Case Report: Invasive Fungal Infection and Daratumumab: A Case Series and Review of Literature.

Life expectancy of multiple myeloma (MM) patients has improved in last years due to the advent of anti-CD38 monoclonal antibodies in combination with immunomodulators and proteasome inhibitors. However, morbidity and mortality related to infections remain high and represent a major concern. This paper describes the "real life" risk of invasive fungal infections (IFI) in patients treated with daratumumab-based therapy and reviews the relevant literature. In a series of 75 patients we only observed three cases of fungal pneumonia. Unfortunately, the early signs and symptoms were not specific for fungal infection. Diagnostic imaging, microbiology and patient history, especially previous therapies, are critical in the decision to start antifungal treatment. Recognising the subgroup of MM patients with high risk of IFI can increase the rate of diagnosis, adequate treatment and MM-treatment recovery.

Hemopoietic stem cell transplantation in thalassemia: a report from the European Society for Blood and Bone Marrow Transplantation Hemoglobinopathy Registry, 2000-2010.

Allogeneic hemopoietic stem cell transplantation (HSCT) is the only method currently available to cure transfusion-dependent thalassemia major that has been widely used worldwide. To verify transplantation distribution, demography, activity, policies and outcomes inside the European Group for Blood and Marrow Transplantation (EBMT), we performed a retrospective non-interventional study, extracting data from the EBMT hemoglobinopathy prospective registry database. We included 1493 consecutive patients with thalassemia major transplanted between 1 January 2000 and 31 December 2010. In total, 1359 (91%) transplants were performed on patients <18 years old, 1061 were from a human leukocyte Ag-identical sibling donor. After a median observation time of 2 years, the 2-year overall survival (OS) and event-free survival (EFS; that is, thalassemia-free survival) were 88 ± 1% and 81 ± 1%, respectively. Transplantation from a human leukocyte Ag-identical sibling offered the best results, with OS and EFS of 91 ± 1% and 83 ± 1%, respectively. No significant differences in survival were reported between countries. The threshold age for optimal transplant outcomes was around 14 years, with an OS of 90-96% and an EFS of 83-93% when transplants were performed before this age. Allogeneic HSCT for thalassemia is a curative approach that is employed internationally and produces excellent results.

Influence of glutathione S-transferase gene polymorphisms on busulfan pharmacokinetics and outcome of hematopoietic stem-cell transplantation in thalassemia pediatric patients.

Hematopoietic stem-cell transplantation (HSCT) is currently the only curative therapeutic option for the treatment of thalassemia. In spite of the high cure rate, HSCT can lead to life-threatening adverse events in some patients. Busulfan (Bu) is a key component of the conditioning regimen prior to HSCT. Inter-individual differences in Bu pharmacokinetics (PK) are hypothesized to influence Bu efficacy and toxicity. Since Bu is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship of GSTA1 and GSTM1 genotypes with first-dose PK and HSCT outcomes in 44 children with thalassemia intermedia and thalassemia major. All children received a myeloablative conditioning regimen with IV Bu. Association analysis revealed a relationship between GSTA169C>T (or haplotype *A/*B) and first Bu dose PK that was dependent on sex and Pesaro risk classification (PRC). Among female patients and patients with PRC I-II, homozygous individuals for the GSTA1T-69 allele defining haplotype *B, had higher Bu exposure and lower clearance (P⩽0.01). Association with HSCT outcomes showed that patients with the GSTM1 null genotypes had higher occurrence of regimen-related toxicity (P=0.01). These results suggest that GST genotypes could be useful to tailor the first Bu dose accordingly to improve HSCT outcome.

Incidence, risk factors and clinical outcome of leukemia relapses with loss of the mismatched HLA after partially incompatible hematopoietic stem cell transplantation.

