Absence of Granzyme B positive tumour-infiltrating lymphocytes in primary melanoma excisional biopsies is strongly associated with the presence of sentinel lymph node metastasis.

BACKGROUND: Sentinel Lymph Node (SLN) status is strongly related to clinical outcome in melanoma patients. In this study we investigated the possible association between the presence of activated and/or suppressive Tumour Infiltrating Lymphocytes (TILs) and SLN status in clinically stage I/II melanoma patients. METHODS: Diagnostic primary melanoma samples from 20 patients with a sentinel lymph node metastasis were compared to melanoma samples from 20 patients with a negative sentinel lymph node, who were matched for gender, age and Breslow thickness. Presence of activated Granzyme B positive (GrB+) TILs, presence of suppressive (FoxP3+) TILs and MHC class I antigen expression on tumour cells were analysed by immunohistochemistry. RESULTS: FoxP3 and MHC-I expression had no direct bearing on the presence of melanoma metastases in the SLN. Whereas the presence of activated GrB+ TILs in the primary melanoma had no predictive value for SLN status either, their absence was strongly associated with the presence of metastasis in the SLN (p=0.001). While both GrB+ and FoxP3+ TILs could be detected in SLN metastases, a majority did not display MHC-I expression. CONCLUSION: These data support a role for cytotoxic T cells in the prevention of early metastasis of melanoma to the draining lymph nodes.

Activity-based differentiation of pathologists' workload in surgical pathology.

Adequate budget control in pathology practice requires accurate allocation of resources. Any changes in types and numbers of specimens handled or protocols used will directly affect the pathologists' workload and consequently the allocation of resources. The aim of the present study was to develop a model for measuring the pathologists' workload that can take into account the changes mentioned above. The diagnostic process was analyzed and broken up into separate activities. The time needed to perform these activities was measured. Based on linear regression analysis, for each activity, the time needed was calculated as a function of the number of slides or blocks involved. The total pathologists' time required for a range of specimens was calculated based on standard protocols and validated by comparing to actually measured workload. Cutting up, microscopic procedures and dictating turned out to be highly correlated to number of blocks and/or slides per specimen. Calculated workload per type of specimen was significantly correlated to the actually measured workload. Modeling pathologists' workload based on formulas that calculate workload per type of specimen as a function of the number of blocks and slides provides a basis for a comprehensive, yet flexible, activity-based costing system for pathology.

Profiling of apoptosis genes identifies distinct types of primary cutaneous large B cell lymphoma.

Two distinct primary cutaneous large B cell lymphomas are recognized: primary cutaneous follicle centre lymphoma (PCFCL), characterized by an excellent prognosis, and primary cutaneous large B cell lymphoma, leg-type (PCLBCL leg-type), with an unfavourable prognosis. To determine whether inhibition of the apoptosis pathways may underlie the difference in clinical outcome between PCFCL and PCLBCL leg-type, we investigated the expression of only apoptosis-related genes by microarray expression profiling. Unsupervised cluster analysis was carried out using 169 genes involved in apoptosis on a group of 21 previously untreated patients diagnosed with primary cutaneous large B cell lymphoma. Cluster analysis resulted in two separate groups which showed large overlap with the PCFCL and PCLBCL leg-type. One group was characterized by high expression levels of pro- and anti-apoptotic genes. The other group was characterized by high expression levels of apoptosis-inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. Our results suggest that the clinically favourable PCFCLs are characterized by a relatively intense cellular cytotoxic immune response and that PCLBCL leg-types are characterized by constitutive activation of the intrinsic mediated apoptosis pathway, with concomitant downstream inhibition of this apoptosis pathway. Thus, strategies neutralizing the function of apoptosis-inhibiting proteins might be effective in PCLBCL leg-type.

