Michiel van den Brand

Activated leukocyte cell adhesion molecule expression predicts lymph node metastasis in oral squamous cell carcinoma.

Lymphatic metastasis of oral squamous cell carcinoma (SCC) is important for prognosis and clinical decision making concerning the treatment of the neck but may be difficult to detect. Activated leukocyte cell adhesion molecule (ALCAM), has been shown to correlate with prognosis or tumor grade in different tumor types and may be a predictor of lymphatic metastasis. ALCAM expression at the invasive front in fresh frozen tissue samples of oral SCCs (n=41) was studied immunohistochemically, using a polyclonal antibody directed against ALCAMs extracellular domain. Membranous expression of ALCAM at the invasive front was significantly related to lymph node metastasis (p=0.001, sensitivity 69%, specificity 84%) and tumor grade (p=0.035). There was no significant relationship with tumor thickness (p=0.394). Lymph node status (p=0.030), correlated with 5-year overall survival. A significant relation between ALCAM and prognosis could not be established, due to an insufficient sample size. However, ALCAM expression does appears to increase risk of early death. Membranous ALCAM expression at the invasive front serves as a molecular marker for lymphatic metastasis and may facilitate better treatment choices concerning the neck in patients with oral SCC.

Sequential immunohistochemistry: a promising new tool for the pathology laboratory

Current immunohistochemical methods to study the expression of multiple proteins in a single tissue section suffer from several limitations. In this article, we report on sequential immunohistochemistry (S‐IHC), a novel, easy method that allows the study of numerous proteins in a single tissue section, while requiring very limited optimization.

Tetraspanin CD37 protects against the development of B cell lymphoma

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.

Angiosarcoma in a patient with immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome

The Immunodeficiency, Centromeric region instability, and Facial anomalies (ICF) syndrome (OMIM #242860) is a rare autosomal recessive disorder caused by defective DNA methylation. Hematological disease and malignancy (macrophage activation syndrome, myelodysplastic syndrome, and Hodgkin lymphoma) have been reported in three patients. To date, there have been no reports of either epithelial or mesenchymal malignancies. We present a patient with all clinical and laboratory findings of the ICF syndrome who died of a metastatic angiosarcoma of the liver. This is the first report of a non‐hematological malignancy in the ICF syndrome. The young age at which our patient developed an angiosarcoma suggests an effect of the defective DNA methylation observed in the ICF syndrome. Therefore, with improvement of recognition and treatment of the ICF syndrome, malignancy could become more common in this condition.

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-κBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.

Immunohistochemical differentiation between follicular lymphoma and nodal marginal zone lymphoma – combined performance of multiple markers

