Do NPM1 and FLT3-ITD mutations modify prognosis in patients treated with non-intensive regimens?

FLT3-ITD and NPM1 mutations are key to defining the genetic risk profile of acute myeloid leukemia (AML). We aimed to assess the prognostic features of the FLT3-ITD and NPM1 mutations in old and/or unfit individuals with AML treated with non-intensive therapies in the era before azacitidine-venetoclax approbation. The results of various non-intensive regimens were also compared. We conducted a retrospective analysis that included patients treated with different non-intensive regimens, between 2007 and 2020 from PETHEMA AML registry. We compiled 707 patients with a median age of 74 years and median follow-up time of 37.7 months. FLT3-ITD patients (N = 98) showed a non-significant difference in overall survival (OS) compared to FLT3-ITD negative-patients (N = 608) (P = 0.17, median OS was 5 vs 7.3 months respectively). NPM1-mutated patients (N = 144) also showed a non-significant difference with NPM1 wild type (N = 519) patients (P = 0.25, median OS 7.2 vs 6.8 respectively). In the Cox regression analysis neither NPM1 nor FLT3-ITD nor age were significant prognostic variables for OS prediction. Abnormal karyotype and a high leukocyte count showed a statistically significant deleterious effect. Azacitidine also showed better survival compared to FLUGA (low dose cytarabine plus fludarabine). NPM1 and FLT3-ITD seem to lack prognostic value in older/unfit AML patients treated with non-intensive regimens other than azacitidine-venetoclax combination.

An increased percentage of myeloid CD34+ bone marrow cells stratifies intermediate IPSS-R myelodysplastic syndrome patients into prognostically significant groups.

INTRODUCTION: The Revised International Prognostic Scoring System (IPSS-R) for myelodysplastic syndromes (MDS) has established an intermediate category where a disease-modifying intervention is a matter of debate. Flow cytometry allows us to determine a fraction of immature myeloid cells in a semiautomated procedure. The aim of this study, mirroring IPSS-R study inclusion criteria, was to test whether bone marrow (BM) CD34+My percentage has independent prognostic value in the MDS setting. METHODS: BM CD34+My cells were quantified, at diagnosis, selecting CD34+/CD45+/CD11b±/CD13+. Patients were excluded when receiving treatment for altering the natural course of the disease and when IPSS-R could not be calculated due to the lack of metaphases. Finally, Cox analyses were performed, on a series of 260 patients, for overall survival (OS) and time to acute myeloid leukemia (AML) transformation. RESULTS: By analyzing ROC curves, the most accurate prognostic variable, regarding blasts by cytology and CD34+ by cytometry, was the percentage of blasts by microscopy. The percentage of CD34+My in BM showed an AUC of 0.767 and 0.576 for time to AML transformation and OS, respectively. When performing a multivariate regression including the IPSS-R and the percentage of BM CD34+My cells >1%, both factors predicted for a shorter time to AML transformation. In addition, CD34+My percentage successfully stratified the intermediate IPSS-R category into two prognostic groups with a relative risk of 5.73 (95% CI [1.2-27.8]; P = .03). CONCLUSION: We found that BM CD34+My percentage has an independent value concerning the IPSS-R, especially relevant for the prediction of transformation to AML and within the intermediate group.

Effectiveness of azacitidine in unselected high-risk myelodysplastic syndromes: results from the Spanish registry.

The benefit of azacitidine treatment in survival of high-risk myelodysplastic syndromes (MDS) patients compared with conventional care treatment (CCT) has not been established outside clinical trials. To assess its effectiveness, we compared overall survival (OS) between azacitidine and conventional treatment (CCT) in high-risk MDS patients, excluding those undergoing stem cell transplantation, submitted to the Spanish MDS registry from 2000 to 2013. Several Cox regression and competing risk models, considering azacitidine as a time-dependent covariate, were used to assess survival and acute myeloblastic leukemia (AML) progression. Among 821 patients included, 251 received azacitidine. Median survival was 13.4 (11.8-16) months for azacitidine-treated patients and 12.2 (11-14.1) for patients under CCT (P=0.41). In a multivariate model, age, International prognostic scoring system and lactate dehydrogenase were predictors of OS whereas azacitidine was not (adjusted odds ratio 1.08, 95% confidence interval 0.86-1.35, P=0.49). However, in patients with chromosome 7 abnormalities, a trend toward a better survival was observed in azacitidine-treated patients (median survival 13.3 (11-18) months) compared with CCT (median survival 8.6 (5-10.4) months, P=0.08). In conclusion, our data show that, in spite of a widespread use of azacitidine, there is a lack of improvement in survival over the years. Identification of predicting factors of response and survival is mandatory.

