Jettie J.F. Muris

Immunohistochemical profiling based on Bcl-2, CD10 and MUM1 expression improves risk stratification in patients with primary nodal diffuse large B cell lymphoma.

Clinical outcome in patients with diffuse large B cell lymphomas (DLBCL) is poorly predictable. Expression of proteins related to germinal centre B (GCB) cell or activated B cells (ABC) and expression of apoptosis‐regulating proteins Bcl‐2 and XIAP have been found previously to be strongly associated with clinical outcome. In this study we aimed to develop an algorithm based on expression of GCB/ABC‐related proteins CD10, Bcl‐6 and MUM1 and apoptosis‐inhibiting proteins Bcl‐2, XIAP and cFLIP for optimal stratification of DLBCL patients into prognostically favourable and unfavourable groups. Expression of CD10 and cFLIP was associated with better overall survival (both p = 0.03), whereas expression of MUM1, Bcl‐2 and XIAP was associated with poor clinical outcome (p = 0.01, p = 0.0007 and p = 0.03, respectively). Multivariate analysis revealed that Bcl‐2 was the strongest prognostic marker followed by CD10 and MUM1. Stratification of patients according to a new algorithm based on expression of these three markers improved patient risk stratification into low and particularly high clinical risk groups (p = 0.04 and p < 0.0001, respectively). We conclude that, in our group of primary nodal DLBCLs, a new algorithm, based on expression of the apoptosis‐inhibiting protein Bcl‐2 and the GCB/ABC‐related proteins CD10 and MUM1, strongly predicts outcome in International Prognostic Index (IPI)‐low and ‐high patients. Its predictive power is stronger than previously published algorithms based on only GCB/ABC‐ or apoptosis‐regulating proteins. Copyright

Expression of the apoptosis inhibitor protease inhibitor 9 predicts clinical outcome in vaccinated patients with stage III and IV melanoma

Purpose: There have been reports of successful treatment of metastatic melanoma patients with active specific immunotherapy (ASI) using irradiated autologous tumor cell vaccination. It is still unknown why some patients respond and others do not. Tumor cells can evade the immune system, for example through interference with antigen presentation by down-regulation of MHC molecules or expressing proteins interfering with cytotoxic lymphocyte–induced apoptosis like the granzyme B antagonist protease inhibitor 9 (PI-9). Experimental Design: PI-9 expression was detected in melanoma cell lines. To investigated if PI-9 is important in the response to ASI, paraffin-embedded tissues from stage III or IV melanoma patients were stained. Results: PI-9 is expressed in melanoma cells and expression in metastasized melanoma cells is, in this group of patients, an adverse prognostic marker with regard to overall and disease-free survival. Moreover, loss of MHC-1 expression frequently occurs during tumor progression but is not associated with poor clinical outcome. Interestingly, melanoma patients with a favorable clinical outcome after ASI therapy usually have high percentages of activated (granzyme B–positive) tumor-infiltrating lymphocytes at time of first diagnosis and low percentages of activated lymphocytes at time of recurrent tumor. Conclusions: Expression of PI-9 in metastatic melanoma cells is associated with unfavorable clinical outcome whereas MHC-1 down-regulation is not. Although it cannot be proven that PI-9 expression is directly responsible for failure of immunotherapy, these data suggest that expression of PI-9 could be an important immune escape mechanism and that modulation of this inhibitor may enhance the efficacy of immunotherapy.

Profiling of apoptosis genes allows for clinical stratification of primary nodal diffuse large B-cell lymphomas.

Intrinsic resistance of lymphoma cells to apoptosis is a probable mechanism causing chemotherapy resistance and eventual fatal outcome in patients with diffuse large B cell lymphomas (DLBCL). We investigated whether microarray expression profiling of apoptosis related genes predicts clinical outcome in 46 patients with primary nodal DLBCL. Unsupervised cluster analysis using genes involved in apoptosis (n = 246) resulted in three separate DLBCL groups partly overlapping with germinal centre B‐lymphocytes versus activated B‐cells like phenotype. One group with poor clinical outcome was characterised by high expression levels of pro‐and anti‐apoptotic genes involved in the intrinsic apoptosis pathway. A second group, also with poor clinical outcome, was characterised by high levels of apoptosis inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. The third group showing a favourable outcome was characterised by low expression levels of genes characteristic for both other groups. Our results suggest that chemotherapy refractory DLBCL are characterised either by an intense cellular cytotoxic immune response or by constitutive activation of the intrinsic mediated apoptosis pathway with concomitant downstream inhibition of this apoptosis pathway. Consequently, strategies neutralising the function of apoptosis‐inhibiting proteins might be effective as alternative treatment modality in part of chemotherapy refractory DLBCL.

