Genetic mutations and leukapheresis in acute myeloid leukemia: is there a link?

Acute myeloid leukemia is the most common acute leukemia in adults and up to 20% of patients present with hyperleukocytosis at the onset of the disease. The therapeutic approach involves medical support, cytoreductive treatment, and/or leukapheresis. Despite WBC count greater than 100.000/µL, not all patients develop symptoms. To clarify the role of leukapheresis in the setting of hyperleukocytotic AML, we aimed to find associations between AML morphologic subtypes and molecular alterations on presence or absence of leukostasis symptoms (and hence therapeutic vs prophylactic leukapheresis) and clinical outcomes in the cohort of 41 patients at our single center who underwent leukapheresis for hyperleukocytotic AML. There was a trend for increased WBC count, 30-day mortality, M4-M5 AML subtypes, and number of leukapheresis procedures performed in symptomatic hyperleukocytotic pts. No molecular marker was significantly associated with presence or absence of leukostasis symptoms due to small sample size, though there was a trend for increased NPM1-mutated and NPM1 + FLT3-mutated AML in asymptomatic patients and a greater proportion of symptomatic patients who were negative for all assessed molecular alterations. In conclusion, leukapheresis combined with cytoreductive treatment represents a synergic and efficient approach in the management of hyperleukocytosis especially in symptomatic patients considering the higher mortality independently from the presence of specific clonal markers whose distribution among the two groups may result more considerable with a higher number of patients.

Successful stem cell collection for atypical teratoid rhabdoid tumor in an extremely low-body weight child: A case report.

The use of peripheral blood hematopoietic stem cells for bone marrow reconstitution after myeloablative therapy is well established in children with malignant disorders. However, the peripheral blood hematopoietic stem cells collection in very low-body weight (≤10 kg) children remains a significant challenge because of technical and clinical issues. A male newborn affected by atypical teratoid rhabdoid tumor, diagnosed prenatally, received two cycles of chemotherapy following surgical resection. After an interdisciplinary discussion, it was decided to intensify the treatment with high-dose chemotherapy followed by autologous stem cell transplantation. After 7 days of G-CSF administration the patient underwent hematopoietic progenitor cells-apheresis collection. The procedure was performed in the pediatric intensive care unit, using two central venous catheters and Spectra Optia device. The cell collection procedure was completed in 200 min, during which time 3.9 total blood volumes were processed. During apheresis we did not observe electrolyte alterations. No adverse events were recorded during or immediately following the cell collection procedure. Our report describes the feasibility of performing large volume leukapheresis without complications in an extremely low-body weight patient weighing 4.5 kg using the Spectra Optia apheresis device. No catheter-related problems occurred, and apheresis was completed without any adverse event. In conclusion, we believe that very low-body weight pediatric patients need a multidisciplinary approach to manage central venous access, hemodynamic monitoring, cell collection, prevention of metabolic complications to improve safety, feasibility, and efficiency of stem cell collection procedures.

Successful "on-demand" plerixafor for autologous peripheral blood stem-cells transplantation for relapsed/refractory germ cell tumors.

BACKGROUND: Germ cell tumors represent, among solid cancers, a potentially curable disease even if up to 20% to 30% of patients (pts) relapse after first-line treatment especially considering intermediate and poor prognosis groups. In this scenario, patients are candidates for high-dose chemotherapy and autologous stem-cells transplantation as second-line treatment even though stem-cells mobilization potential can be affected by several cycles and regimens of chemotherapy. To date, plerixafor is authorized in poor mobilizer adult pts diagnosed with lymphoma or multiple myeloma and in pediatric solid tumors or lymphoma. Therefore, the use of plerixafor in adult pts with relapsing/refractory GCT is still off label. MATERIALS AND METHODS: In our study, we describe mobilization and collection of peripheral blood stem cells for 10 pts with germ cell tumors. Six patients underwent plerixafor administration since classified as poor mobilizers based on WBC count (>5.000/µL) and CD34+ cell count (<15/µL) the day before apheresis procedure. RESULTS: On the first day of apheresis, plerixafor administration in poor mobilizers made possible a remarkable boost of CD34+ cells in such a way to overlap that of good mobilizers' (32/µL vs 35/µL, respectively, P > .05). CONCLUSION: Therefore, in our experience, plerixafor made a good fraction of poor mobilizer patients eligible for mobilization and collection and able to undergo the predicted autologous stem-cells transplantation; thus, the lack of access to the use of plerixafor in this setting of patients risks jeopardizing an effective treatment, especially in case of poor prognosis.

