Lymphoproliferative disorders associated with hypereosinophilia.

Hypereosinophilia, defined as peripheral blood eosinophil counts >1,500/μL, may complicate the course of various lymphoproliferative disorders. Among these, Hodgkin lymphoma (HL) and certain peripheral T-cell lymphomas (PTCLs) derived from CD4 cells, including Sezary syndrome (SS), adult T-cell leukemia/lymphoma (ATLL), and angioimmunoblastic T-cell lymphoma (AITL), are most commonly associated with increased reactive eosinophilopoiesis. Rarely, marked hypereosinophilia (HE) may occur in the setting of acute B-cell lymphoblastic leukemia, with a substantial impact on disease course. The mechanisms leading to blood and tissue eosinophilia in the setting of lymphoproliferative disorders, as well as the clinical complications and prognostic implications of hypereosinophilia, are discussed in this review.

Cancer mortality in Europe, 2005-2009, and an overview of trends since 1980.

BACKGROUND: After a peak in the late 1980s, cancer mortality in Europe has declined by ∼10% in both sexes up to the early 2000s. We provide an up-to-date picture of patterns and trends in mortality from major cancers in Europe. METHODS: We analyzed cancer mortality data from the World Health Organization for 25 cancer sites and 34 European countries (plus the European Union, EU) in 2005-2009. We computed age-standardized rates (per 100 000 person-years) using the world standard population and provided an overview of trends since 1980 for major European countries, using joinpoint regression. RESULTS: Cancer mortality in the EU steadily declined since the late 1980s, with reductions by 1.6% per year in 2002-2009 in men and 1% per year in 1993-2009 in women. In western Europe, rates steadily declined over the last two decades for stomach and colorectal cancer, Hodgkin lymphoma, and leukemias in both sexes, breast and (cervix) uterine cancer in women, and testicular cancer in men. In central/eastern Europe, mortality from major cancer sites has been increasing up to the late 1990s/early 2000s. In most Europe, rates have been increasing for lung cancer in women and for pancreatic cancer and soft tissue sarcomas in both sexes, while they have started to decline over recent years for multiple myeloma. In 2005-2009, there was still an over twofold difference between the highest male cancer mortality in Hungary (235.2/100 000) and the lowest one in Sweden (112.9/100 000), and a 1.7-fold one in women (from 124.4 in Denmark to 71.0/100 000 in Spain). CONCLUSIONS: With the major exceptions of female lung cancer and pancreatic cancer in both sexes, in the last quinquennium, cancer mortality has moderately but steadily declined across Europe. However, substantial differences across countries persist, requiring targeted interventions on risk factor control, early diagnosis, and improved management and pharmacological treatment for selected cancer sites.

Molecular Analysis of the PTEN Gene in a Choroidal Schwannoma in the Context of a Hamartomatous Syndrome

Purpose: To investigate the molecular involvement of PTEN, a tumor suppressor gene, in a case of cellular pigmented choroidal Schwannoma in a patient with hamartomatous syndrome due to heterozygous PTEN germline mutation. Methods: Histopathological, immunohistochemical, and electron microscopy analyses were performed by standard procedures. Paraffin-embedded samples of normal and tumor eye tissues were collected and DNA was extracted. A 145 bp region flanking the heterozygous c.406T>C mutation in exon 5 of PTEN was amplified by PCR and sequenced. To evaluate the allelic status of PTEN in the tumor sample, we cloned different PCR products in E. coli using a TA cloning procedure. Results: Histopathology demonstrated a posterior choroidal mass measuring 1.3 x 1.6 x 1.4 cm. The tumor was composed by fascicles of spindle cells with wavy cytoplasm. No Verrocay bodies could be identified. Scattered histiocytes with clear cytoplasm were present. By immunohistochemistry, the cells were expressing S100 and focally Melan A proteins. Pericellular type IV collagen could be demonstrated. Interlacing cytoplasmic processes covered by thick basement membrane could be found by electron microscopy as well as few premelanosomes. Moderate PTEN expression by immunohistochemistry was identified in some cells. As expected, the germline mutation could be detected by DNA sequencing in both the paraffin-embedded normal and tumor eye tissues. Analysis of 33 E. coli colonies bearing clones from the tumor eye tissue DNA surprisingly revealed that most of them contained the PTEN wild-type allele (29 vs. 4, Fisher's test p-value = 0.002). Conclusions: This is the first reported case of choroidal cellular Schwannoma arising in the context of a PTEN hamartomatous syndrome. Allelic analysis of PTEN in the tumor suggests a statistically-significant partial loss of heterozygozity in favor of the wild-type allele. Our findings are in clear contrast with what is usually observed in cancer tissues, for which mutated alleles of tumor suppressor genes are usually brought to homozygosity. Similar results were previously reported in human non-Hodgkin's lymphomas, displaying an overexpression of the wild-type form of the tumor suppressor gene p53. We are in the process of investigating additional DNA derived from other fresh and paraffin-embedded tissues from the patient, in order to gain insights on the molecular bases of PTEN involvement in this rare choroidal Schwannoma.

