Lymphoma Biopsies Research Articles

Predicting Outcome in Follicular Lymphoma by Using Interactive Gene Pairs

Follicular lymphoma is a common lymphoma of adults. Although its course is often indolent, a substantial proportion of patients have a poor prognosis, often due to rapid progression or transformation to a more aggressive lymphoma. Currently available clinical prognostic scores, such as the follicular lymphoma international prognostic index, are not able to optimally predict transformation or poor outcome. Gene expression profiling was done on primary lymphoma biopsy samples. Using a statistically conservative approach, predictive interaction analysis, we have identified pairs of interacting genes that predict poor outcome, measured as death within 5 years of diagnosis. The best gene pair performs >1,000-fold better than any single gene or the follicular lymphoma international prognostic index in our data set. Many gene pairs achieve outcome prediction accuracies exceeding 85% in extensive cross-validation and noise sensitivity computational analyses. Many genes repeatedly appear in top-ranking pairs, suggesting that they reproducibly provide predictive capability. The evidence reported here may provide the basis for an expression-based, multi-gene test for predicting poor follicular lymphoma outcomes.

Protein Profiling of B-Cell Lymphomas Using Tissue Biopsies: A Potential Tool for Small Samples in Pathology

Non-Hodgkin’s lymphoma comprises many related but distinct diseases and diagnosis and classification is complex. Protein profiling of lymphoma biopsies may be of potential value for use in this lymphoma classification and the discovery of novel markers. In this study, we have optimized a method for SELDI-TOF MS based protein profiling of frozen tissue sections, without dissection of tumour cells. First we have compared chip surfaces and lysis buffers. Also, we have determined the minimal input using laser dissection microscopy. Subsequently, we have analyzed and compared protein profiles of diffuse large B-cell lymphoma (n=8), follicular lymphoma (n=8) and mantle cell lymphoma (n=8). Benign, reactive lymph nodes (n=14) were used as a reference group.CM10 chip surface in combination with urea lysis buffer and an input of approximately 50,000 lymphocytes allowed the detection of many differential peaks. Identification of the diffuse large B-cell lymphoma cases was reliably made in the supervised classification. Unsupervised clustering showed segregation into a benign/indolent cluster predominantly formed by benign, reactive lymph nodes and follicular lymphoma cases and into a more aggressive cluster formed by diffuse large B-cell lymphoma and mantle cell lymphoma cases. In conclusion, our protocol enables protein profiling of protein lysates derived from small histological samples and the subsequent detection of many differentially expressed proteins, without the need of tumour cell dissection. These results support further evaluation of protein profiling of small lymphoma biopsies as an additional tool in pathology.

Secretion of Epstein-Barr virus-encoded BARF1 oncoprotein from latently infected B cells.

Epstein-Barr virus (EBV) encodes two oncogenes, LMP1(Latent Membrane Protein-1) and BARF1 (BamH1-A Reading Frame-1). LMP1 belongs to latent gene family and BARF1 is considered so far as one of early gene family. However BARF1 oncogene was expressed highly in Nasopharyngeal (NPC) and gastric (GC) carcinoma as a type II latency, and in EBV-positive Akata cell and primary epithelial cell infected in vitro by EBV as type I latency. Its expression was also reported in Burkitt's lymphoma's biopsy frequent in Malawi in Africa as well as in nasal NK/T-cell lymphoma. We recently observed a massive secretion of BARF1 protein in serum and saliva of NPC patients. NPC-derived c666-1 epithelial cells also expressed and secreted BARF1 protein without other lytic genes expression. We asked whether this oncogene belongs to latent gene family. To investigate, we examined its transcriptional and translational expression in IB4 and Akata B cells where both cell lines belong to latent cell family. Transcriptional expression was analyzed by RT-PCR. As BARF1 protein is one of secreted proteins, its translational expression was analyzed by immunoblot after concentration of culture medium. Secreted BARF1 protein was futher purified by concanavalin A affinity column. BARF1 was transcribed in both EBV-positive AKATA and IB4 cells, and BARF1 protein was secreted from these latently infected human B cells. Its secretion does not depend EBV genome form in infected cells. Both episomal and integrated form of EBV genome were capable of expressing BARF1 gene. These results suggests that BARF1 is expressed in latent stage and increases its expression during lytic stage.