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)-kappaB 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. METHODS AND RESULTS: TRAF2 was expressed in nine of 20 tested ABC-like DLBCLs and in only one of 13 tested germinal centre B-lymphocyte (GCB)-like DLBCLs. High TRAF2 expression was correlated with high International Prognostic Index at time of presentation, high chance of relapse and short progression-free survival time in 44 tested DLBCLs. Furthermore, when analysis was restricted to ABC-like DLBCL only, TRAF2 expression was significantly associated with poor progression-free survival time. CONCLUSIONS: TRAF2 might be involved in activation of NF-kappaB in a subset of ABC-like DLBCL, and its expression is associated with a particularly poor outcome in primary nodal DLBCL patients. Because of its possible effect on to chemotherapy resistance, resistance, TRAF2 might be an attractive candidate as a molecular target for TRAF2+ DLBCL.

Expression of c-FLIP is primarily detected in diffuse large B-cell lymphoma and Hodgkin's lymphoma and correlates with lack of caspase 8 activation.

AIMS: Inhibition of apoptosis is important in the pathogenesis of lymphomas. c-FLIP, a regulator of caspase 8-mediated apoptosis, plays an important role in protecting normal B and T cells from apoptosis and possibly also in lymphomas. Because of contradictory reports about immunohistochemical detection of c-FLIP expression, the aim was to test the specificity of four antibodies in c-FLIP-transfected cells and subsequently to investigate expression of c-FLIP in different types of lymphoma. METHODS AND RESULTS: Two of four antibodies were specific. In primary lymphomas c-FLIP expression was restricted to Hodgkin's lymphomas (> 90%) and diffuse large B-cell lymphomas (44%). Burkitt lymphomas and indolent B-cell lymphomas were negative in all cases. No expression was detected in primary T-cell lymphomas, although expression was observed in one relapsed ALK+ anaplastic large cell lymphoma. Expression of c-FLIP was inversely correlated with caspase 8 activation. CONCLUSIONS: c-FLIP is important in escape of B cells from apoptosis during normal follicle centre cell reaction and may thus be an important early event in the development of B-cell-derived lymphomas. Moreover, non-specific staining of frequently used antibodies might explain discrepancies in different reports of c-FLIP expression.

Disappointing outcome of autologous stem cell transplantation for enteropathy-associated T-cell lymphoma.

BACKGROUND: Despite treatment, enteropathy-associated T-cell lymphoma has a very poor outcome. Chemotherapy can be complicated by small bowel perforation, gastrointestinal bleeding and development of enterocolic fistulae. Here we report on the feasibility, safety and efficacy of high-dose chemotherapy followed by autologous stem cell transplantation in patients with enteropathy-associated T-cell lymphoma (three upfront and one at relapse), with or without prior partial small bowel resection. METHODS: Four patients [two males, two females, mean age 65 years (range 60-69 years)] received high-dose chemotherapy followed by autologous stem cell transplantation. Partial small bowel resection has been performed in three patients. RESULTS: All four patients completed the mobilization and leucopheresis procedures successfully and subsequently received conditioning chemotherapy and transplantation. Engraftment occurred in all patients. No major non-haematological toxicity or transplantation-related mortality was observed. One patient has ongoing complete remission 32 months after transplantation. Three patients died from relapse within few months after autologous stem cell transplantation. CONCLUSIONS: Autologous stem cell transplantation seems unsatisfactory for patients with enteropathy-associated T-cell lymphoma. More intensive conditioning and aggressive chemotherapy with/or without targeted immunotherapy as well as allogenous stem cell transplantation needs to be explored.

Cross-platform array comparative genomic hybridization meta-analysis separates hematopoietic and mesenchymal from epithelial tumors.