Immunociblage des tumeurs: situation et perspectives en 2000

Following 15 years of experimental studies, tumor immunotargeting using monoclonal antibodies directed against tumor associated antigens shows now important monoclonal antibodies directed against tumor associated antigens shows now important clinical developments. This is mainly due to encouraging therapeutic results which have obtained using humanized antibodies such as the anti-CD20 rituximab in follicular B lymphomas and the anti-DrbB2 herceptin in breast carcinomas. Thanks to genetic engineering it is possible to graft variable or hypervariable regions from murine antibodies to human IgG, and even to obtain fully human antibodies by using either transgenic mice containing a large part of the human repertoire of human IgG, or selection of human antibody fragments expressed by phages. Radiolabeling of antibodies played a major role to demonstrate the tumor immunotargeting specificity and remains attractive for the diagnosis by immunoscintigraphy as well as for the treatment by radioimmunotherapy of some cancers. In this review, the current results and the prospects of diagnostic and therapeutic uses of anti-tumor antibodies and their fragments will be described. Concerning diagnosis, 123-iodine or 99m-technetium labeled Fab fragments allowed very demonstrative tumor images but this technique has a limited effect upon the therapeutic attitude. Immuno-PET (positron emission tomography) could enhance the sensitivity of this imaging method. Radio-immunoguided surgery and immunophotodetection are attractive techniques still under evaluation. Concerning therapy, 131-iodine labeled anti-CD20 antibodies gave spectacular results in non-Hodgkin's B lymphomas. In solid tumors which as less radiosensitive, radioimmunotherapy could concern small tumors and need the use of two-steps targeting and/or alpha emitters radioisotopes. Some other strategies will be described such as bispecific antibodies directed against tumors and immune effector cells, some antibody fragments expressed on T cells called T-bodies or some biological studies using intrabodies. Published data and works in progress demonstrate that immunotargeting of tumors will have a growing place in the treatments of cancer patients.

Ivory vertebra: imaging findings in different diagnoses

Abstract Low back pain is often managed at all levels of healthcare. In general, diagnostic investigation begins with radiography of the lumbar spine. In addition to the most common findings, radiologists can identify increased density of a vertebral body, referred to as ivory vertebra. The objective of this study was to describe the main diseases that can present with this radiologic sign, such as Hodgkin lymphoma, Paget's disease, metastatic prostate cancer, breast cancer, and osteomyelitis. It is extremely important that radiologists be aware of this finding in order to inform the requesting physician of the possible etiologies, given that it can be the initial radiologic presentation for these diseases.

Human regulatory protein Ki-1/57 has characteristics of an intrinsically unstructured protein

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1; in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Small-angle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein. Experimental curves together with ab initio modeling approaches revealed an extended and flexible molecule in solution. An elongated shape was also observed by analytical gel filtration. Furthermore, sedimentation velocity analysis suggested that Ki-1/57 is a highly asymmetric protein. These findings may explain the functional plasticity of Ki-1/57, as suggested by the wide array of proteins with which it is capable of interacting in yeast two-hybrid interaction assays.

