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To the editor: Peripheral T-cell lymphomas (PTCLs) represent a group of rare hematological cancers of mature T-cell or natural killer cell origin accounting for 10% to 15% of all lymphomas.[1][1] Although many patients have poor outcomes, some achieve long-term survival.[2][2],[3][3] Thus

Introduction Follicular lymphoma (FL) is an indolent lymphoma derived from germinal center B cells, covering approximately 20% of all lymphomas. The malignancy is generally considered an incurable condition, hence with a median survival time exceeding 10 years. A portion of patients experience early progression or histological transformation (HT) to a more aggressive lymphoma, typically diffuse large B cell lymphoma which occurs in up to 45% of FL patients, reducing the median survival after transformation to 1-2 years. Early detection of reliable predictors of HT would allow pre-emptive therapeutic intervention strategies and aim at improved survival for patients with transformed FL. In this study, we focus on the two glycolytic enzymes, Aldolase A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both proteins have previously been found to be upregulated at diagnosis in various solid cancers and associated with poor survival. In addition to its glycolytic effects, GAPDH has been recognized for its non-glycolytic effects including regulation of DNA replication and repair, RNA nuclear export and apoptosis. Aim The aim of this study was to identify biomarkers predictive of HT in patients with FL, by investigating the correlation between intratumoral aldolase A and GAPDH expression levels with the risk of transformation in pre-therapeutic tumor samples from FL patients. Materials and Methods Immunohistochemical aldolase A and GAPDH expression levels were quantified using digital image analysis in pre-therapeutic tumor tissue from FL patients at time of diagnosis for patients without (n=51) or with (n=41) subsequent HT, as well as in sequential samples at time of transformation (n=41). Staining intensities were assessed as area fractions (AFs) of the stained area normalized to the region of interest on whole biopsy sections. Results At time of initial FL diagnosis, FL patients with subsequent transformation had significantly higher levels of aldolase A and GAPDH expression compared to patients with no subsequent transformation, (p Conclusion These results suggest that at time of initial FL diagnosis, aldolase A expression and GAPDH expression, maybe as indicators of high metabolic turnover, may be predictive of the patient's risk of subsequent histological transformation. Disclosures No relevant conflicts of interest to declare.

Introduction: Recent results from two independent patient series have shown that chromosomal rearrangements of DUSP22 (DUSP22r+) and TP63 (TP63r+) can predict outcome in ALK-negative anaplastic large cell lymphoma (ALK-ALCL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) with morphologic features resembling ALK-ALCL (Parilla-Castellar E, Blood 2014; Pedersen MB, Blood 2017). While DUSP22r+ is predictive for excellent survival similar to that of ALK+ALCL after CHOP/CHOP-like therapy, the rarely occurring TP63r+ is associated with an aggressive clinical behavior and poor outcome. The largest subgroup, i.e. patients with neither ALK nor DUSP22 nor TP63 rearrangements (triple negative), show a 5 year (yr) overall survival (OS) intermediate between that of ALK-/DUSP22r+ and