A series of studies have been published that evaluate the chromosomal copy number changes of different tumor classes using array comparative genomic hybridization (array CGH); however, the chromosomal aberrations that distinguish the different tumor classes have not been fully characterized. Therefore, we performed a meta-analysis of different array CGH data sets in an attempt to classify samples tested across different platforms. As opposed to RNA expression, a common reference is used in dual channel CGH arrays: normal human DNA, theoretically facilitating cross-platform analysis. To this aim, cell line and primary cancer data sets from three different dual channel array CGH platforms obtained by four different institutes were integrated. The cell line data were used to develop preprocessing methods, which performed noise reduction and transformed samples into a common format. The transformed array CGH profiles allowed perfect clustering by cell line, but importantly not by platform or institute. The same preprocessing procedures used for the cell line data were applied to data from 373 primary tumors profiled by array CGH, including controls. Results indicated that there is no apparent feature related to the institute or platform and that array CGH allows for unambiguous cross-platform meta-analysis. Major clusters with common tissue origin were identified. Interestingly, tumors of hematopoietic and mesenchymal origins cluster separately from tumors of epithelial origin. Therefore, it can be concluded that chromosomal aberrations of tumors from hematopoietic and mesenchymal origin versus tumors of epithelial origin are distinct, and these differences can be picked up by meta-analysis of array CGH data. This suggests the possibility of prospectively using combined analysis of diverse copy number data sets for cancer subtype classification.

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.

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.

Prognostic significance of activated cytotoxic T-lymphocytes in primary nodal diffuse large B-cell lymphomas.

Clinical outcome in diffuse large B-cell lymphoma (DLBCL) remains unpredictable, despite the identification of clinical prognostic parameters. Here, we investigated in pretreatment biopsies of 70 patients with DLBCL whether numbers of activated cytotoxic T-lymphocytes (CTLs), as determined by the percentage of CD3-positive lymphocytes with granzyme B (GrB) expression, have similar prognostic value as found earlier in Hodgkin's lymphoma and anaplastic large-cell lymphoma and whether loss of major histocompatibility complex (MHC)-I molecules or expression of the GrB antagonist protease inhibitor 9 (PI9) may explain immune escape from CTL-mediated cell death. Independent of the International Prognostic Index (IPI), the presence of >/=15% activated CTLs was strongly associated with failure to reach complete remission, with a poor progression-free and overall survival time. Downregulation of MHC-I light- and/or heavy-chain expression was found in 41% of interpretable cases and in 19 of 56 interpretable cases PI9 expression was detected. We conclude that a high percentage of activated CTLs is a strong, IPI independent, indicator for an unfavorable clinical outcome in patients with primary nodal DLBCL. Although in part of DLBCL expression of PI9 and loss of MHC-I expression was found, providing a possible immune-escape mechanism in these cases, no correlation with clinical outcome was found.

ALK-negative anaplastic large-cell lymphoma demonstrates similar poor prognosis to peripheral T-cell lymphoma, unspecified.

AIMS: Anaplastic large cell lymphoma (ALCL) is classically considered a clinicopathological entity separate from other nodal mature T-cell lymphomas (TCL). Recently, the anaplastic lymphoma kinase (ALK) protein was shown to identify a subgroup of nodal ALCL with an excellent prognosis, whereas ALK-negative ALCLs are more heterogeneous. The aim of this study was to investigate the clinicopathological parameters in relation to clinical behaviour of ALK-negative ALCL compared with other nodal mature TCL, i.e. peripheral TCL, unspecified (PTCL-NOS) and angioimmunoblastic lymphoma (AILT). METHODS AND RESULTS: Clinicopathological data of ALK-positive (n = 28) and ALK-negative (n = 46) ALCL; PTCL-NOS (n = 47); and AILT (n = 12) were analysed for their prognostic significance. While ALK-positive ALCL shows favourable clinical features and a good prognosis, ALK-negative ALCL, PTCL-NOS and AILT are all associated with high age groups, advanced disease stage, and poor prognosis (<45% 5-year survival). In multivariate analysis of overall survival time, performed in the combined group of ALK-negative nodal mature T-cell lymphomas, only age and the International Prognostic Index (IPI) remained independent prognostic parameters, while lymphoma subtype (ALCL versus PTCL-NOS versus AILT) gave no additional information. CONCLUSIONS: The distinction between ALK-negative ALCL and PTCL-NOS or AILT is of limited clinical relevance as they show comparable poor prognosis. In these lymphoma subtypes, only age and the IPI are of significant prognostic value.

Analysing gene expressions with GRANK.

