Hodgkin Lymphoma, Classical
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Classical Hodgkin lymphoma is a clonal lymphoproliferative disorder of B-cell origin (Kuppers 2009) composed by a relatively small number of malignant cells occurring in a reactive benign inflammatory background.
CHL accounts for approximately 0.6% of all new cancer cases and 15–30% of all malignant lymphomas (Nakatsuka and Aozasa 2006). The incidence of CHL is higher in industrialized nations than in developing countries. CHL shows a bimodal age of distribution, with a peak in young adult (between 15 and 35 years old) and a second peak in late life after 55 years old. CHL represents the most common subtype of malignant lymphoma in young people in the Western world. CHL is rare in children and exceptional before 3 years old. Male/female ratio is 1.5:1 in children and older adults and 1:1 in younger adult patients. The disease is more frequent in white than hispanic or black people. The incidence is also higher in higher socioeconomic population. The distribution of histologic subtypes varies with age.
Persons with previous infectious mononucleosis or immunodeficiency such as HIV have a high risk for developing CHL, currently associated with EBV (Flavell and Murray 2000). HIV infection is associated with 6–20 fold increase in risk of developing CHL (Biggar et al. 2006). Increased incidence of Hodgkin’s disease after allogeneic bone marrow transplantation has been reported (Garnier et al. 1996). Familial cases have been described, in particular in identical twins (Mack et al. 1995). Polymorphism on 6p21.32 has been associated with the risk of developing CHL nodular sclerosis (Cozen et al. 2012).
CHL is a nodal disease in more than 90% of cases (Harris 1999). The most frequent site is the cervical lymph node, followed by axillary and inguinal lymph nodes. Mediastinal involvement is common. Retroperitoneal lymphadenopathy and splenic involvement are frequent. Bone marrow is involved in only 5% of patients at diagnosis but may occur more frequently in advanced disease. Liver involvement can also be observed more frequently in advanced stage. Any site of the body can be involved in the evolution of the disease, exceptionally at the onset of the disease. Mucosa-associated lymphoid tissue and gastrointestinal tract are very rarely involved. Anecdotal cases of skin involvement or central nervous system disease have been reported.
It may be asymptomatic, especially with isolated mediastinal disease. The symptoms are related to site of organ involvement. B symptoms consisting of fever, night sweats, and weight loss are present in approximately 30–40% of patients, more frequently in advanced stages. Generalized pruritis and pain in involved lymph nodes subsequently alcohol ingestion are very rare symptoms. Hypereosinophilia can be observed in few cases.
The anatomical distribution of the disease and the clinical presentation vary according to the histological subtype (see below).
Treatment and Evolution
Classical HL is a highly curable disease with about 90% of cured patients. At diagnosis, the realization of positron emission tomography (PET) to optimize the staging of the disease is currently mandatory. At the PET era, bone marrow biopsy could be withdrawn. This staging allows the definition of early and disseminated stages which is important for treatment decisions. Important prognostic factors should be defined for early stage to decide the number of cycles of ABVD regimen (Adriamycin, Bleomycin, Vinblastine, Dacarbazine) and the dose of radiotherapy. These factors are the age at diagnosis, the number of involved nodal areas, the presence of a mediastinal bulky disease, and the erythrocyte sedimentation rate. These prognostic factors allow the stratification of patients with favorable and unfavorable early stages. The lymphoma study association (LYSA) recommends three cycles of ABVD followed by an involved field (IF) radiotherapy of 20 Gy for favorable disease. For unfavorable early stages, four cycles of ABVD with 30 Gy IF radiotherapy are recommended. The 5-year progression-free survival is 93% and 88% for, respectively, favorable and unfavorable early stages (Behringer et al. 2016; von Tresckow et al. 2012). The reduction of RT field size is currently under investigation with the realization of nodal field to reduce the long-term secondary effect of radiotherapy (Raemaekers et al. 2014). For disseminated diseases characterized by Ann Arbor stages III and IV, treatment is mainly based on chemotherapy. With ABVD regimen, 5- and 10-year PFS rate are 68% and 69%, respectively. More intensive regimens were developed