ALK-/TP63r+ patients. The aim of the present study was to assess the impact of upfront high-dose therapy with autologous stem cell transplant (HDT/ASCT) on outcome in adult ALCL and PTCL-NOS patients according to their ALK, DUSP22 and TP63 status. The survival results from the two published series were pooled with those of a Nordic Lymphoma Group trial, the NLG-T-01 (d'Amore et al, JCO 2012), where patients were treated with 1st line CHOEP/CHOP followed, in chemosensitive cases, by upfront HDT/ASCT. Methods: Fluorescence in situ hybridization was performed on sections of previously constructed tissue microarrays using break-apart probes for the DUSP22-IRF4 and TP63 loci and a dual-fusion probe for TBL1XR1/TP63 fusion [inv(3)(q26q28)]. Evaluation of DUSP22 and TP63 rearrangements was performed in a blinded fashion without knowledge of PTCL subtype, clinical course, or outcome. Three independent patient cohorts were included: (i) one from Mayo Clinic consisting of 31 DUSP22r-, ALK-ALCL and PTCL-NOS (triple negative: 25; TP63r+: 6); (ii) one from Denmark consisting of 93 DUSP22r-, ALK-ALCL and PTCL-NOS (triple negative: 90; TP63r+: 3); and one from the NLG-T-01 trial consisting of 46 ALK-ALCL and PTCL-NOS (triple negative: 37; TP63r+: 1; DUSP22r+: 8), leading to a total study population of 170 patients. ALK+ ALCL was not included in the analysis, since no patients with this histology entered the NLG-T-01 trial. Association of genetic subtype with OS was assessed using Kaplan-Meier curves and Cox proportional models for hazard rate ratios (HR). Significant differences were defined as P Results: The eight DUSP22r+ patients (7 ALK-ALCL and 1 PTCL-NOS) from NLG-T-01 had a 5-yr OS of 83%, (95%CI 27-97), similar to that reported for DUSP22r+ in the Mayo and Danish cohorts (90% and 80%, respectively). No lymphoma-related events were observed in this subset. The only event was a septic death due to HDT-induced cytopenias in a patient who was in complete remission (CR). Among the 162 patients with DUSP22r-, ALK-ALCL and PTCL-NOS, those consolidated with HDT/ASCT (n=47) had a significantly better outcome (5-yr OS: 45%) than those treated with induction chemotherapy alone (5-yr OS: 30%) (n=115) (P=0.01). The patients in the HDT/ASCT group were younger (P Conclusion: In ALK-ALCL and PTCL-NOS patients from the NLG-T-01 trial, DUSP22r+ was associated with a very good outcome, similar to that seen in DUSP22r+ patients who had not undergone upfront autologous transplant. This observation supports the impression that upfront HDT/ASCT may not be of benefit in these patients. TP63r+ predicted poor outcome in non-transplanted patients. The impact of HDT/ASCT in the TP63r+ setting could not be adequately evaluated, since only one patient from the NLG-T-01 trial cohort was found to be TP63r+. Notably, this patient was the only survivor of the TP63r+ subset. For DUSP22r-, ALK-ALCL and PTCL-NOS patients taken as one group, those who received upfront HDT/ASCT had a superior survival compared to their age- and IPI-matched non-transplanted counterparts. Disclosures Ellin: ROCHE: Consultancy, Research Funding; CTI: Consultancy. Mannisto: Roche: Honoraria, Other: Travel expence; Takeda: Honoraria, Other: Travel expence; Amgen: Other: Travel expence; Novartis: Other: Travel expence; Celgene: Other: Travel expence; Gilead: Other: Travel expence; Pfizer: Honoraria; SOBI: Honoraria. Cerhan: Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering . Toldbod: Takeda Pharma: Honoraria.

We previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

Introduction. HIV infected individuals have an increased risk of developing lymphoma even in the era of combined antiretroviral therapy. Galectin-1 (Gal-1) is known to promote various immunomodulatory functions, including Treg expansion (Dalotto-Moreno et al, Cancer Res 2013), promotion of tolerogenic dendritic cells (Ilarregui et al, Nat Immunol 2009) and apoptosis of fully-differentiated effector T-cells (Toscano et al, Nat Immunol 2007). In the context of cancer, Gal-1 is expressed on both tumor cells and cells in the tumor microenvironment, and is usually associated with immune privilege, tumor escape and hypoxia-driven angiogenesis (Juszczynski et al, Proc Natl Acad Sci 2007; Cedeno-Laurent et al, Blood 2012). Previously, high intratumoral Gal-1 levels have been suggested as an unfavorable outcome predictor in patients with classical Hodgkin lymphoma (cHL) (Kamper et al, Blood 2011). Furthermore, several in vitro studies revealed the benefit of Gal-1 inhibition with regards to overcoming treatment resistance e.g., after anti-VEGF and anti-CD20 therapy (Croci et al, Cell 2014; Lykken et al, Blood 2016). Thus, Gal-1 inhibition may prospectively be an important tool in lymphoma treatment. In this study, we have investigated the Gal-1 expression in pre-therapeutic tumoral tissue samples from patients with HIV-associated lymphoma and its correlation to clinicopathological features at lymphoma diagnosis. Methods. Adequate pre-treatment formalin-fixed paraffin embedded samples from 40 HIV-positive lymphoma patients were included in a tissue micro array. The study samples were immunohistochemically characterized by a panel of monoclonal antibodies including CD3, CD4, CD8, CD10, CD20, CD79a (MRQ-48), CD30 (Ber-H2) and MUM1, granzyme B, CD68 and CD163. Epstein-Barr virus (EBV) proteins were visualized by latent membrane protein 1 and Epstein-Barr nuclear protein 2. Expression of EBV-encoded smallRNAs was analyzed by in-situ hybridization. Immunohistochemical cell-of-origin (COO) evaluation was carried out according to the Hans classifier. Gal-1 expression was digitally quantified as an area fraction (AF) of the total core area. Estimates of differences between groups were evaluated using Students t test, Pearsons χ2, Fisher's exact test or Spearman correlation where appropriate. Optimal cut-off values of the AF were established by a ROC analysis and calculated using Youden's index for diffuse large B-cell lymphoma (DLBCL) (n=22). Outcome was estimated by Kaplan-Meier time-to-event analyses and compared using the log-rank test. Independent prognostic values were tested by Cox-regression analysis in a multivariate model for factors showing a crude association with p Results. High intratumoral Gal-1 expression in HIV-associated DLBCL tissue correlated with improved outcome (p=0.041), Figure 1. Patients with high Gal-1 expression had a higher occurrence of nodal disease and B-symptoms (p=0.006). Gal-1 expression did not vary according to tumoral EBV status, latency type, International prognostic index (IPI), clinical stage or COO signature. In multivariate analysis adjusted for rituximab treatment, both Gal-1 expression and IPI retained independent prognostic value. Furthermore, intratumoral Gal-1 expression correlated positively with a Th1-signature of the tumor microenvironment including the macrophage marker CD68 (p Conclusion. In HIV-associated DLBCL, intratumoral Gal-1 expression positively correlated with a Th1-signature of the tumor microenvironment. In addition, high pre-therapeutic intratumoral Gal-1 expression was found to be an independent predictor of improved outcome in HIV-associated DLBCL. Interestingly, this is the reverse of our previous findings in non-overtly immunocompromised patients with cHL (Kamper et al, Blood 2011). Further investigations on the potential clinical application of intratumoral Gal-1 expression as a predictive marker in different lymphoma types of the immunocompromised vs immunocompetent host are warranted. Figure. Figure. Disclosures d'Amore: CTI LIfe Sciences: Honoraria, Other: Advisory Boards; Servier: Honoraria, Other: Advisory Boards.

Sequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which include loss of a Xho I restriction site (position 169425) and a C-terminal 30-base pair (bp) deletion (position 168287-168256), define EBV genotypes associated with increased tumorigenicity or with disease among particular geographic populations. To determine the frequency of LMP-1 variations in European wild-type virus isolates, we sequenced the LMP1 promoter and gene in EBV from lymphoblastoid cell lines from healthy carriers and patients without EBV-associated disease. Sequence changes were often present, and defined at least four main groups of viral isolates, which we designate Groups A through D. The widespread prevalence of LMP-1 sequence variations, particularly the Xho I polymorphism and the 30-bp deletion, indicate that they cannot be used as simple markers for oncogenic viruses related to particular forms of EBV-associated tumor. Several of the structural changes detected occur, however, at sites where they may affect transcription, translation, or function of LMP-1. Future in vitro studies should aim to establish the functional importance of variations at these sites.

