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.

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.

Reflexiones sobre investigación biomédica en España / Reflections on biomedical research in Spain

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Bone marrow changes in anorexia nervosa are correlated with the amount of weight loss and not with other clinical findings.

The clinical history and biochemical and hematologic variables for 44 consecutive patients diagnosed with anorexia nervosa were recorded. Bone marrow aspirates and biopsy specimens were analyzed by standard morphologic procedures, and bone marrow adipocytes were studied morphometrically. The bone marrow of the 44 patients was classified as normal (5 cases [11%]), hypoplastic or aplastic (17 [39%]), with partial or focal gelatinous degeneration (13 [30%]), or with complete gelatinous degeneration of the bone marrow (GDBM; 9 [20%]). These patterns correlated with amount of weight loss (P = .005) but not other clinical findings. WBC counts were lower in patients with GDBM (P = .0189), but this and other peripheral blood variables did not always reflect the severity of bone marrow damage. Hypoplastic or aplastic bone marrow showed an increase in bone marrow fat fraction due to an increase in adipocyte diameters, while in GDBM, fat fraction and adipocyte diameters decreased. Morphologic changes in bone marrow and stereologic alterations in bone marrow adipocytes may be observed in anorexia nervosa. The extent of damage is related to the amount of weight loss, not to other factors. Peripheral blood cell counts may not reflect the extent of damage. In some patients, this process may be reversible with reestablishment of adequate nutritional intake.

Allogeneic transplantation of CD34+-selected cells from peripheral blood in patients with myeloid malignancies in early phase: a case control comparison with unmodified peripheral blood transplantation.

An allogeneic transplantation of CD34(+)-selected cells from peripheral blood (allo-PBT/CD34(+)) from HLA-identical sibling donors was performed in 50 adult patients with acute myeloid leukemia in first complete remission (AML CR1) (n = 29), myelodysplastic syndrome (MDS) (n = 4), or chronic myeloid leukemia in first chronic phase (CML CP1) (n = 17). Clinical results were compared to a concurrent group of 50 patients transplanted with unmodified peripheral blood progenitor cells (allo-PBT), matched for age, diagnosis, and disease stage. The median follow-up period was 29 months (range 1-69). The actuarial probability of developing acute GVHD clinical grade II to IV was 16% (95%CI: 6-26) for the allo-PBT/CD34(+) group and 41% (95%CI: 29-57) for the allo-PBT group (P = 0.002). The actuarial probability of developing extensive chronic GVHD was 22% (95%CI: 8-36) for the allo-PBT/CD34(+) group and 47% (95%CI: 31-63) for the allo-PBT group (P = 0.02). Recipients of allo-PBT/CD34(+) had less toxicity associated with the transplant and better Karnofsky index at the last follow-up. For AML/MDS patients, the actuarial probability of disease-free survival (DFS) for recipients of allo-PBT/CD34(+) and allo-PBT was 65% (95%CI: 45-85) vs43% (95%CI: 28-58) (P = 0.05), respectively. These data provide a rationale for a randomised trial of allo-PBT/CD34(+) vs allo-PBT in AML/MDS patients in early stage of the disease.

Favourable effect of the combination of acute and chronic graft-versus-host disease on the outcome of allogeneic peripheral blood stem cell transplantation for advanced haematological malignancies.

To assess the influence of graft-versus-host disease (GVHD) on the outcome of patients with advanced haematological malignancies (AHM) who received a primary, unmodified allogeneic peripheral blood progenitor cells transplant (allo-PBT) from a human leucocyte antigen (HLA) identical sibling donor, we analysed 136 patients with myeloid neoplasms (n = 70) or lymphoproliferative disorders (n = 66), transplanted at 19 Spanish institutions. Median age was 35 years (range 1-61). The cumulative incidence of relapse for all patients was 34% (95% CI, 26-42%), 41% (95% CI, 33-49) for patients without GVHD and 14% (95% CI, 3-25) (P = 0.001) for patients with acute and chronic GVHD. After a median follow-up of 11 months (range 2-49), 60 (44%) patients remained alive with an actuarial probability of overall survival and disease-free survival (DFS) at 30 months of 31% (95% CI, 21-41%) and 28% (95% CI, 17-39%) respectively. In patients surviving > 100 d, the low incidence of relapse in those with acute and chronic GVHD led to a DFS of 57% (95% CI, 38-76%) compared with a DFS of 34% (95% CI, 17-51%) in the remaining patients (P = 0.03). Our results indicate a reduced incidence of relapse for patients with AHM receiving an unmodified allo-PBT and developing acute and chronic GVHD, which results in an improved DFS.

