Rocio Hassan

Human Natural Killer Cells Prevent Infectious Mononucleosis Features by Targeting Lytic Epstein-Barr Virus Infection

Primary infection with the human oncogenic Epstein-Barr virus (EBV) can result in infectious mononucleosis (IM), a self-limiting disease caused by massive lymphocyte expansion that predisposes for the development of distinct EBV-associated lymphomas. Why some individuals experience this symptomatic primary EBV infection, whereas the majority acquires the virus asymptomatically, remains unclear. Using a mouse model with reconstituted human immune system components, we show that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis mainly because of a loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies.

Geographic variation in Epstein-Barr virus-associated Burkitt's lymphoma in children from Brazil

In developing countries, BL has a strong association with EBV infection during childhood. In South America, the data have shown an EBV association intermediate between that reported in the United States (30%) and that in equatorial Africa (95%). Early age at EBV infection and lower socioeconomic status have been related to increased EBV‐associated BL in developing countries. In Brazil, there are not enough data on childhood BL related to EBV infection. Our aim was to evaluate the clinicopathologic features and EBV association of 44 children with NHL from the state of Rio de Janeiro, situated in the southeast of Brazil. EBV was detected using RNA in situ hybridization in 36 biopsy specimens. DNA from fresh tumor samples and from paraffin‐embedded tissues of patients were analyzed by PCR, in which the first reaction included primers for an EBNA‐2 common region while the nested reaction amplified the region discriminating between EBV types 1 and 2 in separate reactions. EBV was detected in 21 of 29 BLs (72%), and type 1 virus infected the majority of EBV‐positive BLs (18/21). There was a trend for younger age in children with EBV‐positive BL compared to EBV‐negative BL (median age 4 compared to 6 years, respectively; p = 0.056). Our study confirmed that in the southeast of Brazil BL had an intermediate association with EBV. A higher rate of EBV‐associated BL was described in the northeast of Brazil. These differences are probably related to regional socioeconomic status. In conclusion, our study suggests that early infection with EBV in the background of a low socioeconomic condition associated with other environmental factors could contribute to BL in Brazil.

Tumor-Associated Macrophages in Pediatric Classical Hodgkin Lymphoma: Association with Epstein-Barr Virus, Lymphocyte Subsets, and Prognostic Impact

Purpose: Tumor-infiltrating macrophages are associated with adverse outcome in adult classical Hodgkin lymphoma (cHL). We have previously shown age-related changes in the lymphocyte composition of pediatric cHL. We therefore hypothesized that the number, function, and prognostic impact of macrophages in pediatric cHL would be different from adult cases. Experimental Design: We analyzed the number of macrophages and dendritic cells (DC) in the tumor microenvironment of pediatric cHL by immunohistochemistry. Results were analyzed in context of age, histologic characteristics, Epstein-Barr virus (EBV) status, clinical follow-up, and our previous study of T-cell populations in these cases. Results: One hundred cHL cases were studied, including 69% nodular sclerosis and 23% mixed cellularity cases. A total of 44.8% of cases were EBV-positive. Patients ≤10 years displayed more CD14+ cells (P = 0.025). In comparison with nodular sclerosis, mixed cellularity was characterized by higher numbers of CD14+, (P = 0.003) and CD163+ cells (P = 0.027). EBV+ cases exhibited higher numbers of CD14+ (P < 0.0005), CD68+ (P = 0.005), and CD163+ cells (P = 0.02). CD68-positive cells did not display an effect on outcome. Worse overall survival was observed in cases with CD163/CD8 ratio ≥2 (P = 0.007). High numbers of CD163+ cells were associated with worse progression-free survival (PFS; P = 0.015). Furthermore, high numbers of CD163+ and granzyme B+ cells were associated with worse PFS in EBV-negative (P = 0.005) but not in EBV-positive cases. Conclusion: Our results suggest that macrophage composition in pediatric cHL is distinct from adults. Functional status of macrophages and their value as prognostic indicators in pediatric cHL may depend on EBV status. Clin Cancer Res; 18(14); 3762–71. ©2012 AACR.

Tumor microenvironment composition in pediatric classical Hodgkin lymphoma is modulated by age and Epstein‐Barr virus infection

Classical Hodgkin lymphoma (cHL) is characterized by a small number of neoplastic cells in a background of reactive cells. Children and adults differ in constitution and functionality of the immune system and it is possible that there may be age‐related differences in tumor microenvironment composition in cHL. One hundred children with pediatric cHL were studied. Tumor‐infiltrating lymphocytes were analyzed by immunohistochemistry (IHC) and image analysis. Epstein‐Barr virus (EBV) status was determined by EBER‐specific in situ hybridization and IHC. Results were analyzed in the context of age‐group, histological characteristics and clinical follow‐up. EBV‐status was not associated with age‐group. Children <10 years and EBV+ cases were characterized by a more intense T cell infiltrate, exhibiting a cytotoxic/Th1 profile, characterized by higher numbers of CD3+, CD8+, TIA1+ and TBET+ lymphocytes. Extranodal disease (p = 0.016) and high number of GranzymeB+ lymphocytes (p = 0.04) were independently associated with reduced progression‐free survival (PFS). Yet, in EBV+ cases, improved outcome was observed in cases with low numbers of FOXP3+ lymphocytes (p = 0.046), FOXP3/CD8 ratio < 1 (p = 0.021) and TBET/CMAF ratio < 1 (p = 0.017). By contrast, in EBV− cases, poor survival was observed in cases with extranodal disease (p = 0.028), MC subtype (p = 0.009) and high numbers of TIA1+ (p = 0.044) and GranzymeB+ (p = 0.04) lymphocytes. The results suggest that in EBV+ cHL an effective immune response directed against viral or tumor antigens may be triggered in the tumor microenvironment and that physiological and age‐related changes of the immune system may also modulate the tumor microenvironment in pediatric cHL.

