Hematopathology Diagnoses Too Easy to Miss!
Malignant Lymphomas Mimicking Reactive Processes (continued)
Marsha Kinney, James Cook and Steven Swerdlow
Malignant lymphoma, lymphocyte rich classical Hodgkin type
57 year old male with a mass in the vallecula (base of
tongue), suspicious for malignancy. After his diagnosis, he was treated with radiation. He apparently
remains in a complete remission about 4 years later with negative CT scans done March, 2005. No further
The tonsillar type tissue demonstrates many
reactive-appearing follicles, some of which are closely packed, with germinal centers and often widened
mantle zones. In some follicles the germinal centers are eccentrically placed. In other areas, large
nodules composed of numerous small lymphocytes with scattered large atypical mononuclear and
multinucleate cells with prominent nucleoli are seen. These latter cells are also found in the widened
mantle zones as well as in some interfollicular areas. Some are present in lacunar spaces.
Subepithelial areas have some plasma cells.
Flow cytometric immunophenotypic studies
Polyclonal B-cells, some CD5+ B-cells, and heterogeneous T-cells.
Paraffin section immunohistochemistry:
- CD20 – Follicles positive. A minority of the large cells are
- CD3 – Moderately numerous positive cells outside of follicles.
Highlights rosettes of positive cells around negative large atypical cells.
- CD30 – Large cells positive.
- CD15 – Large cells positive.
- CD45 (LCA) – Large cells, at least outside of germinal centers
- CD57 – Moderate number of scattered positive cells in germinal
centers, otherwise few.
- J chain – Large cells negative.
- CD5 – B-cells appear negative.
- Bcl-2 – Germinal centers negative.
Genotypic & Cytogenetic studies:
IGH@ gene rearrangement not detected by Southern blot analysis nor by PCR (done because of increased CD5+
B-cells in the flow cytometric studies and widened mantle zones).
Arriving at a diagnosis in this case required
recognizing that the histopathology suggested Hodgkin lymphoma even though there were many reactive
features, knowing about lymphocyte rich classical Hodgkin lymphoma, recognizing that the flow cytometric
studies and genotypic studies did not suggest a specific diagnosis and then appreciating how to use
paraffin section immunohistochemistry to make a confident and precise diagnosis.
This case illustrates the following:
- Hodgkin lymphoma can be associated with extensive follicular
hyperplasia and might then get missed.
- The most important thing in this case is (1) to recognize that
there is a lymphoma and (2) to classify it as one of the types of classical Hodgkin lymphoma. It is only
of secondary importance to recognize that this best fits the description of the lymphocyte rich
At least some of these cases have been called "follicular Hodgkin disease" in the
- Don't get mislead by negative flow cytometric immunophenotypic
studies – they don't necessarily translate to benign. They are not going to be helpful in making the
diagnosis of Hodgkin lymphoma, but can be useful in saving you from misdiagnosing a non-Hodgkin lymphoma
as one of Hodgkin type.
- In addition to morphologic features that strongly support the
presence of some type of Hodgkin lymphoma here, the basic tried and true immunostains are of great help
in recognizing that this is classical Hodgkin lymphoma – CD15+, CD30+, LCA-. Some classical
Reed-Sternberg cells can be CD20 positive even though we think of them as usually negative. Even if all
you did was a CD20 and CD3 stain in this case, the presence of CD3 positive rosettes around large cells
that were CD3 and CD20 negative should force you to think about diagnosing classical Hodgkin lymphoma
here. The lack of fairly uniform CD20 expression by the abnormal cells would be a major strike against
nodular lymphocyte predominant Hodgkin lymphoma.
- Lymphocyte rich classical Hodgkin lymphoma shows some clinical
overlap with nodular lymphocyte predominant Hodgkin lymphoma.
- This case also makes us think about the newer ways in which one
can use immunohistochemistry to help diagnose classical Hodgkin lymphoma and distinguish it from other
Hodgkin or non-Hodgkin lymphomas.
