Memory B cell dysregulation in HIV-1-infected individuals.

OBJECTIVE: To characterize the effect of the HIV-1 infection and antiretroviral treatment (ART) in the human memory B (MEB)-cell compartment. DESIGN: A cross-sectional study was designed to analyze MEB cells of HIV-1 ART treated and ART-naive study participants, and uninfected individuals. METHODS: Frequency and absolute counts of MEB cell subsets in blood were determined by multicolor flow cytometry. Spontaneous cell death and B-cell proliferative capacity was evaluated in vitro by cell culture and flow cytometry. Splenic function was determined by pitted erythrocytes quantification in HIV-1 ART-treated study participants. RESULTS: HIV-1 ART-treated individuals did not show functional hyposplenism despite the lack of recovery IgMIgDCD27 marginal zone-like B cells. Moreover, two germinal center-dependent MEB cells subsets were also dysregulated in HIV-1 individuals: IgMIgDCD27 (IgM only) cells were increased, whereas the switched subset (IgMIgD) was reduced in viremic individuals. Althought ART restored the numbers of these populations; the switched MEB cells were enriched in CD27 cells, which showed the highest susceptibility to spontaneous cell death ex vivo. In addition, B cells from viremic individuals showed a poor response to B-cell receptor and toll-like receptor 9 stimulation that was circumvented when both stimuli were used simultaneously. CONCLUSION: B cells from HIV-1 study participants show a poor stimulation capacity, that may be bypassed by the proper combination of stimuli, and a dysregulated MEB cell pool that suggest an affectation of the germinal center reaction, only partially normalized by ART. Interestingly, hyposplenism does not explain the lack of recovery of the marginal zone-like B cells in ART-treated HIV-1 individuals.

Usefulness of bronchoalveolar lavage and flow cytometry in patients with hematological malignancies and respiratory failure. / Utilidad del lavado broncoalveolar y la citometría de flujo en pacientes con hemopatías malignas e insuficiencia respiratoria.

BACKGROUND AND OBJECTIVES: Strategies to improve the efficiency of bronchoalveolar lavage (BAL) are needed. We conducted a study to establish the diagnostic value of BAL in patients with hematological malignancies and pulmonary infiltrates. PATIENTS AND METHODS: The correlation of cytologic and flow cytometric study of BAL with the microbiological findings and the clinical evolution was determined. RESULTS: Seventy BAL were performed and flow cytometric study was analyzed in 23 of them. Fifty-three patients did not present any adverse event attributable to BAL. Anti-infectious therapy was modified in 64 (91%) patients. T lymphocyte count >0.3×109/l in peripheral blood was associated with longer OS at 3 years (53 vs. 22%, p=.009). Higher CD4 (>20/µL) and CD8 (>35/µL) lymphocyte counts in the BAL were associated with a longer OS at 3 years: 82 vs. 21% (p=.030) and 80 vs. 23% (p=.059). CONCLUSIONS: Our study confirms the clinical value of BAL for treatment decision making in patients with hematological malignancies and acute respiratory failure.

Relapse risk after autologous stem cell transplantation in patients with lymphoma based on CD34+ cell dose.

It is unclear whether higher CD34 + cell doses infused for ASCT have any influence on survival or relapse in patients with lymphoma. We analyzed the correlation of infused CD34 + cell dose with relapse, survival, and hematopoietic recovery in 146 consecutive patients undergoing ASCT for lymphoma. Higher doses (>5 × 106/kg) were significantly correlated with earlier hematopoietic recovery, fewer infectious episodes, lower transfusion needs. No differences were observed in lymphoma outcomes (4-year relapse incidence of 38% [95%CI: 29%-48%] in the lower dose group versus 51% [95%CI: 30%-69%] in the higher dose group, 10-year OS probabilities of 58% [95%CI: 48%-68%] versus 75% [95%CI: 59%-91%], 10-year DFS probabilities of 47% [95%CI: 37%-57%] versus 42% [95%CI: 23%-61%], p = NS for all outcomes). In this series, a higher infused CD34 + cell dose did not correlate with survival or relapse but correlated with earlier hematopoietic recovery and lower resource consumption.

