S. T. Pals

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets: Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Loss of Ep-CAM (CO17-1A) expression predicts survival in patients with gastric cancer

Preoperative staging of gastric cancer is difficult and not optimal. The TNM stage is an important prognostic factor, but it can only be assessed reliably after surgery. Therefore, there is need for additional, reliable prognostic factors that can be determined preoperatively in order to select patients who might benefit from (neo) adjuvant treatment. Expression of immunohistochemical markers was demonstrated to be associated with tumour progression and metastasis. The expression of p53, CD44 (splice variants v5, v6 and v9), E-cadherin, Ep-CAM (CO17-1A antigen) and c-erB2/neu were investigated in tumour tissues of 300 patients from the Dutch Gastric Cancer Trial, investigating the value of extended lymphadenectomy compared to that of limited lymphadenectomy). The expression of tumour markers was analysed with respect to patient survival. Patients without loss of Ep-CAM-expression of tumour cells (19%) had a significantly better 10-year survival (P<0.0001) compared to patients with any loss: 42% (s.e.=7%) vs 22% (s.e.=3%). Patients with CD44v6 (VFF18) expression in more than 25% of the tumour cells (69% of the patients) also had a significantly better survival (P=0.01) compared to patients with expression in less than 25% of the tumour cells: 10 year survival rate of 29% (s.e.=3%) vs 19% (s.e.=4%). The prognostic value of both markers was stronger in stages I and II, and independent of the TNM stage. Ep-CAM and CD44v6-expression provides prognostic information additional to the TNM stage. Loss of Ep-CAM-expression identifies aggressive tumours especially in patients with stage I and II disease. This information may be helpful in selecting patients suitable for surgery or for additional treatment pre- or postoperatively.

High prevalence of oncogenic MYD88 and CD79B mutations in diffuse large B-cell lymphomas presenting at immune-privileged sites

Activating mutations in CD79 and MYD88 have recently been found in a subset of diffuse large B-cell lymphoma (DLBCL), identifying B-cell receptor and MYD88 signalling as potential therapeutic targets for personalized treatment. Here, we report the prevalence of CD79B and MYD88 mutations and their relation to established clinical, phenotypic and molecular parameters in a large panel of DLBCLs. We show that these mutations often coexist and demonstrate that their presence is almost mutually exclusive with translocations of BCL2, BCL6 and cMYC, or Epstein–Bar virus infection. Intriguingly, MYD88 mutations were by far most prevalent in immune-privileged site-associated DLBCL (IP-DLBCL), presenting in central nervous system (75%) or testis (71%) and relatively uncommon in nodal (17%) and gastrointestinal tract lymphomas (11%). Our results suggest that MYD88 and CD79B mutations are important drivers of IP-DLBCLs and endow lymphoma-initiating cells with tissue-specific homing properties or a growth advantage in these barrier-protected tissues.

CD44 Splice Variants as Prognostic Markers in Colorectal Cancer

BACKGROUND Splice variants of CD44 play a causal role in the metastatic spread of pancreatic carcinoma in the rat. In previous studies we have shown that homologues of these CD44 isoforms (CD44v6) are overexpressed during colorectal tumorigenesis in man and that CD44v6 overexpression is associated with an unfavorable prognosis in this disease. In the present study we have assessed the prognostic significance of CD44 variants containing exon v5. In addition, we have used a panel of different antibodies against CD44v6 and applied a combined scoring system to improve its value as prognosticator. METHODS Expression of CD44 variants was studied by immunohistochemistry on frozen tissue sections, and the prognostic value of the CD44 variant expression was assessed using univariate and multivariate analysis. RESULTS Our studies show that expression of CD44v6, but not CD44v5, has significant prognostic value. Analysis of CD44v6 expression by means of a combined scoring system, on the basis of a panel of three different monoclonal antibodies (mAbs), makes CD44v6 a highly significant prognostic marker that is independent of Dukes stage, tumor grade, or tumor localization. CONCLUSION Assessment of CD44v6 expression by a combination of mAbs yields an independent prognosticator that may be of value in identifying patients with a high propensity to develop distant metastasis.

