Hodgkin Lymphoma: Diagnostic and Biological Insights
Moderators: Dr. Philippe Gaulard and Dr. Nancy Lee Harris
Section 4 -
Mixed Cellularity and Lymphocyte Depleted Hodgkin Lymphoma: Athology, Epidemiology, Clinical Features, and the Role of Epstein-Barr Virus
Pierre Brousset, MD, PhD
Department of Pathology
1) Morphology and Immunophenotype of the Histopathologic Subtypes of Classical Hodgkin Lymphoma
A neoplasm composed of mononuclear Hodgkin cells and multinucleated Reed-Sternberg cells in a background
containing a variable mixture of reactive small lymphocytes, eosinophils, neutrophils, histiocytes,
plasma cells, fibroblasts and collagen fibres. Some Hodgkin and Reed-Sternberg cells may have a
condensed cytoplasm and pyknotic nuclei (mummified cells). The Hodgkin and Reed-Sternberg cells
represent only a minority of the cellular infiltrate with a frequency ranging from 1 % to 10 %.
Four histological subtypes have been distinguished: nodular sclerosing HL, mixed cellular HL,
lymphocyte rich classical HL, and lymphocyte depleted HL. The immunophenotype of the mononuclear Hodgkin
cells and multinucleated Reed-Sternberg cells is identical in these histological subcategories. They are
positive for CD30 in nearly all cases, and for CD15 in the majority of cases (80% des cas) (Chittal et
al.). They are usually negative for CD45 but the staining is difficult to interpret because of the
surrounding small lymphocytes. In approximately 30 % of cases, B cell antigens such CD79a and/or CD20
are detected. However, their expression is extremely variable with some Hodgkin and Reed-Sternberg cells
being clearly positive and others being completely negative. Transcription factors involved in B-cell
differentiation can be used to phenotype HL (Browne et al.). Thus, PAX5+/Oct-2-/BOB.1- phenotype is in
favor of classical HL whereas PAX5+/Oct-2+/BOB.1+ is predictive of Nodular LPHL. Expression of T cell
antigen by a minority of Hodgkin and Reed-Sternberg cells may be encountered in some cases. Expression
of epithelial membrane (EMA) is rare (less than 5 % of the cases) and is usually weak. In 50% to 70% of
cases, neoplastic cells are latently infected by EBV and positive for latent membrane protein 1 (LMP-1)
and negative for EBNA2 (latency type 2). Most Hodgkin and Reed-Sternberg cells express the
proliferation-associated nuclear antigen Ki-67. Reed-Sternberg cells express several types of cytokines
and interleukins (IL1a, IL5, IL6, TGFβ…) which influence greatly the microenvironment of the tumor
cells. For example, in cases of HL with eosinophilia, Reed-Sternberg cells strongly express IL5.
Hodgkin and Reed-Sternberg cells contain monoclonal immunoglobulin (Ig) gene rearrangements in most
cases. There is a high load of somatic mutations in the variable region of the Ig heavy chain genes (VH)
and Ig mRNA transcripts are absent (Kuppers et al).
Clinical features: Classical Hodgkin lymphoma
most often presents with involved lymph nodes of the cervical region (75 % of cases) followed by the
axillary and inguinal regions. Primary extranodal involvement is rare. 55 % of patients have localized
disease (stage I and II). Approx. 60 % of patients (the majority of them with nodular sclerosing Hodgkin
lymphoma) have large mediastinal masses. Systemic symptoms, present in approx. 25 % of patients, consist
of fever, drenching night sweats, and significant body weight loss. There is a bimodal age specific
incidence curve with a peak at 15 to 40 years and a second peak late in life. Without therapy the
clinical course is moderately aggressive. With modern therapy, 70 to 80 % of patients show long term
survival. Before the progresses of polychemotherapy there was a strong association between histologic
type and overall survival. An excellent response to modern therapy has greatly diminished the
A) Mixed Cellularity Hodgkin Lymphoma (MCHL)
This entity corresponds to 20-30% of all cases of classical HL and predominates in male
(SR:3:1) and in HIV-positive patients. MCHL is diagnosed frequently in an advanced stage with clinical
and biological B and b symptoms respectively. Contrary to NS, MCHL does not involve the mediastinum but
more frequently infiltrates the spleen (25%) and the bone marrow (5-10%). The incidence of bone marrow
involvement reaches 15-30% in HIV+ patients (WHO).