Validação de anticorpo monoclonal anti-CD45 obtido in house para utilização em citometria de fluxo e imuno-histoquímica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sarcoma histiocítico: análise molecular pela técnica de hibridação genômica comparativa, microRNA e imunoistoquímica

Pós-graduação em Patologia - FMB

Loss of Interleukin-10 Signaling and Infantile Inflammatory Bowel Disease: Implications for Diagnosis and Therapy

BACKGROUND & AIMS: Homozygous loss of function mutations in interleukin-10 (IL10) and interleukin-10 receptors (IL10R) cause severe infantile (very early onset) inflammatory bowel disease (IBD). Allogeneic hematopoietic stem cell transplantation (HSCT) was reported to induce sustained remission in 1 patient with IL-10R deficiency. We investigated heterogeneity among patients with very early onset IBD, its mechanisms, and the use of allogeneic HSCT to treat this disorder. METHODS: We analyzed 66 patients with early onset IBD (younger than 5 years of age) for mutations in the genes encoding IL-10, IL-10R1, and IL-10R2. IL-10R deficiency was confirmed by functional assays on patients' peripheral blood mononuclear cells (immunoblot and enzyme-linked immunosorbent assay analyses). We assessed the therapeutic effects of standardized allogeneic HSCT. RESULTS: Using a candidate gene sequencing approach, we identified 16 patients with IL-10 or IL-10R deficiency: 3 patients had mutations in IL-10, 5 had mutations in IL-10R1, and 8 had mutations in IL-10R2. Refractory colitis became manifest in all patients within the first 3 months of life and was associated with perianal disease (16 of 16 patients). Extraintestinal symptoms included folliculitis (11 of 16) and arthritis (4 of 16). Allogeneic HSCT was performed in 5 patients and induced sustained clinical remission with a median follow-up time of 2 years. In vitro experiments confirmed reconstitution of IL-10R-mediated signaling in all patients who received the transplant. CONCLUSIONS: We identified loss of function mutations in IL-10 and IL-10R in patients with very early onset IBD. These findings indicate that infantile IBD patients with perianal disease should be screened for IL-10 and IL-10R deficiency and that allogeneic HSCT can induce remission in those with IL-10R deficiency.

Analisi genetico-molecolare dei linfomi a cellule T periferiche

Molecular profiling of Peripheral T-cell lymphomas not otherwise specified Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of tumors that the WHO classification basically subdivides into specified and not otherwise specified (NOS). In Western countries, they represent around 12% of all non-Hodgkin's lymphomas. In particular, PTCL/NOS is the commonest subtype, corresponding to about 60-70% of all T-cell lymphomas. However, it remains a complex entity showing great variety regarding either morphology, immunophenotype or clinical behavior. Specially, the molecular pathology of these tumors is still poorly known. In fact, many alteration were found, but no single genes were demonstrated to have a pathogenetic role. Recently, gene expression profiling (GEP) allowed the identification of PTCL/NOS-associated molecular signatures, leading to better understanding of their histogenesis, pathogenesis and prognostication. Interestingly, proliferation pathways are commonly altered in PTCLs, being highly proliferative cases characterized by poorer prognosis. In this study, we aimed to investigate the possible role in PTCL/NOS pathogenesis of selected molecules, known to be relevant for proliferation control. In particular, we analyzed the cell cycle regulators PTEN and CDKN1B/p27, the NF-kB pathway, and the tyrosin kinase PDGFR. First, we found that PTEN and p27 seem to be regulated in PTCL/NOS as in normal T-lymphocytes, as to what expression and cellular localization are concerned, and do not present structural abnormalities in the vast majority of PTCL/NOS. Secondly, NF-kB pathway appeared to be variably activated in PTCL/NOS. In particular, according to NF-kB gene expression levels, the tumors could be divided into two clusters (C1 and C2). Specially, C1 corresponded to cases presenting with a global down-regulation of the entire pathway, while C2 showed over-expression of genes involved in TNF signaling. Notably, by immunohistochemistry, we showed that either the canonical or the alternative NK-kB pathway were activated in around 40% of cases. Finally, we found PGDFRA to be consistently over-expressed (at mRNA and protein level) and activated in almost all PTCLs/NOS. Noteworthy, when investigating possible causes for PDGFRA deregulation, we had evidences that PDGFR over-expression is due to the absence of miR-152, which appeared to be responsible for PDGFRA silencing in normal T-cells. Furthermore, we could demonstrate that its aberrant activation is sustained by an autocrine loop. Importantly, this is the first case, to the best of our knowledge, of hematological tumor in which tyrosin kinase aberrant activity is determined by deregulated miRNA expression and autocrine loop activation. Taken together, our results provide novel insight in PTCL/NOS pathogenesis by opening new intriguing scenarios for innovative therapeutic interventions.

