Gene expression profiling of microdissected lymphoma cells
As the present work represents one of the first global transcriptome studies with microdissected lymphoma cells, the reliability of the results obtained is a critical issue. Certainly, when microdissecting L&H cells from tissue sections, a low level contamination with RNA from rosseting CD4+ T cells cannot be excluded. However, for several reasons it is evident that the results obtained in the present analysis are not compromised by such a contamination. First, the immunohistochemical study of genes identified in the comparison of microdissected L&H cells and cytometrically isolated GC B cells revealed that all markers were indeed consistently expressed in L&H cells ( and ). Second, several typical markers of activated CD4+ T cells (e.g., GATA3, CTLA4, and IL7-R), which were clearly detected in tonsillar-activated CD4+ T cells, gave signals only in the range of the background noise in L&H cells (unpublished data). Third, CD4+ T cells do not show an NF-κB signature (unpublished data), ruling out that the strong NF-κB signature detected in L&H cells could derive from contaminating T cells. Finally, a low level contamination with RNA from T cells should furthermore largely be averaged out in the differential gene expression studies, as T cells are also surrounding the other lymphoma cells microdissected as single cells (lymphoma cells from cHL and TCRBL), and T cells are also present at low frequency in the lymphoma microenvironment of the lymphomas in which lymphoma cell–rich areas were microdissected (DLBCL, FL, and BL).
L&H and HRS cells are very similar in their gene expression patterns
NLPHL and cHL are classified as two subtypes of HL, and several genes were already identified as being aberrantly expressed by both L&H and HRS cells (18
). However, several studies stressed many clinical, phenotypic and genetic differences between L&H and HRS cells and also pointed to distinct histogenetic origins of the tumor cells (1
). On this background, it was unexpected to find only a few genes differentially expressed between L&H and HRS cells. Our approach is, however, partly underestimating the differences between L&H and HRS cells, as HRS cells may be quite heterogeneous in gene expression, and we focused on genes with consistent differential expression. Many of the phenotypic differences between L&H and HRS cells seen in our analysis and also described in the literature (4
) are related to the strong down-regulation of B cell markers in HRS cells, which is not as extensive in L&H cells (discussed in the third following section). Nevertheless, we identified a few additional differences in the gene expression of L&H and HRS cells (). Future studies should reveal which of the differences reflect or are responsible for distinct pathogenetic mechanisms in NLPHL and cHL.
Based on many similarities in their histological picture (a nodular growth pattern, and an association with CD57+CD4+ GC T helper cells and follicular dendritic cells), the phenotype of the lymphoma cells (CD20+, BCL6+, activation-induced cytidine deaminase+, centerin+, human GC-associated lymphoma+), and genetic features (selection for functional IgV genes and ongoing somatic hypermutation), one might have expected that L&H cells are actually more similar to FL cells than to HRS cells. However, our study shows that at the level of global gene expression, L&H cells are much more closely related to HRS than to FL cells.
Only a few genes distinguish L&H cells and the lymphoma cells of TCRBL
L&H cells turned out to be most closely related to the neoplastic cells of TCRBL and a subgroup of DLBCL, thus expanding on a genome-wide view of the reported biological, phenotypical, and clinical relatedness of these lymphomas. Despite their morphological and phenotypical similarities, the clinical presentation and treatment strategies of TCRBL and NLPHL are different. Therefore, the differential diagnosis between NLPHL and TCRBL has important therapeutic implications. So far, only the transcription factor PU.1 has been reported to be expressed more frequently in L&H cells than in the lymphoma cells of TCRBL. We identified 42 differentially expressed genes (Fig. S2 and Table S1) that might be suitable for establishing new immunohistochemical markers for differential diagnosis.