Expression of the polycomb-group gene BMI1 is related to an unfavourable prognosis in primary nodal DLBCL.

Background: : Clinical outcome in patients with diffuse large B cell lymphomas (DLBCL) is highly variable and poorly predictable. Microarray studies showed that patients with DLBCL with a germinal centre B cell-like (GCB) phenotype have a better prognosis than those with an activated B cell-like (ABC) phenotype. The BMI1 proto-oncogene was identified as one of the genes present in the signature of the ABC type of DLBCL, associated with a poor prognosis. Objectives: : (1) To investigate, in primary nodal DLBCL, the expression of BMI1 and its association with clinical outcome and DLBCL signature; (2) to look for an association between BMI1 expression and the expression of its putative downstream targets p14ARF and p16INK4a. Results: : BMI1 expression was found to be associated with poor clinical outcome, but not clearly with an ABC-like phenotype of DLBCL. Expression of BMI1 was frequently, but not always, related to low levels of expression of p14ARF and p16INK4a. Conclusion: : Expression of BMI1 is associated with an unfavourable clinical outcome of primary nodal DLBCL.

High numbers of tumour-infiltrating activated cytotoxic T lymphocytes, and frequent loss of HLA class I and II expression, are features of aggressive B cell lymphomas of the brain and testis

Loss of both HLA class I and class II expression in B cell lymphomas is a mechanism of escape from a cytotoxic T lymphocyte (CTL) immune response and will therefore give a strong selective survival advantage in tumours expressing strong immunogenic antigens. We investigated loss of HLA expression using specific antibodies on tissue sections from 254 B cell lymphomas originating from nodal and different extranodal sites in relation to numbers of tumour‐infiltrating T cells. Complete loss of HLA class I and II was observed in a minority of the nodal, stomach, and skin lymphomas but in the majority of the lymphomas originating from the testis and the CNS. Interestingly, relatively high percentages of activated CTLs were detected in both primary testicular and CNS lymphomas compared to lymphomas at other sites, with highest percentages in the testis (p < 0.0001). We conclude that loss of both HLA class I and II expression occurs very frequently in lymphomas originating from the testis and the CNS as compared to nodal and some other extranodal sites. The presence of high percentages of activated CTLs in the testicular and CNS lymphomas suggests that loss of HLA expression provides a strong growth advantage for lymphoma cells in these immune‐privileged sites. Copyright

Urinary granzyme A mRNA is a biomarker to diagnose subclinical and acute cellular rejection in kidney transplant recipients

The distinction between T-cell-mediated rejection (TCMR) and other causes of kidney transplant dysfunction such as tubular necrosis requires biopsy. Subclinical rejection (SCR), an established risk factor for chronic allograft dysfunction, can only be diagnosed by protocol biopsy. A specific non-invasive biomarker to monitor immunological graft status would facilitate diagnosis and treatment of common transplantation-related complications. To identify possible markers, we measured urinary mRNA levels of several cytolytic proteins by quantitative PCR. Our cohort of 70 renal transplant recipients had biopsy proven type I and type II TCMR, acute tubular necrosis, SCR, calcineurin inhibitor-toxicity, cytomegalovirus infection, and stable graft function with normal histology. Granzyme A (GzmA) mRNA was significantly higher in subclinical and acute cellular rejection compared to patients with stable grafts or those with tubular necrosis with 80% sensitivity and up to 100% specificity. Granzyme B and perforin mRNA levels could significantly discriminate acute rejection from stable or tubular necrosis, but were not significantly elevated during SCR. Importantly, only GzmA mRNA remained below detection limits from grafts that were stable and most with tubular necrosis. Hence, the presented data indicate that urinary GzmA mRNA levels may entail a diagnostic non-invasive biomarker to distinguish patients with subclinical and acute cellular rejection from those with tubular necrosis or stable grafts.

