IntroductionThere is increasing evidence of the importance of the immune system in cancer development and control. 1,2 Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) in children and adolescents represents an ideal tumor model to study the existence and impact of a tumor-specific immune response. First, all tumor cells express high levels of ALK in the form of ALK fusion proteins resulting from oncogenic chromosomal translocations. In contrast to many other tumor-associated antigens (TAAs), ALK fusion proteins exert an essential role in lymphomagenesis and the survival of the tumor cells, therefore representing real oncoantigens. 3 The most common ALK fusion protein is nucleophosmin (NPM)-ALK resulting from the t(2;5)(p23;q35) translocation occurring in 90% of ALK-positive ALCLs. 4 Second, the normal tissue distribution of ALK is restricted to a few scattered neurons in the central nervous system, 5 thus permitting specific targeting of ALK while minimizing problems with autoimmunity. Third, immunity to ALK may be implicated in the control of ALK-positive ALCLs. Antibodies against ALK and cytotoxic T-cell and CD4 T-helper responses to ALK have been detected in patients with ALK-positive ALCLs both at the time of diagnosis and during remission. 6-10 Furthermore, murine studies have identified ALK as an ideal tumor antigen for vaccination-based therapies. 11The current study was therefore performed in a large cohort of uniformly treated children and adolescents with ALK-positive ALCLs to investigate (1) the prevalence of a preexisting antibody response to ALK and (2) whether the strength of the antibody response correlates with parameters of tumor dissemination and the risk of relapse. Methods PatientsA total of 236 patients from the Berlin-Frankfurt-Muenster group study NHL-BFM95 study with a diagnosis of ALCL and German patients enrolled in the European inter-group trial ALCL99 between April 1996 and November 2007 were eligible for this study. Both studies were approved by the institutional Ethics committee of the primary investigator of the BFM group (A.R.) at Justus-Liebig-University. NPM-ALK positivity of the ALCL was confirmed by at least one of the following: presence of NPM-ALK mRNA, positive 2-color fluorescence in situ hybridization for the t(2;5)(p23;q35) NPM-ALK translocation, and/or immunolabeling studies to show the presence of nuclear and cytoplasmic ALK in the tumor cells.The treatment strategy was based on protocol NHL-BFM90, as described previously. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From system) and the involvement of at-risk organs. 14 Staging procedures included bone marrow (BM) aspiration cytology and a spinal tap. Detection and quantification of submicroscopic amounts of circulating tumor cells in BM or blood at diagnosis were carried out by quantitative real-time polymerase chain reaction (PCR) for NPM-ALK transcripts as previously described. 15 Serum and/or plasma samples were obtained at the time of diagnosis from 95 patie...

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) has a good prognosis compared to ALK-negative ALCL, possibly as a result of the immune recognition of the ALK proteins. The aim of our study was to investigate the presence of both a B and cytotoxic T cell (CTL) response to ALK in ALK-positive ALCL. We confirmed the presence of an antibody response to ALK in all 9 ALK-positive ALCL patients investigated. An ELISpot assay was used to detect a c-interferon (IFN) T cell response after short term culture of mononuclear blood cells with 2 ALK-derived HLA-A*0201 restricted peptides: ALKa and ALKb. A significant c-IFN response was identified in all 7 HLA-A*0201-positive ALK-positive ALCL patients but not in ALKnegative ALCL patients (n 5 2) or normal subjects (n 5 6). CTL lines (>95% CD8-positive) raised from 2 ALK-positive ALCL patients lysed ALK-positive ALCL derived cell lines in a MHCClass I restricted manner. This is the first report of both a B cell and CTL response to ALK in patients with ALK-positive ALCL. This response persisted during long-term remission. The use of modified vaccinia virus Ankara (MVA) to express ALK is also described. Our findings are of potential prognostic value and open up therapeutic options for those ALK-positive patients who do not respond to conventional treatment. ' 2005 Wiley-Liss, Inc.

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A meningococcal genomic expression library was screened for potent CD4+ T‐cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto‐transporter A) as its peptide sequence shares molecular characteristics of the auto‐transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T‐cell responses in PBLs of patients and some healthy donors, and induced strong primary T‐cell responses in healthy donors. The human B‐cell immunogenicity and cross‐reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross‐reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB‐specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross‐react with AutA. Thus, AutA, being a potent CD4+ T‐cell and B‐cell‐stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.