F18 fluorodeoxyglucose uptake in progressive transformation of germinal centres

FDG-PET/CT is a widely established imaging modality for staging, restaging and monitoring therapy response in lymphoma patients. Progressive transformation of germinal centres (PTGC) is a benign condition presenting characteristically as asymptomatic lymphadenopathy. This paper presents a case of a 53-year-old man with a history of Hodgkin’s disease (HD) whose F18 FDG-PET/CT scan showed high uptake in left axillary lymph nodes (SUV 3.8). A subsequent, left axillary lymph node biopsy revealed PTGC. PTGC can present as a false positive finding on FDG-PET/CT in lymphoma patients and biopsy should be done in HD patients in clinical remission but have a positive FDG-PET/CT scan.

B Cell Lymphomas Escape Bone Morphogenetic Protein (BMP) Induced Growth Inhibition: Downregulation of BMP Receptors and Upregulation of Inhibitory Smads.

Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and mediate their effects mainly through the Smad signaling pathway. TGF-β is one of the most potent negative regulators in hematopoietic cells, and many cancers develop reduced sensitivity towards TGF-β induced growth inhibition by several mechanisms, including functional loss of TGF-β receptors and Smad proteins. We have previously shown that BMP-6 inhibits the growth of normal peripheral blood B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that reduced sensitivity towards BMP-induced growth inhibition might contribute to lymphomagenesis. In the current study, 10 B lymphoma cell lines (representing Burkitt, DLBCL and FL) and tumor material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We found that 5 – 7 out of 10 lymphoma cell lines were resistant towards BMP-2, -4, -6 and -7 induced growth inhibition. In comparison, only 3 of the cell lines were resistant towards TGF-β. Analysis of BMP receptor expression by FACS analysis showed that all lymphoma cell lines and the malignant B cells from primary lymphoma biopsies expressed the BMPR type I Alk-2, whereas the expression of Alk-3 and Alk-6 was variable. Interestingly, the expression of BMPRII was low or undetectable in BMP-6 resistant cell lines, whereas it was highly expressed in 3 out of 4 sensitive cell lines. Also, malignant B cells from lymphoma biopsies showed reduced levels of BMPRII, suggesting that downregulation of BMPRII is a mechanism for evading BMP induced growth inhibition. Interestingly, upregulation of Smad6 or Smad7 was seen in 3 of the BMP-6 resistant cell lines and might represent another way of escaping the inhibitory effects of BMP. The lymphoma cell lines were investigated for endogenous production of BMPs by real-time RT-PCR, and 2 out of 10 cell lines had detectable BMP-6 mRNA, whereas 7 cell lines expressed BMP-7 mRNA. Analysis of purified malignant B cells or normal tumor infiltrating T cells from patient biopsies, confirmed the expression of BMP-6 and BMP-7 in the malignant B cells. Altogether, the data suggest that escape from BMP induced growth inhibition might contribute to increased tumor growth in B cell lymphomas.

Ultrasound Guided Core Biopsy of the Spleen in Lymphoma Diagnosis.

