Section 3 -

Cutaneous Lymphomas Composed of Large B-cell Lymphocytes Beatrice Vergier French Study Group on Cutaneous Lymphomas

Introduction The classification of primary cutaneous large B-cell lymphomas has been the subject of much debate in the past between dermatopathologists and hematopathologists. The former observed that cutaneous large B-cell lymphomas arising in the leg (often elderly patients) had a very bad prognosis, so they used the term "large B-cell lymphoma of the leg" in the European Organization for Research and Treatment of Cancer (EORTC) classification [1, 2] . The latter discussed the use of an anatomic term (leg) for a histological classification [3] . Fortunately the consensus World Health Organization-European Organization for Research and Treatment of Cancer (WHO-EORTC) meetings led to a classification useful for histological, clinical and therapeutical purposes [4] . In the WHO-EORTC classification, primary cutaneous B-cell lymphoma with a histology of diffuse large B-cell lymphoma are classified in three categories: Primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL leg-type) defined as a cutaneous B-cell lymphoma with a predominance or confluent sheets of large round cells (centroblasts and immunoblasts). Primary cutaneous follicle center lymphoma (PCFCL) including large B-cell lymphomas with a predominant cleaved cell morphology (large centrocytes) whatever their diffuse or follicular and diffuse growth pattern. Primary cutaneous diffuse large B-cell lymphoma, other including variants of diffuse large B-cell lymphoma not included in PCLBCL leg type or in PCFCL. This includes also anaplastic or plasmablastic, T-cell/histiocyte-rich large B-cell lymphomas and intra-vascular large B-cell lymphoma defined by an accumulation of large neoplastic B cells within blood vessels. This classification is very useful even if there are always unanswered questions like: do PCLBCL other truly represents a distinct group of cutaneous large B-cell lymphomas or do they consist of phenotypic and/or morphologic variations of both PCLBCL leg type and PCFCL? which parameters allow differentiation between PCLBCL leg typewith a poor or worse prognosis? is bcl2 expression essential for including a large round cell B-cell lymphoma in PCLBCL leg type? In the event of cutaneous diffuse large B-cell lymphoma, the distinction between PCLBCL leg type and PCFCL (predominant large centrocytes) is clinically important because of its impact on the first choice of treatment (radiotherapy vs chemotherapy) and the prognosis (5-year survival 52% and > 95% respectively) [4] . Clinical features [5, 6, 7] PCLBCL, leg-type predominantly affects patients older than 70 years. Women are more often affected than men. Patients present rapidly growing erythematous or violaceous nodules most frequently on one leg (70% of cases [5] ). This lymphoma can involve both legs (17%) and/or less frequently other parts of the body: trunk (8 to 12%), head and neck (2 to 4%), arm (2%). There is often more than one skin lesion localized on the same anatomic site (45% of cases) but dissemination to several sites at the diagnosis is not rare (20 to 30% of cases). The duration of the skin lesions before diagnosis is short. The outcome is poor with a 5-year survival rate of about 50 to 61.7%. This poor prognosis is due to many factors: age, frequent extracutaneous spreading (observed in 50% of cases), a high incidence of cutaneous relapses (40 to 50% of cases, often multiple) and a low rate of cutaneous complete response after first therapy. Among the clinical prognostic factors, old age (more than 70 years) is recognized by all authors to be the worst. The poor prognostic value of multiple lesions (versus single lesion) has been debated [6, 8] . The choice of treatment depends on the age of the patient and the extent of the lesions. For very old patients with few cutaneous lesions, radiotherapy remains the first line of treatment and intralesional rituximab (anti-CD20) may be used as a second line after relapse. In other cases, systemic chemotherapy (usually CHOP) is proposed sometimes associated with rituximab [9] . PCFCL with a diffuse infiltration of large centrocytes has a good prognosis with a 5-year survival of 86.7 to 95% [5, 6] .This type of lymphoma is most often situated on the trunk (40%), on the head and neck (40%) but cases on the legs have been described (10%). The lesion is more frequently single (more than 50% of cases). Extracutaneous spreading is quite rare (10%) but cutaneous relapses are relatively frequent (40 to 50%). The first line therapy depends on the number of lesions. Radiotherapy is the main treatment for patients with single, few or localized lesions. In patients with extensive cutaneous lesions or in those relapsing after radiotherapy, Interferon (IFN-a) or Rituximab may be proposed. Among the PCLBCL others, the extremely rare intravascular large B-cell lymphoma is characterized by the presence of violaceous plaques or tumor situated on the trunk or lower extremities. The central nervous system and lung are frequently involved and this spreading is associated with a worse prognosis. Chemotherapy is used for the first line of treatment alone or combined with Rituximab. Histopathological and immunohistochemical features In this part, we summarize the different steps useful to perform a diagnosis of primary cutaneous large B-cell lymphoma and to allow its WHO-EORTC classification as PCLBCL leg type or PCFCL. First of all, a large specimen excised by an electric knife (and not a punch biopsy) is essential to make a diagnosis of primary cutaneous large B-cell lymphoma because it allows the analysis of the architecture of the lymphoid proliferation. At the morphological step, the architecture of PCLBCL leg type is diffuse with an infiltration of the whole dermis and often the subcutis. A presence of tumoral follicular growth pattern even in a small part of the lesion excludes this diagnosis. Cytologically,in PCLBCL leg type, the lymphoid proliferation is monotonous composed of round (centroblasts and/or immunoblasts) large B-cells with prominent nucleoli and clumped chromatin. On the other hand, the architecture of PCFCL with large centrocytes can be diffuse but there is frequently nodular reinforcement especially around the pilosebaceous units. There is either a monotonous proliferation of large cleaved centrocytes or a polymorphous proliferation with a mixture of large centrocytes and centroblasts. The distinction between round and cleaved large B-cell only with morphological criteria is not always easy and its reproducibility is often insufficient [5] . This underlines the usefulness of immunohistochemistry. The immunohistochemical step is essential for classification and also for prognosis. To classify these B-cell lymphomas, the following antibodies are used: bcl2, bcl6, CD10, follicular dendritic cell markers (CD21 and/or CNA42), MiB1(Ki67) and 2 transcription factors: MUM-1/IRF4 and FOX-P1. The expression of the anti-apoptotic Bcl2 protein contributes to the diagnosis of PCLBCL leg type, in presence of large round B-cells and is very useful in borderline cases (hesitation between large round or cleaved cells). This expression (with a cutoff of more than 50% of bcl2 positive large B-cells) is also reported to be an independent prognostic factor in multivariate analysis [8] . It is a very reproducible factor and often more than 80% of large cells are stained in PCLBCL leg type. Most PCLBCL leg type (80%) are bcl2+ but rare bcl2 negative cases exist [8] . In the WHO-EORTC classification, bcl2 expression is not an obligation for the diagnosis of PCLBCL leg type, but some authors consider large round cells and bcl2 positivity necessary for the diagnosis 6 . Unfortunately, rare cases of bcl2 positive PCFL (with predominance of large cells) have been reported (23% of cases) so bcl2 cannot be used as a gold standard [6, 8] . Moreover, in these PCFL cases the bcl2 expression seems to have no significant prognostic value [6] . Using follicular dendritic cell markers (CD21) is helpful to exclude in border line cases a diagnosis of PCLBCL leg type, if it shows the presence of remnants of tumoral lymphoid follicles. Bcl6 expression is reported as frequent (50% of cases) in PCLBCL leg type [6, 10] , suggesting a follicle center cell origin for some cases. However, CD10 expression is rare (2%). The impact of bcl6 and/or CD10 expression on PCLBCL leg type prognosis is much debated: one study reported no impact on prognosis overall [6] while another microarray study showed an overall prognosis of 176 months in the bcl6+ group compared with 26 months in the bcl6- group [11] . A very useful immunohistochemical distinction between PCLBCL leg type and PCFCL is the high expression of a B-cell transcription factor Mum/IRF4in PCLBCL leg type (> 80% positive-staining cells) compared with low expression in PCFCL [12] . In the study by Kodama et al, the specificity and sensitivity of Mum-expression for