The number of donor CD3(+) cells is the most important factor for graft failure after allogeneic transplantation of CD34(+) selected cells from peripheral blood from HLA-identical siblings.

This study analyzed the characteristics of 257 HLA-identical sibling transplants of granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells depleted of T cells by CD34(+) positive selection (allo-PBT/CD34(+)) for their effect on the incidence of graft failure. Twenty-four patients developed graft failure (actuarial probability, 11%; 95% confidence interval, 7.1-14. 9). Prognostic factors considered were sex and age of donor and recipient, donor-recipient blood group compatibility, diagnosis, disease status at transplant, conditioning regimen, cytomegalovirus serology, number of CD34(+) and CD3(+) cells infused, and cryopreservation. The major factor associated with graft failure was the number of CD3(+) cells in the inoculum. Twenty-three of 155 patients receiving a T-cell dose in the graft less than or equal to 0.2 x 10(6)/kg experienced graft failure, compared with only one of 102 patients receiving more than 0.2 x 10(6)/kg (actuarial probability 18% vs 1%, respectively; P =.0001). The actuarial probability of graft failure progressively increased as the number of CD3(+) cells in the graft decreased, which was determined by grouping the number of CD3(+) cells in quartiles (log-rank P =.03; log-rank for trend P =.003). In the multivariate analysis by the proportional hazard method, 2 covariates entered into regression at a significant level: CD3(+) cells less than or equal to 0.2 x 10(6)/kg (risk ratio = 17; P <.0001), and patients with chronic myelogenous leukemia (CML) conditioned with busulphan-based regimens (risk ratio = 4.8; P =.001). From these results it appears that the number of CD3(+) cells in the inoculum-with a threshold of 0.2 x 10(6)/kg or less-is the most critical factor in maintaining a sustained engraftment in allo-PBT/CD34(+) from HLA-identical siblings. In addition, for patients with CML receiving 0.2 x 10(6)/kg or less CD3(+) cells, total body irradiation might be better than busulphan-based regimens.

Increased conventional chemotherapy does not improve survival in multiple myeloma: long-term results of two PETHEMA trials including 914 patients.

BACKGROUND: Melphalan and prednisone (MP) has been the standard treatment for multiple myeloma (MM) for the last 30 years. Combination chemotherapy at conventional doses has not shown a significant prolongation of survival when compared to MP. There are few data comparing conventional chemotherapy at standard doses with conventional treatment at higher doses. We present the long-term outcome of 914 patients from two randomized trials comparing three different dose intensity regimens. METHODS: From 1 January, 1985 to 31 December, 1989, 487 patients were randomized between MP (melphalan 9 mg/m(2) p.o. and prednisone 60 mg/m(2) days 1-4) and alternating VCMP (vincristine 1 mg i.v. on day 1, cyclophosphamide 500 mg/m(2) i.v. on day 1, melphalan 6 mg/m(2) p.o. on days 1-4, and prednisone 60 mg/m(2) on days 1-4) and VBAP (vincristine 1 mg i.v. on day 1, BCNU and doxorubicin 30 mg/m(2) i.v. each on day 1, and prednisone 60 mg/m(2) on days 1-4). From 1 January, 1990 to 31 May, 1994, 427 patients were randomized between VCMP/VBAP at the above detailed doses (VCMP/VBAP 'SD') and the same regimen increasing the doses of cyclophosphamide and doxorubicin from 500 to 1200 mg/m(2) and from 30 to 50 mg/m(2), respectively (VCMP/VBAP 'HD'). RESULTS: Increasing dose intensity produced a significantly higher partial response rate (31% vs 45% vs 51% for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P < 0.01). However, a significantly early death rate was observed in the HD arm (7.7, 7.5 and 12.1% for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = 0.05). Median duration of response (20 vs 18 vs 19 months for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = NS) and median survival (25 vs 31 vs 29 months for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = NS) were similar in the three groups. MP produced a higher degree of thrombocytopenia than combination chemotherapy at standard (P = 0.002) or high dose (P = 0.01), this leading to a significantly higher dose reduction in the MP arm (P < 0.001 and P = 0.003 for VCMP/VBAP 'SD' and VCMP/VBAP 'HD', respectively). CONCLUSION: In these trials the response rate significantly correlated with the regimen intensity. However, no significant differences in response duration and survival were found. This highlights the limited role of conventional chemotherapy in MM and the need for further trials, aimed at determining the impact of new treatment approaches such as high-dose therapy/autotransplantation.