Patients with clinical or radiological splenic abnormalities are frequently referred to the lymphoma service for assessment. Previously diagnosis would require open biopsy or diagnostic splenectomy, with significant associated morbidity. Cytological examination of fine needle aspiration biopsy material is insufficient for accurate diagnosis and classification of lymphoma. Concerns regarding the risk of splenic core biopsy have limited its application. We report our experience with an ultrasound (US) guided technique, with particular reference to diagnostic yield, accuracy and complication rate. Sixty-two procedures were performed in 57 patients, 28 males and 34 females, median age 61 years (range 18–84). Biopsy was requested for diagnosis of isolated splenic abnormalities (n=28) or where the spleen represented the primary clinical or radiographic abnormality (n=34). Splenic abnormalities evaluated by US or computed tomography were defined as diffuse (n=26), multiple focal (n=24) or solitary focal (n=12) lesions. Using a subcostal or intercostal approach, a cutting needle was passed under US guidance during tidal respiration. Seven patients received platelet transfusion prior to biopsy for platelet counts <100 × 109/L. Coagulation screening tests were normal in all cases. A median of two cores (range 1–4) was obtained. The complication rate was very low. Only 1 patient suffered a significant haemorrhage despite a normal platelet count. This was managed conservatively and no patient required surgical intervention. Eight patients experienced local discomfort requiring analgesia before discharge. Pathological samples adequate for diagnosis were obtained from 59/62 (95%) procedures. Of these 59 specimens, malignant diagnoses were reached in 34 (31 lymphoma, 3 metastatic adenocarcinoma), from patients with both diffusely abnormal spleens (n=14) and focal splenic lesions (n=20). Fourteen specimens revealed benign pathology (including extramedullary hemopoiesis, granulomas and chronic inflammation) whilst 11 were normal. Sufficient material was obtained for immunohistochemistry to be performed on all 31 lymphoma biopsies allowing sub-classification in all but one case. Lymphoma diagnoses were diffuse large B cell lymphoma (DLBCL) (n=17), splenic marginal zone lymphoma (n=8), nodular lymphocyte predominant hodgkin's disease (n=2), hodgkin's lymphoma (n=1), mantle cell lymphoma (n=1), lymphoplasmacytoid lymphoma (n=1), low grade non-hodgkin's lymphoma (n=1). In 5 patients with DLBCL the spleen was the only site of disease. Subsequent therapeutic splenectomy was performed in 14 patients (17 prior biopsies) and demonstrated no false positives and 1 false negative core biopsy result. The 20 remaining patients with normal or benign spleen histology on core biopsy have reached a median follow-up of 21 months (range 3–157). No further false negative results have been identified by alternative tissue biopsy (n=10) or clinical evaluation. The 1 false negative core biopsy was followed immediately by splenectomy due to high clinical suspicion and a diagnosis of hepatosplenic T cell lymphoma was reached. US guided core biopsy of the spleen is a safe procedure with high accuracy. It allows a spleen conserving approach and we recommend it as a technique in the diagnosis or exclusion of splenic lymphoma.

Expression of two markers of germinal center T cells (SAP and PD-1) in angioimmunoblastic T-cell lymphoma

In the present paper we report that SAP, an intracytoplasmic molecule that is involved in cell signaling, is an immunohistologic marker for germinal center T cells in paraffin-embedded tissue. We document its expression, and also that of PD-1 (another recently described marker of germinal center T cells to which a new antibody has been raised), in normal and neoplastic lymphoid tissue to evaluate the suggestion that helper T cells within the germinal centers of human lymphoid tissue are the cell of origin of angioimmunoblastic T-cell lymphoma (AITL), and to assess the diagnostic value of these two markers. Expression of SAP and PD-1 was investigated by immunohistochemistry in paraffin-embedded tissue sections and in cell lines. Western blotting was performed on cell lines, and antibody specificity was confirmed by immunostaining of transfected cells. RESULTS Screening on more than 500 lymphoma biopsies showed that 95% (40/42) of cases of AITL expressed at least one of these markers. SAP was also expressed on many cases (15/21) of acute T lymphoblastic leukemia, in keeping with its presence in cortical thymocytes. However, PD-1 and SAP were also found in a minority of cases of peripheral T-cell lymphoma other than AITL, in contrast to a report that the former marker is specific for AITL. This observation raises the possibility that such non-angioimmunoblastic cases may be related to germinal center helper T cells. These two markers provide additional evidence that AITL arises from germinal center T cells. They may also prove of value in the diagnosis of this disease since a negative reaction was rarely observed in this disorder.

Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma

Single-nucleotide polymorphism (SNP) array analysis was performed using the 10K GeneChip array on a series of 26 paired follicular lymphoma (FL) and transformed-FL (t-FL) biopsies and the lymphoma cell lines SCI-1, DoHH2 and RL2261. Regions of acquired homozygosity were detected in 43/52 (83%) primary specimens with a mean of 1.7 and 3.0 aberrations in the FL and t-FL, respectively. A notable feature was the occurrence of recurring sites of acquired uniparental disomy (aUDP) on 6p, 9p, 12q and 17p in cell lines and primary samples. Homozygosity of 9p and 17p arose predominantly in t-FL and in three cases rendered the cell homozygous for a pre-existing mutation of either CDKN2A or TP53. These data suggest that mutation precedes mitotic recombination, which leads to the removal of the remaining wild-type allele. In all, 18 cases exhibited abnormalities in both FL and t-FL samples. In 10 cases blocks of homozygosity were detected in FL that were absent in the subsequent t-FL sample. These differences support the notion that FL and t-FL may arise in a proportion of patients by divergence from a common malignant ancestor cell rather than by clonal evolution from an antecedent FL.