L&H cells resemble GC B cells at the transition to memory B cells
Although L&H cells clearly resemble GC B cells in many phenotypic and genetic aspects, their gene expression profile () suggests that they might originate from transformed late GC B cells in the process of differentiation toward memory B cells. This hypothesis would be consistent with our finding of constitutive NF-κB activity in L&H cells, as a subset of centrocytes in the GC light zone presumably developing toward memory B cells shows active NF-κB, whereas the majority of GC B cells lacks NF-κB activity (41
). However, it cannot be excluded that NF-κB activation is acquired after neoplastic transformation of a GC B cell into a L&H cell, thus being unrelated to the cell of origin of NLPHL.
L&H cells show a partial loss of their B cell phenotype
Previous immunohistochemical studies reported that L&H cells usually express general B cell markers and GC B cell–specific molecules (3
). However, at the level of sensitivity of immunohistochemistry, L&H cells also lack expression of some B lineage markers (65
). These data are, however, not quantitative and are restricted to a relatively small number of proteins. Analyzing a comprehensive set of 61 B lineage–specific genes, we identified 60 genes that were not expressed or transcribed at reduced levels in L&H cells in comparison to normal GC B cells, including many already described.
As plasma cells down-regulate many B cell genes, it has to be considered whether the low expression of multiple B cell genes reflects a (partial) differentiation of L&H cells or their precursors toward a plasma cell phenotype. However, the PCA suggested that L&H cells are more similar to GC and memory B cells than to plasma cells. Moreover, for key plasma cell markers (i.e., BLIMP1, XBP1, and CD38), the expression levels of L&H cells were similar to the ones of GC B cells and lower than in plasma cells (unpublished data). Thus, the down-regulated B cell phenotype of L&H cells cannot be explained by a plasma cell differentiation.
The diminished expression of the B cell–specific transcription factors PAX5, E2A, EBF, Ikaros, and BCL11A, and of the Notch1 inhibitor deltex 1, is likely to result in a reduced expression of multiple B cell genes and, hence, is presumably a major cause of the partial loss of the B cell phenotype in L&H cells. Furthermore, the aberrant expression of ID2, a negative regulator of E2A, in L&H cells (66
) may also contribute to the diminished expression of several B lineage–specific genes in these cells. This could also be caused by epigenetic silencing, but promoter methylation does not explain the loss of expression of CD10, CD19, or LCK in L&H cells (65
Only a single B cell–associated gene, IL21R
, was up-regulated in L&H cells, and it also showed a tendency for increased expression at the protein level. As follicular T helper cells are the main producers of IL21 and as IL21R triggering improves the survival of normal GC B cells, sustained IL21R expression may be important for the survival of L&H cells and their interaction with surrounding T helper cells (67
In cHL, the global loss of the B cell phenotype may be of selective advantage for the HRS (precursor) cells to escape the apoptotic program in GC B cells with destructive IgV mutations (2
). However, L&H cells express functional B cell receptors, rendering this concept unlikely for NLPHL. Moreover, L&H cells still express many genes at immunohistochemically detectable levels, and they are closely associated with normal constituents of GC, indicating microenvironmental interactions like those of normal GC B cells. Therefore, it appears that (some) B cell functions are still essential for the survival and proliferation of those cells. Perhaps the diminished expression and/or function of B cell genes in L&H cells is a side effect of the so far largely unknown transforming event in these cells. Indeed, for PAX5
, mutations were recently described in L&H cells, although their functional consequences are still unclear (14
Identification of L&H cell–specific genes
We identified 49 genes as being significantly up-regulated in L&H cells in comparison to normal B cells and B-NHL. By immunohistochemistry, we showed protein expression of MMP12, CTSB, and EOMES () in L&H cells of primary NLPHL, thereby confirming our microarray data. Indeed, the genes showing deregulated expression in L&H cells may represent interesting new diagnostic markers and might be of special interest for the pathogenesis of NLPHL. Protein kinase C ζ is involved in cell growth, proliferation, and differentiation, as well as in the activation of NF-κB (68
). KISS1 receptor activates ERK signaling (46
). High levels of ubiquitin D (UBD) were shown to increase mitotic nondisjunction and chromosome instability promoting tumorigenesis (69
). Up-regulated UBD in L&H cells might therefore contribute to chromosomal abnormalities in NLPHL.