such as escalated BEACOPP regimen (6 cycles) (Bleomycin, Etoposide, Adriamycin, Cyclophosphamide, Vincristine, and Procarbazine) to improve the results with ABVD regimen (Viviani et al. 2011). In addition, patients with a stage II disease with B- symptoms and the presence of risk factors (bulky disease or an extranodal involvement) are currently treated as advanced stages without radiotherapy. The evaluation of the response by PET during treatment is important for adapting therapy. A PET is currently performed after two cycles of chemotherapy. For early stages, for patients with a positive PET after two cycles of ABVD, a more intensive treatment with escalated BEACOPP (2 cycles) could be proposed before radiotherapy (Raemaekers 2015). For advanced stages treated by escalated BEACOPP, a negative TEP according to international response criteria using Deauville scale after two cycles could allow the realization of less intensive chemotherapy such as ABVD regimen (Casasnovas et al. 2019). Despite the efficacy of first-line therapy, 10% of localized and 20% of disseminated diseases relapse. At relapse, the main prognostic factors are the presence of a refractory disease, an early relapse before 12 months since the end of first-line treatment, and a stage III–IV. For relapsed patients, salvage regimens are proposed followed by high-dose chemotherapy (HDT) and autologous stem cell transplantation (ASCT). For relapses after HDT + ASCT, about 34% of patients were in complete response with an anti-CD30 monoclonal antibody drug conjugate to an anti-microtubule agent (brentuximab vedotin, BV) (Younes et al. 2012). This target therapy is now used in consolidation treatment after HDT + ASCT for some specific clinical situations. Regarding the important role of the microenvironment in HL pathogenesis, new therapies were developed to target the exhaustion of immune response from HRS cells (Hodgkin Reed Sternberg cells). Two anti-PD1 monoclonal antibodies, nivolumab and pembrolizumab, were developed for HL patients who relapsed after HDT + ASCT and BV. For these refractory patients, these checkpoint inhibitors allow 80% of objective responses rates ORR (Ansell et al. 2015).
Treating HL in elderly patients remains also a therapeutic challenge. These patients represent 20% of all HL. As compared with younger patients, they presentea more aggressive disease. Toxicities with current treatments such as ABVD regimen are frequent with about 5–9% of toxic deaths. The 5-year PFS and OS rates with ABVD are 50% and 60%, respectively. Finally, long-term toxicities are a major concern in cured HL patients with an excess of cardiovascular diseases, secondary and infectious events as compared to general population. A specific follow-up of HL survivors should be performed to prevent or early detect the occurrence of these long-term side effects. Specific programs should be developed to support HL survivors and improve their quality of life.
Lymph nodes are enlarged with a fish-flesh tumor on cut section. In scleronodular variant, macroscopic examination shows sometimes a nodularity with nodule circumscribed by dense fibrosis. In spleen involvement When spleen is involved scattered nodules with fibrosis can be observed. Necrosis can be observed.
Tumor Cell Cytology
Nodular Sclerosis Classic Hodgkin Lymphoma (NSHL)
NSHL is the most common histological variant, representing more than 70% of cases of CHL (Swerdlow 2016). It is the most frequent type in young patients and higher socioeconomic population. It presents currently with cervical, supraclavicular, and mediastinal lymph nodes. Bulky disease is seen in about half of cases. Clinical presentation is often with B symptoms and with stage II disease.
Mixed Cellularity Classic Hodgkin Lymphoma
Lymphocyte-Rich Classic Hodgkin Lymphoma
Lymphocyte-Depleted Classic Hodgkin Lymphoma
Lymphocyte-depleted CHL is the rarest subtype of CHL (<1% of cases) (Swerdlow 2016). Patients are more older males with HIV infection. It presents more frequently with stage II and B symptoms. Histologically it is characterized by sheets of large atypical cells with less inflammatory background and a diffuse fibrosis (Fig. 5). The cells can be more pleomorphic- or anaplastic-like than in other subtypes.
In several cases, CHL remains unclassified, especially with the use of small microbiopsy at diagnosis. Interfollicular involvement of CHL can occur. Relapse CHL retains usually the initial histologic subtype. A diagnosis of CHL in extranodal site should be done with caution. The immunohistochemical study is mandatory in every situation to confirm the diagnosis of CHL suspected by morphology in any site.