CD34 expression and the outcome of nucleophosmin 1-mutated acute myeloid leukemia.

CD34 positivity has been considered as an adverse prognostic factor in acute myeloid leukemia (AML). Although nucleophosmin 1-mutated (NPM1m) AML is usually CD34 negative, this marker may be expressed at diagnosis or acquired at relapse in a variable number of cases. Our objective was to ascertain if CD34 expression has any influence on the general outcome of this form of acute leukemia. Analysis of clinical outcome (complete remissions, relapses, disease-free survival, and overall survival) was performed depending on the degree of expression of CD34 determined by flow cytometry, in 67 adult patients with NPM1m AML. CD34 expression did not have any influence on the variables analyzed whatever the percentage of blasts expressing this marker. In contrast to other forms of AML, CD34 expression is not an unfavorable prognostic factor in NPM1m AML, neither at diagnosis nor at relapse.

Targeted deep sequencing improves outcome stratification in chronic myelomonocytic leukemia with low risk cytogenetic features.

Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.

Prognostic significance of copy number alterations in adolescent and adult patients with precursor B acute lymphoblastic leukemia enrolled in PETHEMA protocols.

BACKGROUND: Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL). METHODS: This study analyzed via multiplex ligation-dependent probe amplification the frequency and prognostic impact of CNAs of 12 genetic regions in 142 adolescents and adults with de novo precursor B-cell ALL. RESULTS: The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion (59 of 142 or 42%) was the most frequent CNA, and it was followed by Ikaros family zinc finger 1 (IKZF1) losses (49 of 142 or 35%). IKZF1 deletions were more prevalent in Philadelphia chromosome (Ph)-positive ALL and were associated with advanced age and high white blood cell (WBC) counts. The multivariate analysis showed that advanced age and early B-cell factor 1 (EBF1) deletions were associated with chemotherapy resistance in both the whole series (hazard ratios, 0.949 and 0.135, respectively) and the Ph-negative subgroup (hazard ratios, 0.946 and 0.118, respectively). High WBC counts and focal IKZF1 deletions correlated with disease recurrence (hazard ratios, 1.005 and 1.869, respectively), whereas advanced age and CDKN2A/B losses influenced overall survival in both the whole series (hazard ratios, 1.038 and 2.545, respectively) and the Ph-negative subgroup (hazard ratios, 1.044 and 2.105, respectively). CONCLUSIONS: Deletions of EBF1, IKZF1, and CDKN2A/B have an independent adverse prognosis for adolescents and adults with B-precursor ALL, and this suggests that these CNAs should be included in the initial risk assessment of ALL.

A thirty-five nucleotides BCR-ABL1 insertion mutation of controversial significance confers resistance to imatinib in a patient with chronic myeloid leukemia (CML).

Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients does not achieve the optimal response or are resistant to TKI. ABL1 kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Although deletion or insertion of nucleotides in BCR-ABL1 has rarely been described, we identified a CML patient with an already described 35 nucleotides insertion (BCR-ABL1(35INS)) of controversial significance, that confers resistance to imatinib but sensitivity to dasatinib.

Acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): Clinical and biological features and comparison with other acute myeloid leukemias with cytogenetic aberrations involving long arm of chromosome 3.

OBJECTIVES: To compare, from a biological and clinical perspective, a significant group of patients with AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) with another group of AML carrying different abnormalities of 3q at q21 or q26, the latter named as the AML abn(3q) group. METHODS: We developed a national survey with the participation of 13 Spanish hospitals, and retrospectively reviewed (from 1990 to 2010) these subtypes of AML. Fifty-five patients were collected: 35 with AML inv(3)/t(3;3) and 20 with AML abn(3q). A data collecting page that included main features at diagnosis, therapeutic approach and response, and survival variables, was distributed and completed. RESULTS: We did not find significant differences in sex, age, history of myelodysplastic syndrome or chemo-/radiotherapy, clinical presentation, WBC and platelet counts, hemoglobin level, blasts immunophenotype, serum lactatedehydrogenase, peripheral blood and bone marrow cellular dysplasia, and bone marrow biopsy findings. Although the association with monosomy 7 was significantly more frequent in AML inv(3)/t(3;3), this did not seem to influence outcome. The lack of response to the different modalities of treatment and the aggressive course of the disease were the standard in both cohorts of patients. DISCUSSION: Although not yet recognized by the World Health Organization classification, our results are in agreement with the findings of other authors, who include both subsets of AML together in the same group of adverse prognosis. CONCLUSION: In an attempt to simplify and bound entities with similar genetic background and clinical behavior, it would be desirable to bring together both subgroups of AML in a single section.

