Donor specific anti-HLA antibodies in hematopoietic stem cell transplantation. Single Center prospective evaluation and desensitization strategies employed.

BACKGROUND: In the setting of mismatched-hematopoietic stem cells transplantation, the detection of antibodies directed against donor-specific HLA allele(s) or antigen(s) (DSA) represents a barrier for engraftment. It is thus necessary to plan an immunosuppressive strategy, or to select an alternative donor. This prospective study aimed at evaluating the efficacy of our strategy for testing DSAs and the efficacy of the desensitization strategy (DS) employed between November 2017 and November 2020. MATERIALS AND METHODS: The anti-HLA antibody search was performed using the Luminex bead assays (Lifecode ID and LSA I/II-Immucor) and expressed as mean fluorescence intensity (MFI >1,000 positive). If the patient had DSAs and no alternative donors, a DS was employed with rituximab (day -15), 2 single volume plasmaphereses (PP; days -9 and -8), intravenous immunoglobulins (day -7) and infusion of HLA selected platelets, if persistent DSAs were directed against class I HLA. DS was scheduled with or without PP, according to the DSA MFI (>1,000 or <5,000) and FCXM (flow cytometry crossmatch). RESULTS: Twenty-two out of 126 patients (17.46%) showed anti-HLA antibodies, 5 of them DSAs (3.97% of total); 3 patients underwent DS obtaining engraftment. Female gender (p=0.033) and a history of previous pregnancies or miscarriages (p=0.009) showed a statistically significant impact on alloimmunization. Factors associated with a delayed neutrophil engraftment were patient's female gender (p=0.039), stem cell source (p=0.025), and a high HSCT-specific comorbidity index (p=0.028). None of the analyzed variables, including the DSA detection, influenced engraftment. CONCLUSIONS: Our study confirms the importance to test DSAs in mismatched-hematopoietic stem cells transplantation The DS used proved successful in removing DSAs. Prospective multicenter studies are needed to better define and validate consensus strategies on DSA management in HSCT.

Managing leukapheresis in adult and pediatric patients eligible for chimeric antigen receptor T-cell therapy: suggestions from an Italian Expert Panel.

Chimeric antigen receptor (CAR) T-cell therapy relies on T cells engineered to target specific tumor antigens such as CD-19 in B-cell malignancies. In this setting, the commercially available products have offered a potential long-term cure for both pediatric and adult patients. Yet manufacturing CAR T cells is a cumbersome, multistep process, the success of which strictly depends on the characteristics of the starting material, i.e., lymphocyte collection yield and composition. These, in turn, might be affected by patient factors such as age, performance status, comorbidities, and previous therapies. Ideally, CAR T-cell therapies are a one-off treatment; therefore, optimization and the possible standardization of the leukapheresis procedure is critical, also in view of the novel CAR T cells currently under investigation for hematological malignancies and solid tumors. The most recent Best Practice recommendations for the management of children and adults undergoing CAR T-cell therapy provide a comprehensive guide to their use. However, their application in local practice is not straightforward and some grey areas remain. An Italian Expert Panel of apheresis specialists and hematologists from the centers authorized to administer CAR T-cell therapy took part in a detailed discussion on the following: 1) pre-apheresis patient evaluation; 2) management of the leukapheresis procedure, also in special situations represented by low lymphocyte count, peripheral blastosis, pediatric population <25 kg, and the COVID-19 outbreak; and 3) release and cryopreservation of the apheresis unit. This article presents some of the important challenges that must be faced to optimize the leukapheresis procedure and offers suggestions as to how to improve it, some of which are specific to the Italian setting.

Autologous stem cell transplantation in favorable-risk acute myeloid leukemia: single-center experience and current challenges.