Linfomas no hodgkinianos: aspectos actuales / Non-hodgkin lymphomas: current aspects

Objetivo. Revisar brevemente los conocimientos actuales sobre los linfomas no hodgkinianos (LNH), con especial énfasis en los aspectos de interés en oftalmología, otorrinolaringología y neurología. Desarrollo. Se analiza la evolución histórica de la clasificación de los LNH y se refiere la desarrollada recientemente por la Organización Mundial de la Salud. Entre las numerosas entidades que forman parte de este grupo de afecciones se refieren los principales datos (clínicos, inmunofenotípicos, citogénicos y de biología molecular) relativos a los cinco tipos más importantes: 1. Linfoma molecular; 2. Linfoma difuso de células grandes; 3. Linfoma de células del manto; 4. Linfoma MALT (tejido linfoide asociado a mucosas), y 5. Linfoma T periférico. A continuación, se procede a la descripción de los tipos y formas clínicas de LNH en oftalmología, otorrinolaringología y neurología. Por último, se señalan los recientes avances en el pronóstico (índice internacional, marcadores biológicos) y tratamiento (nuevos tipos de poliquimioterapia, erradicación del Helicobacter pylori, trasplante de progenitores hemopoyéticos, anticuerpos monoclonales, terapia antisentido) de estos procesos. Conclusiones. Los LNH constituyen un grupo de afecciones, cuya incidencia ha experimentado en los últimos años un incremento anual superior al 4 por ciento. Con una frecuencia no despreciable, estos tumores asientan de modo primitivo o en el sistema nervioso; en este último caso, aparte de la expresión tumoral directa, pueden dar lugar también a manifestaciones paraneoplásicas (AU)

Immune reconstitution following allogeneic peripheral blood progenitor cell transplantation.

Delayed immune reconstitution following allogeneic stem cell transplantation remains a major clinical problem, resulting in significant transplant-related mortality from infectious complications. The recovery of immunity after bone marrow transplantation (BMT) is a complex process dependent on a large number of pre- and post-transplant factors. It has been suggested that the use of peripheral blood instead of bone marrow as stem cell source may accelerate immune reconstitution after allogeneic transplantation. Some authors have recently reported a more rapid recovery of the number and function of T and B cells after allogeneic peripheral blood progenitor cell transplant (allo-PBPCT) in comparison with conventional BMT, results which would reflect the high number of lymphocytes infused to the patients. Such a rapid immune recovery could indeed contribute to the apparent therapeutic advantage of PBPCT when compared with BMT. However, there is limited knowledge on this issue and randomized trials are required to prove whether allo-PBPCT is indeed superior to BMT in terms of immune reconstitution post-transplant. A review of some quantitative and functional aspects of immune recovery after allo-PBPCT is presented in this article.

Roquinimex (Linomide) vs placebo in AML after autologous bone marrow transplantation.

Roquinimex, Linomide, a quinoline derivative with pleiotropic immunomodulatory activity, has previously been shown to enhance natural killer (NK) cell number and activity after ABMT in patients with AML. In this study 278 AML patients in remission were randomized to receive Roquinimex 0.2 mg/kg body weight or placebo twice weekly for 2 years following ABMT. Out of 139 patients in each group, 109 Roquinimex patients and 108 placebo patients were in their first CR. Median age at inclusion was 41 years for Roquinimex patients and 39 years for placebo patients. Twelve patients in each group had their marrow purged prior to reinfusion. Relapse and death were study endpoints. Surviving patients were followed for 2.6 to 6. 9 years. The total number of relapses was 60 in the Roquinimex group and 63 in the placebo group (not significant). Leukemia-free and overall survivals were similar in the two groups. Recovery of platelet counts was significantly delayed in the Roquinimex group as compared to placebo. No other significant differences regarding toxicity parameters were recorded. In conclusion, previous findings on NK cells could not be confirmed and the study showed no benefit for Roquinimex over placebo regarding relapse or survival following ABMT for AML in remission.