UNLABELLED: Unravelling overlapping clusters of functionally linked genes is a major challenge to current clustering programs. Algorithm GRANK permits systematic study of overlap between clusters of genes of a similar expression profile and large variation across a series of microarrays. AVAILABILITY: GRANK is freely available from http://www.bio.vu.nl/microb/research/tumor.html provided one adheres to the end-user license agreement in the associated manual. SUPPLEMENTARY INFORMATION: Above website also supplies the in- and output files used in the example run.

MUC1 (EMA) is preferentially expressed by ALK positive anaplastic large cell lymphoma, in the normally glycosylated or only partly hypoglycosylated form.

AIMS: To investigate whether MUC1 mucin, a high molecular weight transmembrane glycoprotein, also known as epithelial membrane antigen (EMA), differs in its expression and degree of glycosylation between anaplastic large cell lymphoma (ALCL) and classic Hodgkin's disease (HD), and whether MUC1 immunostaining can be used to differentiate between CD30 positive large cell lymphomas. METHODS/RESULTS: Using five different monoclonal antibodies (E29/anti-EMA, DF3, 139H2, VU-4H5, and SM3) that distinguish between various MUC1 glycoforms, high MUC1 expression (50-95% of tumour cells positive) was found in 13 of 17 anaplastic lymphoma kinase (ALK) positive systemic nodal ALCLs, and in one of 20 cases of classic HD. Scattered or focal staining (< 25% of tumour cells) was seen in two additional ALK positive systemic ALCLs, two additional classic HD cases, and in three of 20 cases of ALK negative systemic nodal ALCL. Primary cutaneous ALCL showed no staining with the anti-MUC1 antibodies. Antibodies detecting hypoglycosylated MUC1 were found to be absent in all lymphomas (SM3) or present in only six of 15 ALK positive ALCLs (VU-4H5). CONCLUSIONS: MUC1 is preferentially expressed by a subtype of systemic nodal ALCL, characterised by ALK expression, but is found in only a few cases of classic HD and ALK negative ALCL. Therefore, although MUC1 could be used in a panel of markers for CD30 positive lymphomas, it is probably not a valuable tool to differentiate between ALK negative CD30 positive large cell lymphomas. Finally, the degree of MUC1 glycosylation in lymphomas is relatively high, compared with the aberrant hypoglycosylation found in adenocarcinomas.

Percentage of activated cytotoxic T-lymphocytes in anaplastic large cell lymphoma and Hodgkin's disease: an independent biological prognostic marker.

Recently, we demonstrated that the presence of high percentages of activated cytotoxic T-lymphocytes (CTLs) in biopsy specimens of both Hodgkin's disease (HD) and ALK negative anaplastic large cell lymphoma (ALCL) is associated with a poor prognosis. To test whether this biological prognostic factor is more important in predicting clinical outcome than histological diagnosis or clinical factors, we compared the prognostic value of these parameters in an expanded group of classical HD and ALK negative ALCL. Tumor biopsies of classical HD (n = 83) and ALK negative systemic nodal ALCL (n = 43) were investigated for the presence of activated CTLs by immunohistochemistry, using a monoclonal antibody directed against granzyme B. Percentages of activated CTLs were quantified using Q-PRODIT, and their prognostic value was compared to that of histological diagnosis and clinical parameters, including age and stage. Both in classical HD and ALK negative ALCL, a high percentage of activated CTLs (ie > or = 15%) identified a group of patients with poor overall and progression-free survival time, even when adjusted for stage. In multivariate analysis, percentage of activated CTLs remained a strong independent prognostic marker, and was more sensitive than histological diagnosis or clinical factors in predicting overall survival time. We conclude that a high percentage of activated CTLs in the reactive infiltrate of ALK negative ALCL and classical HD is a strong indicator for an unfavorable clinical outcome, regardless of histological diagnosis or clinical parameters. As such, this biological parameter may be an especially helpful tool to determine therapeutic strategies in cases in which the differentiation between ALK negative ALCL and HD remains difficult.