Giovanna Roncador

Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level.

The transcription factor FOXP3 plays a key role in CD4+CD25+ regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4+CD25+ cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti‐FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow‐cytometric analysis showed that FOXP3 was expressed by the majority of CD4+CD25high T cells in peripheral blood. By contrast, less than half of the CD4+CD25int population were FOXP3+, providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4+CD25high pool. Although FOXP3 expression was primarily restricted to CD4+CD25+ cells, it was induced following activation of both CD4+ and CD8+ T cell clones. These findings indicate that the frequency of FOXP3+ cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4+ and CD8+ T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells.

Purpose: Recent studies of Hodgkins lymphoma (HL) have suggested that the presence of regulatory T cells in the reactive background may explain the inhibition of the antitumoral host immune response observed in these patients. This study aimed to assess the relevance of regulatory T cells and CTLs present in the background of HL samples in the prognosis of a series of classic HL (cHL) patients. Experimental Design: Expression of granzyme B and TIA-1 (markers for CTL) and FOXP3 (a marker for regulatory T cells) were evaluated independently by immunohistochemistry in tissue microarrays of 257 cHL patients and correlated with patient outcome. Results: The combined influence of the presence of FOXP3+ and TIA-1+ cells distinguished three risk groups of patients with 5-year overall survival of 100%, 88%, and 73%. The presence of a small number of FOXP3+ cells and a high proportion of TIA-1+ cells in the infiltrate represent an independent prognostic factor that negatively influenced event-free survival and disease-free survival in cHL. Compared with the features at diagnosis, relapsed samples tended to have more TIA-1+ cells and a lower proportion of FOXP3+ cells in the reactive background. Conclusions: These data suggest that low infiltration of FOXP3+ cells in conjunction with high infiltration of TIA-1+ cells in cHL may represent biological markers predicting an unfavorable outcome. Moreover, the variation of these markers over the course of the disease implies a possible role for them in the progression of HL cases.

Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma

Hereditary pheochromocytoma (PCC) is often caused by germline mutations in one of nine susceptibility genes described to date, but there are familial cases without mutations in these known genes. We sequenced the exomes of three unrelated individuals with hereditary PCC (cases) and identified mutations in MAX, the MYC associated factor X gene. Absence of MAX protein in the tumors and loss of heterozygosity caused by uniparental disomy supported the involvement of MAX alterations in the disease. A follow-up study of a selected series of 59 cases with PCC identified five additional MAX mutations and suggested an association with malignant outcome and preferential paternal transmission of MAX mutations. The involvement of the MYC-MAX-MXD1 network in the development and progression of neural crest cell tumors is further supported by the lack of functional MAX in rat PCC (PC12) cells and by the amplification of MYCN in neuroblastoma and suggests that loss of MAX function is correlated with metastatic potential.

Antigen retrieval techniques in immunohistochemistry: comparison of different methods.

Routine sections of normal and pathological samples fixed in 10 per cent buffered formalin or B5, including EDTA‐decalcified bone‐marrow biopsies, were tested with 61 antibodies following heating in three different fluids: 0·01 m citrate buffer (pH 6·0), 0·1 m Tris–HCl (pH 8·0), and 1 mm EDTA–NaOH solution (pH 8·0). The sections underwent either three cycles of microwave treatment (5 min each) or pressure cooking for 1–2 min. The alkaline phosphatase/anti‐alkaline phosphatase (APAAP) technique was used as the standard detection method; with 16 antibodies a slightly modified streptavidin–biotin complex (SABC)‐immunoperoxidase technique was applied in parallel. The results obtained were compared with those observed without any antigen retrieval (AR), or following section digestion with 0·05 per cent protease XIV at 37°C for 5 min. Chess‐board titration tests showed that all antibodies but one profited by AR. Protease XIV digestion represented the gold standard for five antibodies, while 55 produced optimal results following the application of heat‐based AR. By comparison with the other fluids, EDTA appeared to be superior in terms of both staining intensity and the number of marked cells. These results were independent of tissue processing, immunohistochemical approach, and heating device. Pressure cooking was found to be more convenient on practical grounds, as it allowed the simultaneous handling of a large number of slides and a time saving of 1 min 30 s, representing the proper time for the treatment.

High Numbers of Tumor-Infiltrating Programmed Cell Death 1–Positive Regulatory Lymphocytes Are Associated With Improved Overall Survival in Follicular Lymphoma

PURPOSE Tumor microenvironment influences the behavior of follicular lymphoma (FL), although the specific cell subsets involved are not well known. The aim of this study was to determine the impact of programmed cell death 1 (PD-1) -positive inhibitory immunoregulatory lymphoid cells in the clinicobiologic features and outcome of patients with FL. PATIENTS AND METHODS We examined samples from 100 patients (53 men and 47 women; median age, 54 years) at diagnosis, as well as in 32 patients at first relapse, with a recently generated monoclonal antibody against PD-1. The cells were quantified using computerized image analysis. Additional analysis consisted of double immunofluorescence and flow cytometry. RESULTS PD-1 expression was alternative to FOXP3 in lymphoid cells from both reactive tonsils and FL. At diagnosis, the median percentage of PD-1-positive cells was 14% (range, 0.1% to 74%). Patients with grade 3 FL, poor performance status, and high serum lactate dehydrogenase showed lower numbers of PD-1-positive cells. After a median follow-up of 6.2 years, patients with PD-1-positive cells <or= 5% (n = 25), 6% to 33% (n = 50), and more than 33% (n = 25) had a 5-year progression-free survival rate of 20%, 46%, and 48% (P = .038) and overall survival (OS) of 50%, 77%, and 95% (P = .004), respectively. PD-1 and FL International Prognostic Index maintained prognostic value for OS in multivariate analysis. Patients with PD-1-positive cells <or= 5% showed a higher risk of histologic transformation. At that time, transformed diffuse large B-cell lymphomas had lower percentage of PD-1-positive cells than FL. CONCLUSION A high content of PD-1-positive cells predicted favorable outcome of FL patients, whereas a marked reduction is observed in transformation.