La hora del relevo / Change of pace

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[Non-Hodgkin's lymphomas: current aspects]. / Linfomas no hodgkinianos: aspectos actuales.

OBJECTIVE: We wish to briefly review current knowledge of non-Hodgkin lymphomas (NHL) with special emphasis on aspects of interest in ophthalmology, otorhinolaryngology and neurology. DEVELOPMENT: We analyze the historical evolution of the classification of the NHL and refer to that recently devised by the World Health Organization. Amongst the numerous conditions forming part of this group of disorders we refer to the main data (clinical, immunophenotype, cytogenetics and of molecular biology) relative to the five most important types: 1. Follicular lymphoma; 2. Diffuse large cell lymphoma; 3. 'Mantle' cell lymphoma; 4. MALT lymphoma (mucosa associated lymphoid tissue), and 5. Peripheral T lymphoma. Then we describe the types and clinical forms of NHL in ophthalmology, otorhinolaryngology and neurology. Finally we consider the recent advances in prognosis (international index, biological markers) and treatment (new types of polychemotherapy, eradication of Helicobacter pylori, transplant of hemopoietic progenitors, monoclonal antibodies and anti-sense treatment of these conditions. CONCLUSIONS: The NHL form a group of conditions in which the annual increase in recent years has been over 4%. With considerable frequency these tumors are found from the beginning or during their course in otorhinological and ophthalmic tissues, or in the nervous system; in the latter case, apart from direct tumour expression they may also cause paraneoplastic manifestations.

Bone marrow transplantation for severe aplastic anemia: the Barcelona Hospital Clinic experience.

BACKGROUND AND OBJECTIVE: The outcome of patients with severe aplastic anemia (SAA) has improved considerably over the last decades. Bone marrow transplantation (BMT) is the treatment of choice in young patients who have an HLA-identical sibling donor. This study analyzes the outcome and factors related to survival in patients with SAA receiving BMT in our institution. DESIGN AND METHODS: Between March 1978 and December 1996, 49 consecutive patients received an HLA-identical sibling marrow transplant for SAA. Median age was 21 years (range, 4 to 47) and 15 (31%) were women. Median interval from diagnosis to transplant was 2.6 months (range, 0.5 to 159). Between 1978 and 1982 all patients were conditioned with cyclophosphamide (CY) alone and received methotrexate (MTX) until day 102 as graft-versus-host disease (GvHD) prophylaxis. From 1983 most patients received CY and thoraco-abdominal irradiation (TAI) as the conditioning regimen and cyclosporin A (CSA) as GvHD prophylaxis. RESULTS: Survival probability at 10 years was 55 +/- 7% with a median follow-up for the surviving patients of 8.5 years. The incidences of graft failure, grade II to IV acute GvHD, and chronic GvHD were 21%, 39.5% and 31%, respectively. In multivariate analysis three factors adversely influenced survival: a) age > or = 30 years (p = 0.05); b) > or = 10 transfusion units pre-BMT (p = 0.008); and c) use of long course MTX for GvHD prophylaxis (p = 0.01). One case of squamous-cell carcinoma occurred in a TAI-treated patient 13 years post-transplantation. INTERPRETATION AND CONCLUSIONS: BMT is effective in young patients with SAA who have an HLA-identical sibling donor, particularly if minimally transfused pre-transplant. The introduction of TAI and CSA to our preparative regimen has led to a remarkably increased survival.

Immune reconstitution following allogeneic peripheral blood progenitor cell transplantation: comparison of recipients of positive CD34+ selected grafts with recipients of unmanipulated grafts.