Inline and offline extracorporeal photopheresis: Device performance, cell yields and clinical response.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective treatment for graft-vs-host-disease (GvHD). Photopheresis can be performed in offline or inline method. The first uses a conventional cell separator for collection of mononuclear-cells that are photoactivated by a separate device and manually reinfused; the second one involves a dedicated device performing the entire procedure (collection, photoactivation and reinfusion). STUDY DESIGN AND METHODS: The objective was to compare the two methods and cell product features to highlight key process, devices performance, and to evaluate ECP clinical response. Patients developing steroid-resistant GvHD underwent ECP as second-line treatment using either inline (Therakos CellEx) or offline system (Terumo BCT Spectra or Optia and UVA PIT system). Data about patients' features, pre-apheresis blood-count, cell product characteristics and clinical response were collected for analysis. RESULTS: We evaluated 494 procedures performed on 28 patients from April 2018 to March 2019. The offline procedure allows to achieve greater cell yield, it is characterized by larger processed blood volume, longer runtime, and higher ACD consumption. The inline procedure shows shorter runtime, high mononuclear-cells percentage and low percentage of granulocytes in cell product. We observed a significant difference in cell yields between inline and offline system; furthermore we did not find a significant relationship between cell dose and clinical response. CONCLUSION: Inline ECP is fast, highly automated and productive, making it particularly suitable for ECP treatments. Offline ECP collects high cell yields implying longer procedure and greater operator intervention. Our study did not find a significant relationship between cell dose and GVHD response.

Inline extracorporeal photopheresis: evaluation of cell collection efficiency.

BACKGROUND: Extracorporeal photopheresis (ECP) therapy has proved to be an effective and safe treatment for graft-versus-host-disease (GvHD), an important complication after hematopoietic stem cell transplantation. In 2016, we acquired Therakos CellEx, a dedicated inline ECP device to accomplish a significant increase in ECP activity. In literature, we found few data reporting CellEx performance evaluated in terms of collection efficiency to qualify the device. Hence, we decided to collect and analyze our data in order to build a reference in terms of expected results of the procedure. Here we report our data of ECP performed using CellEx in a 12-month period focusing on collection efficiency assessment, as well as procedural and apheretic product characteristics. STUDY DESIGN AND METHODS: We collected data of patients undergoing ECP from April 2018 to March 2019 using CellEx in order to evaluate collection efficiency. RESULTS: Between April 2018 and March 2019 we treated 28 adult patients affected by GvHD performing 319 ECP using CellEx. CellEx mononuclear cell product was characterized by high mononuclear cell percentage and low percentage of granulocytes, resulting particularly suitable for ECP treatments. Median collection efficiency for total nucleated cells and for mononuclear cells was 31.2% and 62.3%, respectively. CONCLUSION: Collection efficiency of CellEx was comparable to that usually obtained by cell separators designed for cell collection and was comparable to that of offline systems. Our results provide a detailed performance evaluation for inline ECP system users.

Mononuclear cell collection for extracorporeal photopheresis: Concentrate characteristics for off-line UV-A irradiation procedure.