L&H cells show deregulation of apoptosis regulators
L&H cells show up-regulated expression of genes encoding antiapoptotic molecules and down-regulation of most proapoptotic genes. The pattern is not only a characteristic feature of L&H cells in comparison to the apoptosis-prone GC B cells, but also in comparison to the other normal B cell subsets, indicating a tumor-specific deregulation of molecules regulating apoptosis (Fig. S4). Notably, HRS cells and most TCRBLs and DLBCLs show a similar expression pattern of pro- and antiapoptotic genes as L&H cells, whereas FLs and BLs behave differently in this regard. This indicates similar pathogenic mechanisms in NLPHLs, TCRBLs, and some DLBCLs to escape apoptosis, further supporting the similarity of these malignancies.
L&H cells are characterized by a strong constitutive NF-κB activity
Constitutive NF-κB activity is involved in deregulated proliferation, malignant transformation, and resistance to apoptosis in a variety of tumors, including cHL (70
). In previous studies, active RelA was detected in L&H cells in a small collection of 6 NLPHL cases (71
), and cREL was found in only 3 out of 15 NLPHLs (72
). These studies did not address whether these NF-κB factors indeed cause expression of NF-κB target genes. To clarify this, we analyzed the expression of a large set of NF-κB target genes in L&H cells and found it to be at least as high as in HRS cells (). Strong immunostaining for the active form of p65 in L&H cells in 19 out of 19 NLPHLs () further confirms constitutive NF-κB activity in L&H cells.
The mechanisms underlying the strong NF-κB activity in L&H cells are still unclear. Genomic gains of the REL
gene, as frequently seen in cHL, are presumably not involved, because L&H cells rarely show active c-REL (72
). A contribution by EBV, as in cHL, does not play a role in NLPHL, as L&H cells are virtually never EBV infected. Remarkably, whereas T cells rosetting around HRS cells in cHL frequently express CD40L and may thus cause NF-κB activation by triggering CD40 on the HRS cells, rosetting T cells surrounding L&H cells rarely express CD40L, rendering a contribution of this signaling pathway in L&H cells also unlikely (73
). Whether inactivating mutations of the IκBα or the IκBε genes as in cHL are involved in the pathogenesis of NLPHL remains to be clarified.
Implications for NLPHL pathogenesis, treatment, and diagnosis
The present work revealed important novel insights into the nature and pathogenesis of the L&H cells in NLPHL. Although L&H cells clearly resemble GC B cells in many phenotypic and genetic aspects, their gene expression profile seems to place them at the transition of GC B cells to memory B cells. We identified several pathogenetic mechanisms in NLPHL, including strong constitutive activity of NF-κB and activation of the ERK signaling pathway. Activation of the ERK pathway is critical for a large number of Ras-induced cellular responses and has been implicated in the pathogenesis of several cancers. Its aberrant activation in L&H cells was previously unknown and might be of pathogenic relevance. Several pharmacological agents that inhibit NF-κB or ERK are currently under clinical investigations (74
) and may become suitable for therapy in NLPHL. Further pathogenetic mechanisms in NLPHL involve the suppression of apoptosis and the creation of an immunosuppressive environment to escape from immune surveillance. The role of other genes that emerged to be specifically deregulated in L&H cells requires further investigation.
Because HRS and L&H cells show marked differences in immunohistochemical analysis (1
), their close relatedness in terms of gene expression profile appears surprising. Similarities among these tumor cells include constitutive NF-κB activity and ERK signaling, which suggest that NLPHL and cHL may share similar pathogenetic mechanisms. The gene expression profile of HRS and L&H cells also resemble each another because of down-regulation of B lineage–specific genes, although this event is not as severe in L&H cells as in HRS cells. L&H cells turned out to be even more similar to TCRBL (and a subset of DLBCL) in terms of gene expression. Because the distinction between NLPHL and TCRBL is, however, clinically important (76
), the genes discriminating between L&H cells and TCRBL identified in this paper may become valuable markers for the differential diagnosis of NLPHL and TCRBL.