SummaryThe identification of immunogenic cancer testis antigens (CTAs) as immunotherapeutic targets represents one approach to improve treatment options for diffuse large B-cell lymphoma (DLBCL). We previously identified PASD1 [PAS (Per ARNT Sim) domain containing 1 (PASD1)], a DLBCLassociated CTA that was expressed in a range of hematopoietic malignancies. The aim of the present study was to investigate the presence of a cytotoxic T-cell (CTL) response to PASD1 in DLBCL patients. A significant cinterferon (IFN) release was detected in 21/29 HLA-A*0201-positive DLBCL patients (18 de novo DLBCL, two transformed DLBCL and one T-cell rich B-cell lymphoma) following short-term culture of their peripheral blood mononuclear cells stimulated with five HLA-A*0201-restricted PASD1 peptides. No significant responses were detected in 21 HLA-A*0201-negative DLBCL patients (12 de novo DLBCL, seven transformed DLBCL, two T-cell rich B-cell lymphoma) or six normal subjects. CTL cell lines were able to lyse PASD1-positive tumour cells in a major histocompatibility complex-Class I dependent manner. The presence of a c-IFN response correlated with PASD1 protein expression in the tumour cells in 12/15 cases studied. This is the first report of a CTL response to a CTA in DLBCL. Our results provide additional valuable evidence supporting PASD1 as a potential immunotherapeutic target for the treatment of DLBCL and other malignancies.

We have previously shown both humoral and CTL responses to anaplastic lymphoma kinase (ALK) in patients with ALKpositive anaplastic large-cell lymphoma (ALCL). However, because CD4 + T-helper (Th) cells also play a vital role in developing and maintaining tumor immunity, we investigated the presence of a CD4 + Th response in ALK-positive ALCL. Using an IFN-; ELISPOT assay, we identified two ALK-derived DRB1-restricted 24-mer promiscuous peptides, ALK1 278-301 and ALK2 , as being immunogenic in six ALK-positive ALCL patients but not in two ALK-negative ALCL patients or five normal subjects. A significant interleukin-4 response to the ALK peptides was detected in only one ALK-positive patient.

naplastic large-cell lymphoma (ALCL) makes up approximately 15% of pediatric non-Hodgkin's lymphoma (NHL) and the vast majority of them is associated with the t(2;5) translocation that results in the expression of a hybrid oncogenic tyrosine kinase. It consists of the amino terminus of the nuclear protein nucleophosmin (NPM) fused to the intracytoplasmic region of the anaplastic lymphoma kinase (ALK) that impacts many cellular responses including proliferation, growth and apoptosis.1 In addition to its direct effects of ALK on signal transduction pathways, ALK overexpression may also induce a host immune reaction, giving rise to autologous anti-ALK antibodies in patients with ALK-positive ALCL. Circulating antibodies against NPM-ALK were originally reported in 11 ALK-positive ALCL patients using an immunocytochemical approach.2 In that study, no conclusions concerning the clinical significance of anti-NPM-ALK antibodies could be drawn, due to the small number of patients; however, a tendency to higher antibody levels in pretreatment blood samples was observed. Recently, we demonstrated that minimal bone marrow (BM) involvement at diagnosis is a common event in pediatric ALCL and that minimal BM disease monitoring can identify patients at risk of relapse.3 In addition, Damm-Welk et al.4 showed that patients with more than 10 normalized copy numbers (NCNs) NPM-ALK in BM had a cumulative incidence of relapse (CI-R) of 71plusminus14% compared with CI-R of 18plusminus6% for patients with 10 or fewer NCNs (P<0.001) and that quantitative PCR for NPM-ALK in BM and peripheral blood (PB) is highly concordant. In this study, we assessed the levels of anti-NPM-ALK antibodies at diagnosis and at stop therapy in plasma of children with ALCL treated according to the ALCL-99 protocol5 and evaluated the possible correlation with minimal residual disease (MRD) status during chemotherap

In the 1997-98 academic year, we conducted a longitudinal study of meningococcal carriage and acquisition among first-year students at Nottingham University, Nottingham, United Kingdom. We examined the dynamics of long-term meningococcal carriage with detailed characterization of the isolates. Pharyngeal swabs were obtained from 2,453 first-year students at the start of the academic year (October), later on during the autumn term, and again in March. Swabs were immediately cultured on selective media, and meningococci were identified and serologically characterized. Nongroupable strains were genetically grouped using a PCR-based assay. Pulsed-field gel electrophoresis was used to determine the link between sequential isolates. Of the carriers initially identified in October, 44.1% (98 of 222) were still positive later on in the autumn (November or December); 57.1% of these remained persistent carriers at 6 months. Of the index carriers who lost carriage during the autumn, 16% were recolonized at 6 months. Of 344 index noncarriers followed up, 22.1% acquired carriage during the autumn term and another 13.7% acquired carriage by March. Overall, 43.9% (397 of 904) of the isolates were noncapsulated (serologically nongroupable); by PCR-based genogrouping, a quarter of these belonged to the capsular groups B and C. The ratio of capsulated to noncapsulated forms for group B and C strains was 2.9 and 0.95, respectively. Sequential isolates of persistent carriers revealed that individuals may carry the same or entirely different organisms at different times. We identified three strains that clearly switched their capsular expression on and off at different times in vivo. One student developed invasive meningococcal disease after carrying the same organism for over 7 weeks. The study revealed a high rate of turnover of meningococcal carriage among students. Noncapsulated organisms are capable of switching their capsular expression on and off (both ways) in the nasopharynx, and group C strains are more likely to be noncapsulated than group B strains. Carriage of a particular meningococcal strain does not necessarily protect against colonization or invasion by a homologous or heterologous strain.