BACKGROUND AND OBJECTIVE: Extracorporeal photopheresis (ECP) is the most represented cell therapy for treatment of cutaneous T-cell lymphoma, graft-versus host disease and organ rejection. We analyzed our experience in ECP using 2 cell separators (Cobe Spectra and Spectra Optia) focusing on leukapheretic product characteristics, UV-A irradiation procedure and entire ECP process. MATERIALS AND METHODS: We collected data of patients undergoing ECP between January 2012 and February 2015 in order to evaluate collection procedures performed using Cobe Spectra and Spectra Optia, mononuclear cell product, UV-A photoirradiation procedure by Pit System. RESULTS: We performed 484 ECP procedures in 27 patients. Cobe-derived mononuclear cell products were characterized by higher cell yields while Optia-derived mononuclear cell products were characterized by smaller volume, comparable mononuclear cell content but lower erythrocytes, granulocytes, and platelets contamination. CONCLUSION: Our study confirms good results for both cell separators. Blood volume processed being equal, Cobe collects a number of total nucleated cells significantly higher than Optia. Optia, collecting only target cells without significant erythrocytes, granulocytes and platelets contamination, is able to collect a leukapheretic product particularly suitable for ECP.

Red Cell Alloantibody Screening: Comparative Analysis of Three Different Technologies.

BACKGROUND: The detection of irregular antibody is a critical issue in the management of red blood cell transfusion according to the Type & Screen (T&S) practice. In order to implement the T&S procedure at our blood bank, we compared three different automated analyzers based on column agglutination technique (CAT) or solid phase red cell adherence assay (SPACA) methods. METHODS: Pre-transfusion antibody screening was performed in 986 patients candidate to elective surgery at low risk for red blood cell transfusion. We tested the following kits: the three-cell panel micro-CAT system ID-DiaCell I-II-III (DiaMed), the four-cell panel solid-phase system Capture-R Ready Screen-4 (Immucor), and the four-cell panel micro-CAT system Serascan Diana-4 (Grifols). Positive results were further investigated using corresponding identification panels, and discrepant results were investigated with all the antibody identification systems. RESULTS: Among 986 samples, we observed 967 concordant negative results (98.1%), 8 concordant positive results (0.8% of cases), and 11 discrepant results (1.1%). Among discrepant samples, an alloantibody could been identified in two patents (anti-M, detected by Serascan Diana-4 and ID-DiaCell I, II, III; anti-Kpa, detected by Capture-R Ready Screen-4 and Serascan Diana-4). CONCLUSION: Among the evaluated technologies, the four-cell panel micro-CAT system displayed the highest sensitivity and specificity with an optimal negative predictive value. These features might be relevant to the routine implementation of the T&S transfusion strategy.

The role of molecular typing and perfect match transfusion in sickle cell disease and thalassaemia: An innovative transfusion strategy.

Chronic red blood cell transfusions remain an essential part of supportive treatment in patients with thalassaemia and sickle cell disease (SCD). Red blood cell (RBC) transfusions expose patients to the risk of developing antibodies: RBC alloimmunization occurs when the immune system meets foreign antigens. We created a register of extensively genotyped donors to achieve a better matched transfusion in order to reduce transfusion alloimmunization. Extended RBC antigen typing was determined and confirmed by molecular biology techniques using Human Erythrocyte Antigen (HEA) BeadChip (BioArray Solutions Ltd., Warren, NJ) in periodic blood donors and in patients with thalassaemia and SCD. During 3 years, we typed extensively 1220 periodic blood donors, 898 male and 322 female. We also studied 10 hematologic patients affected by thalassaemia and sickle cell disease referred to our institution as candidate to periodic transfusions. Our patients (8 females and 2 males with a median age of 48 years, range 24-76 years), extensively typed using molecular techniques and screened for RBC alloantibodies, were transfused with a median of 33.5 RBC units. After three years of molecular typing, the "perfect match" transfusion strategy avoided new alloantibodies development in all studied patients.

Weak D Type 4.2.2 (DAR1.2) in an African child: Serology and molecular characterization.

The weak D phenotype is represented by a group of RHD genotypes that code for alterated RhD proteins associated with a reduced RhD expression on red blood cell. By routine serology, some partial D variants are likely to be missed. In this report we describe the case of a three-year-old Black African child with a "unclear" reaction with monoclonal anti-D. We analyzed the blood sample of the child with different methods to conclude that it is a case of DAR 1.2 (weak D 4.2.2) and that it must be transfused with D negative erithrocytes.