Results of allogeneic stem cell transplantation in the Spanish MDS registry: prognostic factors for low risk patients.

Although new agents have been approved for the treatment of MDS, the only curative approach is allogeneic hematopoietic stem cell transplantation (HSCT) and thus, in particular circumstances this procedure has been proposed as a treatment option for low risk patients. We have retrospectively analyzed the results of HSCT in 291 patients from the Spanish MDS registry with special attention to low risk MDS (LR-MDS) in order to define the variables that could impact their clinical evolution after transplantation. At 2 years OS was 51% and EFS was 50% (95% CI 0.7-4.5 years for OS and 95% CI 0.1-3.9 years for EFS). Among 43 LR-MDS, transplant-related mortality was 28%. At 3 years, OS was 67% (95% CI 264.7-8927.2 days for OS) and EFS was 64% (95% CI 0-9697.2 days for EFS). In the multivariate analysis only cytogenetics retained statistical significant effect on both OS (p=.047) and EFS (p=.046). Conditioning regimen could improve outcome among this subset of patients (OS 86% and RFS 100% for patients receiving RIC regimen). The present study confirms that specific disease characteristic as well as transplant characteristics have a significant impact on transplant outcome. Regarding low risk patients a non-myeloablative conditioning would be preferable especially in cases without high-risk cytogenetics.

Factors that influence long-term hematopoietic function following autologous stem cell transplantation.

The aim of the present study was to assess which factors influence hematopoietic function long term after transplantation. For this purpose, we have analyzed a series of 79 patients who underwent autologous transplantation. None of them received any further chemotherapy or radiotherapy after transplant. All patients were disease-free 1 year after autologous transplantation. Late impairment of hematopoietic function was defined as the presence of non-transient peripheral blood cytopenias, detected 6 and 12 months after autografting. Before transplantation, 38.7% of patients showed peripheral blood cytopenias. Six and 12 months after transplantation, cytopenias presented in 44.2% and 42.4% of patients, respectively. Cases displaying cytopenias 6 months after transplantation had received a significantly lower dose of CFU-GM and CD34+ cells than patients without cytopenias (P = 0.012 and P = 0.04, respectively). The same correlation, with even higher statistical significance, was observed 12 months after transplant (P = 0.007 and P = 0.005). Alkylating agents and radiotherapy administered prior to transplantation and age did not seem to influence the presence of permanent cytopenias. The incidence did not vary significantly according to the stem cell source (bone marrow or peripheral blood). The number of CFU-GM and CD34+ cells infused was the most important factor for maintenance of adequate hematopoiesis.

Hematopoietic damage prior to PBSCT and its influence on hematopoietic recovery.

BACKGROUND AND OBJECTIVE: Patients with malignancies receive chemotherapy to induce tumor remission which could damage hematopoiesis and adversely influence hematopoietic reconstitution after transplantation. In the present study we used a long-term culture (LTBMC) system and clonogenic assays to evaluate the marrow damage in patients selected to receive peripheral blood stem cell transplantation (PBSCT). DESIGN AND METHODS: Thirty-five patients - 20 with breast cancer (BC), 9 with non-Hodgkin's lymphoma (NHL) and 6 with Hodgkin's disease (HD) - were included. Bone marrow aspiration was performed one day prior to the initiation of the conditioning therapy. CFU-GM were cultured in methylcellulose with PHA-LCM. Delta assays of plastic adherent progenitor cells (PD) were performed according to Gordon's method. LTBMC were established for 5 weeks. RESULTS: There were fewer CFU-GM from all patient groups than from normal BM (p<0.05). In contrast, the number of immature progenitor cells (PD) was not decreased. The total number of CFU-GM produced by LTBMC patients was significantly reduced (p<0.05). The adherent layer from patients was often qualitatively different. In order to know whether the hematopoietic damage could affect hematopoietic reconstitution, we correlated culture data with time taken to reach peripheral cell counts. A negative correlation (r= - 0.71) was found between percentage of stromal layer and time taken to reach 20x10(9) platelets/L (tplat= 20x3-0.08% stromal layer). INTERPRETATION AND CONCLUSIONS: We can conclude that prior to PBSCT, hematopoietic function is impaired at both the level of committed progenitor cells and that of BM stroma. This damage could influence platelet recovery.