A case of CD138-/CD19+/CD4+ IgD plasma cell leukemia.

BACKGROUND: Plasma cell leukemia (PCL) is an uncommon and aggressive disease caused by the clonal proliferation of atypical plasma cells with phenotypical abnormalities similar to those seen in multiple myeloma (MM), although at different rates. Here, we report a case of IgD PCL with a very unusual CD138-/CD19+/CD4+ phenotype. METHODS: Peripheral blood and bone marrow samples from a 37-year-old patient afflicted by an aggressive plasma cell dyscrasia were examined and analyzed by conventional morphology, flow cytometry, and immunohistochemistry. RESULTS: Analysis of peripheral blood fulfilled criteria for PCL (more than 20% and more than 2 × 10e9 cells/L). However, flow cytometry and immunohistochemistry phenotyping revealed that the cells were CD138-/CD38+/CD19+/CD4+/CD56-/CD117-. CONCLUSIONS: PCL is diagnosed on peripheral blood smear. Immunophenotyping is a tool that can be helpful in diagnosing difficult cases but its atypical findings should not prevent the appropriate PCL diagnosis in clinically and morphologically unquestionable cases. © 2014 International Clinical Cytometry Society.

Multilineage dysplasia is associated with a poorer prognosis in patients with de novo acute myeloid leukemia with intermediate-risk cytogenetics and wild-type NPM1.

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1. Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n = 16, 9 %), absent MLD (n = 80, 45 %), or non-evaluable MLD (n = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p = 0.03; HR, 95 % CI = 2.3, 1.08-4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact (p = 0.087, HR, 95 % CI = 1.426, 0.95-2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026-2.492; p = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1.

Correlation of CD11b and CD56 expression in adult acute myeloid leukemia with cytogenetic risk groups and prognosis.

Among other phenotypic markers, CD11b expression has been considered as an unfavorable prognostic factor, both in terms of overall survival (OS), disease-free survival (DFS), and attainment and duration of complete remissions (CRs) in adult patients with acute myeloid leukemia (AML). Recently, some groups have restricted its prognostic impact to poor prognostic karyotypic risk groups. The aim of this study was to retrospectively analyze the prevalence of CD11b and of CD56 expression in blast cells of 158 AML patients [excluding those with t(15;17)] stratified according to their cytogenetic risk and to correlate these phenotypic characteristics with OS, DFS, and CR. CD11b was more frequently expressed in intermediate and unfavorable cytogenetic prognostic groups (38.9 and 35.5 %, respectively) than in the favorable group (9.5 %). No differences were observed in CD56 expression according to the cytogenetic risk groups. When OS, DFS, and CR were analyzed according to these two markers, no statistical differences were recorded in any cytogenetic risk group. In conclusion, although CD11b was more frequently expressed in blast cells of patients with intermediate and unfavorable cytogenetic risk groups, this feature did not translate into different clinical outcome. Similarly, CD56 positivity did not have any influence on the prognosis of these patients.

A case of CD138-/CD19+/CD4+ IgD plasma cell leukemia.