Day -1 CD34+ Cells and Platelet Count Predict the Number of Apheresis in Poor-Mobilizer Patients Rescued by Plerixafor.

Plerixafor is widely used as up-front treatment with G-CSF to enhance peripheral blood hematopoietic stem cell output in patients failing previous mobilizations. Less frequently, plerixafor is used to rescue an unsatisfactory mobilization following chemotherapy (CT) and G-CSF. This study investigates if pre-collection factors affect the CD34+ cell harvest in chemotherapy and G-CSF mobilizations rescued by plerixafor. Clinical and hematological data relative to patients, mobilization, and apheresis products were retrospectively examined. The outcome was completing a target cell dose ≥ 2 × 106 CD34+ cells/kg at first apheresis. The effect exerted on the outcome by patient- and disease-related factors was investigated by univariate and multivariate logistic regression analysis. The analysis included data from 42 patients affected by hematological (39 patients) and non-hematological malignancies (three patients). Twenty-nine patients (69%) attained the target cell dose at first apheresis. Twelve out of the remaining 13 patients received an additional plerixafor administration, and all accomplished the transplant dose at a second apheresis procedure. Day -1 CD34+ PB count (OR1.46, 95% CI 1.1-1.9, p = 0.008) and platelet count (OR1.0, 95% CI 1.0-1.0, p = 0.033) predicted the achievement of the target dose at first apheresis, independently of pre-mobilization CT, radiation therapy, and disease status at mobilization. At ROC curve analysis, the best cut-off value predicting the successful collection at first apheresis was 7.5/µL for Day -1 CD34+ cell count (AUC 0.830, 0.69 sensitivity, and 0.92 specificity) and 75 × 109/L for Day -1 platelet count (AUC = 0.736, 0.65 sensitivity and 0.85 specificity). In conclusion, on-demand plerixafor rescue allows a successful stem cell collection, irrespectively of disease type and status, prior CT lines, and radiation exposure. Pre-apheresis CD34+ cells and platelet count predict the need for one or two aphereses.

Peripheral Blood Allogeneic Stem Cell Mobilization: Can We Predict a Suboptimal Mobilization?

Allogeneic peripheral blood stem cells mobilization is now the basis of most stem cell transplants. In a very limited number of cases, mobilization is suboptimal leading to further collection procedures, to suboptimal cell doses infusion with delayed engraftment time, increased risks of transplant procedure and of related costs. To date we have no recognized and shared criteria for early estimating the probability of poor mobilization in healthy donors. We then analyzed allogeneic peripheral blood stem cell donations performed at the Fondazione Policlinico Universitario A.Gemelli IRCCS Hospital from January 2013 to December 2021 in order to identify premobilization factors associated with successful mobilization. The following data were collected: age, gender, weight, complete blood cell count at baseline, G-CSF dose, number of collection procedures, CD34+ cell count in peripheral blood on the first day of collection, CD34+ cell dose per kg body weight of recipient. Mobilization efficacy was defined according to the number of CD34+ cells in peripheral blood on day +5 of G-CSF administration. We classified donors as sub-optimal mobilizers or good mobilizers according to the achievement of the 50 CD34+ cell/µL threshold. We observed 30 suboptimal mobilizations in 158 allogeneic peripheral blood stem cell donations. Age and baseline white blood cell count were factors significantly associated with negative or positive impact on mobilization, respectively. We did not find significant differences in mobilization based on gender or G-CSF dose. Using cut-off values of 43 years and 5.5×109/L WBC count, we built a suboptimal mobilization score: donors who reach 2, 1 or 0 points have a 46%, 16% or 4% probability of suboptimal mobilization, respectively. Our model explains 26% of the variability of mobilization confirming that most of the mobilization magnitude depends on genetically determined factors; however, suboptimal mobilization score is a simple tool providing an early assessment of mobilization efficacy before G-CSF administration begins in order to support allogeneic stem cells selection, mobilization and collection. Through a systematic review, we looked for confirmation of our findings. According to the published articles, all the variables we included in our model are confirmed to be strongly related to the success of mobilization. We believe that score system approach could be applied in clinical practice to assess the risk of mobilization failure at baseline allowing for a priori intervention.