Background: PTLD comprises a diverse spectrum of hematological conditions, ranging from early lesions, characterized by reactive-like proliferations, to monomorphic lesions, resembling overt lymphoma. Â In most cases, the lesions are believed to arise as the result of reduced immune surveillance secondary to the use of immunosuppressive drugs post-transplant. This view is supported by the observation that PTLDs, particularly those characterized by early or polymorphic lesions, may regress spontaneously upon reduction of the immunosuppressive treatment. Studies in sporadic lymphomas have identified distinct microenvironmental characteristics, predictive of the clinical behavior, but data is scarce in the immunocompromised setting. Therefore, the aim of this study was to investigate the tumor microenvironment in a population-based cohort of PTLD. Methods: We identified 108 PTLD patients diagnosed in the period 1994-2011. Of these, 62 cases had adequate tissue for tissue microarray construction. All biopsies were reviewed and classified according to the WHO 2008 criteria. Immunohistochemically stained sections were digitally quantified using Tissuemorph (Visiopharm Integrator System 4.0.3.0, Visiopharm, Denmark). Determination of optimal cut-off values of the area fraction (AF) was established by a ROC-curve. ROC analyses were performed in every disease entity and used for endpoint analyses. Results: Themedian age was 45 yrs (range 2-77 yrs) with a M/F ratio of 3:1. The majority presented in stage I-II (61%), and B symptoms and extranodal disease were common features (40% and 42%, respectively). Most tissue samples were EBV-positive (85%). The EBV latency pattern was predominantly latency II (41%) or III (43%). The AF of galectin-1 (gal-1) positive cells was clearly correlated to latency type (p=0.0001), whereas FOXP3 and programmed death-1 (PD-1) did not show such correlation (fig 1). Overall survival (OS) was significantly higher in cases with high levels of PD-1 expression, with 5-yrs OS of 39% (23-55%) and 69% (47-83%) for low and high levels, respectively (p=0.01). Expression levels of FOXP3 and gal-1 had no impact on OS in the total cohort (fig 1). In the monomorphic setting, a low AF of FOXP3 positive cells was associated with an increased risk of progression (OR 6.1; 95% CI: 1.4-26.0). This did not, however, translate into an inferior OS (p=0.163). To specifically characterize the tumor microenvironmental features of DLBCL-type PTLD with respect to cell of origin (COO), 30 evaluable cases were analyzed according to the Hans classifier; 10 (33%) were germinal center (GC) type and 20 (67%) of non-GC type. Cases of non-GC subtype had a significantly shorter time to PTLD of 1.16 yrs (CI: 0.64- 2.11), than those of GC-subtype (3.66 yrs; CI: 1.64- 8.16) (p=0.023) and a lower AF of gal-1 positive cells (p=0.042). The 5-yrs OS was 58% (32-77%) and 0% for non-GC versus GC-tumors, respectively (p=0.075). High levels of FOXP3 expression were associated with superior OS in the non-GC sub-group (p=0.04); gal-1 and PD-1 had no influence in the COO setting. Conclusion: The present study is one of the few attempts to describe tumor microenvironmental features in PTLD and relate them to outcome. In a population-based PTLD cohort, we found that specific immune cell subsets were variably expressed in different PTLD subtypes, and that high expression of PD-1 (whole cohort) and FOXP3 (non-GC DLBCL only) correlated with significantly better outcome. Â | | N(%) pts in TMA | | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------- | | Organ tx(%) | | | Kidney Heart Lung | 47(75.8) 9(14.5) 6(9.7) | | Type PTLD(%) | | | Early/polymorphic lesions Monomorphic PTLD Diffuse large B-cell lymphoma Peripheral T-cel lymphomal, NOS* T-Anaplastic large cell lymphoma Burkitt lymphoma Hodgkin lymphoma-type PTLD Marginal zone lymphoma | 18(29.0) 38(61.2) 32(51.6) 1(1.6) 3(4.8) 2(3.2) 4(6.5) 2(3.2) | ![Figure 1][1] Figure 1 Disclosures No relevant conflicts of interest to declare. [1]: pending:yes