CHL shows a EBV latency type II pattern of viral gene expression (EBERS+, LMP1+, EBNA1+, LMP2A+, EBNA2-) (Kapatai and Murray 2007). Immunohistochemistry for latent membrane protein 1 (LMP1) and nonradioactive in situ hybridization for EBV-encoded early RNAs (EBERS) are the methods of choice for the detection of EBV in FFPE samples in routine diagnosis (Isaacson et al. 1992). LMP1 and EBER are expressed much more frequently in MCCHL (about 75%) than in other subtypes. Children and older patients are more likely to have EBV+ CHL. As in healthy patient, EBERs-positive small B cells can be observed in the inflammatory background of CHL, the so-called bystander lymphocytes.
Immune checkpoint ligands, PD-L1 and PD-L2, are expressed on the surface of malignant cells in 65–100% of CHL (Savage and Steidl 2016) resulting from chromosome 9p24.1 abnormalities identified in 97% of CHL. PD1 are frequent in the inflammatory background.
The inflammatory background lymphocytes of CHL are predominantly CD4-positive T cells and correspond to immunosuppressive regulatory T cells. The nodular pattern in LRCHL is characterized by nodule of small B cells.
Pathogenesis and Molecular Features
HRS cells show a deregulated activation of numerous signaling pathways, which is mediated by cellular interactions with the lymphoma microenvironment and by genetic lesions (Kuppers 2012).
Postulated Cell of Origin
HRS cells are derived from mature B cells. Most cases demonstrated hypermutated Ig genes consistent with post-germinal center B-cell origin. However despite a B-cell origin, the HRS cells have lost most of the B-cell markers and show a very unusual expression of many markers of other hematopoietic cell lineages. The bi- or multinucleated HRS cells most likely derive from the mononuclear Hodgkin cells through a process of incomplete cytokinesis (Rengstl et al. 2013).
Genetic Lesion and Molecular Pathogenesis
Hyperploidy and various cytogenetic abnormalities are detected in nearly 100% of RS cells. Recurrent genetic lesions in HRS cells often involve members of the nuclear factor-κB (NF-κB) and JAK/STAT pathways and genes involved in major histocompatibility complex expression. HRS cells show strong constitutive activity of the transcription factor NF-kappaB (Steidl et al. 2010; Liu et al. 2014). Many mutations and structural alterations in the genes involved in these pathways leading to constitutive activity, including chromosomal gains, overexpression of pathway genes, and inactivating mutations of tumor suppressor genes have been described. Multiple mechanisms likely contribute to this deregulated activation, including signaling through particular receptors and genetic lesions. Recently, receptor tyrosine kinase signaling has been reported to play an important role in the pathogenesis of HL. In particular, the surface receptors mesenchymal-epithelial transition factor (c-MET) (Neagu et al. 2015), tyrosine receptor kinase A (TRKA), and discoidin domain receptor tyrosine kinase 2(DDR2) are expressed and mediate survival signaling in HRS cells through paracrine signaling.
Importance of the Microenvironment
Although the malignant HRS cells gain a survival advantage by deregulated transcription factor networks and genetic lesions altering intrinsic signaling, the fully developed proliferation and antiapoptotic phenotype of HRS cells are critically dependent on their interaction with the microenvironment (Tudor et al. 2014). HL is particular and unique among all cancers because malignant cells are outnumbered by reactive cells in the tumor microenvironment and make up only approximately 1% of the tumor. Expression of a variety of cytokines and chemokines by the HRS leads to an abnormal immune response, perpetuated by additional factors secreted by reactive cells in the microenvironment. The reactive cells in the microenvironment produce specific cytokines and chemokines that help to maintain and even to amplify the intense inflammatory reaction. The malignant HRS manipulates the microenvironment, permitting them to develop fully their malignant phenotype and to evade host immune attack. The attracted cells enhance the pro-survival and growth stimulating signals for the tumor cells. To escape from an effective antitumor response, tumor cells avoid recognition by T and NK cells, by downregulating HLA molecules and modulating NK and T-cell receptors. In addition, the tumor cells produce immunosuppressive cytokines that inhibit cytotoxic responses. In this process HRS cells are reprogrammed to cells resembling undifferentiated hematopoietic progenitor cells. HL is thus a unique example to demonstrate the plasticity of human lymphoid cells.