Phosphorylated STAT5 represents a new possible prognostic marker in Hodgkin lymphoma.

An important pathogenetic mechanism in Hodgkin lymphoma (HL) is the interaction between the neoplastic and reactive cells mediated by a complex network of cytokines with activation of cytokine signal transduction (STAT) pathways. We studied the prognostic impact of the phosphorylation status of STAT5 in HL. By using immunohistochemical analysis, we found phosphorylated STAT5 (pSTAT5) in 35 (38%) of 93 lymph node biopsy specimens of patients with HL. The detection of pSTAT5 in Hodgkin and Reed-Sternberg (HRS) cells in classical HL (cHL) was not associated with any clinical and biologic features evaluated, including Epstein-Barr virus status. The primary end point for analysis of clinical outcome was freedom from treatment failure (FFTF). At a median follow-up of 5 years, pSTAT5+ patients with cHL had a better FFTF than pSTAT5-patients (77% vs 56%; P = .03), which translated into a reduced risk for failure for pSTAT5+ patients with a hazard ratio of 0.2 (95% confidence interval, 0.06-0.73; P = .015). Our data suggest that the phosphorylation status of STAT5 of HRS cells in cHL could be a prognostic marker in HL.

Glutathione-S-transferase genotypes influence prognosis in follicular non-Hodgkin's Lymphoma.

Polymorphisms in detoxification enzymes of the glutathione S-transferase (GST) family have been associated with risk and prognosis of several cancer types. We studied deletions of GSTM1 and GSTT1, and the GSTP1 Ile(105)Val polymorphism in 89 patients with follicular lymphoma (FL). Patients with a GSTM1 or GSTT1 deletion had a significantly worse event-free survival, when compared with patients with undeleted genotype (p = 0.03 and p = 0.03, respectively). Outcome was even worse in patients with a double negative genotype, in comparison with patients with only one GST deletion or normal genotype (p = 0.01). In the multivariate analysis, the GSTM1/GSTT1 genotype tended to have a prognostic significance independent from the Follicular Lymphoma International Prognostic Index (FLIPI) score. In particular, GSTM1/T1 deletions identified patients with negative prognosis in the low (<3) FLIPI score group (p = 0.01). Larger prospective studies including homogeneously treated patients will be needed to confirm these results.

DAP-kinase hypermethylation in the bone marrow of patients with follicular lymphoma.

We studied whether DAP-kinase hypermethylation plays a role as a prognostic marker in patients with follicular lymphoma (FL). We found that DAP-kinase was frequently hypermethylated in bone marrow (BM) samples of 52 FL patients at diagnosis (71%) and identified patients with worse progression-free survival (p=0.06). In particular, patients with histologically proven BM infiltration and DAP-kinase hypermethylation had a poorer outcome (p=0.037). In a total of 170 BM samples obtained at diagnosis or during follow-up, DAP-kinase hypermethylation and the bcl2/IgH rearrangement gave concordant results in 67% of samples (48% both positive, 19% both negative). Both mrakers were independent predictors of the disease status (p<0.001).

Glutathione S-transferase P1 genotype and prognosis in Hodgkin's lymphoma.