diagnosis of PCLBCL leg type were 85.7% and 75.9% respectively [6] . Moreover, in the same study, a worse prognosis of Mum-positive PCFCL was found. Another transcription factor, FOX-P1, expressed in the non-germinal center of nodal diffuse LBCL, is demonstrated predominantly in PCLBCL leg type (72,4% versus 10% in PCFCL) [6] . This Fox P1 expression seems to be linked to round cell morphology but failed to reach a statistically significant prognostic value. The expression of these 2 proteins (Mum 1 and Fox P1) confirms the active B-cell phenotype of the majority of PCLBCL leg type. The frequent coexpression of bcl6 could suggest an active GC profile as described in nodal B-cell lymphoma. Finally, combined histopathological and immunohistochemical features are very useful to classify primary cutaneous large B-cell lymphoma. In Figure 1 we propose an algorithm based on histological and immunohistochemical criteria in order to improve classification of such B-cell primary cutaneous lymphomas. Figure 1: Proposed Algorithm Based on Histopathological and Immunohistochemical Criteria for Classification of Primary Cutaneous Large B-cell As indicated above, histopathological and immunohistochemical criteria do not always provide clear-cut arguments to classify large B-cell lymphoma as PCLBCL, leg-type or PCFCL (large centrocytes). Molecular and cytogenetic features Despite a strong expression of bcl2, PCLBCL leg type are reported to lack t(14;18) translocation [13, 14] . This strong expression is reported to result from chromosomal amplification of the Bcl2 gene in some cases [15] . The frequent immunostaining of Mum1 in PCLBCL leg type suggesting an active B-cell-like (ABC) phenotype and explaining their poor prognosis, is confirmed by tissue and oligonucleotide microarray studies. In a gene expression profiling study performed on 21 primary cutaneous large B-cell lymphoma (including cases with more than 80% of large cells), Hoefnagel and al showed that several highly expressed genes in the 13 PCLBCL leg type samples (genes encoding for IgM heavy chain, MUM1/IRF4, Pim-kinases (PIM1 and PIM2) were members of the activated B-cell (ABC) gene profile [12] . On the other hand, several genes highly expressed in the germinal center B-cell (GCB) profile and especially SPINK2 were highly expressed in the 8 PCFCL samples but not in those of PCLBCL leg type. Only 8 of their 11 PCLBCL leg type showed a strong expression of MUM1 by immunohistochemistry, so gene expression profiling could be useful and more sensitive for distinguishing PCLBCL leg type with an ABC profile. The clinical and therapeutical relevance of the WHO-EORTC classification for primary cutaneous large B-cell lymphomas has a molecular basis, as shown by the results of fluorescence in situ hybridization (FISH), comparative genomic hybridization (classical and array-based CGH) analyses. All these studies demonstrated a higher number of chromosomal aberrations in PCLBCL leg type compared with PCFCL [16, 17] . Hallerman et al showed that translocations involving c-MYC, BCL6 and IgH genes are found only in PCLBCL leg type. Dijkman et al identified clear-cut differences between PCLBCL leg type and PCFCL : high level amplification of chromosome 2p16.1 and deletion of 14q32.33 in the group of PCFCL. Interestingly, the 2p16.1 region contains BCL11A and c-REL genes and c-REL amplification is characteristically found in the GCB profile. high level amplification of chromosome 18q21 and deletion of chromosome 9p21.3 in PCLBCL leg type. The 18q21 region amplified in 8/12 PCLBCL leg type involves the BCL-2 and MALT1 genes. In the same study, Dijkman et al provided parameters for identify PCLBCL leg type with worse prognosis. The deletion of a small region on chromosome 9p21.3 containing the CDKN2A (p16 INK4A) gene locus was associated with shorter survival in PCLBCL leg type: 5/5 PCLBCL leg type died compared to 1/6 PCLBCL leg type without this deletion. Finally the use molecular and cytogenetic analyses could be helpful in primary cutaneous large B-cell lymphoma, not only to confirm the concordance between the WHO-EORTC classification and molecular pathogenesis but also to provide a sensitive method to distinguish PCLBCL leg type from PCFCL (large centrocytes) especially in difficult border line cases, and to specify the behavior of the PCLBCL leg type. References Vermeer MH, Geelen FA, van Haselen CW et al. Primary cutaneous large B-cell lymphomas of the legs. 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