CD44 expression predicts disease outcome in localized large B cell lymphoma

Diffuse large B cell non-Hodgkin’s lymphomas (DLCL) form a heterogeneous group of tumors with diverse morphology, clinical features, treatment response and prognosis. The biological variables underlying this heterogeneity are unknown. In the present study, we explored the value of the lymphocyte homing receptor CD44, a putative determinant of lymphoma dissemination, in predicting prognosis in DLCL. Expression of the standard form of CD44 (CD44s) and of CD44 isoforms containing exon v6 (CD44v6) on tumor cells was assessed by immunohistochemistry in a cohort of 276 DLCL patients from a population based lymphoma registry. We observed that CD44s as well as CD44v6 expression correlated with tumor dissemination in patients with primary nodal DLCL. Importantly, in patients with localized nodal disease, CD44s was a strong prognosticator predicting tumor related death independent of the other parameters of the International Prognostic Index (IPI). Incorporation of CD44s in the IPI parameter ‘stage’, increased the prognostic value of this parameter in nodal DLCL. Our data identify CD44 as a biological prognosticator, which can be used to ‘fine-tune’ the IPI for nodal DLCL.

High prevalence of oncogenic MYD88 and CD79B mutations in primary testicular diffuse large B-cell lymphoma

High prevalence of oncogenic MYD88 and CD79B mutations in primary testicular diffuse large B-cell lymphoma

Expression of oncoproteins and the amount of eosinophilic and lymphocytic infiltrates can be used as prognostic factors in gastric cancer. Dutch Gastric Cancer Group (DGCG).

Preoperative staging of gastric cancer is difficult. Several molecular markers associated with initiation and progression of cancer seem promising for obtaining preoperative prognostic information. To investigate whether these markers are indicative especially for the presence of lymph node metastases in patients with gastric cancer, we have examined primary tumour specimens from 105 patients with primary adenocarcinoma of the stomach entered in a surgical trial. In this trial, lymph node status was determined by strictly quality-controlled lymph node dissection and examination. The selected markers were growth regulators (p53, Rb and myc), metastasis-suppressor gene product (nm23), adhesion molecules (Ep-CAM, E-cadherin, CD44v5 and CD44v6) and urokinase-type plasminogen activator (u-PA). Also, the amount of eosinophilic and lymphocytic infiltrates available post-operatively was analysed with respect to its prognostic value for lymph node status. Moreover, the association of these parameters with survival and disease-free period (DFP) was evaluated. Of all molecular markers investigated, only Rb expression had a significant association with the presence of lymph node metastasis in both univariate and multivariate analysis. For curative resectability, a significant association was found with Rb and E-cadherin expression, while in multivariate analysis Rb and myc were selected as the combination with additional independent prognostic value, and E-cadherin had no additional independent value. For overall survival in univariate analysis, the amount of both eosinophilic and lymphocytic infiltrates and Rb and myc expression were of significant prognostic value. Only the amount of lymphocytic infiltrate had a prognostic significance for DFP. In stepwise multivariate analysis, TNM stage (I + II) and marked lymphocytic infiltrate were associated with better overall survival and longer DFP. We conclude that, if these results are confirmed in a larger series of patients, molecular markers can provide useful prognostic information.

Diffuse large B cell lymphomas relapsing in the CNS lack oncogenic MYD88 and CD79B mutations

Diffuse large B cell lymphomas relapsing in the CNS lack oncogenic MYD88 and CD79B mutations