Background: Plasma cell leukemia (PCL) is an uncommon and aggressive disease caused by the clonal proliferation of atypical plasma cells with phenotypical abnormalities similar to those seen in multiple myeloma (MM), although at different rates. Here we report a case of IgD PCL with a very unusual CD138-/CD19+/CD4+ phenotype Methods: Peripheral blood and bone marrow samples from a 37 year old patient afflicted by an aggressive plasma cell dyscrasia were examined and analyzed by conventional morphology, flow cytometry and immunohistochemistry. Results: Analysis of peripheral blood fulfilled criteria for PCL (more than 20% and more than 2x10e9 cells/L). However, flow cytometry and immunohistochemistry phenotyping revealed that the cells were CD138-/CD38+/CD19+/CD4+/CD56-/CD117- Conclusion: PCL is diagnosed on peripheral blood smear. Immunophenotyping is a tool that can be helpful in diagnosing difficult cases but its atypical findings should not prevent the appropriate PCL diagnosis in clinically and morphologically unquestionable cases. © 2014 Clinical Cytometry Society.

Adult T-cell leukemia/lymphoma with an unusual CD1a positive phenotype.

BACKGROUND: Acute T-cell leukemia lymphoma (ATLL) tumor cells generally express CD2/CD3/CD5, but lack CD7. These T cells are usually CD4+CD8- and strongly express CD25, although some variability in this basic pattern may be found. Here we report a case with a very unusual CD1a positive phenotype. METHODS: Samples from peripheral blood, bone marrow aspirate, lymph node, and cerebrospinal fluid obtained from a 45-year-old male patient with a T-cell lymphoproliferative disorder were immunophenotyped by multiparametric flow cytometry. Analysis of HTLV-I genome integration in tumoral cells was performed by PCR. RESULTS: Neoplastic T cells were cCD3, CD2/CD5/CD30/CD25, and CD1a positive, but CD3/CD7/CD4/CD8/CD34/CD10/TdT negative. Serology and integration of HTLV-I were positive. CONCLUSION: To the best of our knowledge, CD1a expression has not been previously described in this entity. Its detection raised the differential diagnosis with acute T lymphoblastic leukemia. The rest of the phenotypic markers, the morphology of the neoplastic cells, and the demonstration of HTLV-I genome integration provided the final diagnosis.

Mobilization and engraftment of peripheral blood stem cells in healthy related donors >55 years old.

BACKGROUND AIMS: The increasing scarcity of young related donors has led to the use of older donors for related allogeneic hematopoietic stem cell transplantation (HSCT). This study analyzed the influence of age on the results of mobilization of peripheral blood stem cells (PBSCs) in healthy donors as well as on the engraftment and outcome of HSCT. METHODS: A retrospective analysis from a single center was performed comparing the results of PBSC mobilization from related healthy donors according to their age. RESULTS: The study included 133 consecutive related donors. The median age was 50 years (range, 4-77 years); 70 (53%) donors were males, and 44 (33%) were >55 years old. All donors were mobilized with granulocyte colony-stimulating factor for 5 days. The peak CD34(+) cell count in peripheral blood was higher in younger than in older donors (median, 90.5 CD34(+) cells/µL [range, 18-240 CD34(+) cells/µL] versus 72 CD34(+) cells/µL [range, 20-172.5 CD34(+) cells/µL], P = 0.008). The volume processed was lower in younger than in older donors (16,131 mL [range, 4424-36,906 mL] versus 18,653 mL [range, 10,003-26,261 mL], P = 0.002) with similar CD34(+) cells collected (579.3 × 10(6) cells [range, 135.14 × 10(6)-1557.24 × 10(6) cells] versus 513.69 × 10(6) cells [range, 149.81 × 10(6)-1290 × 10(6) cells], P = 0.844). There were no differences in time to recovery of neutrophils and platelets or in the incidences of acute and chronic graft-versus-host disease, overall survival, non-relapse mortality and relapse incidence. CONCLUSIONS: Donors >55 years old mobilized fewer CD34(+) cells and required a greater volume to collect a similar number of CD34(+) cells. The outcome of HSCT was not influenced by donor age. Donor age should not be a limitation for related allogeneic HSCT.

Usefulness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

In up to 5-15% of studies of lymphoproliferative disorders (LPD), flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, two clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and nine TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (four IGH, 10 TCRγ), albeit nonconclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In eight IGH samples, the results of PCR techniques were noninformative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and nonconclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data.