Blastic Plasmacytoid Dendritic Cell Neoplasm: A Single-Center Experience. Clinical Characterization, Mutational Landscape, and Clinical Outcome of Patients Undergoing Hematopoietic Stem Cell Transplantation Intensive Therapy.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematodermic neoplasm usually involving the skin. In this retrospective case series, 10 cases of BPDCN were identified, 90% of which had skin involvement and exhibited predominantly violaceous nodules and/or bruise-like plaques. Skin lesions showed diffuse or nodular dermal-based infiltrates of intermediate sized blasts with a grenz zone. Tumor immunophenotyping was CD4(+), CD56(+), CD123(+) and CD303(+). The most frequently mutated genes according to targeted next-generation sequencing were TET2 (3/7) and NRAS (2/7). Multiagent chemotherapy (CT) was administered as first-line therapy, and a total of 5 patients underwent allogenic hematopoietic stem cell transplantation (allo-HSCT). Better outcomes were observed in younger patients and those treated with acute lymphoblastic leukemia (ALL)-like CT followed by allo-HSCT. This study shows the clinical range of cutaneous lesions of BPDCN. Despite the absence of a gold standard therapy, patients treated with myeloablative intensive regimens and allo-HSCT seems to have a more favorable prognosis.

Model of charge transfer collisions between C60 and slow ions.

A semiclassical model describing the charge transfer collisions of C60 fullerene with different slow ions has been developed to analyze available observations. These data reveal multiple Breit-Wigner-like peaks in the cross sections, with subsequent peaks of reactive cross sections decreasing in magnitude. Calculations of charge transfer probabilities, quasi-resonant cross sections, and cross sections for reactive collisions have been performed using semiempirical interaction potentials between fullerenes and ion projectiles. All computations have been carried out with realistic wave functions for C60's valence electrons derived from the simplified jellium model. The quality of these electron wave functions has been successfully verified by comparing theoretical calculations and experimental data on the small angle cross sections of resonant C60+C60 + collisions. Using the semiempirical potentials to describe resonant scattering phenomena in C60 collisions with ions and Landau-Zener charge transfer theory, we calculated theoretical cross sections for various C60 charge transfer and fragmentation reactions which agree with experiments.

Acquired erythropoietic uroporphyria secondary to myelodysplastic syndrome with chromosome 3 alterations: a case report.

Congenital erythropoietic porphyria is a rare autosomal recessive disease caused by a deficiency of uroporphyrinogen III synthase, owing to mutations in UROS in chromosome 10. Occasionally, patients show a mild, late-onset disease, without germline UROS mutations, associated with haematological malignancies. We report a 65-year-old patient with photosensitivity, overexcretion of porphyrins and thrombocytopenia. Bone marrow analysis gave a diagnosis of myelodysplastic syndrome (MDS) with the presence of a derivative chromosome 3, possibly due to an inversion including 3q21 and 3q26 break points. After allogeneic stem-cell transplantation, complete remission of MDS and uroporphyria was achieved. To our knowledge, this is the first reported case of acquired erythropoietic uroporphyria associated with MDS, with chromosome 3 alterations.

Central nervous system involvement in mantle cell lymphoma.

BACKGROUND: Extranodal involvement, including central nervous system (CNS), is a frequent event in patients with mantle cell lymphoma (MCL). However, the incidence, risk factors, and impact on outcome remain controversial. PATIENTS AND METHODS: Main clinical, biological, and evolutive features of 82 patients (60 males/22 females; median age: 61 years) diagnosed with MCL (blastoid, 26%) in a single institution were analyzed for risk of CNS involvement and prognosis. RESULTS: Most patients had advanced stage and intermediate or high-risk International Prognostic Index (IPI). Eleven patients eventually developed CNS involvement with an actuarial 5-year risk of 26% (95% confidence interval 10% to 42%). In one asymptomatic patient, cerebrospinal fluid infiltration was detected at staging maneuvers (1/62; 1.6%). The remaining 10 patients developed neurological symptoms during the course of the disease (median time from diagnosis, 25 months). Initial variables predicting CNS involvement were blastoid histology, high proliferative index measured by Ki-67 staining, high lactate dehydrogenase (LDH) and intermediate- or high-risk IPI. Histological subtype and serum LDH maintained significance in multivariate analysis. Treatment of CNS infiltration consisted of intrathecal chemotherapy (two cases), and intrathecal chemotherapy plus systemic treatment (seven cases). Median survival after CNS involvement was 4.8 months, patients with this complication having shorter survival than those with no CNS disease. CONCLUSION: This study confirms the high incidence of CNS involvement in MCL patients. Treatments aimed at preventing this complication are warranted.

Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Sequential study of myeloid differentiation antigens of neutrophil granulocytes in different phases of chronic myeloid leukaemia: natural history and prognostic significance.

In an attempt to contribute to the knowledge of the natural history of Philadelphia-chromosome-positive chronic myeloid leukaemia (CML) and its prognosis, we analyzed sequentially the myeloid differentiation antigens of peripheral blood neutrophil granulocytes (NG) in different evolutive stages of the disease. Four monoclonal antibodies (CD15, CD24, 31D8, and 13F6) were used, and a total number of 116 sequential studies were performed in 43 patients. At diagnosis, there is a significant decrease of NG expressing myeloid differentiation antigens, which recover to nearly normal levels after initial control of the disease. The onset reduction is probably due to the circulation of incompletely mature NG. In accelerated/blastic phase NG expressing myeloid differentiation antigens decrease again, probably due to a true antigen loss. This reduction could herald by a few months the development of accelerated/blastic phase. In such a case, its predictive strength is higher than that of the well recognized initial prognostic parameters in CML. These results indicate that the sequential study of NG myeloid differentiation antigens may contribute to both a better understanding of the natural history of CML and the evolutive prognosis of this disease.

Giant parallel tube arrays (PTAs), a new type of lymphocyte inclusion.

In a 56-year-old male patient, receiving chemotherapy after radical surgery for bladder carcinoma, an unusual type of cytoplasmic inclusion was discovered in about 30% of peripheral blood lymphocytes. This was a single, large (about 2 microns in diameter), round or ovoid body, darker than the nucleus and reddish-violet in May-Grünwald-Giemsa stain. The examination with transmission electron microscope demonstrated that such inclusions were made up of giant parallel tube arrays (PTAs). The absolute lymphocyte count was normal, but there was an expansion of CD3+, CD4-, CD8+, CD11b, TCR alpha beta lymphocytes. The lymphocytes bearing the inclusion were CD3+ and CD8+. DNA studies suggested an expansion of T-cell population with clonal rearrangement of TCR beta and TCR gamma. This case can be classified as an asymptomatic disorder of large granular lymphocytes, with unusual morphology. Giant PTAs should be taken into account in the differential diagnosis of lymphocyte cytoplasmic inclusions.

Leukemia after true histiocytic lymphoma: another type of acute monocytic leukemia with histiocytic differentiation (AML-M5c)?

The case of a 44-year-old man diagnosed of a true histiocytic lymphoma who, after autologous bone marrow transplantation, developed leukemia with histiocytic cells is reported. Morphologic, cytochemical, immunophenotypic and genotypic characteristics of malignant cells are described, and the literature about this and related entities is reviewed. In addition, comparison with a recent report of malignant histiocytosis with leukemic involvement is established and its inclusion in the recently proposed subtype of monocytic leukemia with histiocytic differentiation (M5c), suggested.

Acute myeloblastic leukemia with minimal myeloid differentiation: phenotypical and ultrastructural characteristics.

AML-M0 is an infrequent form of acute myeloblastic leukemia characterized by negative reaction with myeloperoxidase (MPO), Sudan Black and lymphoid antigens and positivity for CD13 or CD33. In the present study we describe the immunophenotypical and ultrastructural characteristics of a group of AML-M0 in adult patients. Nine out 218 AML leukemias (4.1%) fulfilled the AML-M0 criteria. CD13 or CD33 were positive in eight out nine cases, with two or more positive myeloid antigens being present in 82% of the cases. Immunological MPO was positive in 57% of the cases and CD68 in 33%. In no case megakaryocytic and erythroid markers present. Four cases (44%) expressed CD7 and TdT but only two coexpressed both antigens. In none of the cases was CD3 or CD22 cytoplasmic expression found. Ultrastructurally, a low number of granules was seen in all cases whereas ferritin particles or rhopheocytosis were not observed. Ultrastructural MPO was positive in one out of five cases and platelet peroxidase (PPO) was negative in the four cases studied. Two out of six cases showed karyotypic abnormalities (hypotetraploidy and a complex karyotype, respectively). In two out three cases a rearranged pattern for JH gene was observed. TCR (Cbeta and Jgamma) rearrangements were not detected in any case. AML-M0 is an infrequent form of acute myeloblastic leukemia. A large panel of myeloid monoclonal antibodies (MoAb) and the study of the cytoplasmic expression of myeloid antigens is necessary to diagnose this form of leukemia. AML-M0 usually coexpress lymphoid markers. Ultrastructural studies may be of help to discard an immature erythroid proliferation.