MiRNA profiling in B non-Hodgkin lymphoma

Several studies have indicated the importance of miRNAs in B cell maturation and in the development of B cell lymphomas. The oncogene MYC plays an important role in B cell lymphomagenesis, particularly in Burkitt lymphoma (BL). Several recent publications have shown that MYC regulates expression of up to 60 miRNAs (Tables I and SI). The impact of the translocation and overexpression of MYC on the miRNA profile in BL has not yet been explored. We determined the miRNA expression profile of paediatric t(8;14) positive and high MYC expressing BL in comparison to MYC translocation negative mantle cell lymphoma (MCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). As a control we included normal B cell subsets obtained from hyperplastic tonsils. Hierarchical clustering showed that the B cell subsets and the non-Hodgkin lymphomas (NHLs) formed two distinct sub-clusters (Fig 1). Unsupervised clustering of the 23 miRNAs significantly differentially expressed between the four B cell subsets (>4-fold) revealed one cluster for the naı̈ve and memory B cells and two additional clusters for the germinal centre (GC) B cells and plasma cells. These results were consistent with previously published data (Appendix S1). 76 miRNAs were differentially expressed (>4-fold) between the four NHL subtypes. Most differences were observed between BL and the other NHLs (CLL n = 58, FL n = 32 and MCL n = 36 miRNAs). Unsupervised hierarchical clustering analysis revealed a unique miRNA profile in BL (Appendix S2). In contrast, a maximum of eight miRNAs were differently expressed between MCL, FL and CLL. A list of validated target genes of the differentially expressed miRNAs is presented in Table SII. Comparing each malignancy to its normal counterpart (MCL with naı̈ve, FL and BL with GC B cells and CLL with memory cells), 54–77 miRNAs were differentially expressed (Figure S1). MYC expression has been shown to be a dominant factor in the regulation of many miRNAs. In the present series of lymphomas, quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed a much higher expression in BL than all other NHL (Fig 1C). Comparison of the MYChigh BL to all other MYC-low lymphomas revealed 122 differentially expressed miRNAs (Table SIII), including 39 of the 50 evaluable (78%) known targets of MYC (10 miRNAs were not expressed in our cases) (Fig 1D and Table I). The expression level of these 39 miRNAs was always consistent with the expected upor downregulation, with most miRNAs being downregulated. This indicates a dominant MYC-induced miRNA profile in primary BL. This signature included 39 of the known MYC-regulated miRNAs. The 83 other miRNAs were also differentially expressed between MYC-high BL and MYC-low NHL samples. At present, there are no data supporting a direct MYC-dependent regulation for those 83 miRNAs. A MYC-dependent miRNA signature has also been suggested for diffuse large B cell lymphoma based on a differential expression of MYC targets (Li et al, 2009). Four BL cases (B4, B5, B6 and B7) and two MCL cases (M2 and M3) harboured a 13q31.3 amplification (determined by fluorescence in situ hybridization, results not shown). These six cases showed high expression of the miR-17-92 cluster (Fig 1D). Both in BL and MCL, this association was independent of the MYC expression level (Fig 1C). In line with the findings of Tagawa et al (2007) this indicates that genomic amplification and not MYC overexpression is instrumental in the expression of the miR-17-92 cluster. The miR-17-92 cluster is positively regulated by MYC and acts with MYC to accelerate tumour development (He et al, 2005). MiR-17 and miR-20, two members of this cluster, promote cell cycle progression via E2F1 (O’Donnell et al, 2005). One interesting MYC-repressed gene is MIRLET7A. As MYC is a direct target of MIRLET7A this suggests a positive stimulatory loop for MYC (Sampson et al, 2007). Induction of MIRLET7A in the Namalwa BL cell line resulted in reduced MYC expression and reduced proliferation, whereas downregulation of MYC resulted in increased expression of MIRLET7A. In addition to a significant downregulation of MIRLET7A, we also observed a significant downregulation for MIRLET7E in BL compared to the three other NHL subtypes. MiR-150 was also significantly downregulated in BL and targets MYB, which has an essential role in haematopoietic and lymphoid development and apoptosis (Xiao et al, 2007). Overexpression of miR-150 in BL cells resulted in reduced MYB levels and increased apoptosis (Tan et al, 2009). Interestingly miR-15a has recently also been shown to repress the MYB oncogene in leukemic cell lines (Zhao et al, 2009). Low expression of miR-150 in BL compared to the other three NHL subtypes and of miR-15a in comparison to CLL might thus result in enhanced MYB levels in BL. Ectopic expression of miR-26a in BL cell lines impaired cell cycle progression via its target EZH2, a member of the polycomb-group of genes (Sander et al, 2008). The low expression of miR-26a in BL is thus consistent with the previously observed high expression of correspondence