Diagnosis of secondary myelodysplastic syndromes (MDS) following autologous transplantation should not be based only on morphological criteria used for diagnosis of de novo MDS.

Secondary myelodysplastic syndromes (MDS) are increasingly being reported after autologous transplantation. Transient dysplastic changes have also been observed after this type of treatment. However, to the best of our knowledge no systematic morphological analysis has been performed to determine the influence of stem cell transplantation on bone marrow morphology. In 53 patients undergoing autologous transplantation, we evaluated the bone marrow, before and 6 and 12 months after the transplant, in order to analyze the appearance of dyshemopoietic changes, assessed according to a pre-established score. We also studied 25 bone marrow samples obtained at the time of diagnosis, prior to treatment, but we did not find morphological atypia. Six months after transplant, cellularity and thrombopoiesis had decreased in 38% and 49% of patients respectively, although 1 year after the process they were normal in most cases. Myelodysplasia was already present in bone marrow before transplantation and continued to be in evidence for a long time afterwards. This suggests that chemotherapy and radiotherapy used prior to transplantation are responsible for dysplastic changes. The myeloid line was the most affected with 100% of patients showing dysgranulopoiesis 1 year after autografting. Cytopenias were observed in 51% and 44% of patients 6 and 12 months after transplantation. Moreover, concomitant presence of cytopenia and myelodysplasia was observed in 37.7% of patients at 6 months after transplantation and 25% at 12 months, and therefore they could be diagnosed with MDS. These data contrast with the incidence of secondary MDS reported in earlier publications. According to these findings, the value of the French-American-British Co-operative Group criteria for the diagnosis of MDS following autologous transplantation is questionable. Moreover, since dyshemopoietic features are almost always present after autologous transplant, morphological criteria are not useful for early recognition of patients with secondary MDS after transplantation.

Minimal number of circulating CD34+ cells to ensure successful leukapheresis and engraftment in autologous peripheral blood progenitor cell transplantation.

BACKGROUND: The number of peripheral blood (PB) CD34+ cells has been widely used to monitor the timing of leukapheresis for autologous transplantation. However, no cutoff value for CD34+ cells in PB has been defined as a guideline for the identification of patients in whom the harvest would be effective and those in whom there was a high probability of failure. STUDY DESIGN AND METHODS: The present study investigated the best threshold of CD34+ cells in PB for successful harvesting and engraftment, using 263 PB samples with their corresponding leukapheresis components. In addition, that measure has been compared to other commonly used criteria such as the white cell count, the number of mononuclear cells, and the number of colony-forming units-granulocyte macrophage in PB. RESULTS: Time to engraftment of both granulocytes and platelets was significantly influenced by the number of CD34+ cells transfused, but all patients receiving > or = 0.75 x 10(6) CD34+ cells per kg achieved engraftment within a reasonable number of days (> 0.5 x 10(9)/L granulocytes by Day 11 and > 20 x 10(9)/L platelets by Day 13). A clear correlation between the number of CD34+ cells per microL in PB and of CD34+ cells per kg collected was found at each apheresis (r = 0.9, p < 0.0001). Moreover, the number of CD34+ cells per microL measured in PB the day the first leukapheresis was initiated displayed an excellent correlation with the total amount of CD34+ cells per kg finally collected (r = 0.81, p < 0.0001). On the basis of the regression curve obtained and the clinical engraftment results, it was found that the presence of > 5 CD34+ cells per microL in PB ensured a good yield from the harvest in 95 percent of patients and would avoid an unsuccessful harvest in 81 percent of cases. CONCLUSION: A dose of only 0.75 x 10(6) CD34+ cells per kg guarantees hematopoietic recovery within a reasonable number of days. To initiate a leukapheresis from which enough progenitor cells may confidently be obtained, a minimum of 5 CD34+ cells per microL in PB is required.

Alternating mini-BEAM/ESHAP as salvage therapy for refractory non-Hodgkin's lymphomas.