Background: T-cell malignancies originating from regulatory T (Treg) cells are almost exclusively confined to human T-cell leukemia virus 1 (HTLV-1) associated adult T-cell leukemia/lymphoma (ATLL), although sporadic cases of other peripheral T-cell lymphomas (PTCLs) with hypothesized Treg derivation have been reported. Patients and methods: We investigated a series of 169 well characterized PTCLs for the expression of Treg-cell phenotypic markers FOXP3, CD25 and CD4 by immunohistochemistry (IHC) using tissue microarray on formalin-fixed paraffin embedded tissue. Clinico-pathological data for patients enrolled were retrieved from the Danish Lymphoma Registry and medical records. Treg tumors were further invetsigated by Affymetrix OncoScan analysis and Illumina Infinium HumanMethylation450 BeadChips. Results: Variable amounts of FOXP3-positive Treg cells were often (99% of the cases) found as part of the non-neoplastic cellular infiltrate. However, in five cases (3%) classified as PTCL, not otherwise specified (PTCL-NOS) the vast majority of what morphologically appeared to be the neoplastic cell population displayed a strong positivity for FOXP3, CD4, CD25 and CD3 suggesting a probable Treg origin of these cells. Cases were HTLV-1 negative and showed monoclonal rearrangements of the T-cell receptor genes. All cases were male older than 60 years and showed an aggressive clinical course with overall survival less than two years, despite all patients had low IPI-risk profile at the time of diagnosis. The PTCL with Treg phenotype showed a complex pattern of chromosomal imbalances. Moreover, as compared to normal T-cell subsets their DNA methylation profiles were mostly related to that of normal Treg cells but significantly differed from that. Conclusions: We suggest that FOXP3-positve PTCL, in the absence of HTLV-1 infection, constitute a distinct entity separated from PTCL-NOS, occurring predominantly in elderly male patients and characterized by a highly aggressive clinical behavior. Further genomic analyses are ongoing. The identification and characterization of these cases may be useful to guide upfront therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Introduction: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous group of mature T-cell lymphomas, probably composed by different biologically related subsets that have not yet been conclusively identified. In the WHO classification, PTCL-NOS accounts for 25-30% of all mature T-/NK-cell malignancies. The clinical outcome is generally poor with a 5-yr overall survival of 30-35% after conventional treatment strategies. The aim of the study was to apply proteomic analysis in PTCL-NOS and to use the protein expression profiles to characterize clinically relevant subsets within this heterogeneous entity by means of unsupervised cluster analysis. Methods: Archival frozen tumor tissue samples from 20 patients diagnosed with PTCL-NOS from 1991 to 2010 were analyzed for protein expression by high-resolution two-dimensional gel electrophoresis. Individual protein spots were visualized with fluorescence staining and the expression profiles were identified. All patients were homogeneously treated with curatively intended anthracycline-containing combination regimens. Clinico-pathological features were obtained from the Danish Lymphoma Registry (LYFO) and from patient records. Hyperplastic tonsils from healthy adults were included as reference tissue (n=8). Principal component analysis and unsupervised hierarchical cluster analysis were performed on the basis of the protein expression profiles. Differentially expressed (two-fold or higher, Mann-Whitney U-test) proteins between the detected clusters were identified by liquid chromatography - tandem mass spectrometry. Results: Unsupervised cluster analysis defined three distinct clusters: one containing all reference samples and two additional ones further subdividing the PTCL-NOS cases in two separate subsets. Patients from these two PTCL-NOS subsets had significantly different responses to treatment and survival (p = 0.001). The differentially expressed proteins were primarily involved in (i) promotion of tumor growth, (ii) regulation of cellular metabolism, and (iii) immune responses. Conclusion : Proteomic analysis identified shared protein expression patterns and potential prognostic markers in subsets of PTCL-NOS. Disclosures No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma (DLBCL) is clinically and molecularly heterogeneous and portends a poor prognosis in more than half the affected patients. We developed a multivariate model based on the RNA expression of six genes – LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2 – that independently predicts survival in DLBCL