Detection of aberrant transcription of major histocompatibility complex class II antigen presentation genes in chronic lymphocytic leukaemia identifies HLA-DOA mRNA as a prognostic factor for survival.

In human B cells, effective major histocompatibility complex (MHC) class II‐antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA‐DM (DM) and HLA‐DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA‐DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B‐CLL). Real‐time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In‐depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon‐γ‐inducible promoter CIITA‐PIV in B‐CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B‐CLL.

Human soluble TRAIL/Apo2L induces apoptosis in a subpopulation of chemotherapy refractory nodal diffuse large B‐cell lymphomas, determined by a highly sensitive in vitro apoptosis assay

Resistance to chemotherapy in therapy‐refractory diffuse large B‐cell lymphomas (DLBCL) is related to inhibition of the intrinsic apoptosis pathway. Human soluble tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (hsTRAIL/Apo2L) induces apoptosis via the alternative, death‐receptor mediated apoptosis pathway and might be an effective alternative form of therapy for these lymphomas. This study investigated whether hsTRAIL/Apo2L could actually induce apoptosis in isolated lymphoma cells of DLBCL biopsies of patients with chemotherapy‐refractory DLBCL. Twelve out of a total of 22 DLBCL samples were sensitive to hsTRAIL/Apo2L. These sensitive lymphomas included seven clinically chemotherapy‐refractory lymphomas. Furthermore, hsTRAIL/Apo2L induced apoptosis in DLBCL cells and in B‐cell lines that showed high expression levels of inhibitors of the intrinsic apoptosis pathway: Bcl‐2 and/or X‐linked inhibitor of apoptosis (XIAP). hsTRAIL/Apo2L‐sensitive lymphoma cells showed expression of the TRAIL receptors R1 and/or R2 and absence of R3 and R4. We conclude that hsTRAIL/Apo2L induced apoptosis in a subpopulation of chemotherapy‐refractory nodal DLBCL and that disruption of the intrinsic apoptosis‐mediated pathway and expression of Bcl‐2 and XIAP did not confer resistance to hsTRAIL/Apo2L‐induced apoptosis in DLBCL. Thus, based on our results, further exploration of hsTRAIL/Apo2L as an alternative treatment for patients with chemotherapy‐refractory DLBCL should be considered.

Inhibition of caspase 9 and not caspase 8 mediated apoptosis may determine clinical response to chemotherapy in primary nodal diffuse large B-cell lymphomas.

Clinical response to chemotherapy in patients with diffuse large B-cell lymphomas is poorly predictable. We demonstrated that an expression profile consistent with inhibition of the stress-induced, caspase 9 mediated apoptosis pathway predicts poor clinical response to chemotherapy, whereas an expression profile consistent with inhibition of only the death-receptor induced, caspase 8 mediated pathway was associated with an excellent response to chemotherapy and favorable outcome. Furthermore, functional analysis of apoptosis sensitivity showed that lymphomas with a caspase 9 inhibition profile were indeed relatively resistant to Etoposide induced apoptosis. Determining the functional status of both apoptosis signaling pathways may accurately identify patients at high risk for fatal outcome and may have implications for alternative therapies triggering the death receptor mediated apoptosis pathway.

Expression of TNF-receptor associated factor 2 correlates with poor progression-free survival time in ABC-like primary nodal diffuse large B-cell lymphomas.

Aims:  Tumour necrosis factor (TNF)‐receptor associated factor 2 (TRAF2) is an adaptor molecule involved in nuclear factor (NF)‐κB activation, which is characteristic of in vitro activated B‐cell (ABC)‐like diffuse large B‐cell lymphomas (DLBCL) and may result in expression of anti‐apoptotic genes and poor response to chemotherapy. TRAF2 also has direct anti‐apoptotic properties via interference with the apoptosis signalling cascade. The aim was to determine whether TRAF2 is preferentially expressed in ABC‐like DLBCL, and whether expression correlates with clinical outcome.