EBV infection is associated with CHL in about 40% of cases in Western countries and 90% in African countries (Kapatai and Murray 2007). The EBV infection is clonal and occurs probably early in the pathogenesis, with a latency type II (expression of EBNA1, LMP1, and LMP2a). EBNA1 is important for episomal replication in proliferating cells. LMP1 simulates CD40 receptor B. LMP2a presents a cytoplasmic domain mimicking signalization pathway of the BCR.
Major Molecular Features
The genomic landscape of Hodgkin lymphoma is not extensively studied considering the low number of tumor cells in a stroma-rich microenvironment (Liu et al. 2014; Liang et al. 2019; Jardin et al. 2016). Ten to 20% of patients have a high mutation burden, which in cancers may indicate sensitivity to immune checkpoint inhibitors. The most commonly mutated genes reported were TP53 (tumor protein p53), B2M (béta-2 microglobulin), and XPO1 (exportin 1). Components of JAK/STAT signaling pathway are affected by frequent mutations of SOCS1 (suppressor of cytokine signaling 1) and STAT6 (signal transducer and activator of transcription 6) as well as copy number gains of JAK2. Involvement of nuclear factor-κB (NF-κB) pathway compounds is represented by recurrent gains of the locus containing the REL gene and mutations in TNFAIP3 (tumor necrosis factor, alpha-induced protein 3) and CARD11. Finally, genetic alterations of PD-L1 and B2M suggested immune evasion as mechanisms of oncogenesis in some patients.
Mutations in XPO1 and SOCS1 have also been reported in primary mediastinal B-cell lymphoma especially a recurrent hotspot mutation of XPO1 E571K. Emerging evidence suggests that the mutant XPO1 E571K plays a role in carcinogenesis, and this variant is quantifiable in tumor and plasma cell-free DNA of patients using highly sensitive molecular biology techniques, such as digital PCR and next-generation sequencing (Jardin et al. 2016). The mutation can be detected in plasma cell-free DNA, and patients with a detectable XPO1 mutation at the end of treatment seem to display a tendency toward shorter progression-free survival, as compared to patients with undetectable mutation. Therefore, it was proposed that the XPO1 E571K variant may serve as a minimal residual disease tool in this setting. In addition patients with SOCS1 major mutations seem to suffer from early relapse and significantly shorter overall survival.
Hodgkin or Reed-Sternberg-Like Cells in Reactive Lymphadenopathies or Non-Hodgkin Lymphomas
Large atypical cells with morphologic and immunophenotypic features resembling RSC can be seen in the background of reactive lymphadenopathies as well as non-Hodgkin lymphomas or non-hematopoietic tumor (Gomez-Gelvez and Smith 2015). The presence of these cells is an important diagnostic pitfall that must be recognized by pathologists. A thorough evaluation of the morphologic and immunophenotypic features of these cells and the cellular milieu is crucial to achieve the correct diagnosis. These cells are commonly seen in high-grade lymphomas, rare cases of low-grade B-cell and T-cell lymphomas. Among low-grade B-cell lymphomas, RS-like cells can be seen in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma, mantle cell lymphoma, or marginal zone lymphoma. Sometimes proof of clonal relationship between HRS cells and small or large cell lymphoma is useful to distinguish these cases from true composite non-Hodgkin lymphoma and CHL. Both CD30 and CD15 can also be expressed in a non-hematopoietic tumor.
In reactive condition, infectious mononucleosis can be difficult to distinguish with Hodgkin lymphoma and affect usually teenagers and young patients. However cases after 40–50 years old are reported. In this condition the lymph node architecture is partially preserved with extensive proliferation of immunoblast occasionally mononucleated or polynucleated with prominent nucleoli resembling Hodgkin or Reed-Sternberg cells and expressing CD30 without CD15 and expressing B-cell markers. The activated B cells are also EBV associated expressing EBNA-2+, LMP1+, and EBERS+. Some large immunoblastic cells are also of T-cell phenotype. Most of the small lymphocytes in the background are T cytotoxic CD8+ lymphocytes.
In chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), scattered RS-like cells can be observed in the background of neoplastic cells. They may express CD20 and CD30 and are typically negative for CD15. However, in some cases, they may show expression of CD30 and CD15 as well as EBV positive, making them virtually indistinguishable from the true RS cell of CHL. When RS-like cells are seen in the context of CLL/SLL, this is commonly referred to as CLL/SLL with HRS cells. These cases should not be diagnosed as Hodgkin lymphoma. The diagnosis of Hodgkin lymphoma (Richter syndrome) in the setting of CLL requires classical RS cells in an appropriate background. But CLL/SLL with scattered HRS cells may represent precursor lesions for CHL (Xiao et al. 2016).
In follicular lymphoma, RS-like cells may be seen between or within the neoplastic follicles and can express CD30 (Son and Huh 2014). As opposed to CLL/SLL cases, the RS-like cells seen in follicular lymphomas are not associated with Epstein-Barr virus infection.
In T-cell lymphomas, RS-like cells can be observed especially in angioimmunoblastic T-cell lymphoma but also in peripheral T-cell lymphomas (Nicolae et al. 2013). The RS-like cells demonstrate expression of CD30, CD20, and occasionally CD15 and are commonly associated with EBV.
The differential diagnosis with NLPHL can be difficult, especially with lymphocyte-rich CHL variant. The morphology and cytology can be similar in both entity, but the immunohistochemistry is very helpful for the differential diagnosis. Both show usually a nodular small B CD20+ lymphocyte-rich pattern. In contrast to CHL, the lymphoma cells of NLPHL express CD20, CD79a, CD45, and EMA, sometimes IgD, but are usually negative for CD15, CD30, and EBV (Savage et al. 2016). Nonneoplastic CD30+ immunoblasts can be observed in the perifollicular area that will be not misinterpreted as Hodgkin cells.
Primary Mediastinal Large B-Cell Lymphoma (PMBL)
It has long been recognized that there is distinct clinical and pathologic overlap between PMBL and NSCHL. Indeed a molecular gene expression signature reminiscent of nodular sclerosis subtype of CHL has been identified in PMBL (Savage et al. 2003). Both PMBL and NSCHL commonly present in young patients as localized, mediastinal tumors. PMBL shows a diffuse growth pattern of large cells with a pale or “clear” cytoplasm, sometimes RS-like, and variable degrees of sclerosis. PMBL tumors often lack surface Ig, a feature that also typifies the HRS cell. CD30 can be expressed but is usually weak and inhomogeneous in contrast to the uniform and strong expression seen in CHL or anaplastic large cell lymphoma. MAL (lipid raft component) has been identified as being differentially expressed in PMBL and found in only few cases of NSCHL (Copie-Bergman et al. 1999). PMBL malignant cells and HRS cells also exhibit common genetic abnormalities, including gains of chromosome 2p and 9p, the latter being unique to these two diseases. In addition to these striking clinical, immunologic, and molecular similarities, there are rare reported cases of composite or sequential NSCHL and PMBL (Sarkozy et al. 2017; Traverse-Glehen et al.2005a). However the differential diagnostic is often easy as, unlike HRS cells, PMBL tumor cells retain several B-cell differentiation markers (e.g., CD20, CD79a), and, histologically, they appear more similar to other DLBCLs and usually lack HRS cells. The brisk inflammatory background seen in CHL is usually absent in PMBL. The case with difficulties in the differential diagnosis should be classified as mediastinal gray zone lymphoma or unclassified large B-cell lymphoma with intermediate feature between PMBL and CHL (Sarkozy et al. 2019). This category has been included in the 2008 WHO classification as a provisional entity and has been approved as an entity in the updated 2016 WHO classification.
Mediastinal gray zone lymphoma shows intermediate morphology and phenotype between CHL and PMBL (Sarkozy et al. 2019; Traverse-Glehen et al. 2005b): CHL-like with tumoral cells resembling Hodgkin lymphoma cells with less inflammatory background and sheet of large cells or PMBL-like with larger and more pleomorphic cells than in typical PMBL and some cells resembling bi- or multinucleated Reed-Sternberg cells. Cases with CHL-like morphology had a strong and diffuse CD30 and CD20 expression. They also expressed one or several B-cell transcription factors (OCT2, BOB1, PAX5) (O’Malley et al. 2016). Cases with PMBL-like morphology are CD30 positive usually with and high and diffuse expression. CD30 weak cases occur but with CD15 positive. CD20-negative cases had an expression of CD79a or of B-cell transcription factors with an expression of CD30 or CD15.