Lack of an association between Chlamydia psittaci and ocular adnexal lymphoma

The objective of this study was to assess whether there is PCR evidence for C. psittaci DNA in ocular adnexal lymphoma specimens collected in an academic institution in the U.S. This was a retrospective, single-center study of patients from 1994 – 2004. We used 28 ocular adnexal lymphoma biopsy specimens from adult patients, 16 control lymphoma specimens from patients with systemic lymphomas not involving the ocular adnexa, and five control benign adnexal tissue samples. The presence of C. psittaci DNA was investigated by polymerase chain reaction (PCR) in each group. Two different assays were utilized: (1) conventional PCR/gel based assay targeting a 111-bp fragment of the 16S gene and (2) a real-time PCR assay amplifying a 148-bp portion of the 16S gene with detection via a specific fluorescent probe. Amplification was carried out to 60 cycles. Positive controls consisted of isolated DNA from C. psittaci strains VS1, CP3, and FP. A human DNA internal control was used to assess sample DNA quality and amplification success. Mean outcome measure was the presence of C. psittaci DNA. Using both assays, all patient samples in all categories yielded negative results. Both assays detected C. psittaci DNA from isolated strains. Internationally, Chlamydia psittaci has been associated with ocular adnexal lymphomas with great variability. Similar to several other recent studies in the USA, our study could not confirm the presence of C. psittaci in ocular adnexal lymphomas. Differences in the prevalence of C. psittaci infection in various geographic regions or technical differences in the application of the assays may underlie the variability in the association between C. psittaci and ocular adnexal lymphoma.

Tumor-Infiltrating T Cells Are Not Predictive of Clinical Outcome in Follicular Lymphoma.

Background: Follicular lymphoma (FL) has variable clinical outcomes. It has been suspected that tumor-infiltrating immune cells affect the biology and outcome of this disease. Using gene expression profiling and immunoperoxide tissue staining techniques, T cells and macrophages have been related to the survival outcome in some studies, but not others. In the current study, we used flow cytometry to analyze T cells and their subsets in follicular lymphoma biopsy specimens and determined whether these cell populations correlated with clinical features and outcomes.Methods: Two hundred and eighty-nine follicular lymphoma patients (pt) presented from 1997 to 2003 underwent an excisional lymph node biopsy prior to any treatment. The median age of pt at diagnosis was 45.7 yrs, median follow-up was 8.6 yrs for living pts, and median survival was 15.7 yrs. All but 8 patients had stage III/IV disease, 5 had stage I/II, and 3 were unknown. The histological grades were: 162 (56%) grade 1, 112 (39%) grade 2, 13 (4.5%) grade 3 and 2 (0.5%) unknown. Among the 289 patients, 41(17%) had low FLIPI score, 150 (63%) intermediate, 48 (20%) high and 50 unknown. All biopsies were analyzed for CD20, CD3, CD4, CD8 and HLA-DR expression by single-parameter flow cytometry. The 289 pts were divided into a training set of 147 and a validation set of 142, stratified by age and era of diagnosis. We used these two factors to stratify the pts because age at diagnosis is the most important prognostic factor for survival, and, in our data set, the era of diagnosis had an impact on survival and on the time from diagnosis to first treatment. For our analysis, we began with the training set and used the percentages of each immune cell population as a continuous variable in a univariate analysis in relation to clinical features and outcomes. We chose 8 phenotypic variables: CD20, CD3, CD4, CD8, HLA-DR, CD4/CD3 ratio, CD8/CD3 ratio, and activated T cells [defined as (HLA-DR-CD20)/CD3]. Five parameters were used as clinical endpoints: overall survival, FLIPI score at diagnosis, the time from diagnosis to first treatment (defined as the time from the first treatment to second treatment), response to CVP as the first treatment and the duration of the benefit from the first treatment (defined as the time interval between initiation of first treatment and initiation of second treatment).Results: The number of pt evaluable for each of the outcome parameters was as follows: 289 for time to first treatment and for overall survival, 239 for FLIPI scores, 164 for response to CVP and 129 for duration of the benefit from the first treatment., Of the 8 variables tested in the training set, only CD4/CD3 ratio and CD8/CD3 ratio were marginally significant for the survival endpoint, with p 0.034 and 0.088, respectively. None of the variables was significant for any of the other endpoints. A multivariate analysis yielded CD4/CD3 as the only significant predictor for survival. When CD4/CD3 was tested in the validation set, it yielded a p value of 0.48.Conclusion: We find no evidence that the percentage of tumor-infiltrating T cells or their subsets is predictive of clinical outcome in follicular lymphoma. Any gene expression signature involving T cells that does relate to clinical outcome could therefore be a property of the activity of the cells rather than a simple reflection of their numbers.