Classification of Hodgkin lymphoma according to the WHO:
classification recognizes two major types of Hodgkin lymphoma (HL) - nodular lymphocyte predominant and
classical Hodgkin lymphoma. The latter is further subdivided into four categories, the three old
standards (Frank Sinatra, Dean Martin and Sammy Davis, Jr – or for those not needing a humorous break,
nodular sclerosis, mixed cellularity and lymphocyte depleted) plus one newer entity, the lymphocyte-rich
type.  The biologic features of Hodgkin lymphoma and current therapeutic concepts have been
recently reviewed for those interested. 
Lymphocyte-rich classical Hodgkin lymphoma
Lymphocyte-rich classical HL is defined by the WHO as a
"...subtype of classical Hodgkin lymphoma (CHL) with scattered Hodgkin and Reed-Stenberg [SIC] (HRS)
cells and a nodular or less common diffuse cellular background consisting of small lymphocytes and with
an absence of neutrophils and eosinophils."  The "Hodgkin/Reed-Sternberg" cells have the
features of those of classical rather than L&H type. A debatable proportion of these cases (perhaps
most) have the features of what has also been termed "follicular" Hodgkin's disease . 
Lymphocyte-rich classical HL demonstrates B-cell follicles
with classic RS cells within the mantles as well as in interfollicular areas. The germinal centers, when
present, are often eccentrically placed. Fibrosis that would suggest the diagnosis of nodular sclerosis
HL is not present. These are the type of classic HL most easily confused with NLPHL. In fact, many
cases will be considered NLPHL until immunostains are performed. 
Classic RS cells are unlike L&H RS variants because
they should be CD45 (LCA) negative (beware of large histiocytes staining), are usually CD20 negative (and
when positive usually only a proportion are positive), are usually but not invariably CD15 (LeuM1)
positive and should be CD30 (Ki-1) positive. CD15 negativity in no way rules out the possibility of
classical HL and is a reason why one needs to use a panel of immunostains in evaluating cases of
potential HL. In one very large series, CD15 was only positive in 74% of the LRCHL cases! 
Overall, about half of classic HL are EBV positive with the highest proportion of positive cases found in
those of mixed cellularity or lymphocyte depleted type. EMA should usually be negative as should J
chain. CD57 positive cells are not frequent with CD57 positive rosettes not usually identified. One
must be aware of the lack of a perfectly typical phenotype in a significant minority of cases of
classical HL including the lymphocyte-rich type (see table below). 
| ||Lymph Rich Classical HL ||(Nodular) Lymph Pred HL|
|CD20 ||32% ||98%|
|J chain ||0% ||91.5%|
|CD15 ||74% ||0%|
|CD30 ||98% ||0%*|
|EMA ||6% ||54%|
|EBER (EBV) ||47% ||0%|
*Non-neoplastic CD30+ cells may be seen and some also report the presence of some CD30 expression in
LRCHL is most similar to LPHL compared to either
nodular sclerosis or mixed cellularity classical HL.  It demonstrates a male predominance,
70% of patients are stage I/II and only 11% have B symptoms. Compared to patients with LPHL, a slightly
greater proportion of those with LRCHL are greater than 50 years of age (32 vs 18%), more are stage III
(24 vs 14%) and more have mediastinal involvement although it is not very common(15 vs 7%). Survival and
failure free survival are not significantly different between LRCHL and LPHL; however, multiple relapses
are more common in LPHL. A similar proportion relapse in the two groups but survival after relapse is
worse for those patients with LRCHL when one restricts the analysis to those under 45 years of age.
Newer markers being used in the evaluation of potential cases of Hodgkin lymphoma, especially of classical type:
Antibodies to B-cell specific activator protein (BSAP) encoded by the PAX-5 gene are good
pan-B-cell markers and will be positive in most B-cell lymphomas, NLPHL and classical HL.
