Bcl6 for identification of germinal centres in salivary gland biopsies in primary Sjögren's syndrome.

Histopathological assessment of salivary gland biopsies is an important element of the diagnostic work-up of Sjögren's syndrome (SS) (Fox, 2017; Kroese et al., 2018). Microscopic evaluation of salivary glands of primary SS (pSS) patients reveals characteristic periductal lymphocytic infiltrates (foci), which mainly consist of T- and B-lymphocytes, as well as a variety of non-lymphoid cells, including dendritic cells and macrophages. Over time, these infiltrates may become organised to ectopic lymphoid tissue with T/B cell compartmentalisation, presence of CD21+ follicular dendritic cell (FDC) networks and high endothelial venules (Fisher et al., 2017; Kroese et al., 2014; Kroese et al., 2018; Salomonsson et al., 2003).

Microscopic evaluation of salivary glands of primary SS (pSS) patients reveals characteristic periductal lymphocytic infiltrates (foci), which mainly consist of T and B lymphocytes, as well as a variety of non-lymphoid cells, including dendritic cells and macrophages. Over time, these infiltrates may become organised to ectopic lymphoid tissue with T/B cell compartmentalisation, presence of CD21 + follicular dendritic cell (FDC) networks and high endothelial venules (Fisher et al., 2017;Kroese et al., 2014Kroese et al., , 2018Salomonsson et al., 2003). Germinal centres (GCs) are present within this ectopic lymphoid tissue in roughly one-quarter of the salivary gland biopsies of pSS patients and their presence is associated with more severe disease compared to GC-negative pSS patients (Risselada, Looije, Kruize, Bijlsma, & Roon, 2013). These glandular ectopic GCs express mRNA encoding for activation-induced deaminase, an enzyme critical for the induction of somatic hypermutation and essential for the main function of GCs, the generation of high-affinity memory B cells (Bombardieri et al., 2007;Le Pottier et al., 2009;Muramatsu et al., 2000).
Presence of GCs in biopsies taken for the diagnosis of pSS has been suggested to be a risk factor for lymphoma development (Nishishinya et al., 2015;Sène et al., 2018;Theander et al., 2011), a finding recently disputed by us (Haacke et al., 2017(Haacke et al., , 2019. Detection of GCs in routine haematoxylin and eosin (H&E)-stained sections can be challenging because small GCs may be overlooked and distinction between GCs and lymphoepithelial lesions may be difficult (Fisher et al., 2017;Haacke et al., 2018). Therefore, immunohistochemical identification using antibodies directed against CD21, expressed by FDCs (but also by B cells) or Bcl6, a transcription factor highly expressed by GC-B cells, has been used (Bombardieri et al., 2007;Carubbi et al., 2019;Haacke et al., 2017), but consensus criteria regarding identification of GCs are lacking . Hence, the aim of this study was to asses which staining is most suitable to unequivocally identify GCs in diagnostic salivary gland biopsies of pSS patients by comparing H&E, CD21 and Bcl6 stainings.
In our study, we restricted ourselves to these three markers, which can be easily applied in an automated fashion in diagnostic pathology laboratories. For this reason, we did not consider staining for other GC-associated markers, such as activation-induced deaminase, as potential candidates for identification of GCs in biopsies.  Table 1). Four-micrometre-thick serial sections of salivary gland biopsies were stained with H&E and immunohistochemically for CD21 and Bcl6. For detailed ethical approval information, staining characteristics and statistical analysis see Appendix S1.
In H&E-stained sections, a GC was defined as a clearly visible lighter area in a lymphocytic infiltrate containing centrocytes, centroblasts, FDCs and macrophages. In CD21-stained sections, a network of positive staining within a focus was classified as a FDC network. In Bcl6-stained sections, a cluster of ≥5 adjacent positive cells within a focus was classified as a GC (Haacke et al., 2017). Even though Bcl6 is also expressed by follicular helper T cells, this expression does not interfere with detection of GCs as these cells are not organised in clusters as GCs, but lie scattered throughout the tissue (Figure 1).
Consequently, the number of CD21 + FDC networks/mm 2 was significantly higher than the number of H&E + -and Bcl6 + -defined GCs/mm 2 in both labial and parotid salivary glands (Figure 1b and c). We observed a significant correlation between CD21 + FDC networks/mm 2 in parotid and labial salivary gland biopsies (Figure 1d, r = .60, p = .001), indicating comparability in lymphoid organisation at these two anatomical sites. Such a correlation was not seen for the presence of H&E-or Bcl6-defined GCs.
In a recent study, Carubbi et al. analysed the usage of CD3/ CD20 as well as CD21 and Bcl6 as markers for the detection of GCs (Carubbi et al., 2019). While they conclude that combination of CD3/CD20 and CD21 should be recommended for assessment of GCs, we clearly show here that usage of CD21 as surrogate marker for GCs significantly overestimates GC counts. The reason for this is that formation of B-cell follicles and presence of CD21 + FDC networks (which are also present in primary B-cell follicles) do not also imply presence of GCs (MacLennan, 1994). On the other hand, staining with H&E revealed fewer GCs compared to staining for Bcl6, most likely because small GCs can easily be overlooked on H&E.
Although staining for CD21 is thus less appropriate for detection of GCs, staining for CD21 is still valuable. FDCs play an essential role in the spatial orientation and B/T-cell compartmentalisation in ectopic lymphoid tissues due to their CXCL13 producing property. Presence of FDC networks suggests a more advanced stage of ectopic lymphoid development and may therefore be a useful marker for classification of the organisation of glandular tissue (Jonsson & Skarstein, 2008).
In conclusion, we propose to use Bcl6 as a simple, sensitive and specific marker for unequivocal identification of GCs in salivary gland biopsies of (suspected) pSS patients. Large prospective studies are now needed to evaluate whether presence of GCs in diagnostic salivary gland biopsies for pSS is a risk factor for non-Hodgkin lymphomas or not, and whether it can be used for stratification of pSS patients for personalised medicine (Delli et al., 2019;Kroese et al., 2018).

CO N FLI C T O F I NTE R E S T
There are no competing interests for any author.