Multiple cell cycle regulator alterations in Richter's transformation of chronic lymphocytic leukemia.

To investigate the role of the cell cycle regulators p21(Waf1), p27(Kip1), retinoblastoma (Rb), and cyclin D1 in Richter's transformation of chronic lymphocytic leukemia (CLL), we analyzed 19 CLL and eight Richter's syndrome (RS) tumors, previously characterized for p53 and ARF/INK4a abnormalities. p21(Waf1)immunohistochemical expression was negative in 12 of 15 CLL (80%), whereas it was moderate or strong in three of seven RS (43%). p21(Waf1) gene was in germline configuration in all the tumors analyzed. Four immunohistochemical patterns of p53 and p21(Waf1) expression were observed: (1) p53-/p21- in 10 of 15 CLL (67%), but only in two of six RS (33%); (2) p53+/p21+ in three CLL (20%) and two RS (33%); (3) p53-/p21+ in one RS; and (4) p53++/p21- in two CLL and one RS. Two p53+/p21+ CLL evolved into RS. p53 mutations clustered around the p53++/p21- (two CLL and one RS) and p53-/p21- (one CLL and one RS) tumors. While the majority of CLL displayed strong p27 immunoreactivity, RS tumors were constantly p27-negative. p27(Kip1) gene was in germline configuration in all the tumors analyzed. Most CLL cases were negative for Rb expression. In contrast, all RS exhibited strong Rb expression. Cyclin D1 overexpression was only detected in one CLL evolving into RS and one RS. In conclusion, a p53+/p21- immunohistochemical pattern is shown exclusively by p53-mutated CLL/RS. Additionally, our results suggest a possible implication of moderate/strong p21(Waf1) expression, loss of p27 expression, and cyclin D1 overexpression in the Richter's transformation of CLL.

Simultaneous diagnosis of hairy cell leukemia and chronic lymphocytic leukemia/small lymphocytic lymphoma: a frequent association?

The association of hairy cell leukemia (HCL) with other neoplasms, mainly non-Hodgkin's lymphomas, is well known. However, the simultaneous diagnosis of HCL and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is rare, with only few cases of such an association having been reported. We describe three patients with a well-characterized HCL in whom a CLL/SLL population was detected. Of note, these cases represent a significant proportion (11.5%; 95% CI: 0% to 24%) of the total number of HCL cases diagnosed in our institution during the same period of time. All three patients were treated with deoxycoformycin. They achieved a complete response of the HCL, whereas the CLL/SLL population persisted in all cases. The immunoglobulin gene rearrangement analysis, in two informative cases, suggested that the HCL and CLL/SLL populations arose from different B cell clones. This study indicates that the association of HCL and CLL/SLL might be much more frequent than previously recognized. Therefore, a large panel of monoclonal antibodies, including those necessary to detect CLL/SLL, should be employed when studying patients with HCL.

Analysis of the clinical relevance of the breakpoint location within M-BCR and the type of chimeric mRNA in chronic myelogenous leukemia.

It has been suggested that the breakpoint location within the M-BCR segment of chromosome 22 and the type of chimeric mRNA BCR/ABL (b2a2 or b3a2) are associated with differences in the clinical and hematological characteristics of chronic myelogenous leukemia (CML). To assist in clarifying this matter, in a series of Ph-positive CML patients the relationship of both the breakpoint location within M-BCR (n = 71) and the type of chimeric mRNA BCR/ABL (n = 40) with the chronic phase duration, patients' survival, and thrombopoietic activity was analyzed. Median survival for patients with breakpoints in zones 1+2+3 (n = 38) and zones 4+5 (n = 31) was 62 and 75 months, respectively, the difference being not significant; patients with breaks in zones 1+2 (n = 19) and zones 3+4+5 (n = 50) had a median survival of 50 and 67 months, respectively (P also not significant). Moreover, no significant differences were found in the survival of patients with b2a2 (n = 16) and b3a2 (n = 24) mRNA junctions. Finally, no differences were observed in the platelet or megakaryocyte counts between patients with breakpoints in extremes 5' and 3' nor between patients with b2a2 and b3a2 mRNA. The above results are in agreement with those reported in most recent studies, confirming the lack of clinical relevance of molecular pattern in CML.