Genomic loss of the mismatched human leukocyte antigen (HLA) is a recently described mechanism of leukemia immune escape and relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Here we first evaluated its incidence, risk factors and outcome in 233 consecutive transplants from partially HLA-mismatched related and unrelated donors (MMRD and MMUD, respectively). We documented 84 relapses, 23 of which with HLA loss. All the HLA loss relapses occurred after MMRD HSCT, and 20/23 in patients with acute myeloid leukemia. Upon MMRD HSCT, HLA loss variants accounted for 33% of the relapses (23/69), occurring later than their 'classical' counterparts (median: 307 vs 88 days, P<0.0001). Active disease at HSCT increased the risk of HLA loss (hazard ratio (HR): 10.16; confidence interval (CI): 2.65-38.92; P=0.001), whereas older patient ages had a protective role (HR: 0.16; CI: 0.05-0.46; P=0.001). A weaker association with HLA loss was observed for graft T-cell dose and occurrence of chronic graft-versus-host disease. Outcome after 'classical' and HLA loss relapses was similarly poor, and second transplantation from a different donor appeared to provide a slight advantage for survival. In conclusion, HLA loss is a frequent mechanism of evasion from T-cell alloreactivity and relapse in patients with myeloid malignancies transplanted from MMRDs, warranting routine screening in this transplantation setting.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

Early molecular diagnosis of aspergillosis in a patient with acute myeloid leukaemia.

Diagnosis of invasive fungal infection remains challenging. Here we report a case of early diagnosis of invasive aspergillosis in a neutropenic patient affected by acute myeloid leukaemia, achieved through the detection of Aspergillus fumigatus species-specific ribonucleic acid sequences by a sensitive multiplex real-time polymerase chain reaction-based molecular assay. Thanks to the early diagnosis, targeted therapy was promptly established and the severe fungal infection controlled, allowing the patient to subsequently receive allogeneic hematopoietic stem cell transplantation from a haploidentical donor, her only curative option. Also in this instance, targeted secondary antifungal prophylaxis with voriconazole avoided any other fungal infection afterwards. This report suggests how the implementation of molecular assays in combination with routine diagnostic procedures, can improve microbiological diagnosis in sepsis, particularly in case of fungal infection, difficult to detect with standard microbiological culture methods.

Genotypes and haplotypes in the 3' untranslated region of the HLA-G gene and their association with clinical outcome of hematopoietic stem cell transplantation for beta-thalassemia.

Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.

Escalating doses of donor lymphocytes for incipient graft rejection following SCT for thalassemia.

Mixed chimerism (MC) and secondary graft failure are frequent events following SCT for thalassemia. There is limited information regarding the outcome of donor lymphocyte infusion (DLI) to prevent rejection, mainly from case reports describing only successful cases. We describe a series of seven children affected by beta-thalassemia treated with escalating doses of DLI for level 2-3 MC (donor<90%) following myeloablative SCT from a matched family donor. The infusions were safe and no acute or chronic GVHD were documented; five patients experienced neutropenia and thrombocytopenia resolving spontaneously. DLI was successful in converting to full donor chimerism two patients stratified in the low-risk class (Pesaro class II). Conversely, for five high-risk patients, DLI was not effective in preventing secondary graft failure. This limited series suggests that escalating doses of DLI are safe in thalassemia patients post myeloablative therapy but efficacy may be jeopardized by rapidly growing anti-donor alloimmunity in high-risk patients. We suggest giving escalating doses of donor T cells to attempt a graft-versus-thalassemia as soon as level 2-3 MC is detected.

Multiple BM harvests in pediatric donors for thalassemic siblings: safety, efficacy and ethical issues.

Allogeneic BMT represents the only chance of cure for beta-thalassemia. Occasionally, two affected individuals from the same family share a matched healthy sibling. Moreover, a high incidence of transplant rejection is still observed in Pesaro class III patients, requiring a second BMT procedure. In these settings, one option is to perform a second BM harvest from the same donor. Although BM harvest is a safe procedure in children, ethical issues concerning this invasive practice still arise. Here, we describe our series of seven pediatric, healthy donors, who donated BM more than once in favor of their beta-thalassemic HLA-identical siblings between June 2005 and January 2008. Three donors donated BM twice to two affected siblings and four donors donated twice for the same sibling following graft rejection of the first BMT. All donors tolerated the procedures well and no relevant side effects occurred. There was no significant difference between the two harvests concerning cell yield and time to engraftment. Our experience shows that for pediatric donors, a second BM donation is safe and feasible and good cellularity can be obtained. We suggest that a second harvest of a pediatric donor can be performed when a strong indication for BMT exists.