MAX mutations cause hereditary and sporadic pheochromocytoma and paraganglioma.

Purpose: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest–derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL. Design: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics. Results: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine. Conclusions: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients. Clin Cancer Res; 18(10); 2828–37. ©2012 AACR.

Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors.

HLA‐F is a human non‐classical MHC molecule. Recombinant HLA‐F heavy chain was refolded with β2‐microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high‐affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA‐F. HLA‐F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA‐F could be immunoprecipitated from B cell lines and from HUT‐78, a T cell line. HLA‐F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA‐F tetramers stain peripheral blood monocytes and B cells. HLA‐F tetramer binding could be conferred on non‐binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA‐F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA‐F, suggest that HLA‐F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.

Primary cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma expresses follicular T-cell markers.

Cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma (CSTCL) is a cutaneous T-cell lymphoma defined by a predominance of small-to-medium–sized CD4+ pleomorphic T cells, with a favorable clinical course. Cases are also characterized by the presence of a rich infiltrate of reactive B cells. Recently, it has been reported that follicular helper T cells (TFH cells) display a distinct gene expression profile, positive for PD-1, CXCL13, and BCL-6. We report for the first time the expression of PD-1 and other TFH cell markers in CSTCLs and discuss its biologic significance. Sixteen CSTCLs were included in this study, and also 20 reactive inflammatory conditions, 10 primary cutaneous marginal zone, 10 follicular center lymphomas, and 5 primary CD30+ cutaneous lymphomas. They were immunohistochemically analyzed for a large panel of markers. Double immunoperoxidase labeling of paraffin sections was performed for PD-1, OCT-2, and BCL-6. Clonal Ig and T-cell receptor rearrangements and Epstein-Barr virus-encoded RNA expression were also evaluated. Morphologic and clinical data were reviewed. Histologic examination showed a dense polymorphic lymphoid infiltrate throughout the dermis. Atypical large CD4+ cells were positive for PD-1, CXCL13, and BCL-6 in all cases, and were attached in small clusters, or formed rosettes around CD30/OCT-2+ B blast cells. Epstein-Barr virus was not apparent in any of the cases. A dominant T-cell clone was identified in 14 cases, whereas polymerase chain reaction IgH gene rearrangement studies showed that all cases were polyclonal. None of the patients had lymphadenopathy or showed any evidence of systemic disease, nor did they have any previous history of mycosis fungoides or drug reactions. FTH cell markers are not exclusive to angioimmunoblastic lymphadenopathy but may also be seen in neoplastic cells of CSTCLs. Moreover, these findings suggest that B-cell stimulation by FTH could also take place in some cutaneous T-cell lymphomas.

Molecular Characterization of a New ALK Translocation Involving Moesin (MSN-ALK) in Anaplastic Large Cell Lymphoma

The majority of anaplastic large cell lymphomas (ALCL) are associated with chromosomal abnormalities affecting the anaplastic lymphoma kinase (ALK) gene which result in the expression of hybrid ALK fusion proteins in the tumor cells. In most of these tumors, the hybrid gene comprises the 5′ region of nucleophosmin (NPM) fused in frame to the 3′ portion of ALK, resulting in the expression of the chimeric oncogenic tyrosine kinase NPM-ALK. However, other variant rearrangements have been described in which ALK fuses to a partner other than NPM. Here we have identified the moesin (MSN) gene at Xq11–12 as a new partner of ALK in a case of ALCL which exhibited a distinctive membrane-restricted pattern of ALK labeling. The hybrid MSN-ALK protein had a molecular weight of 125 kd and contained an active tyrosine kinase domain. The unique membrane staining pattern of ALK is presumed to reflect association of moesin with cell membrane proteins. In contrast to other translocations involving the ALK gene, the ALK breakpoint in this case occurred within the exonic sequence coding for the juxtamembrane portion of ALK. Identification of the genomic breakpoint confirmed the in-frame fusion of the whole MSN intron 10 to a 17 bp shorter juxtamembrane exon of ALK. The breakpoint in der(2) chromosome showed a deletion, including 30 bp of ALK and 36 bp of MSN genes. These findings indicate that MSN may act as an alternative fusion partner for activation of ALK in ALCL and provide further evidence that oncogenic activation of ALK may occur at different intracellular locations.

FOXP3, a selective marker for a subset of adult T-cell leukaemia/lymphoma.

FOXP3 is a forkhead transcription factor family member, implicated in T-cell regulation, activation and differentiation. FOXP3 has been shown to be a master control gene for the development and function of CD4+/CD25+ regulatory T-cells (Treg). In this study, FOXP3 protein expression has been analysed using a new anti-FOXP3 monoclonal antibody in 172 paraffin-embedded lymphoma samples. FOXP3 expression in tumour cells was confined to adult T-cell leukaemia/lymphoma (ATLL) cases (17/25, 68%), with some variability in the intensity of the staining and the proportion of positive cells. No other lymphoma types studied exhibited FOXP3 expression in the malignant population. The selective expression of FOXP3 by tumour cells in ATLL makes this antibody a potentially useful diagnostic tool.