In classical HL, the RS cells typically show weak staining and may be negative. This stain is
especially useful if an ALCL is in the differential diagnosis. Antibodies to the MUM1/IRF4 transciption factor that is found in late germinal center
cells/post-germinal center B-cells, plasma cells and a small T-cell subset show positivity in the
neoplastic cells of almost all cases of classical Hodgkin lymphoma compared to only a minority of cases
of NLPHL and with variable staining in DLBCL (see above).
There appear to be a growing
number of laboratories following the recommendation of Browne, et al that antibodies to the Oct2 B-cell transcription factor and its co-activator Bob.1 "should be a welcome
addition to any standard Hodgkin lymphoma panel."  Both of these factors are expressed
strongly in germinal center B-cells and more weakly in mantle zone B-cells. Virtually all NLPHL and
DLBCL are positive for both factors but one or both are negative in many cases of classical Hodgkin
While some have reported no cases of classical Hodgkin lymphoma to
be positive for both, other studies have. Strong staining with both, however should usually be absent.
The extreme variation in the proportion of cases reported to be positive does mean that added caution is
advised in the interpretation of these markers. PU.1 is another transcription
factor that has been used since it is only rarely positive in classical HL and usually but not
always positive in NLPHL; however, it shows more variable staining in DLBCL and may be difficult to
interpret because histiocytes are positive.
- Anagnostopoulos I, Hansmann ML, Franssila K, et al. European Task Force on Lymphoma project on lymphocyte predominance Hodgkin disease: histologic and immunohistologic analysis of submitted cases reveals 2 types of Hodgkin disease with a nodular growth pattern and abundant lymphocytes. Blood 96: 1889-1899, 2000.
- Stein H, Delsol G, Pileri SA, et al., Classical Hodgkin Lymphoma, introduction. in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Swerdlow SH, Campo E, Harris NL, et al., [Eds.] IARC: Lyon. 2008, 326-329.
- Ashton-Key M, Thorpe PA, Allen JP, et al. Follicular Hodgkin's disease. American Journal of Surgical Pathology 19: 1294-1299, 1995.
- Diehl V, Sextro M, Franklin J, et al. Clinical presentation, course, and prognostic factors in lymphocyte-predominant Hodgkin's disease and lymphocyte-rich classical Hodgkin's disease: report from the European Task Force on Lymphoma Project on Lymphocyte-Predominant Hodgkin's Disease. J Clin Oncol 17: 776-783, 1999.
- Re D, Thomas RK, Behringer K, et al. From Hodgkin disease to Hodgkin lymphoma: biologic insights and therapeutic potential. Blood 105: 4553-4560, 2005.
- Anagnostopoulos I, Issacson PG, Stein H, Lymphocyte-rich classical Hodgkin lymphoma. in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Swerdlow SH, Campo E, Harris NL, et al., [Eds.] IARC: Lyon. 2008, 332-333.
- Browne P, Petrosyan K, Hernandez A, et al. The B-cell transcription factors BSAP, Oct-2, and BOB.1 and the pan-B-cell markers CD20, CD22, and CD79a are useful in the differential diagnosis of classic Hodgkin lymphoma. Am J Clin Pathol 120: 767-777, 2003.
- Loddenkemper C, Anagnostopoulos I, Hummel M, et al. Differential Emu enhancer activity and expression of BOB.1/OBF.1, Oct2, PU.1, and immunoglobulin in reactive B-cell populations, B-cell non-Hodgkin lymphomas, and Hodgkin lymphomas. J Pathol 202: 60-69, 2004.
- Falini B, Fizzotti M, Pucciarini A, et al. A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood 95: 2084-2092, 2000.
- Steimle-Grauer SA, Tinguely M, Seada L, et al. Expression patterns of transcription factors in progressively transformed germinal centers and Hodgkin lymphoma. Virchows Arch 442: 284-293, 2003.
- Jundt F, Kley K, Anagnostopoulos I, et al. Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease. Blood 99: 3060-3062, 2002.
- Torlakovic E, Tierens A, Dang HD, et al. The transcription factor PU.1, necessary for B-cell development is expressed in lymphocyte predominance, but not classical Hodgkin's disease. Am J Pathol 159: 1807-1814, 2001.