patients treated with anthracycline-containing regimens (Lossos et al, NEJM 2004). Since the transcription factor LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to document the tissue expression pattern of LMO2 protein and to establish its prognostic significance. Immunohistological analysis of 1200 normal tissues and hematolymphoid neoplasms showed that LMO2 protein is expressed as a nuclear marker in normal germinal center (GC) B-cells and in a subset of B-cell lymphomas. It is rarely expressed in mature T, NK and plasma cell neoplasms. Immature precursors of all bone marrow hematopoietic lineages and a significant proportion of acute lymphomphoblastic and myeloid leukemias express LMO2 protein. Apart from endothelial cells, no other non-hematolymphoid tissues we tested showed LMO2 protein expression. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of LMO2 protein is similar to that of other GC-associated proteins (HGAL, BCL6 and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). LMO2 protein expression paralleled its RNA expression in B-cell lymphoma cell lines and was found in GC B, but not in non-GC, B-cell lines. To test the prognostic significance of LMO2 protein we analyzed an independent cohort of 203 DLBCL patients (mean age 63, range 18–93), uniformly treated with anthracycline-containing chemotherapy not containing rituximab, from four medical centers. No significant difference in response to therapy (CR and CRu) was observed between patients with LMO2-positive and LMO2-negative lymphomas (77% and 59%, respectively, p 0.05). However, Kaplan- Meier curves demonstrated a statistically significant difference in overall survival (OS) between LMO2-positive and LMO2-negative cases (p 0.035; median OS of 74 and 20 months, respectively). Similarly, event-free survival (EFS) was also significantly longer in patients with LMO2-positive compared to LMO2-negative lymphomas (p 0.01, median EFS of 48 and 12 months, respectively). The predictive power of LMO2 expression was IPI-independent. Multivariate analyses with protein expression profiles of HGAL, BCL6, CD10, JAW1, MUM1/IRF4 and BCL2 on this cohort of patients are underway to construct a clinically applicable immunohistologic algorithm for predicting survival. The capacity of LMO2 protein to identify DLBCL patients with improved outcome in an IPI-independent manner indicates its important role in risk stratification in this disease.

Recent effort in the molecular characterization of diffuse large B-cell lymphoma (DLBCL) has led to the recognition that patients with DLBCL of germinal center origin exhibit a better overall survival. Thus, identification and characterization of markers of germinal center derivation are of importance in dissecting prognostic subclasses of DLBCL. The VICKZ (Vg1 RBP/Vera, IMP1, 2, 3, CRD-BP, KOC, ZBP-1) family members are RNA-binding proteins that recognize specific RNA targets and have been implicated in diverse cellular functions including cell polarity, migration, proliferation and tumorigenesis/metastasis. We generated an antibody that recognizes all three isoforms of VICKZ protein and characterized its expression in normal lymphoid tissue and in lymphoma subtypes. In normal tonsils, VICKZ protein showed a germinal center-specific pattern of expression with staining localized to the cytoplasm. Among 868 non-Hodgkin and Hodgkin lymphomas tested by immunohistochemistry on tissue microarrays, staining for VICKZ protein was present in 76% (126/165) of follicular lymphoma, 78% (155/200) of DLBCL, 90% (9/10) of mediastinal large B-cell lymphoma, and 100% (2/2) of Burkitt lymphoma. A subset of mantle cell lymphoma (11%, 2/19), extranodal (8%, 2/25), and nodal (20%, 1/5) marginal zone lymphoma and lymphoblastic lymphoma (25%, 4/13), showed VICKZ staining. The majority of lymphocyte predominant Hodgkin (92%, 12/13) and classical Hodgkin (94%, 101/108) lymphoma were found to be positive. Among T cell lymphoma, anaplastic large cell lymphoma were positive (75%, 6/9). Additional work is in progress to correlate VICKZ protein expression with other germinal center markers such as HGAL, BCL6 and CD10 as well as with prognostic subclasses of DLBCL. The differential expression pattern of VICKZ protein in lymphoma subtypes suggests a potential utility for VICKZ in the identification of subgroups of DLBCL associated with different prognoses.