BACKGROUND: Autologous stem cell transplantation (ASCT) has gained growing consideration as a treatment option for favorable-risk acute myeloid leukemia (FR-AML) in first complete remission (CR1), compared with chemotherapy. MATERIALS AND METHODS: We report the long-term outcomes of 117 consecutive patients with FR-AML fit for intensive chemotherapy diagnosed in our center between 1999 and 2020, who underwent ASCT. RESULTS: Sixty-five of the 117 were eligible for intensive post-remission treatment, and 42 of those 65 received ASCT. Median follow up was 132 months. Overall survival (OS) and disease-free survival (DFS) were 75% and 76%. Higher doses of CD34 + stem cell infusions negatively impacted DFS in multivariate analysis. Core-binding factor (CBF) leukemia was an independent prognostic factor for improved DFS. No differences based on pre-transplant measurable residual disease (MRD) were observed. In CBF leukemia, 10-year DFS is 72% for MRD-positive patients versus 100% for MRD negative patients. CONCLUSIONS: ASCT is effective and safe in FR-AML patients. In CBF leukemia, ASCT provides excellent results regardless of achievement of bone marrow MRD negativity. In NPM1-mutated/FLT3-wild type (mNPM1) AML, early molecular response seems to have more impact on prognosis. Prospective investigation of the role of gemtuzumab ozogamicin in this setting is ongoing.

Immuno-hematological monitoring after allogeneic stem cell transplantation: a single-center, prospective study of 104 patients.

BACKGROUND: The impact of ABO incompatibility on the outcome of hematopoietic stem cell transplantation (HSCT) is still debated. We report the results of a prospective, single-center study evaluating the impact of ABO mismatch on the development of immediate and late immuno-hematological complications, and the efficacy of the protocol used at the "Sapienza" University (Rome, Italy) to manage ABO incompatibility in patients undergoing HSCT. MATERIALS AND METHODS: From January 2013 to December 2016, we prospectively analyzed all patients undergoing HSCT. Graft manipulation or desensitization strategies were used according to ABO incompatibility, donor sex and donor transfusion history. Red blood cell and platelet transfusions were given based on immunohematological features. RESULTS: From January 2013 to December 2016, 104 consecutive patients underwent HSCT from a matched related donor (29.81%), matched unrelated donor (53.58%), cord blood (1.9%) or haploidentical donor (14.42%). Forty-nine patients (47%) were ABO-identical and 55 (53%) ABO-incompatible (23 major, 25 minor, 7 bidirectional). Donor engraftment, graft failure or other complications did not differ between ABO compatible or incompatible patients. ABO incompatibility did not show a significant impact on graft-versus-host disease, overall survival or disease-free survival. Factors associated with the need for prolonged red blood cell support were ABO incompatibility (p=0.0395), HLA disparity between donor and recipient (p=0.004) and the onset of hemorrhagic cystitis (p=0.015). In multivariate analysis HLA disparity was the only statistically significant condition (p=0.004). DISCUSSION: ABO incompatibility does not represent a barrier to allogeneic HSCT. It is, however, associated with prolonged transfusion requirements. Close immunohematological monitoring, as a shared standard procedure, allows appropriate transfusion support to be provided and limits post-HSCT immuno-hematological complications.

Therapeutic Apheresis in Pregnancy: Three Differential Indications With Positive Maternal and Fetal Outcome.

Therapeutic apheresis (TA) is a complex extracorporeal procedure for the treatment of several acute and chronic diseases. TA in pregnancy is considered safe for both mother and fetus and has the same indications of non-pregnant patients. TA can be used during the entire course of the pregnancy with the following purposes: (i) to treat several maternal acute and chronic conditions; (ii) to treat fetal conditions; (iii) to avoid administration of drugs potentially harmful to the fetus; and (iv) to reach a more advanced gestational age in order to prevent fetal prematurity. We report three successfully treated patients throughout pregnancy, for differential indications: thrombotic thrombocytopenic purpura, red blood cells alloimmunization and ulcerative colitis. Multiple courses of TA have been performed without any complications for the mother and the fetus. A review and a discussion on the particular TA implications related to maternal-fetal medicine have been reported. When approaching TA in pregnancy, clinicians have to consider the severity of disease, the strength of the indications, and the gestational age. Each case must be evaluated individually on the basis of existing evidence since, despite the increasing use, specific guidelines for apheresis in pregnancy are still lacking.