Mini-BEAM and ESHAP are two non-cross-resistant salvage regimens that have been used separately in patients with lymphoma. The aim of the present study was to investigate the efficacy of the combination of these two regimens, administered in alternating cycles, as salvage therapy for refractory non-Hodgkin's lymphoma (NHL) patients. A total of 28 patients were included in the study: 14 patients were primary refractory, seven were partial responders, and seven were in relapse. The alternating cycles of mini-BEAM and ESHAP were given until there was maximum response or progression. The overall response rate to mini-BEAM/ESHAP was 39%; 25% of patients achieved a complete response and 14% a partial response. Nevertheless, it should be noted that none of the primary refractory patients responded to this protocol. Nine of the 11 patients who responded to mini-BEAM/ESHAP were consolidated with autologous transplantation using BEAM as a conditioning regimen. The survival at 3 years in this group of 11 patients who responded to the salvage regimen is 64%, with a disease-free survival of 67% at 2 years. No major toxic effects were observed with mini-BEAM/ESHAP. Myelosuppression was the most frequent complication, especially with the mini-BEAM cycles. Other toxicities were infrequent and no treatment-related deaths were observed. These results suggest that alternating mini-BEAM/ESHAP chemotherapy is a safe regimen that is effective in partial responders or relapsing patients with NHL who have sensitive disease, but not in primary refractory patients. Moreover, although this therapy has a potential advantage, combining as it does two non-cross-resistant regimens, it does not seem superior to ESHAP alone.

Immune thrombocytopenic purpura secondary to endometriosis.

In this paper, we describe a patient with immune thrombocytopenic purpura (ITP) secondary to endometriosis. To our knowledge, this is the first reported case presenting such an association. Surgical eradication of the endometriosis was the only effective treatment for the thrombocytopenia. The pathogenic connection between both disorders seems to be an altered immune function.

Multiparametric cell-cycle analysis of peripheral blood-activated lymphocyte subsets using staining based on the TdT method for incorporated BrdUrd.

This study describes a new method for the simultaneous assessment of the distribution of a cell population in the G0/G1, S, and G2/M cell-cycle phases by using multiparameter flow cytometry and single staining based on BrdUrd incorporation. Both the K562 cell line and PHA-stimulated peripheral blood lymphocytes (PBL) were analyzed. Cells were cultured in the presence of BrdUrd for 30 min prior to cell harvesting. Once collected, cells were exposed to ultraviolet light for 5 min and then fixed immediately in 70% ethanol (-20 degrees C) for at least 30 min. Once fixed, the cells were placed for 30 min at 37 degrees C in the presence of terminal deoxynucleotidyl transferase (TdT) and dUTP labeled with digoxigenin; they were then stained with FITC-labeled anti-digoxigenin. Our results show that G0/G1, s, and G2/M cell populations can be clearly discriminated according to FITC fluorescence and light-scatter parameters. In this way, S-phase cells can be identified by their FITC staining. From the cells which were negative for anti-digoxigenin-FITC antibody, two clear populations could be resolved in a forward scatter, side scatter, and fluorescence pulse-width three-dimensional plot; the values obtained for G0/G1 cells were lower than those obtained for G2/M cells in all three parameters. Multiparameter analysis of PBL stained for two surface antigens (CD3 and CD8) and for BrdUrd by direct or indirect TdT method permitted cell-cycle analysis of different subpopulations, including CD3+/CD8+, CD3+/CD8-, CD3-/CD8+, and CD3-/CD8-.

[Spinal cord compression as the presenting form of non-Hodgkin's lymphoma]. / Compresión medular como forma de presentación de linfoma no hodgkiniano.

Spinal cord compression as first presentation of non-Hodgkin's lymphoma (NHL) is an uncommon event. Diagnosis of NHL usually is performed on a laminectomy specimen. Spinal cord compression has been reported in 5% of patients with solid tumors. Although this clinical picture has been considered as very unusual among NHL patients, some series regarding descompresive laminectomy indicate that NHL was the underlying cause in 15% of these cases. We report two cases of patients with NHL who presented paraparesia secondary to spinal cord compression due to lymphomatous mass in the epidural region which was showed by magnetic resonance imaging. The emergency laminectomy and treatment with chemotherapy allowed the clinical recuperation of both patients.