Aim: To develop a multiplex PCR method applicable in a clinical setting for the simultaneous detection of the chromosomal lesions t(11;14)(q13q32), t(14;18)(q32;q21), t(2;5)(p23;q35), t(11;18)(q21;q21), t(3q27;var), and t(8;14)(q24;q32) frequently found in non-Hodgkin lymphoma (NHL). Methods: DNA and RNA were prepared from 50μm lymph node (LN) sections by homogenization on a FastPrep instrument (Qbiogene, Cedex, France) followed by automated nucleic acid purification on a MagNa-Pure LC robot (Roche Diagnostics, Basel, Switzerland). The multiplex PCR was condensed in four PCR tubes. The first covered the MTC and mTCp94 region of BCL1/IGH fusion DNA, the MBR and MCR breakpoint regions of BCL2/IGH fusion DNA together with the control gene TCF20. The second included the API2/MLT and ALK/NPM breakpoints on cDNA along with β-ACTIN as a control gene. The third contained primers amplifying eight different fusions partners of BCL6 (IGH (14q32), IGL (22q11), HSP89α (14q32), HSP90β (6p12), PIM1 (6p21), TFR (3q26), TTF (4p13), and H4 (6p21)) on cDNA together with β-ACTIN as the control gene. The fourth tube harbored a long range PCR with primers detecting the CMYC/IGH breakpoints on genomic DNA (Cμ, Cγ, Cα, and joining region of the IGH (Basso et al., 1999, Am J Pathology)) together with ABL as a control gene. Patient samples and cell lines: One-hundred-and-twelve LN biopsies frozen in Tissue-Tek OCT Compound (Sakura, Vaerloese, Denmark) were randomly selected from consecutive patients referred with suspected hematological malignancy. The following cell lines were used as positive controls: B-CLL line JVM-2 (t(11;14)+), NHL lines DOHH-2 (t(14;18)+, t(8;14)+) and WSU-NHL (t(14;18)+), Burkitt’s lymphoma lines BL-41, BL-70 (t(8;14)+), and MD901 (t(3;22)+), T-NHL line Karpas 299 (t(2;5)+), and ALL line MD903 (t(3;14)+). Results: In pilot experiments employing cell lines and fresh LN material, this optimized multiplex PCR reaction proved to be simple and fast with a short turnover time, considering the large number of genetic aberrations detected. In a retrospective LN material encompassing 112 blinded samples, BCL1/IGH fusion DNA with breakpoint in the MTC region was detected once while BCL2/IGH was found in 20 samples (19 in MBR and one in MCR). BCL6/IGH fusion cDNA was found in three samples while the TTF gene was utilized twice as translocation partner to BCL6. Finally, CMYC/IGH fusion DNA was detected three times (1 IGHCα, 2 IGHCγ). All PCR products apart from CMYC/IGH were sequenced and verified the specific chromosomal lesions. Nineteen were excluded due to weak control bands in the first three PCR tubes, while 38 were excluded in the long range PCR detecting CMYC/IGH. Conclusion: We conclude that the NHL multiplex PCR described is an easy and timesaving method for identifying heterogeneous molecular disease markers in NHL. The standardized DNA- and RNA preparation together with the condensation into four PCR tubes, moreover, makes it convenient to the clinical setting. Application of this assay and identification of positive cases has the added advantage that quantitative real-time PCR monitoring residual disease can be applied.