Morphologically, MCHL is more often diagnosed on a resected lymph node the architecture of which is
destroyed by a granulomatous proliferation. In some cases, residual lymphoid follicles may persist but
in general, the whole lymph node is involved. The background granuloma is composed of an admixture of
different cell types is various proportions (lymphocytes, plasma cells, neutrophils, eosinophils,
histiocytes nore or less epithelioid). One of this cell types may predominate, for instance eosinophils
in case of interleukin-5 production by the tumor cells. In other cases, epithelioid cells may
predominate, giving rise to tuberculoid granulomas. Contrary to NSHL, MCHL does not present
sclero-hyalinosis and lacunar cells but rather diagnostic Reed-Sternberg cells and Hodgkin variants. The
latter cells may be associated to areas of necrosis or apoptosis. In some cases, mummified (mummy) cells
are frequent. . In some cases, there is a nodular pattern due to the presence of B-cell follicles and a
preferential lymphoma involvement in the interfollicular regions (so-called interfollicular classic HL
initially described by Dorfman). The phenotype of RS and H cells is that of lacunar cells (pan B+/-, pan
T-, CD30+, CD15+, EMA-/+, ALK-, PAX5+) (WHO, Zukerberg et al.). In case of expression of CD20 (L26), one
important sign in keeping with the diagnostic of HL is the heterogeneity of the staining. There is a
kind of gradient from cells clearly positive to cells completely unstained.
B) Lymphocyte Depleted Hodgkin Lymphoma (LDHL)
Synonyms: Jackson and Parker:Sarcoma; Lukes and Butler:
Diffuse Fibrosis and Reticular; Rye : Lymphocytic Depletion; REAL: Lymphocyte
The appearance is highly variable, including the degree of lymphocyte depletion. One pattern may
resemble mixed cellularity with increased numbers of Hodgkin and Reed-Sternberg cells. In another
pattern, pleomorphic mononuclear Hodgkin cells predominate, producing a sarcomatous appearance. These
cases may be difficult to differentiate from anaplastic forms of large cell non-Hodgkin's lymphoma.
Another pattern is characterized by a diffuse fibrosis or proliferation of fibroblasts and/or
myofibroblasts with only a few Hodgkin and Reed-Sternberg cells. The morphologic diagnosis of lymphocyte
depleted (LD) subtype is now rarely made (1-5% of HL). There is no difference in phenotype with NS or MC
Some cases previously diagnosed as lymphocyte depletion are now recognized as anaplastic large cell
lymphoma. This form of HL tends to affect elderly patients with systemic symptoms and/or with
involvement retroperitoneal lymph nodes, and bone marrow. The clinical course is aggressive.
2) Detection of Epstein-Barr Virus in HL
The incidence of EBV infection in
HL varies with histological subtypes. Virtually all cases of LPHL are negative for EBV. The most
frequent incidence is observed in MC subtypes with 60-70% of the cases being infected. The incidence in
NS subtypes varies significantly with the different reports between 10 to 40% of the cases (Delsol et
al.). Classically, the mediastinal forms of NS HL are EBV-negative. The incidence of EBV infection in
LRHL looks weak. Of note, virtually all cases of HL in HIV-positive patients are associated with EBV.