Ruolo della proteina arginin-metiltrasferasi-5 (PRMT5) nella linfomagenesi B: implicazioni terapeutiche

Numerosi studi hanno mostrato come i meccanismi epigenetici di regolazione della cromatina svolgano un ruolo centrale nel controllare la trascrizione genica. E’ stato infatti dimostrato che complessi inibitori come SWI/SNF e gli enzimi ad esso associati quali istone deacetilasi (HDAC) e protein arginin-metiltrasferasi (PRMT), siano coinvolti nel controllo della crescita, differenziazione e proliferazione cellulare. Diversi studi hanno mostrato come i meccanismi epigenetici di controllo della trascrizione genica svolgano un ruolo di primo piano nel promuovere la sopravvivenza cellulare in leucemie/linfomi di derivazione dai linfociti B come la leucemia linfatica cronica, il linfoma mantellare ed i linfomi associati al virus di Epstein-Barr (EBV). Tuttavia, molto poco e’ conosciuto circa i meccanismi epigenetici di controllo della trascrizione che divengono operativi e che contribuiscono al processo di trasformazione dei linfociti B. PRMT5 e’ un enzima che di-metila specificamente residui argininici sugli istoni (H) 3 (H3R8) ed 4 (H4R3). PRMT5 ed HDAC lavorano in concerto per reprimere la trascrizione di specifici geni oncosoppressori. In questo progetto sono stati studiati i meccanismi e le conseguenze dell’iperespressione di PRMT5 durante il processo di trasformazione dei linfociti B indotto da EBV, e’ stata dimostrata l’importanza di questo enzima nel processo di trasformazione, e sono stati studiati nuovi metodi per inibirne l’espressione/attivita’. In particolare si e’ dimostrato che l’espressione di PRMT5 e’ ridotta o assente in linfociti B normali (o attivati da stimoli fisiologici) ed elevata in linee cellulari linfoblastoidi immortalizzate o completamente trasformate. Elevati livelli citosolici di PRMT5 sono detectabili dopo 4 giorni dall’infezione di linfociti B normali con EBV, PRMT5 e’ detectabile a livello nucleare, dove esercita la sua funzione repressoria la trascrizione, a partire dal giorno 8. L’utilizzo di specifici small interference RNAs (siRNA) in linee cellulari linfoblastoidi ha permesso di dimostrare la riduzione dell’espressione di PRMT5, la riduzione della metilazione degli istoni target di PRMT5, inibizione della proliferazione cellulare e abbassamento della soglia di sensibilita’ cellulare a stimoli pro-apoptotici. Esperimenti di co-immunoprecipitazione cromatinica hanno permesso di evidenziare che in queste cellule PRMT5 e’ parte di un complesso proteico a funzione inibitoria e che questo complesso si lega alla regione promotrice di specifici geni oncosoppressori quali ST7, GAS e NM23, inibendone la trascrizione. Si e’ inoltre provveduto a sviluppare una categoria di inibitori allosterici di PRMT5: l’attivita’ terapeutica/la specificita’ in vitro e la modalita’ di somministrazione ottimale in modelli murini di linfoma non-Hodgkin, sono in corso di valutazione.

Leukaemia, brain tumours and exposure to extremely low frequency magnetic fields: cohort study of Swiss railway employees

AIMS: To investigate the relationship between extremely low frequency magnetic field (ELF-MF) exposure and mortality from leukaemia and brain tumour in a cohort of Swiss railway workers. METHODS: 20,141 Swiss railway employees with 464,129 person-years of follow-up between 1972 and 2002 were studied. Mortality rates for leukaemia and brain tumour of highly exposed train drivers (21 muT average annual exposure) were compared with medium and low exposed occupational groups (i.e. station masters with an average exposure of 1 muT). In addition, individual cumulative exposure was calculated from on-site measurements and modelling of past exposures. RESULTS: The hazard ratio (HR) for leukaemia mortality of train drivers was 1.43 (95% CI 0.74 to 2.77) compared with station masters. For myeloid leukaemia the HR of train drivers was 4.74 (95% CI 1.04 to 21.60) and for Hodgkin's disease 3.29 (95% CI 0.69 to 15.63). Lymphoid leukaemia, non-Hodgkin's disease and brain tumour mortality were not associated with magnetic field exposure. Concordant results were obtained from analyses based on individual cumulative exposure. CONCLUSIONS: Some evidence of an exposure-response association was found for myeloid leukaemia and Hodgkin's disease, but not for other haematopoietic and lymphatic malignancies and brain tumours.