Although many lymphomas can be reliably classified according to the World Health Organization Classification of 20081, the differentiation between nodal marginal zone lymphoma (NMZL) and follicular lymphoma (FL) is problematic in some cases. In fact, NMZL is often diagnosed by exclusion, resulting in heterogeneity in the diagnostic category of NMZL. New markers for NMZL have been described, but they have not yet been tested in combination.2,3 In this study, we compared multiple immunohistochemical markers for their use in distinguishing NMZL from FL. From the results obtained, we constructed an algorithm that combines these markers to help distinguish between FL and NMZL. Notably, this algorithm also contains a category of “B-cell lymphoma, unclassifiable”, thus underlining the difficulty that remains in distinguishing NMZL from FL. For the initial test series, we selected 47 patients with FL with a chromosomal rearrangement of BCL2, and 44 patients with a diagnosis of NMZL or probable NMZL, from the archive of the Department of Pathology at the Radboud university medical center (Nijmegen, the Netherlands). For all NMZLs, BCL2 translocations were excluded using fluorescent in-situ hybridization with split-signal probes. Patient characteristics are described in Online Supplementary Table S1. For a diagnosis of NMZL, the following diagnostic criteria were used: 1) effaced architecture of the lymph node, due to a small B-cell proliferation with a follicular, marginal zone, or diffuse growth pattern, 2) either centrocytoid or more round cells with intermingled centroblasts, 3) a mature B-cell immunophenotype with expression of BCL2, 4) in cases with a follicular/ nodular growth pattern, signs of follicular colonization (presence of BCL2 negative cells and high Ki67 staining), 5) not fitting a diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, or lymphoplasmacytic lymphoma. Expression of germinal center markers was not considered an exclusion criterion for a diagnosis of NMZL in this study. As expected, immunohistochemistry showed significant differences between NMZL and FL (Table 1). Overall, FLs were positive for germinal center markers (CD10, BCL6, LMO2, HGAL) and negative for MNDA and IRTA1 (Online Supplementary Figure S1). NMZLs mostly showed an opposite pattern with positivity for MNDA in approximately two thirds of cases, IRTA1 staining in approximately one fifth of cases and usually no staining with germinal center markers. However, all germinal center markers were positive in a subset of NMZLs, and similarly, FLs with expression of MNDA were also identified (Online Supplementary Figure S2). Table 1. Immunohistochemistry results. Based on the immunohistochemistry results, a combination of markers were used to design an algorithm that helped to distinguish NMZL from FL (Figure 1). This algorithm was built empirically, allowing inclusion of a category of “B-cell lymphoma, unclassifiable” to prevent contamination of the NMZL category. As expected, this algorithm classified most lymphomas according to their original diagnosis (Table 2). However, in the initial test series, one case of FL was classified as NMZL, and 6 cases of NMZL were classified as FL by the algorithm; a significant proportion of cases (13%) were considered “B-cell lymphoma, unclassifiable” by the algorithm. Most (75%) of these unclassifiable cases had an original diagnosis of NMZL. Figure 1. Immunohistochemical algorithm for separation of nodal marginal zone lymphoma (NMZL) from follicular lymphoma (FL). The algorithm starts at the top with a lymphoma that is considered to be either FL or NMZL. If all four germinal center markers (BCL6, CD10, ... Table 2. Algorithm results. To validate the algorithm, a second validation group of 21 FLs and 13 NMZLs, collected from the archive of the Department of Pathology at the Hospital del Mar (Barcelona, Spain) was stained for the same markers as the initial group. Overall, staining results were comparable to those in the test group, with a high sensitivity of BCL6 for FL and a high specificity of IRTA1 for NMZL (Table 1); CD10 expression had a higher sensitivity in comparison to the test group, but a lower specificity. LMO2 and HGAL were less sensitive but more specific. In this validation group, the algorithm gave a concordant classification as either NMZL or FL in 85% of cases (Table 2). No follicular lymphoma was misclassified as NMZL, and only one NMZL was misclassified as FL. Four cases (12%) were considered unclassifiable, three of which had an original diagnosis of FL and one with an original diagnosis of NMZL. The algorithm was designed based on a comparison of NMZLs with FLs with a translocation involving BCL2. However, because a BCL2 translocation can be demonstrated relatively easily, the actual problem we are faced with in daily practice is the separation of NMZL from FL without a BCL2 translocation. FLs with and without a BCL2 translocation might be different from each other, as has been suggested by one gene expression study4, and also by a recent comparative genomic hybridization study.5 In the latter study, genetic aberrations in FLs without a BCL2 translocation bore more resemblance to those in NMZL than those in FLs with a BCL2 translocation. To address this problem, we tested a small series of 6 FLs without a BCL2 translocation, which were all classified as FL by the algorithm. This supports the idea that this algorithm also applies to FLs without a BCL2 translocation, but confirmation of this theory will require the study of a larger series. Thus far, the majority of the markers in the algorithm has only been described in single studies. Kanellis and colleagues reported expression of MNDA in 75% of NMZLs versus 5% of FLs.2 In our series, a less pronounced, but similar difference was observed, with MNDA expression in NMZL and FL in 70% and 15%, respectively. In accordance with Falini and colleagues6, IRTA1 also discriminated between NMZL and FL in our series. However, in the study carried out by Falini et al.,73% of NMZLs expressed IRTA1, compared to only 21% in our study. This difference could be caused by a difference in interpretation. In our case, faint IRTA1 expression was quite frequently observed in NMZLs, but also in some FLs. As the reproducibility of the scores assigned to these cases proved to be very poor amongst different observers, a case was only considered positive if 30% or more of the cells showed moderate or strong expression of IRTA1. This aforementioned approach produced a strong improvement in the diagnostic value of IRTA1 in our series, and explains the small proportion of NMZLs positive for IRTA1. Expression of the germinal center markers HGAL and LMO2 in lymphomas has been described by Natkunam et al., whose work showed expression in the majority of FLs and only very rare expression in NMZL.7–10 For both markers, they detected only a single case of NMZL that was positive.7,8 In our series, we observed more frequent expression of both LMO2 and HGAL in NMZL. We believe this could be caused by differences in inclusion criteria between our study and previous studies. In the study effected by Salama et al., which contains the largest majority of NMZLs previously studied for LMO2 and HGAL, cases were excluded from the NMZL group if they expressed germinal center markers9, which explains why expression of germinal center markers was not detected in NMZLs in their study. A recent study by Dyhdalo and colleagues reported LMO2 staining in 2 out of 25 NMZLs, of which one case also expressed BCL6.11 In our study, expression of germinal center markers was not considered an exclusion criterion. We made this choice because, in our experience, typical cases of NMZL do occasionally express germinal center markers and expression of CD10 and BCL6 in NMZL has been previously reported.12–14 The FLs that were used to build the algorithm were all required to have a BCL2 break, which, together with morphology, ensured that the diagnosis of FL was correct. Thus, misclassification of FL with a BCL2 break as NMZL has been excluded. Unfortunately, no markers for NMZL can compare with the BCL2 translocation for FL. Therefore, the NMZL group can still be expected to be more heterogeneous than the FL group, with some cases representing FL or other lymphomas rather than NMZL. Indeed, when looking at the initial and validation series together, ‘NMZLs’ were quite frequently considered FL (in 12%) or unclassifiable (in 18%) by the algorithm. This illustrates the difficulty that remains in the definition and diagnosis of NMZL; the lack of both an accurate definition and of positive diagnostic markers for NMZL result in a heterogeneous diagnostic category. The ultimate question is: what is the gold standard? For this study we have used the combination of morphology and phenotyping for follicular colonization as defining criteria for NMZL. The addition of extensive immunohistochemistry, including new markers, might help to create a better gold standard for NMZL. At present, however, it is very difficult to compare different strategies to diagnose NMZL, due to the fact that as of yet no perfect positive marker for NMZL is available. Hopefully, elucidation of the pathogenesis of NMZL will provide us with better positive markers for NMZL. The results from this study could assist in achieving this goal; the addition of extensive immunohistochemistry to conventional criteria for NMZL will help to establish smaller, but potentially more homogeneous study groups, facilitating studies into the pathogenesis of NMZL.