This finding is also noted in childhood cases from developing countries. In all cases of EBV+ HL, the
detection of a clonal population of episomes indicates that only one type of EBV is present within RS
Eleven EBV genes are expressed in latent infection. Some proteins encoded by these genes can be
detected by monoclonal antibodies such as those directed against EBV LMP1 and EBNA2. There is usually a
close correlation between the results obtained with in situ hybridization using EBER oligonucleotides and
with immunohistochemistry with anti-LMP1 antibody but the former method is more sensitive. In addition
to LMP1, six nuclear proteins (EBNA-1, 2, 3A, 3B, 3C or LP) are usually expressed in latently infected
B-cells but not in RS cells. Latency type II infection (EBNA1+, EBNA2-, LMP1-) is the usual finding in
RS cells, similar to that observed in nasopharyngeal carcinoma and nasal T/NK cell lymphoma (Delsol et
On the basis of EBV findings, three groups of HL can be distinguished. In the first (50%
to 60%), EBV is present in H-RS cells and commonly found in scarce infiltrating small lymphocytes
(«reservoir lymphocytes» whichare present in all individual who have been infected by
EBV. In the second (10 %), only EBV-positive small lymphocytes are detectable (Meggetto et al.). In the
third group (40 %), EBV is detected neither in H-RS cells nor in small lymphocytes. Comparative analysis
of genome sequences between RS cells and bystander lymphocytes indicates that the strains are distinct,
in particular when looking at the polymorphism of the carboxy terminal region of LMP1. However, several
experiments indicate that, compared to EBV strains observed in lymphocytes from non-neoplastic lesions,
the genome sequence of EBV is strongly divergent. In fact, viral strains detected in HL tissues
including RS cells and bystander B-lymphocytes, are infected by different although related EBV strains,
but are four times more polymorphic than EBV strains infecting bystander B-lymphocytes of reactive lymph
nodes (Faumont et al.). The biological significance of these observations remains open, in particular
regarding the origin and chronology of multiple infections in the same patient.
3) Clinical Implications of EBV Findings in HL
So far, the several studies dealing with the clinical and prognostic implications of EBV
findings have failed to yield clear definitive data. Recently, two conflicting studies have been
published in Blood. The first study by Jarrett et al. conducting a Scottish study, demonstrated an
impact of the presence of EBV in HL on presenting features and outcome in age-defined subgroups of
patients. In response to this study, Herling et al from the MD Anderson in Houston reported negative
results i.e; that EBV has no clear impact in the outcome, even after 10 years of follow up. One of the
main arguments developed in the latter report is the heterogeneity of the treatments received by the
Genomic alterations (deletions and point mutations near the 3'end) of the LMP1 gene were
found to be clustered in about 10% of cases (Knecht et al.). Such molecular abnormalities were suspected
to be associated with an aggressive behaviour but to date larger series of cases based on this parameter
have not been investigated.
There are some points which are now accepted, in particular that the EBV genome persists
throughout the evolution of the disease. In 12 HL cases with relapses (range 14-126 months), there were
seven EBV positive cases (MC= 6; NS=1) and five EBV-negative cases (MC=2; NS=2). Close agreement was
found between the detection of EBV in Reed-Sternberg cells at the time of the first diagnosis and at
relapses in all cases tested. These results suggest that the virus persists throughout the course of the
disease. In addition, the molecular investigations performed in two EBV-positive cases, both at the
onset and at relapses showed that the size of terminal repeat fragments were the same. LMP1 sequence
analysis further confirmed the persistence of a distinctive viral strain in each of the 2 cases with
point mutation and/or 30bp deletion of the LMP1 gene. The persistence of a given strain in early and
late relapses supports the view that in Hodgkin's disease, relapses are related to a single residual
tumor cell clone.
4) Further Readings
- Browne P. Am J Clin Pathol. 2003;120:767-77
- Butler JJ. Semin Diagn Pathol 1992;9:252-256
- Chittal S, et al. Am J Surg Pathol 1988;12: 9-21.
- Delsol G et al. Am J Pathol 1992;140:247-253.
- Faumont N, et al. Virus Res. 2004;101:163-73.
- Herling M, et al. Blood. 2006 ;107:1240.
- Jarrett RF, et al. Blood. 2005;106:2444-51.
- Knecht H. Oncology. 2001;60:289-302
- Kuppers R., Adv. Cancer Res. 2002; 84: 277–312.
- Meggetto F, et al. J Virol. 1997;71:2547-9.
- Vassallo J, et al. Am J Surg Pathol. 2006;30:223-9.
- World Hearth Organization classification of tumours. Pathology and genetics. Tumours of
haematopoietic and lymphoid tissues. Jaffe ES, Harris NL, Stein H, Vardiman JW eds. IARC Press, Lyon
- Zukerberg LR et al. Am J Pathol 1991;139:475-483.