The Role of the Transcription Factor NFAT in the Pathogenesis of Non-Hodgkin's Lymphomas.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT translocates to the nucleus, where it induces the transcription of a large number of genes necessary for a productive immune response. NFAT signalling has also been implicated in lymphocyte homeostasis and its deregulation has been suggested to be involved in the pathogenesis of different malignancies. A recent immunohistochemical evaluation of approximately 300 Non-Hodgkin's Lymphoma biopsy samples showed overexpression of NFAT2 in the majority of specimens with strong nuclear translocation in certain histologic subtypes (DLBCL, Burkitt's Lymphoma) presumably reflecting activation of the NFAT pathway as part of their pathogenesis. Other recent studies have shown that NFAT activation leads to increased expression of different cell survival factors (CD154, BLyS) in several lymphoma subtypes (DLBCL, MCL).To study the role of constitutive activation of the NFAT pathway on lymphomagenesis, we generated several hyperactivable NFAT mutants which will be used to generate transgenic mice expressing the mutant proteins from the ROSA26 locus. Here, we present the in vitro characterization of these hyperactivable NFAT proteins in cell lines and primary lymphocytes. The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (e.g. SPRIEIT in NFAT1 and NFAT2). Substitution of the SPRIEIT sequence of NFAT with HPVIVIT, a higher-affinity version obtained by peptide selection, increases the basal calcineurin sensitivity of the protein significantly. Similarly, a major kinase for NFAT is CK1, and mutation of the CK1 docking site (FSILF to ASILA in NFAT1, FDFEF to ADAEA in NFAT2) also leads to partial nuclear localization of NFAT proteins by decreasing rephosphorylation and nuclear export. In the current analysis we compared the different hyperactivable NFAT proteins with respect to dephosphorylation status and nuclear translocation using western blotting and immunocytochemistry. Whereas the wildtype NFAT protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, we observed an increasing degree of nuclear translocation and dephosphorylation for the different mutant proteins (ASILA 20%, VIVIT 30–40%, ASILA-VIVIT 50–70%). This system will allow us to study the impact of different levels of NFAT activation on the pathogenesis of lymphomas in vivo.

The role of Epstein–Barr virus in cancer

Epstein–Barr virus (EBV), discovered > 40 years ago from a Burkitt’s lymphoma biopsy, was the first virus to be directly associated with human cancer. EBV has two distinct life cycles in the human host; a lytic form of infection that produces new infectious virions, and a latent form of infection that allows the virus to persist in a dormant state for the lifetime of the host. EBV has evolved a life cycle that mimics the natural differentiation pathway of antigen-activated B cells, giving the virus access to its site of latent infection, the resting memory B cell. By steering infected cells through the various stages of lymphocyte differentiation, EBV is able to enter a cell type suitable for long-term latent persistence and periodic reactivation. However, its presence in various stages of B-cell development, and its ability to infect certain epithelial cells, can have pathogenic consequences, and can contribute to the development of a diverse group of lymphomas and carcinomas. The presence of EBV in the tumour cells of EBV-associated cancers might provide a basis for specific therapy. This article focuses on the contributions that the virus may play in different types of human cancer, particularly Burkitt’s lymphoma, Hodgkin’s lymphoma, lymphomas and lymphoproliferative diseases in the immunocompromised, and nasopharyngeal and gastric carcinoma.