HIV-associated malignancies among children attending the Baylor Clinic in Kampala, Uganda

Introduction: HIV-associated malignancies such as Kaposi’s sarcoma and Non-Hodgkin’s lymphoma occur in children and usually lead to significant morbidity and mortality. No studies have been done to establish prevalence and outcome of these malignancies in children in a hospital setting in Uganda. ^ Research question: What proportion of children attending the Baylor-Uganda COE present with HIV-associated malignancies and what are the characteristics and outcome of these malignancies? The objective was to determine the prevalence, associated factors and outcome of HIV-associated malignancies among children attending the Baylor-Uganda Clinic in Kampala, Uganda. Study Design: This was a retrospective case series involving records review of patients who presented to the Baylor-Clinic between January 2004 and December 2008. Study Setting: The Baylor-Uganda Clinic, where I worked as a physician before coming to Houston, is a well funded, well staffed; Pediatric HIV clinic located in Mulago Hospital, Kampala, Uganda and is affiliated to Makerere University Medical School. Study Participants: Medical charts of patients aged 6 weeks to 18 years who enrolled for care at the clinic during the years 2004 to 2008 were retrieved for data abstraction. Selection Criteria: Study participants had to be patients of Baylor-Uganda seen during the study period; they had to be aged 6 weeks to 18 years; and had to be HIV positive. Patients with incomplete data or whose malignancies were not confirmed by histology were excluded. Study Variables: Data on patient’s age, sex, diagnosis, type of malignancy, anatomic location of the malignancy; pathology report, baseline laboratory results and outcome of treatment, were abstracted. Data Analysis: Cross tabulation to determine associations between variables using Pearson’s chi square at 95% level of significance was done. Proportions of malignancies among different groups were determined. In addition, Kaplan Meier survival analysis and comparison of survival distributions using the log-rank test was done. Change in CD4 percentages from baseline was assessed with the Wilcoxon signed rank test. Results: The proportion of children with malignancies during the study period was found to be 1.65%. Only 2 malignancies: Kaposi’s sarcoma and Non-Hodgkin’s lymphoma were found. 90% of the malignancies were Kaposi’s sarcoma. Lymph node involvement in children with Kaposi’s sarcoma was common, but the worst prognosis was seen with visceral involvement. Deaths during follow-up were seen in the first few weeks to months. Upon starting treatment the CD4 cell percentage increased significantly from a baseline median of 6% to 14% at 6 months and 15.8% at 12 months of follow-up.^

Mixture modeling for joint analysis of survival, discrete, and continuous data

Mixture modeling is commonly used to model categorical latent variables that represent subpopulations in which population membership is unknown but can be inferred from the data. In relatively recent years, the potential of finite mixture models has been applied in time-to-event data. However, the commonly used survival mixture model assumes that the effects of the covariates involved in failure times differ across latent classes, but the covariate distribution is homogeneous. The aim of this dissertation is to develop a method to examine time-to-event data in the presence of unobserved heterogeneity under a framework of mixture modeling. A joint model is developed to incorporate the latent survival trajectory along with the observed information for the joint analysis of a time-to-event variable, its discrete and continuous covariates, and a latent class variable. It is assumed that the effects of covariates on survival times and the distribution of covariates vary across different latent classes. The unobservable survival trajectories are identified through estimating the probability that a subject belongs to a particular class based on observed information. We applied this method to a Hodgkin lymphoma study with long-term follow-up and observed four distinct latent classes in terms of long-term survival and distributions of prognostic factors. Our results from simulation studies and from the Hodgkin lymphoma study demonstrated the superiority of our joint model compared with the conventional survival model. This flexible inference method provides more accurate estimation and accommodates unobservable heterogeneity among individuals while taking involved interactions between covariates into consideration.^