Raul Gabus

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

The use of a sequential high dose recombinant interleukin 2 regimen after autologous bone marrow transplantation does not improve the disease free survival of patients with acute leukemia transplanted in first complete remission.

We report the outcome of 50 consecutive patients with CR1 acute leukemia (AML = 22; ALL = 28) treated with autologous BMT, after cyclophosphamide and TBI, followed with a sequential high dose rIL2 regimen. rIL-2 (RU 49637 from Roussel-Uclaf, Romainville, France) was started after hematological reconstitution an average of 72 +/- 22 days post transplant. The schedule consisted of a continuous infusion over 5 cycles (Cycle 1: 5 days starting on day 1; cycle 2-5: 2 days starting on day 15, 29, 43 and 57). Patients were treated at 4 different dosages (12 (N = 40), 16 (N = 3), 20 (N = 2), 24 (N = 5) x 10(6) IU/m2/day). Toxicities were mainly related to capillary leak syndrome and thrombocytopenia. Patients received an average of 122 +/- 49 10(6) IU/m2. Two patients with AML died from toxicity. rIL-2 infusion was associated with very a high level of immune stimu-lation of both T-cells (P < 0.05) and natural killer (NK) cells (P < 0.05) and associated cytolytic functions (P < 0.05). With a minimal and median follow-up of 21 and 46 months, 3 year leukemia free survival is 41 +/- 6% overall, 39 +/- 10% and 43 +/- 8% for AML and ALL respectively. Relapse probabilities at 3 years are 59 +/- 11% for AML and 57 +/- 8% for ALL. We conclude that this short infusion of rIL-2 over 2 months, resulting in an increased immune stimulation, is not associated with a better leukemic control for patients with acute leukemia transplanted early after reaching first complete remission.

T-Cell Lymphomas in South America and Europe

Peripheral T-cell lymphomas are a group of rare neoplasms originating from clonal proliferation of mature post-thymic lymphocytes with different entities having specific biological characteristics and clinical features. As natural killer cells are closely related to T-cells, natural killer-cell lymphomas are also part of the group. The current World Health Organization classification recognizes four categories of T/natural killer-cell lymphomas with respect to their presentation: disseminated (leukemic), nodal, extranodal and cutaneous. Geographic variations in the distribution of these diseases are well documented: nodal subtypes are more frequent in Europe and North America, while extranodal forms, including natural killer-cell lymphomas, occur almost exclusively in Asia and South America. On the whole, T-cell lymphomas are more common in Asia than in western countries, usually affect adults, with a higher tendency in men, and, excluding a few subtypes, usually have an aggressive course and poor prognosis. Apart from anaplastic lymphoma kinase-positive anaplastic large cell lymphoma, that have a good outcome, other nodal and extranodal forms have a 5-year overall survival of about 30%. According to the principal prognostic indexes, the majority of patients are allocated to the unfavorable subset. In the past, the rarity of these diseases prevented progress in the understanding of their biology and improvements in the efficaciousness of therapy. Recently, international projects devoted to these diseases created networks promoting investigations on T-cell lymphomas. These projects are the basis of forthcoming cooperative, large scale trials to detail biologic characteristics of each sub-entity and to possibly individuate targets for new therapies.

Methylation status regulates lipoprotein lipase expression in chronic lymphocytic leukemia.

Abstract Among different prognostic factors in chronic lymphocytic leukemia (CLL), we previously demonstrated that lipoprotein lipase (LPL) is associated with an unmutated immunoglobulin profile and clinical poor outcome. Despite the usefulness of LPL for CLL prognosis, its functional role and the molecular mechanism regulating its expression are still open questions. Interaction of CLL B-cells with the tissue microenvironment favors disease progression by promoting malignant B-cell growth. Since tissue methylation can be altered by environmental factors, we investigated the methylation status of the LPL gene and the possibility that overexpression could be associated with microenvironment signals. Our results show that a demethylated state of the LPL gene is responsible for its anomalous expression in unmutated CLL cases and that this expression is dependent on microenvironment signals. Overall, this work proposes that an epigenetic mechanism, triggered by the microenvironment, regulates LPL expression in CLL disease.

S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.

Treatment of chronic myelogenous leukemia with interleukin-2 : A phase II study in 21 patients

We designed a phase II study to assess the activity of recombinant interleukin-2 (rIL-2) in patients with chronic myelogenous leukemia (CML). Study population included 11 patients in the chronic phase of CML (6 in hematologic remission and 5 with active disease), 6 patients in the accelerated phase, and 4 in blastic phase of CML. Patients received three 5-day cycles administrated every other week. rIL-2 was given as intravenous bolus infusions of 8 x 10(6) IU/m2 three times a day during cycle 1 and twice a day during cycles 2 and 3. Response to rIL-2 was assessed on day 45. No hematologic response was achieved in the patients with evaluable disease. One patient in hematologic remission with rIL-2 achieved a major response (from 72% to 9% Ph+ metaphases), and two patients had some degree of reduction of Ph+ metaphases. Responses were short-lived (< 6 months), but two of these three patients achieved a new cytogenetic response with interferon given post-rIL-2. A significant immune activation was achieved with rIL-2 including a marked increase in CD3+/CD25+ cells, CD56+ cells, and in natural killer/lymphokine activated killer cell cytotoxic activity. These results confirm preclinical studies, which showed that IL-2 has antileukemic activity in CML. However, the responses observed were short lived and restricted to a subgroup of patients with low disease burden. This invites further studies testing its impact in situations of minimal disease or in combination with other cytokines.

