derived neoplasia-associated myofibroblasts, however, were identified in the rectal adenoma and in a gastric cancer. Bone marrow-derived cells can generate myofibroblasts in the setting of human gastrointestinal neoplasia.

Mannose-binding lectin status is associated with risk of major infection following myeloablative sibling allogeneic hematopoietic stem cell transplantation.

Mannose-binding lectin (MBL) is a mediator of innate immunity that influences the risk of infection in a range of clinical settings. We previously reported associations between MBL2 genotype and infection in a retrospective study of myeloablative allogeneic hematopoietic stem cell transplantation (allo-HCT). However, other studies have been inconclusive, and the role of MBL in reduced-intensity conditioning (RIC) transplantation is unknown. Here we report a prospective study examining MBL2 genotype, MBL levels, and risk of major infection following HLA-matched sibling myeloablative (n = 83) and RIC (n = 59) HCT. Baseline MBL levels were higher in recipients than donors (P < .001), and recipient MBL levels increased during the peritransplantation period (P = .001), most notably in MBL2 wild-type individuals receiving myeloablative total body irradiation (mTBI). MBL2 coding mutations were associated with major infection in recipients receiving mTBI. The cumulative incidence of major infection in recipient harboring an MBL2 mutation receiving mTBI was 70.6%, compared with 31.1% of those without mutations not receiving mTBI (P = .01). MBL status was not associated with infection in RIC transplants. These results confirm the association of MBL status with risk of infection in myeloablative, TBI-conditioned transplantation. Studies examining the role of MBL replacement therapy to prevent infection in this setting should be considered.

New directions in the genomics of allogeneic hematopoietic stem cell transplantation.

Despite optimal supportive care and high-resolution HLA matching, complications such as GVHD and infection remain major barriers to the success of allogeneic HCT (allo-HCT). This has led to growing interest in the non-HLA genetic determinants of complications after allo-HCT. Most studies have examined genetic predictors of GVHD, relapse, and mortality and have focused on 3 main areas: minor histocompatibility antigen (miHAs), inflammatory mediators of GVHD, and more recently NK cell-mediated allorecognition. The genetic basis of other outcomes such as infection and drug toxicity are less well studied but are being actively investigated. High-throughput methodologies such as single nucleotide polymorphism arrays are enabling the study of hundreds of thousands of genetic markers throughout the genome and the interrogation of novel genetic variants such as copy number variations. These data offer the opportunity to better predict those at risk of complications and to identify novel targets for therapeutic intervention. This review examines the current data regarding the non-HLA genomics of allo-HCT and appraises the promises and pitfalls for integration of this new genetic information into clinical transplantation practice.

Mannose-binding lectin and maladies of the bowel and liver.

Mannose-binding lectin (MBL) is a pattern-recognition molecule that binds to characteristic carbohydrate motifs present on the surface of many different pathogens. MBL binding stimulates the immune system via the lectin pathway of complement activation. In certain clinical situations, often characterized by pre-existing immune compromise, MBL deficiency increases the risk of infectious and other disease-specific complications. Many of the key pathogenic processes inherent to common gastroenterological diseases, such as infection, immunological damage, and carcinogenesis, have been linked to MBL. This editorial reviews the biology of MBL, outlines key disease associations to document the breadth of influence of MBL, and finally, highlights the relevance of MBL to both gastroenterological health and disease.

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma.

BACKGROUND: The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. METHODS: RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. RESULTS: All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. CONCLUSION: EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target.

Mannose-binding lectin and infection following allogeneic hemopoietic stem cell transplantation.

Major infection remains a major barrier to the success of allogeneic hemopoietic stem cell transplantation (SCT). There is growing interest in the importance of innate immunity in host defense, particularly when adaptive immunity is compromised. Furthermore, many host defense genes are polymorphic, and immunogenetic factors are known to influence the risk of other transplant complications, such as graft-versus-host disease. Mannose-binding lectin (MBL) has emerged as an important innate host defense molecule. MBL binds a wide range of pathogens independently of antibody and activates complement leading to lysis and phagocytosis. Genetically determined MBL deficiency is common and results in an increased risk of infection in a variety of clinical settings, especially in individuals already immunocompromised for other reasons. We conducted a retrospective study examining associations between polymorphisms in the gene encoding MBL, MBL2 and risk of major infection post-SCT in 96 related myeloablative transplants. This showed that "low-producing" MBL2 coding alleles, when present in the donor, were significantly associated with increased risk of major infection in the recipient following neutrophil count recovery. Furthermore, a "high-producing" MBL2 haplotype, HYA, when present in the recipient, was protective against infection. As MBL is under development as a therapeutic agent, these findings suggest that administration of MBL may reduce the risk of infection post-transplant. Prior to embarking upon trials of MBL replacement therapy in SCT, further work is required to confirm these results, to examine the kinetics of MBL synthesis peri-transplant, to correlate MBL2 genotype with blood MBL levels, and to examine the role of MBL in other settings, such as transplantation using reduced intensity conditioning regimens, and unrelated donor transplants. These results are the first report of a genetic determinant of risk of infection post-SCT, and highlight the importance of non-HLA genetic factors in determining the risk of transplant complications. Further studies examining other host defence genes are warranted, and are in progress.

Chronic myelomonocytic leukemia requires granulocyte-macrophage colony-stimulating factor for growth in vitro and in vivo.

OBJECTIVE: Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease with no effective treatments or cure. Several factors have been implicated in its pathogenesis. In the current study, we studied the dependence of CMML on granulocyte-macrophage colony-stimulating factor (GM-CSF). MATERIALS AND METHODS: We used in vitro colony assays in methylcellulose where CMML cells were tested in the presence or absence of the specific GM-CSF antagonist E21R. We also developed an in vivo model in which CMML cells were tested for their ability to engraft into immunodeficient mice transgenic for human GM-CSF. RESULTS: Bone marrow cells from seven of seven patients with CMML formed spontaneous colonies that were sensitive to E21R treatment, with reduction in colony growth by up to 92%. E21R also inhibited colony formation by CMML patient cells stimulated by exogenously added GM-CSF but not interleukin-3. In in vivo experiments we observed engraftment of CMML cells (but not normal cells) in immunodeficient mice transgenic for human GM-CSF. None engrafted in nontransgenic mice. Cell dose escalation showed that the optimal number was 0.5 to 1 x 10(8) peripheral blood mononuclear cells per mouse, which is equivalent to an infusion of 0.2 to 3.6 x 10(6) CD34(+) cells. Time course experiments showed that maximal engraftment occurred 6 weeks after injection. CONCLUSIONS: These results demonstrate that in some CMML patients, GM-CSF produced by either autocrine or paracrine mechanisms is a major growth determinant. The results suggest that therapies directed at blocking this cytokine could control the growth of some CMML patients in vivo.

Isolated cardiac recurrence of acute lymphoblastic leukemia characterized by t(11;19) two years after unrelated allogeneic bone marrow transplantation.

A 33-year-old male presented with acute lymphoblastic leukemia (ALL) characterized by translocation (11;19)(q23;p13.3). He received an allogeneic bone marrow transplant from a matched unrelated donor. Two years later his disease relapsed with an isolated intracardiac mass, presenting as right heart failure. He had no evidence of concomitant relapse in the bone marrow. Tumor cytogenetics revealed clonal evolution with the karyotype 46,XY,t(3;16)(q23;p13),t(11;19)(q23;p13.3), the chromosome 16 breakpoint involving the band where the genes for multidrug resistance-associated protein and CREB binding protein are known to reside. To our knowledge, this is the first report of an isolated extramedullary relapse of ALL in the heart.