Number of CD4+ Cells and Location of Forkhead Box Protein P3–Positive Cells in Diagnostic Follicular Lymphoma Tissue Microarrays Correlates With Outcome

To examine the immune microenvironment in diagnostic follicular lymphoma (FL) biopsies and evaluate its prognostic significance. Immunohistochemistry was used to study numbers and location of cells staining positive for immune cell markers CD4, CD7, CD8, CD25, CD68, forkhead box protein P3 (FOXP3), T-cell intracellular antigen-1, and Granzyme B in tissue microarrays of paraffin-embedded, diagnostic lymph node biopsies taken from 59 FL patients who lived less than 5 years (short-survival group; n = 34) and more than 15 years (long-survival group; n = 25). CD4 and FOXP3 expression were significantly different between the two groups. Samples from the long-survival group were more likely than those from the short-survival group to have CD4+ staining cells and to have FOXP3-positive cells in a perifollicular location. This study has identified differences in immune cell composition of the diagnostic FL lymph node immune microenvironment and these have the potential for use as prognostic biomarkers in a routine histopathology setting.

Chlamydia or Not Chlamydia , That Is the Question: Which Is the Microorganism Associated With MALT Lymphomas of the Ocular Adnexa?

In the mid-1990s, the demonstration that Helicobacter pylori infection is a risk factor for gastric marginal-zone B-cell lymphoma of MALT type (MALT lymphoma) and the fi nding that eradication of these bacteria can result in histological lymphoma regression in most patients made this tumor a popular model of antigen-driven lymphomagenesis ( 1 ) . The association of H. pylori with gastric MALT lymphoma led to the hypothesis that the microorganism provides an antigenic stimulus that sustains the growth of the lymphoma in the stomach, and today it is generally accepted that administration of antibiotics to eradicate H. pylori should be the sole initial treatment of gastric MALT lymphoma that is confi ned to the gastric wall. This approach has been validated in more than 20 reported studies ( 2 ) . These advances in the understanding and treatment of gastric MALT lymphoma pro mpted a search for microorganisms responsible for the growth of MALT lymphomas outside the stomach. Although Borrelia burgdorferi and Campylobacter jejuni have been associated with marginal-zone lymphomas arising in the skin and small intestine, respectively ( 3 , 4 ) , results implicating particular infectious microorganisms in nongastric lymphomas have often been elusive. In 2004, Ferreri et al. ( 5 ) reported in this journal that Chlamydia psittaci DNA was detected in 87% of lymphomas arising in the ocular adnexa (orbital soft tissue, lachrymal glands, and conjunctiva), and therefore, it has been proposed that C. psittaci infection may be a cause of these lymphomas. Furthermore, a very recent epidemiologic paper has shown a possible real increase in the incidence of ocular adnexal lymphomas, suggesting C. psittaci or another microorganism as possible causative agents ( 6 ) . However, subsequent to the report of Ferreri et al., other groups analyzed the prevalence of C. psittaci in these lymphomas, and overall, the reported data did not confi rm a strong link between C. psittaci infection and ocular adnexal MALT lymphomas ( Table 1 ). Thus, our understanding of the role of C. psittaci is far from complete. In this issue of the Journal, Ferreri et al. ( 7 ) report the results of a prospective trial in which patients affected by marginal-zone B-cell lymphoma of the ocular adnexa were treated for 3 weeks with the oral antibiotic doxycycline, with the intent of eradicating C. psittaci. The endpoints of the study were the rate of objective lymphoma response to antibiotic treatment and the presence or absence of C. psittaci DNA in peripheral blood mononuclear cells, the latter endpoint serving as a measure of C. psittaci eradication. Twenty-seven patients were enrolled and evaluated for response to treatment, 15 of them were newly diagnosed with ocular adnexal lymphoma and 12 had relapsed after previous therapies. Three patients had lymphoma involving regional lymph nodes and fi ve had bilateral MALT lymphomas. Preliminary results in the fi rst nine cases of the study have previously been published ( 8 ). C. psittaci was detected, using, as in the previous studies, a touchdown enzyme time release polymerase chain reaction ( 5 , 7 – 8 ) in 11 of 27 or 41% of lymphoma biopsies, a percentage lower than in the fi rst report from the same investigators

Fine-needle aspiration biopsy of non-Hodgkin lymphoma for use in expression microarray analysis