Immunoglobulin heavy chain V-D-J gene rearrangement and mutational status in Uruguayan patients with chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived circulating clonal leukemic B-cells, although the etiopathogenesis remains unclear. The incidence of CLL is variable in different regions around the world. While it is the most frequent chronic leukemia in Western countries, it has a low incidence in Asia. In this work we have investigated the immunoglobulin heavy chain gene rearrangements and mutational status in 80 Uruguayan patients with CLL, and compared these results with those obtained in other geographic regions. Our results demonstrate that Uruguayan patients with CLL display an IGHV gene usage which resembles that observed in Mediterranean countries and exhibits certain differences compared with Brazilian and Asian series, as expected, considering the ethnic basis of the Uruguayan population. This suggests that genetic influences could be important in the development and etiopathogenesis of CLL, but larger studies are necessary to substantiate this possibility.

Mobilization of hematopoietic progenitor cells with granulocyte colony stimulating factors for autologous transplant in hematologic malignancies: a single center experience

Background In 2006 the Hematology Service of Hospital Maciel published its experience with peripheral blood progenitor cell harvesting for autologous stem cell transplantation using Filgen JP (Clausen Filgrastim). After mobilization with a mean filgrastim dose of 78 mcg/Kg, 4.7 x 106 CD34+ cells/Kg were obtained by apheresis. Age above 50, multiple myeloma as underlying disease and a malignancy that was not in remission were identified as frequent characteristics among patients showing complex mobilization. Objective The aim of this study was to compare stem cell mobilization using different brands of filgrastim. Methods One hundred and fifty-seven mobilizations performed between 1997 and 2006 were analyzed. This retrospective analysis comparative two groups of patients: those mobilized with different brands of filgrastim (Group A) and those who received Filgen JP (Clausen Filgrastim) as mobilizing agent (Group B). A cluster analysis technique was used to identify four clusters of individuals with different behaviors differentiated by age, total dose of filgrastim required, number of apheresis and harvested CD34+ cells. Results The mean total dose of filgrastim administered was 105 mcg/Kg, the median number of apheresis was 2 procedures and the mean number of harvested stem cells was 4.98 x 106 CD34+ cells/Kg. No significant differences were observed between Groups A and B regarding the number of apheresis, harvested CD34+ cells and number of mobilization failures, however the total dose of filgrastim was significantly lower in Group B. Conclusions Among other factors, the origin of the cytokine used as mobilizing agent is an element to be considered when evaluating CD34+ cell mobilization results.

Hematology in Latin America: where are we? Analysis of the reports of Societies of Hematology associated organization of the Highlights of ASH in Latin America