Granulocytic sarcoma of the small intestine with CBFβ/MYH11 fusion gene: report of an aleukaemic case and review of the literature

Granulocytic sarcomas (GS) are rare extramedullary tumours composed of immature myeloid cells. Inversion of chromosome 16 [inv(16)] is a cytogenetic marker for M4Eo subtype of acute myeloid leukaemia (AML). The possibility of an association between the development of granulocytic sarcoma of the small intestine (GSSI) and the M4Eo subtype of AML was suggested in nine previous case reports. Here we report an aleukaemic case of GSSI with inv(16) and its molecular equivalent, the CBFbeta/MYH11 fusion gene, detected by reverse transcriptase-polymerase chain reaction (RT-PCR), that after treatment with conventional AML chemotherapy followed by autologous bone marrow transplantation, achieved complete haematological and molecular remission on bone marrow examination. After chemotherapy, a thickened ileum wall positive for CBFbeta/MYH11 on tumour mass samples was still observed on computed tomography (CT) studies, raising the question of residual GS representing a reservoir of malignant cells. This case demonstrates the critical need of multidisciplinary diagnosis and follow-up of this entity combining immunopathologic, cytogenetic and molecular studies, reinforcing the potentiality of risk-adapted therapy strategies, as it is increasingly claimed for patients with overt AML.

miRNA-451: A putative predictor marker of Imatinib therapy response in chronic myeloid leukemia

In a recent issue of Leukemia Research, Lopotova et al. [1] sug-gest theexistenceofareciprocalregulatoryloopbetweenBCR-ABLand microRNA-451 (miR-451) as a maintenance mechanism of theleukemic stateofCMLcells.Theauthorsalsoreportthatdownreg-ulation of miR-451 might be inversely related to BCR-ABL kinaseactivity in chronic myeloid leukemia (CML) cells. MiRNAs are non-coding RNAs of 21–25 nucleotides that have been implicated ina number of biological processes, regulating gene expression bypromoting mRNA degradation or repressing its translation. Aber-rant miRNA expression has been described for a variety of solidtumors andhematologicalmalignancies,includingCML.SomemiR-NAs, as for example miR-150, miR-151, miR-10a and miR-96 wereseen aberrantly expressed in purified CD34+ population and totalleukocytes from peripheral blood of CML patients [2]. The role ofmiRNAs in CML resistance has not been thus far fully evaluated. Byreal-time PCR Lopotova et al. [1] investigated miR-451 expressionin samples of CML patients treated with Imatinib (IM) at the timeof diagnosis (n=14), in major molecular response (MMR; n=14),in hematological relapse (n=17) and in suboptimal response to IM(n =7) and found miR-451 down regulated in most of the diagno-sis and hematological relapse samples in contrast with normal orslightly increasedlevelsobservedinMMRandsuboptimalresponsesamples.

Pediatric Hodgkin Lymphoma in 2 South American Series: A Distinctive Epidemiologic Pattern and Lack of Association of Epstein-Barr Virus With Clinical Outcome

Hodgkin lymphoma (HL) shows a bimodal distribution with a first peak in developing countries during childhood. The causative role and prognostic significance of Epstein-Barr virus (EBV) association in patients with HL is controversial. Our aim was to perform a comparative study of EBV association in 2 Latin American pediatric HL series, and to correlate it with patients survival. Epstein-Barr encoded RNAs in situ hybridization and latent membrane protein 1 immunohistochemistry were performed on formalin-fixed, paraffin-embedded HL biopsies from 176 pediatric patients from 2 public institutions from Argentina and Southeast Brazil. Mixed cellularity subtype was prevalent in Argentine HL (Arg HL) (52%) and nodular sclerosis subtype in Brazilian HL (BR HL) (83%). EBV expression was detected in 52% of cases, namely 54% Arg HL and 48% Br HL. EBV was significantly associated with mixed cellularity subtype in both populations. In Arg HL, EBV positivity was significantly higher in patients ≤10 years (P=0.0011). Event-free survival did not attain statistical significance neither in Arg HL (P=0.5317), nor in Br HL (P=0.8321). Our results do not support EBV association stated for pediatric HL in developing countries. Correlation of younger age with EBV infection only in Argentine patients might be related to a different age background. In our pediatric series, EBV status cannot be used as prognostic factor.

Impact of complex NOTCH1 mutations on survival in paediatric T-cell leukaemia

BackgroundMolecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether NOTCH1 mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases.MethodsT-ALL subtypes, status of SIL-TAL1 fusion, ectopic expression of TLX3, and mutations in FBXW7, KRAS, PTEN and NOTCH1 were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test.ResultsThe frequencies of mutations were 43.5% for NOTCH1, while FBXW7, KRAS and PTEN exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between NOTCH1 mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. SIL-TAL1 was identified as an adverse prognostic factor. NOTCH1 mutation status was not associated with outcome, while the presence of NOTCH1 complex mutations (indels) were associated with a longer overall survival (p = 0.031) than point mutations.ConclusionNOTCH1 mutations alone or in combination with FBXW7 did not impact T-ALL prognosis. Nevertheless, complex NOTCH1 mutations appear to have a positive impact on OS and the SIL-TAL1 fusion was validated as a negative prognostic marker in our series of T-ALL.

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCR&ggr; PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.

Macrophage polarization reflects T cell composition of tumor microenvironment in pediatric classical Hodgkin lymphoma and has impact on survival.

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.