We compared the kinetic recovery of lymphocytes and their subsets in two groups of patients submitted to allogeneic peripheral blood progenitor cell transplantation (allo-PBT): those receiving lymphocyte-depleted leukaphereses by positive selection of CD34+ cells (group 1, n = 18) and those receiving unmanipulated leukaphereses (group 2, n = 15). Patients were conditioned with cyclophosphamide (120 mg/kg) and fractioned total body irradiation (13 Gy, group 1; 12 Gy, group 2). The mean number (x 10(6)/kg) of CD34+ and CD3+ cells infused was 4.0 and 0.67, respectively, in group 1 patients, and 4.7 and 274, respectively, for group 2 patients. Graft-versus-host disease prophylaxis consisted of cyclosporin A + methylprednisolone for group 1 and cyclosporin A + methotrexate for group 2. Median follow-up was 7 months (range 2-8 months) for both groups. During the first 6 months post-transplant, CD4+ cell counts were lower in group 1 as compared with group 2 (p = 0.014, 0.010, 0.011, 0.0003, and 0.052 at 0.5, 1, 2, 3, and 6 months, respectively), whereas there was no difference at 8 months. The number of CD4+CD45RA+ cells was very low throughout the study in both groups, being lower in group 1 than in group 2, especially during the first 3 months post-transplant (p = 0.007 and 0.0006 at 1 and 3 months). Normal levels of CD8+ cells were reached by 1 month post-transplant in both groups. TCR gamma delta + cell counts were lower in group 1 than in group 2 during the first 4 months post-transplant (p = 0.001, 0.004, and 0.04 at 1, 3, and 4 months). A normal number of natural killer cells (CD3-CD56+) was achieved 1 month post-transplant in both groups. B lymphocytes (CD19+) showed low or undetectable counts throughout the first 4 months in both groups, achieving the normal range at 8 months. These results show that, during the first 6 months following allo-PBT with CD34+ selected grafts, the number of CD4+, CD4+CD45RA+, and TCR gamma delta + cells is significantly lower than after unmanipulated allo-PBT; these differences disappeared at 8 months. In contrast, there are no differences between transplant groups in the recovery of CD8+, CD19+, and natural killer cells.

Magnetic resonance imaging in myelofibrosis and essential thrombocythaemia: contribution to differential diagnosis.

To ascertain the value of magnetic resonance (MR) imaging in the differential diagnosis between myelofibrosis (MF) and essential thrombocythaemia (ET), 38 patients were analysed. 20 patients had MF (idiopathic myelofibrosis, 15 cases; post-ET myelofibrosis, four cases; post-polycythaemic MF, one case) and 18 ET. Mean age was 61.5 years (range 30-89) for patients with MF and 60.9 years (range 26-83) for ET patients. MR imaging was performed in the dorsal vertebrae in all cases, and also in both femurs in 2 5 of the patients. In most ET cases the MR signal of the dorsal vertebrae was not modified, whereas it was markedly reduced in MF (P=0.0000001). With regard to femoral marrow, it was usually fatty in ET, with an absent to moderate degree of reconversion seen in the 14 cases analysed, contrasting with the marked degree of reconversion noted in 10/11 patients with MF (P=0.000007). An inverse correlation was demonstrated between the vertebral signal and the degree of femoral reconversion. These differences were due to the fact that in ET the bone marrow adipose tissue is grossly preserved, whereas in MF it is usually markedly decreased or absent. The above results indicate that MR imaging is a useful tool for the differential diagnosis of ET and MF, with the usefulness of this technique increasing when vertebral and femoral bone marrow studies are combined.

Major vascular complications in essential thrombocythemia: a study of the predictive factors in a series of 148 patients.

To determine the clinicohematological factors predictive for the appearance of major vascular complications (MVC) in patients with essential thrombocythemia (ET), 148 consecutive such patients were retrospectively assessed for the development of MVC during a median follow-up of 58.5 months. Seventy-seven patients had vascular risk factors, and 37 a history of MVC at ET diagnosis. Forty-nine MVC were registered in 33 patients during the follow-up period. The actuarial probability of MVC was 27% at 6 years in the whole series, 35.6% for patients above 60 years, and 21.4% for patients younger than 60 years, whereas only one of the 36 patients younger than 45 years had MVC. At multivariate analysis, age >60 years, history of major ischemia and hypercholesterolemia were the variables associated with an increased MVC risk. These results suggest that all ET patients above 60 years should be treated, whereas in younger patients treatment decisions should be primarily based on the existence of risk factors for MVC.