Recurrent mutations in genes involved in nuclear factor-κB signalling in nodal marginal zone lymphoma—diagnostic and therapeutic implications

To investigate the spectrum of mutations in 20 genes involved in B‐cell receptor and/or Toll‐like receptor signalling resulting in activation of nuclear factor‐κB (NF‐κB) in 20 nodal marginal zone lymphomas (NMZLs), 20 follicular lymphomas (FLs), and 11 cases of B‐cell lymphoma, unclassifiable (BCL‐u).

A subset of low-grade B cell lymphomas with a follicular growth pattern but without a BCL2 translocation shows features suggestive of nodal marginal zone lymphoma

In our consultation practice, it was noted that many cases that were considered to represent follicular lymphoma (FL) without a BCL2 translocation were ultimately classified as nodal marginal zone lymphoma (NMZL). This study set out to define recurrent morphological features of these cases. Thirty-three low-grade B cell lymphomas without a BCL2 rearrangement were studied for recurrent morphological features. These features were then applied on 20 randomly selected cases to verify if these criteria are able to distinguish between lymphomas with and without a BCL2 rearrangement, assigning them to one of five categories ranging from “certain FL” to “certain NMZL.” Highly recurrent morphological features were noted in the lymphomas without a BCL2 rearrangement, which were strongly overlapping with the morphological features of NMZL. All six cases that were assigned to the category of certainly FL or most likely FL indeed harbored a BCL2 rearrangement, whereas all 12 cases assigned to the category of most likely NMZL or certain NMZL had no BCL2 break. Of the two cases in the ambiguous category, one had received a final diagnosis of FL and the other of NMZL. This study raises the hypothesis that a subset of low-grade B cell lymphomas with a follicular growth pattern but without a BCL2 translocation actually represents NMZL. This is at present difficult to prove, because no gold standard is available to differentiate between NMZL and FL without a BCL2 rearrangement, so further investigations are needed.

High prevalence of MYD88 and CD79B mutations in intravascular large B-cell lymphoma

TO THE EDITOR: Intravascular large B-cell lymphoma (IVLBCL) is a rare variant of extranodal diffuse large B-cell lymphoma (DLBCL). It is characterized by proliferation of blastic, neoplastic B cells within the lumina of small- or intermediate-sized blood vessels and capillaries.[1][1] IVLBCL may

High prevalence ofandmutations in intravascular large B-cell lymphoma

TO THE EDITOR: Intravascular large B-cell lymphoma (IVLBCL) is a rare variant of extranodal diffuse large B-cell lymphoma (DLBCL). It is characterized by proliferation of blastic, neoplastic B cells within the lumina of small- or intermediate-sized blood vessels and capillaries.[1][1] IVLBCL may