T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) represents a distinct subtype of diffuse large B-cell lymphoma and is characterized by the presence of scattered large neoplastic B cells in a background of abundant T cells and histiocytes (Tousseyn and De Wolf-Peeters 2011). The differential diagnosis of THRLBCL from Hodgkin’s lymphoma is facilitated by the integration of different immunophenotypic, molecular, and clinical findings. Diagnostic criteria for THRLBCL is a predominant diffuse growth pattern without nodular dendritic cell meshwork and scattered atypical large B cells and a reactive population of small T cells with histiocytes. THRLBCL may exhibit HRS-like cells, closely mimicking diffuse variant of lymphocyte-rich CHL or NLPHL. The characterization of the reactive background, IgH gene rearrangement studies by conventional PCR, and clinical features are useful for the differential diagnosis. The scattered large B cells in THRLBCL can be distinguished from Hodgkin’s lymphoma cells by the absence of CD30 and CD15 with a B-cell phenotype CD20+, PAX5+, and CD79a+.
Anaplastic Large Cell Lymphoma (ALCL)
CHL and ALCL share many common features such as CD30 expression and loss of B- and T-cell markers, and the distinction can be difficult especially in ALCL ALK-. The distinction is of major importance since, in contrast to ALCL, CHL can be cured in more than 90% of cases. The expression of PAX5 in HRSC and the expression of cytotoxic markers (granzyme, perforin, or Tia1) on anaplastic cells can help in the differential diagnosis (Doring et al. 2014). However rare cases of CHL can express cytotoxic molecules in tumor cells, and PAX5-negative HRSC can be observed. For difficult cases molecular study for clonality is helpful for the differential diagnosis.
EBV+ Lymphoproliferative Disorders and EBV+ DLBCL
EBV-associated lymphoproliferative disorder or EBV-associated lymphoma frequently presents scattered HRS-like cells. In those cases RS cells express pan-B-cell marker with variable expression of CD30 and negativity of CD15.
Composite lymphomas are defined as two unrelated lymphomas occurring at the same time within the same tissue. The incidence of these tumors is low. Of all possible combinations between lymphomas, concurrent CHL and non-Hodgkin lymphoma may occur and may be genetically related. More commonly composite lymphoma of CHL can occur with follicular lymphoma, marginal zone lymphoma, and mantle cell lymphoma and less frequently peripheral T-cell lymphoma. Sometimes, the two components of CHL and NHL are clonally related and suggest a shared origin from a common B-cell precursor.
References and Further Reading
- Nicolae, A., Pittaluga, S., Venkataraman, G., et al. (2013). Peripheral T-cell lymphomas of follicular T-helper cell derivation with Hodgkin/Reed-Sternberg cells of B-cell lineage: Both EBV-positive and EBV-negative variants exist. The American Journal of Surgical Pathology, 37, 816–826.CrossRefGoogle Scholar
- Raemaekers, J. M. (2015). Early FDG-PET adapted treatment improves the outcome of early FDG-PET positive patients with stage I/II Hodgkin lymphoma (HL): Final results of the randomized Intergroup EORTC/LYSA/FIL H10 trial. 13th International Conference on Malignant Lymphoma (ICML) Lugano Switserlzand.Google Scholar
- Raemaekers, J. M., Andre, M. P., Federico, M., et al. (2014). Omitting radiotherapy in early positron emission tomography-negative stage I/II Hodgkin lymphoma is associated with an increased risk of early relapse: Clinical results of the preplanned interim analysis of the randomized EORTC/LYSA/FIL H10 trial. Journal of Clinical Oncology, 32, 1188–1194.CrossRefGoogle Scholar
- Rengstl, B., Newrzela, S., Heinrich, T., et al. (2013). Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed-Sternberg cells. Proceedings of the National Academy of Sciences of the United States of America, 110, 20729–20734.CrossRefGoogle Scholar
- Swerdlow, S. H. (2008). WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: International Agency for Research on Cancer.Google Scholar
- Swerdlow, S.H. (2016). WHO classification of Tumours of Haematopoietic and lymphoid tissues. Lyon.Google Scholar