In recent years there has been great increase in the relationship between the Latin American Societies of Hematology and the American Society of Hematology. This led to the meeting Highlights of the American Society of Hematology (ASH) in Latin America (HOA-LA), first held in Sao Paulo and Rio de Janeiro, Brazil with its third meeting taking place in 2011 in Punta del Este in Uruguay. With this exchange, it soon proved necessary to know the reality of hematology in Latin America and the context of development in different countries, so that the links between the different scientific societies of the area would become stronger. With this in mind, information was collected using a standardized questionnaire provided to the participants of the 2011 HOA-LA accompanied by guidelines on how to complete the questionnaire. The information provided was discussed during a specific section of the 2011 HOA-LA on April 30 coordinated by Drs. Raul Gabus, Carmino Antonio de Souza and Scott Howard. The session was attended by representatives of Argentina, Brazil, Bolivia, Chile, Colombia, Mexico, Paraguay, Peru, Uruguay and Venezuela as well as those from the United States of America, Canada, Spain and Italy. The aim of the meeting was to prepare a document to evaluate the current status of hematology in Latin America in order to improve interactions between Latin American Societies of Hematology. An additional objective was to encourage the organization of cooperative projects on support and training for the diagnosis and treatment of patients with blood diseases in Latin America. Member societies of the OAH-LA were asked to designate a spokesperson for this activity before 15 December 2010. Questionnaires were distributed to these spokespeople to obtain a representative sample of partners and hematology services of each country. The questionnaires were then sent to the coordinators in January 2011. In February 2011, each representative wrote a brief summary of data on the general situation in their country that reflected the opinion of the respective Society of Hematology. Data were collected and analyzed by the coordinators of the LA-HOA in Uruguay in March 2011 with the reports being divulged to the participating scientific societies. In April 2011 the results were presented and the final text of the document was discussed. The estimated population of the countries involved (Argentina, Bolivia, Brazil, Chile, Colombia, Mexico, Paraguay, Peru, Uruguay and Venezuela) is approximately 475 million people in an area of 19,046,336 km2. There are currently 4,306 hematologists for this population which is a rate of 0.9/100,000 hematologists/inhabitants with a heterogeneous distribution between the countries and between regions within each country. A total of 76.7% hematologists are connected to hematology institutions in their countries (range: 45-100%). In the case of Colombia, the Hematology Society is linked to oncology and in five countries (Bolivia, Brazil, Chile, Paraguay and Venezuela) to transfusion medicine. In seven countries (Argentina, Bolivia, Brazil, Colombia, Paraguay, Uruguay and Venezuela), there are formal medical fellowships programs in hematology. Three countries (Brazil, Colombia and Uruguay) reported that it is the National Health System that regulates funds and supportive care. In the other countries the health care system is divided between the public health insurance, prepayments, loans and others. The participation of the public sector in different countries varies between 25 and 73%. Exclusive private healthcare covers 10 and 20% of care. There are few national records and those that exist are limited to specific diseases. The registries are mainly of isolated Hematology Centers with partial data and with little intervention of Hematology Societies (Figure 1). Figure 1 Registry With regards to healthcare in hematology, although most people can easily have access to referral centers, however there are geographic areas far from these centers, such as the Brazilian Amazon and Bolivian Altiplano (Figure 2). Figure 2 Healthcare in hematology accessibility The availability of cytogenetics, flow cytometry, molecular biology, immunohistochemistry, computed tomography, magnetic resonance imaging (MRI) and positron emission tomography (PET-CT) was found to be heterogeneous across countries and even within the same country. Some complex techniques such as computed tomography or immunophenotyping by flow cytometry (IHC) are generally accessible to the public. Some countries do not have cytogenetics, molecular biology, MRI or PET-CT availability. Moreover, even in countries that rely on these resources, access to them is not universal for all inhabitants (Figure 3). The reports also mention a need to develop IHC to assist diagnosis of hematologic diseases. Figure 3 Healthcare in hematology diagnosis About the availability of blood components and other resources in transfusion medicine, packed red blood cell and platelet concentrates are availability to the entire population and usually at no cost to the patient. The availability of platelets by apheresis and irradiated blood products is generally limited; normally they are only available to patients in Hematopoietic Stem Cell Transplantation (HSCT) units (Figure 4). It was found that in most countries, patients have access to basic hematology-oncology treatment, but only a part of the population has access to high-cost treatment. Monoclonal antibodies, new drugs and access to HSCT are considered high-cost (Figure 5). There are countries where HSCT is not performed or the program is poorly developed. Figure 4 Healthcare in hematology support Figure 5 Access to treatment and medications The financing of regular treatment is guaranteed by the state, by health insurance or medical organization to which the patient is affiliated. However, there are situations that hematological patients lack coverage (Figure 5). There is no guarantee of coverage of high-cost treatments and medications to all populations. In some countries it is the responsibility of the state, parastatal organizations, other health insurance institutions or medical organizations to which the patient is affiliated. Occasionally, this coverage is funded by a foundation or through a support program of the pharmaceutical industry (Figure 5). Regarding the study protocol and / or treatment, some hematology centers with guidelines or consensus on certain conditions, however almost none of them are national protocols. Most treatment programs follow international protocols with little or no adaptation to the country or region (Figure 6). Figure 6 Hemato-oncological protocol treatment This survey showed that the number of hematologists is inadequate and they are unevenly distributed (Figure 7). Figure 7 Human resources Most societies stated that the number of doctors who are currently in hematology specialist courses is insufficient to meet the needs of the population (Figure 7).

Methotrexate pharmacogenetics in Uruguayan adults with hematological malignant diseases

Background: Individual variability is among the causes of toxicity and interruption of treatment in acute lymphoblastic leukemia (ALL) and severe non‐Hodgkin lymphoma (NHL) patients under protocols including Methotrexate (MTX): 2,4‐diamino‐N10‐methyl propyl‐glutamic acid. Methods: 41 Uruguayan patients were recruited. Gene polymorphisms involved in MTX pathway were analyzed and their association with treatment toxicities and outcome was evaluated. Results: Genotype distribution and allele frequency were determined for SLC19A1 G80A, MTHFR C677T and A1298C, TYMS 28 bp copy number variation, SLCO1B1 T521C, DHFR C−1610G/T, DHFR C‐680A, DHFR A‐317G and DHFR 19 bp indel. Multivariate analysis showed that DHFR‐1610G/T (OR = 0.107, p = 0.018) and MTHFR677T alleles (OR = 0.12, p = 0.026) had a strong protective effect against hematologic toxicity, while DHFR‐1610CC genotype increased this toxicity (OR = 9, p = 0.045). No more associations were found. Conclusions: The associations found between gene polymorphisms and toxicities in this small cohort are encouraging for a more extensive research to gain a better dose individualization in adult ALL and NHL patients. Besides, genotype distribution showed to be different from other populations, reinforcing the idea that genotype data from other populations should not be extrapolated to ours. Graphical Abstract Figure. No caption available.