A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.

Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinicobiologic CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to classify CLL patients accurately into the three subgroups, namely naive B-cell-like, intermediate and memory B-cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV (immunoglobulin heavy-chain locus) mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time to first treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinicobiologic features and outcome.

Malignant transformation in IgM monoclonal gammopathy of undetermined significance.

Monoclonal gammopathy of undetermined significance (MGUS) is a frequent disorder characterized by the presence of a small serum M-protein in individuals with no evidence of multiple myeloma (MM), Waldenstrom's macroglobulinemia (WM), or primary amyloidosis (AL). Although one fourth of these individuals will develop a malignant disease, there are no well-established predictors of outcome, particularly in the IgM type MGUS. Among 434 patients diagnosed with MGUS from 1970 to 2001 with a minimum follow-up of 1 year, 52 (27 men and 25 women; median age, 67 years) of IgM type were identified. After a median follow-up of 5 years, five patients (9.6%) have developed WM. The risk of transformation was 13.3% (95% confidence interval [CI], 0 to 27) and 27.7% (95% CI, 0.3 to 55.1) at 10 and 20 years, respectively. The variables significantly associated with transformation were the proportion of bone marrow plasma cells (BMPC) and the percentage of bone marrow lymphocytes (BML). No significant differences in the risk of transformation were found between IgM MGUS and the remaining MGUS types. Thus, in IgM MGUS the rate of transformation was similar to the risk observed in other MGUS types, the percentage of BMPC and BML being the features significantly associated with evolution into WM.

Complete remission of idiopathic myelofibrosis following donor lymphocyte infusion after failure of allogeneic transplantation: demonstration of a graft-versus-myelofibrosis effect.

A patient with idiopathic myelofibrosis (IM) in the osteosclerotic phase received an allogeneic stem cell transplant. Hemopoietic engraftment was rapid, and full donor chimerism was observed on day +70. However, a few months later, replacement of donor hemopoiesis by the patient's 20q- cell clone was observed, followed by reappearance of the blood IM features, marrow fibrosis and osteosclerosis. At 8 months from transplant donor lymphocytes were infused, which induced chronic GVHD. This resulted in normalization of the blood, with disappearance of the fibrosis and osteosclerosis, effects which persisted 20 months later. This case provides evidence for a graft-versus-disease effect in IM.

Effects of short-term administration of G-CSF (filgrastim) on bone marrow progenitor cells: analysis of serial marrow samples from normal donors.

To determine the effect of G-CSF administration on both the total number of CD34+ cells and the primitive CD34+ subsets in bone marrow (BM), we have analyzed BM samples serially obtained from 10 normal donors in steady-state and during G-CSF treatment. Filgrastim was administered subcutaneously at a dosage of 10 microg/kg/day (n = 7) or 10 microg/kg/12 h (n = 3) for 4 consecutive days. Peripheral blood sampling and BM aspirates were performed on day 1 (just before G-CSF administration), day 3 (after 2 days of G-CSF), and day 5 (after 4 days of G-CSF). During G-CSF administration, a significant increase in the total number of BM nucleated cells was observed. The percentage (range) of CD34+ cells decreased in BM from a median of 0.88 (0.47-1.44) on day 1 to 0.57 (0.32-1.87), and to 0.42 (0.16-0.87) on days 3 and 5, respectively. We observed a slight increase in the total number of BM CD34+ cells on day 3 (0.66 x 10(9)/l (0.13-0.77)), and a decrease on day 5 (0.23 x 10(9)/l (0.06-1.23)) as compared with steady-state (0.40 x 10(9)/l (0.06-1.68)). The proportion of primitive BM hematopoietic progenitor cells (CD34+CD38-, CD34+HLA-DR-, CD34+CD117-) decreased during G-CSF administration. In parallel, a significant increase in the total number of CD34+ cells in peripheral blood was observed, achieving the maximum value on day 5. These results suggest that in normal subjects the administration of G-CSF for 5 days may reduce the number of progenitor cells in BM, particularly the most primitive ones.