Second hematopoietic SCT in patients with thalassemia recurrence following rejection of the first graft.

There is a substantial incidence of graft failure in patients with thalassemia after myeloablative conditioning regimens especially in class 3 patients in whom its incidence could be as high as 8-38.5%. Most patients with graft failure have recurrence of thalassemic marrow. Historically, results of second transplants for thalassemia were poor because of a high rejection rate and/or increased TRM. Sixteen patients with thalassemia recurrence following rejection of the first graft and with a median age of 9 years (range, 4-20) were given second transplants using BM (n=7) or PBSC (n=9) after preparation with a new treatment protocol. All but two patients received stem cells from the same donor. The median interval between two transplants was 28 months (range, 8-204). The sustained engraftment rate was high (94%) with only one patient having primary graft failure. The probability of overall survival, event-free survival, TRM and graft failure were 79, 79, 16 and 6%, respectively. There were three transplant-related deaths. Thirteen patients are alive with Lansky/Karnofsky score of 100. This intensified treatment protocol was well tolerated with no significant increase in toxicity. The excellent results obtained with this new preparative regimen allow us to recommend it for second transplantation for patients with thalassemia recurrence.

Response to donor lymphocyte infusions for chronic myeloid leukemia is dose-dependent: the importance of escalating the cell dose to maximize therapeutic efficacy.

Donor lymphocyte infusions (DLI) are an effective treatment for patients with chronic myeloid leukemia (CML) in relapse after allografting but the optimal cell dose has yet to be identified. To address this question, we investigated the factors affecting the dose required to achieve remission (effective cell dose, (ECD)) in 81 patients treated with an escalating dose regimen. The overall proportion of patients who achieved a molecular remission was 88%. The cumulative proportion of remitters increased significantly at each dose level. With a CD3(+) cell dose < or =10(7)/kg, 56% of patients in molecular/cytogenetic relapse obtained molecular remission, whereas only 20% of those in hematologic relapse did so. At the same cell dose, 58% of patients who received lymphocytes from volunteer unrelated donors achieved remission, as compared to 29% of those who received DLI from sibling donors. We conclude that the response to DLI is dose-dependent and that the ECD is influenced by the quantity and phase of CML at relapse and degree of donor/recipient histocompatibility.

Management of chronic myeloid leukaemia in relapse following donor lymphocyte infusion induced remission: a retrospective study of the Clinical Trials Committee of the British Society of Blood & Marrow Transplantation (BSBMT).

Donor lymphocyte infusion (DLI) can restore remission in a high percentage of patients with chronic myeloid leukaemia (CML) who relapse after allogeneic stem cell transplant (SCT). Subsequent relapses after a DLI-induced remission do occur and the optimal management of these patients is not defined. A retrospective study of the practice of UK transplant centres was conducted. In all, 13 patients from seven centres were identified: all were treated for relapse post allogeneic SCT with DLI and achieved either a complete cytogenetic (n=5) or molecular (n=8) remission. All patients subsequently had a second relapse, at molecular (n=7), cytogenetic (n=4) and haematological (n=2) levels. Further DLI was used in the treatment of 11 patients, imatinib mesylate in three and chemotherapy in two. The two patients with haematological relapse died of blastic disease. The remaining 11 patients achieved either a complete cytogenetic (n=2) or molecular (n=9) remission. Nine patients remain in molecular remission at a median follow-up of 29 months, seven of whom had received DLI alone as treatment for second relapse, one DLI plus imatinib and one imatinib alone. Toxicity following DLI for second relapse was low. Longer follow-up will be required to see if these second DLI-induced remissions will be durable.