The majority of patients with lymphoma undergo a single biopsy for diagnosis, and there are few opportunities to acquire posttreatment material. Fine-needle aspiration (FNA) biopsy is a minimally invasive procedure, and acquiring routine posttreatment material would require minimal effort and provide needed material for gene expression profiling. Ex vivo FNA biopsies were performed using a standard clinical technique with 21-gauge, 22-gauge, 23-gauge, and 25-gauge needles for 8 lymph node specimens and were collected in either RNA stabilization reagent (RNAlater) or Trizol. Eight patients with known or suspected Non-Hodgkin lymphoma (NHL) underwent interoperative (in vivo) FNA biopsies based on the best technique derived from the ex vivo aspirates. RNA derived from the in vivo FNA biopsies and the matched, snap-frozen surgical lymph node biopsy remnant was used for gene expression analysis with proprietary U133A chips. The results confirmed the authors' experience, that RNA isolated from FNA biopsies of lymph nodes collected in Trizol is superior both quantitatively and qualitatively to RNA collected in RNAlater. Gene expression profiles of NHL derived from in vivo FNA biopsies and matched, frozen surgical specimens showed good overall correlation. High-throughput gene expression analysis in patients with NHL derived from material acquired by FNA biopsy is a feasible approach to developing a platform for real-time analysis of treatment responses in this group of patients.

Cloning of idiotype immunoglobulin genes in B cell lymphomas by anchored PCR and production of individual recombinant idiotype vaccines in Escherichia coli

Individual immunoglobulins expressed by B-cell lymphomas represent tumor-specific antigens ('idiotypes'). Immunization with idiotype in follicular lymphoma patients may induce specific immune responses, sustained progression-free survival, and disappearance of minimal residual disease. Manufacturing of idiotype vaccines has mostly relied on heterohybridomas established from viable lymphoma cells. This paper describes the feasibility of production of GMP-grade idiotype vaccines as recombinant Fab fragments in Escherichia coli. IgH and IgL transcripts were analyzed by anchored PCR from 106 lymphoma and nine control biopsies. Lymphoma-derived V segments were inserted into prokaryotic expression plasmids. Recombinant idiotype Fab fragments were expressed in E. coli in a fermentation system. Idiotype IgH and IgL transcripts were identified in 95% of 106 lymphoma biopsies according to stringent clonality criteria. Large-scale idiotype expression was successful in 69 of 78 cases (89%) and yielded a median of 17 mg (range: 1.2-250 mg) recombinant Fab protein. After affinity chromatography, median vaccine purity was 99% heterodimeric Fab protein (range: 72-100%). Bacterial protein contamination was detectable in one vaccine only. Fab proteins with IgL lambda chains had a tendency for inferior yield and lesser purity than kappa-type Fabs. Among other structural idiotype features (isotype, V family usage, somatic hypermutation pattern, novel glycosylation sites, CDR III net charge), no consistent influences on Fab yield or purity were detected. Anchored PCR cloning and subsequent protein expression in E. coli provides a reliable technological basis for clinical idiotype vaccination trials.

Retrospective analysis of cutaneous T-cell lymphoma (CTCL) biopsy samples for interleukin-2 receptor (IL2-R) subunit expression by immunohistochemistry (IHC) and real-time quantitative PCR (RT-PCR): Correlation to response to denileukin diftitox

20080 Background: Denileukin diftitox, a chimeric diphtheria toxin-IL2 protein, exerts its cytotoxic effect on cells expressing high or intermediate affinity IL2-R. IL2-R is a complex of 3 distinct polypeptide chains: IL2-Rα (CD25), IL2-Rβ (CD122), and IL2-Rγ (CD132). IL2-R α/β/γ heterotrimer binds with high affinity, IL2-R β/γ with intermediate affinity, and IL2-R α/γ with low affinity. Denileukin diftitox is indicated for patients with persistent or recurrent CTCL whose malignant cells express IL2-Rα protein. There is no currently available methodology to measure expression of the high affinity receptor in patient samples. This study was initiated to discover a correlation between expression of IL2-R components (at the mRNA and protein levels) and clinical response to denileukin diftitox. Methods: Of 33 available frozen clinical CTCL biopsy samples, 27 (10 responders) were sufficient for IHC. IL2-Rβ staining intensity was assigned a value of 0 (no staining relative to background), 1+ (weak), 2+ (moderate), or 3+ (strong). 22 samples (8 responders) were sufficient for RT-PCR. mRNA levels of the 3 IL2-R chains were normalized to 36B4 mRNA or 18S rRNA. Correlation between clinical response and IL2-R chain expression was assessed using Spotfire Decision Site. Results: The population of samples tested was representative of the study population. 26 of 27 samples exhibited 2+, all exhibited 1+, and none 3+ IL2-Rβ staining. The percentage of cells stained varied from 20%–90% for 1+ and 10%–80% for 2+ with no correlation to response. Correlation of levels of individual IL2-R subunit mRNA expression with response could not be established. Conclusions: Neither the IL2-Rβ IHC nor the RT-PCR assay, at current sensitivity levels, appear to identify denileukin diftitox responders. Larger sample datasets from current studies, and further assay refinement, are ongoing to define the merits of these approaches. [Table: see text]