Rapid diagnosis of acute promyelocytic leukemia by analyzing the immunocytochemical pattern of the PML protein with the monoclonal antibody PG-M3.

The fusion protein, promyelocytic leukemia-retinoic acid receptor (PML-RAR)alpha, generated by the t(15;17) translocation has an abnormal cellular distribution with colocalization of RARalpha and PML proteins. We analyzed the immunostaining pattern of PML protein using the PG-M3 monoclonal antibody directed against the amino terminal portion of PML (retained in wild-type PML and PML-RARalpha fusion protein) in the diagnosis of acute promyelocytic leukemia (APL). In addition, we compared this test with other methods for detecting the PML-RARalpha fusion gene. A normal immunostaining pattern was observed in nonmyeloid disorders and in 78 of 111 acute myeloid leukemias (AMLs). A microgranular pattern was observed in 25 AMLs, all corresponding to APL. These results were concordant with the reverse transcriptase-polymerase chain reaction results for PML-RARalpha fusion gene. Only 1 case positive for the PML-RARalpha transcript showed a normal protein pattern by immunocytochemistry. PML immunostaining was helpful to rapidly differentiate 7 cases with borderline characteristics and to obtain the diagnosis in 2 cases with scarce material. The effectiveness and low cost of this technique support its routine use as a first-line procedure in the differential diagnosis of AML.

Usefulness and reproducibility of cytomorphologic evaluations to differentiate myeloma from monoclonal gammopathies of unknown significance.

We attempted to differentiate monoclonal gammopathies of unknown significance (MGUS) and multiple myeloma (MM) on morphologic grounds and to determine interobserver reproducibility of the differentiation. Cytologists blindly evaluated bone marrow smears from 154 patients with bone marrow plasmacytosis for the proportion of plasma cells with predefined cellular atypias. The single morphologic characteristic that most strongly differentiated MM from MGUS was the presence of nucleoli. The percentage of plasma cells, cytoplasmic contour irregularities, and anisocytosis also predicted a diagnosis of myeloma in multivariate analysis. Six cytologists independently evaluated 68 consecutive cases to determine sensitivity and specificity of these cytomorphologic features. The interobserver coefficient of variation for the plasma cell count was 33%. On consideration of the diagnosis, 36 of 41 MGUS cases and all 24 cases of myeloma were classified correctly. The use of a predesigned score system did not present such a bias, although it did not improve overall efficiency. The plasma cell count is the most predictive characteristic of myeloma from a cytologic viewpoint, but the interobserver variability is high. Interobserver variability is also high in the assessment of morphologic atypia, and atypical traits are not uncommon in plasma cells in MGUS.

Prognosis of chronic myeloid leukemia: studies from the Barcelona Group.

Individual and multicenter efforts have facilitated the recognition of different parameters with prognostic value at presentation of chronic myeloid leukemia (CML). Interest is currently focused on the prognostic evaluation of features obtained from molecular analysis, and isolation of data with evolutive predictive value. The site of breakpoint within the M-BCR has been suggested as a prognostic factor in some studies. A recent analysis from our group failed to demonstrate differences in either duration of chronic phase or survival between patients with 5' and 3' breakpoints. We have confirmed in a sequential study that a decrease in the expression of some myeloid differentiation antigens of the blood granulocytes can be a prognostic indicator along CML evolution. Our more recent study, separating blast crises on immunological and molecular grounds, confirms lymphoid cases as a distinct subgroup and shows a trend for an association between megakaryoblastic blast crisis and 3' location of the breakpoint.

Early Pre-B Lymphoblastic Transformation in A Patient with Refractory Anemia.

A 77 year-old male with refractory anemia receiving only supportive therapy presented with an acute transformation six years following diagnosis. The lineage of the blast cells could not be ascertained by morphological and cytochemical studies. Nevertheless, immunophenotypical studies, including monoclonal antibodies against myeloid, monocytic, erythroid, megakaryo-cytic and T and B lymphoid antigens, and analysis of immunoglobulin and T-cell receptors genes demonstrated the early pre-B nature of the blasts. Lymphoblastic transformation is extremely rare in myelodysplastic syndromes (MDS). The case presented here and other similar cases previously reported are consistent with the concept that MDS are clonal disorders arising from a pluripotent stem cell.