Prognostic factors for patients with chronic myeloid leukaemia in chronic phase treated with imatinib mesylate after failure of interferon alfa.

We assessed clinical results in 145 patients with chronic myeloid leukaemia in chronic phase who satisfied criteria for interferon-alpha failure and were thus eligible for treatment with imatinib at the Hammersmith Hospital. We used univariate and multivariate analyses to develop a risk score based on features defined after treatment for 3 months. We identified a low neutrophil count and poor cytogenetic response (<35% Ph-negative marrow metaphases) at 3 months as principal independent predictive factors and incorporated them into a three-tier prognostic scoring system for individual patients. For patients in the low-, intermediate- and high-risk groups, the probabilities of survival at 24 months were 100, 82 and 40% (P<0.0001) and progression-free survival 100, 66 and 15% (P<0.0001), respectively. This Hammersmith prognostic scoring system was validated with an independent cohort of patients treated at another UK centre.

Locoregional intrasplenic chemotherapy for hypersplenism in myelofibrosis.

A 79-year-old patient with post-polycythaemic myelofibrosis presented with severe hypersplenism. After splenic artery catheterization, cytosine arabinoside was given intrasplenically from November 1999 to March 2000 for 5 d/month at 10 mg/m2 and increased each month by 10 mg/m2. It was then administered by continuous infusion until June 2000, starting at 20 mg/m2/d and tapering by 5 mg/m2 every 2 weeks to a final daily dose of 5 mg/m2/d. The drug was then stopped. The spleen had decreased to one third of the initial volume. Clinical conditions and haematological indices improved substantially. Intrasplenic therapy could be a new therapeutic tool for hypersplenism in chronic idiopathic and post-myeloproliferative myelofibrosis.

A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. One strategy to treat GVHD is to equip donor T cells with a conditional suicide mechanism that can be triggered when GVHD occurs. The herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir system used clinically has several limitations, including immunogenicity and cell cycle dependence. An alternative switch based on chemically inducible apoptosis was designed and evaluated. A chimeric human protein was expressed comprising an extracellular marker (DeltaLNGFR), the Fas intracellular domain, and 2 copies of an FK506-binding protein (FKBP). Primary human T lymphocytes retrovirally transduced with this construct could be purified to homogeneity using immunomagnetic beads. Genetic integrity of the construct was ensured by redesigning repetitive sequences. Transduced T cells behaved indistinguishably from untransduced cells, retaining the ability to mount a specific antiallogeneic immune response. However, they rapidly underwent apoptosis with the addition of subnanomolar concentrations of AP1903, a bivalent "dimerizer" drug that binds FKBP and induces Fas cross-linking. A single 2-hour treatment eliminated approximately 80% of T cells, and multiple exposures induced further apoptosis. T cells were eliminated regardless of their proliferation state, suggesting that the AP1903/Fas system, which contains only human components, is a promising alternative to HSV-tk for treating GVHD.

Potential of gene therapy in bone marrow transplantation.

Gene therapy, initiated as a treatment for inherited disorders such as adenosine deaminase deficiency, is now a promising therapeutic strategy for malignancies and other acquired diseases. In particular, in the field of bone marrow transplantation (BMT) for haematological malignancies, the gene transfer of the suicide gene HSV-TK into donor lymphocytes allows control of the severe complication graft-versus-host disease (GvHD). The transfer of the HSV-TK suicide gene confers selective sensitivity to the drug ganciclovir, allowing in vivo elimination of the donor T-cells if severe GvHD occurs. In Italy, the first pilot study on delayed infusion of genetically engineered donor lymphocytes after T-depleted allogeneic BMT documented efficacy of engineered donor lymphocytes in terms of anti-tumour activity and efficiency of the suicide system. GvHD developed in 3 out of 8 patients and was successfully treated by ganciclovir administration.