Thrombotic thrombocytopenic purpura during pregnancy versus imitator of preeclampsia.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a severe disorder affecting the microcirculation of multiple organs due to a systemic endothelial cell injury secondary to a deficiency in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity. TTP is a rare complication of pregnancy with a poor prognosis and high fetal mortality, especially when it occurs during the first trimester. Recent data have supported that effective treatment of TTP is plasma therapy. Unfortunately a major problem remains in the delay in diagnosis due to confounding factors between other "imitators of preeclampsia." Rapid and readily available laboratory testing to quickly diagnose TTP is desperately needed to improve care and to save mother and future child life. CASE REPORT: We describe a rare case of successful pregnancy after TTP manifestations occurring in the first trimester; most importantly, our experience represents the first case of atypical manifestation due to neurologic and kidney manifestations preceding laboratory assay alterations. RESULTS: We treated a patient with plasma replacement of 30 mL/kg/day and daily plasmapheresis in combination with continuous infusion of fresh-frozen plasma 10 mL/kg/day. The response of clinical manifestation immediately improved. At 30 weeks, the patient had multiple episodes of high blood pressure and concomitant decrease of hemoglobin and platelet count, so a cesarean section was immediately performed. She delivered a healthy female baby. CONCLUSION: Early diagnosis by ADAMTS13 activity, occasionally occurring before clinical manifestations, aided us in promptly administering commended and life-saving treatments.

A good manufacturing practice method to ex vivo expand natural killer cells for clinical use.

BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.

A three month-old infant with severe hyperchylomicronemia: molecular diagnosis and extracorporeal treatment.

OBJECTIVE: Chylomicronemia syndrome presenting in childhood is a rare recessive disorder due to mutations of lipoprotein lipase (LPL) and more rarely of APOC2, APOA5, GPIHBP1 or LMF1 genes. It often requires urgent and suitable treatment to avoid acute pancreatitis. The aim of this study was the molecular characterization and treatment of a 3 month-old infant with plasma triglycerides (TG) > 300 mmol/L. METHODS: All candidate genes were sequenced. The patient was submitted to one plasma-exchange (PEX) procedure and subsequently to a rigid lipid-lowering diet (milk: Monogen(®)). RESULTS: The proband was homozygous for a novel LPL mutation (c.242G > A, p.G81D) which in silico results pathogenic. After PEX, which was well tolerated, TG dropped to 64 mmol/L. During 5-month follow-up there was a clear trend towards lower and stable TG values. CONCLUSION: PEX is applicable in subjects with very low body weight when the extreme severity of the clinical picture has no therapeutic alternatives.

[Multimodal treatment for acute antibody-mediated renal transplant rejection: successful rescue therapy with combined plasmapheresis, photopheresis and intravenous immunoglobulin]. / Trattamento multimodale per il rigetto acuto anticorpo-mediato nel trapianto di rene: terapia di salvataggio con plasmaferesi, fotoaferesi e immunoglobuline endovenose.

The fundamental role of antibodies in the development of acute graft rejection has been established recently. Antibody-mediated acute rejection may develop at any time during the post-transplant period. Several therapeutic approaches have been proposed in the last decades. However, there is no standardized therapy. The aim of this study is to report the Sapienza University experience of combined plasma treatment and high-dose intravenous immunoglobulin ± extracorporeal photopheresis. From January 2006 to September 2009, 6 patients were treated at Sapienza University. In 5 cases (83%) complete regression of the acute rejection was observed, followed by stable renal function (median creatinine value at 1-year follow-up: 1.5 mg/dL). No adverse events were reported. Our approach seems to give good results in terms of graft survival and procedure safety. Further studies on a larger number of patients will be needed to confirm the validity of these findings. Moreover, comparison between our protocol and other treatments is necessary.