Transgastric Endoscopic Ultrasound-Guided Fine-Needle Aspiration Biopsy and Flow Cytometry of Suspected Lymphoma of the Spleen

Masses in the spleen can be sampled by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) but the diagnosis of lymphoma using EUS-FNA and flow cytometry has not been reported. We report our experience with transgastric EUS-FNA and flow cytometry in the investigation of patients with suspected lymphoma of the spleen. All patients with splenic lesions that had been detected by computed tomography and who were referred for transgastric EUS-FNA over a 3-year period were enrolled in this study. The tissue obtained by EUS-FNA was evaluated by flow cytometry in all patients. Six patients with splenic masses were enrolled (four men, two women; median age 58.5 years, range 41 - 82 years). The mean size of the short axis of the lesions was 37.8 mm (SD 23.76 mm) and the mean size of the long axis was 45.6 mm (SD 31.72 mm). EUS-FNA was performed successfully in all patients and the tissue obtained was evaluated by flow cytometry. Two patients were diagnosed with lymphoma; no pathology was identified in the other four patients. Lymphoma of the spleen appeared as sharply demarcated echo-poor lesions; benign lesions appeared echo-rich in comparison with the surrounding splenic tissue. The two patients who were diagnosed with lymphoma underwent chemotherapy. Of the four patients in whom no pathology was identified, one patient subsequently underwent splenectomy for evaluation of persistent abdominal pain and was diagnosed with lymphoma; the three other patients had true-negative disease on the evidence of long-term follow-up (mean 8 months; range 6 - 12 months). No complications related to the EUS-FNA procedure were encountered in any patient. EUS-FNA of spleen masses is a safe technique that aids in the diagnosis of lymphoma when used in conjunction with flow cytometry.

Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma

Most non-Hodgkin lymphomas (NHLs) are of B-cell origin, but the tumor tissue can be variably infiltrated with T cells. In the present study, we have identified a subset of CD4(+)CD25(+) T cells with high levels of CTLA-4 and Foxp3 (intratumoral T(reg) cells) that are overrepresented in biopsy specimens of B-cell NHL (median of 17% in lymphoma biopsies, 12% in inflammatory tonsil, and 6% in tumor-free lymph nodes; P = .001). We found that these CD4(+)CD25(+) T cells suppressed the proliferation and cytokine (IFN-gamma and IL-4) production of infiltrating CD4(+)CD25(-) T cells in response to PHA stimulation. PD-1 was found to be constitutively and exclusively expressed on a subset of infiltrating CD4(+)CD25(-) T cells, and B7-H1 could be induced on intratumoral CD4(+)CD25(+) T cells in B-cell NHL. Anti-B7-H1 antibody or PD-1 fusion protein partly restored the proliferation of infiltrating CD4(+)CD25(-) T cells when cocultured with intratumoral T(reg) cells. Finally, we found that CCL22 secreted by lymphoma B cells is involved in the chemotaxis and migration of intratumoral T(reg) cells that express CCR4, but not CCR8. Taken together, our results suggest that T(reg) cells are highly represented in the area of B-cell NHL and that malignant B cells are involved in the recruitment of these cells into the area of lymphoma.

High expression of FMNL1 protein in T non-Hodgkin's lymphomas

We recently cloned a novel Formin-family human gene, denominated human leukocyte formin and posteriorly named as FMNL1 by the HUGO Gene Nomenclature Committee. This gene is overexpressed in lymphoid malignances and is found associated with Akt. Here, we describe the expression of the FMNL1 protein in 54 frozen biopsies of non-Hodgkin's lymphoma (NHL) patients and five reactive lymph nodes. Western blot analysis revealed expression of FMNL1 in all samples studied, although the highest expression was in T-cell NHL ( P < 0.05).