PURPOSE: Glutathione S-transferase P1 (GSTP1) is a member of the GST enzyme superfamily that is important for the detoxification of several cytotoxic drugs and their by-products. A single nucleotide polymorphism results in the substitution of isoleucine (Ile) to valine (Val) at codon 105, causing a metabolically less active variant of the enzyme. We assessed the impact of the GSTP1 codon 105 genotype on treatment outcome in patients with Hodgkin's lymphoma. EXPERIMENTAL DESIGN: The Ile(105)Val polymorphism in the GSTP1 gene was analyzed using a PCR-RFLP technique. Ninety-seven patients with Hodgkin's lymphoma were included and associations with patient characteristics and treatment outcome were analyzed. RESULTS: The GSTP1 Ile(105)Val polymorphism was associated in a dose-dependent fashion with an improved failure-free survival in patients with Hodgkin's lymphoma (P = 0.02). The probability of 5-year survival for patients homozygous for the (105)Val/(105)Val GSTP1 genotype was 100%, for heterozygous patients 74% (95% confidence interval, 56-85), and for patients homozygous for the (105)Ile/(105)Ile genotype 43% (95% confidence interval, 23-61). The Cox multivariate analysis showed that GSTP1 codon 105 genotype was an independent prognostic factor. CONCLUSIONS: The GSTP1 genotype predicts clinical outcome in patients with Hodgkin's lymphoma.

Association between glutathione S-transferase genotypes and Hodgkin's lymphoma risk and prognosis.

PURPOSE: The interplay between genetic susceptibility and exposure to carcinogens has been shown to be involved in the etiology of many solid tumors. We studied the frequency and clinical correlates of polymorphisms resulting in deletions of two genes involved in the detoxification of potentially carcinogenic agents, glutathione S-transferase (GST)-M1 and GSTT1 in patients with Hodgkin's lymphoma (HL). EXPERIMENTAL DESIGN: The prevalence of gene deletions in 90 patients with HL was compared with a case-matched cohort of 176 normal blood donors. GST gene polymorphisms were studied using a multiplex PCR method, including the BCL2 gene as an internal control. RESULTS: Deletions of the GSTT1 gene were more frequent in cases compared with controls (28.9 versus 17.6%, P = 0.04), resulting in an increased risk for HL in individuals with the GSTT1-null genotype (odds risk, 1.9; 95% confidence interval 1.04-3.46). The GSTT1-null genotype particularly increased the HL risk in females aged <45 years (odds risk 6.1, 95% confidence interval 1.6-23, P = 0.008). Correlating patient characteristics to genotype, we found an association between the GSTT1-null genotype and a limited stage of disease (I/IIA versus IIB-IV, 40.6 versus 19.6%, P = 0.047) and an erythrocyte sedimentation rate of <50 mm/h (P = 0.02). Patients with at least one GST deletion (GSTM1- or GSTT1-) had a significant better disease-free survival when compared with those with undeleted GST genes (GSTM1+/GSTT1+; P = 0.012). CONCLUSIONS: The GSTT1-null genotype may increase the risk for HL and is associated with favorable prognostic factors, and the presence of at least one GST deletion indicates an improved disease-free survival.

Polymorphisms of CYP1A1 and glutathione S-transferase and susceptibility to adult acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: The origin of acute myeloid leukemia (AML) may be explained by a combination of genetic susceptibility factors and environmental exposure. We studied the polymorphisms of cytochrome P450 CYP1A1 and glutathione S-transferase (GST), enzymes involved in the metabolism of carcinogens and anti-cancer drugs, as risk factors for adult AML. DESIGN AND METHODS: The prevalence of CYP1A1*2A, *2B and *4 alleles and of GSTM1 and GSTT1 homozygous deletions was examined in 193 patients with AML and 273 normal individuals using polymerase chain reaction (PCR)-based methods. RESULTS: A higher prevalence of the CYP1A1*4 allele was found in AML patients than in controls (19.1% vs 9.9%, OR =2.2, 95% C.I. 1.3-3.7, p=0.006). GSTT1 homozygous deletions were also more frequent in AML patients (29% vs 19%, OR = 1.7, 95% CI 1.1-2.7, p=0.02). The combination of GSTT1 null genotype and CYP1A1 *2B and *4 alleles further increased the risk of AML (OR =10.2, 95% CI 1.2-83.9, p=0.01, and OR =7.0, 95% CI 2.0-24.8, p=0.001, respectively). INTERPRETATION AND CONCLUSIONS: Polymorphic variants in xenobiotic-metabolism genes, including CYP1A1 and GSTT1, may increase the risk of adult AML, particularly when present together.