Standard- versus high-dose lenograstim in adults with hematologic malignancies for peripheral blood progenitor cell mobilization.

BACKGROUND: The aim of this retrospective, multicenter study was to compare high- versus standard-dose lenograstim after chemotherapy in collecting target dose of CD34+ peripheral blood progenitor cells (PBPCs) in adult candidates for autologous transplant. STUDY DESIGN AND METHODS: A total of 166 consecutive patients (28 acute leukemias [ALs], 77 lymphomas, 61 multiple myeloma [MM]) underwent 182 mobilization procedures. Only the first were analyzed. The CD34+ cell target was at least 2×10(6) , 4×10(6) , and 8×10(6) /kg and lenograstim started on days +19, +1, and +5 from the end of chemotherapy for AL, lymphomas, and MM, respectively. Eighty-seven and 79 patients, respectively, received 5 and 10µg/kg/day lenograstim subcutaneously (sc). An analysis to evaluate factors predicting satisfactory procedures and outcome of transplants performed with first-mobilization-procedure PBPCs was conducted. Most patients received 6mg of pegfilgrastim or 5µg/kg/day lenograstim sc after transplant. RESULTS: In multivariate analysis, high-dose lenograstim (p=0.053) in MM and male sex (p=0.028) were positive predictive factors for reaching cell target. Fludarabine negatively influenced stimulation length (p=0.002). Apheresis, CD34+ cells mobilized and collected, blood volume processed, side effects, transplants performed, and engraftment time were similar between lenograstim cohorts. Pegfilgrastim versus lenograstim delayed platelet (PLT) recovery times (13 days vs. 11 days, p=0.036). CONCLUSIONS: High-dose lenograstim more efficiently mobilized MM patients requiring the highest PBPC target but did not influence transplants performed and engraftment time. Male patients mobilized more efficiently. Fludarabine negatively influenced stimulation length. Finally, pegfilgrastim seems to delay PLT recovery.

B-cell concentration in the apheretic product predicts acute graft-versus-host disease and treatment-related mortality of allogeneic peripheral blood stem cell transplantation.

BACKGROUND: The influence of the graft composition on the clinical outcome after allogeneic peripheral blood stem cell (PBSC) transplantation is not well established. METHODS: The cellular composition of the apheretic products obtained from 63 human leukocyte antigen-identical siblings was prospectively correlated with the outcome of patients with hematological malignancies undergoing an allogeneic PBSC transplant after myeloablative conditioning. The concentration of nuclear, mononuclear, CD34+, T-cell subsets, B cells, and natural killer cells in the graft has been analyzed. RESULTS: In univariate analysis, acute graft-versus-host disease (GVHD) correlated with the disease (P=0.002), with the phase of disease at transplant (P=0.01), and with the number of CD20+ cells infused (P=0.05). In multivariate analysis, a dose of CD20+ cells in the graft higher than the median dose remained the only factor negatively affecting the incidence of acute GVHD (P=0.01; 95% confidence interval [CI]: 0.12-0.78). In univariate analysis, treatment-related mortality (TRM) correlated with the disease (P=0.04) and was negatively affected by a dose of infused B cells greater than the median value (28% versus 50%; P=0.02). In multivariate analysis, TRM was close to statistical correlation with the dose of CD20+ cells (P=0.06; 95% CI: 0.02-1.05). No other clinical parameter was influenced by the composition of the graft. CONCLUSIONS: Our results suggest that the concentration of B cells in the apheretic product may predict the incidence of acute GVHD and TRM in patients undergoing an allogeneic PBSC transplantation and open the way to the new preventive and therapeutic strategies for the management of GVHD.