Hematopathology / FOLLICULAR HYPERPLASIA, FOLLICULAR LYSIS, AND PROGRESSIVE TRANSFORMATION OF GERMINAL CENTERS
Follicular Hyperplasia, Follicular Lysis, and Progressive
Transformation of Germinal Centers
A Sequential Spectrum of Morphologic Evolution in Lymphoid
Hyperplasia
Chung-Che Chang, MD, PhD,1 Vladimir Osipov, MD,1 Sue Wheaton, MD,2
Sheryl Tripp, MT(ASCP),3 and Sherrie L. Perkins, MD, PhD 3
Key Words: Hyperplastic follicles; Follicle lysis; Progressive transformation of germinal centers; PTGC; Follicular lymphoma;
Immunohistochemistry
DOI: 10.1309/UUEPLF2PHYXQDJNQ
Abstract
We studied mantle B-cell and T-cell ingression in
hyperplastic follicles (HFs), follicular lysis (FL), and
progressive transformation of germinal centers (PTGC)
in 19 paraffin-embedded, H&E-, bcl-2–, CD20-, and
CD3-stained lymph nodes. We enumerated the T cells
(CD3+) and mantle B cells (bcl-2+/CD3–) per 100
cells in 5 high-power fields of each entity (mean ± SD).
Compared with HF, FL had increased numbers of T
cells migrating into germinal centers (39.8 ± 10.0 vs
25.8 ± 7.8; P < .0001) and a mild increase of mantle B
cells (12.3 ± 11.4 vs 2.1 ± 1.6; P < .001). PTGC
showed an increase of T-cell ingression compared with
HF (36.5 ± 12.1 vs 25.8 ± 7.8; P < .0001) and more
migration of mantle B cells into the follicle than FL
(41.0 ± 22.5 vs 12.3 ± 11.4; P < .0001).
T cells and mantle B cells ingress in FL and PTGC,
although the mantle B-cell component predominates in
the latter, suggesting that follicular hyperplasia, FL,
and PTGC constitute an evolutionary spectrum in
resolution of lymphoid hyperplasia with sequential
ingression of T cells followed by mantle B cells. The
maintenance of bcl-2 expression in mantle B cells in
PTGC may cause differential diagnostic pitfalls in
florid PTGC vs follicular lymphoma, particularly the
so-called floral variant.
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DOI: 10.1309/UUEPLF2PHYXQDJNQ
Progressive transformation of germinal centers (PTGC)
is a morphologic feature of reactive hyperplasia initially
described by Lennert and Müller-Hermelink1 as “germinal
centers that are lost in a mass of lymphocytes.” PTGC is seen
most commonly in lymph nodes in association with reactive
follicular hyperplasia.2-4 Overall, 3.5% to 10% of reactive
lymph nodes with chronic nonspecific lymphadenitis will
contain 1 or more areas of PTGC.2-4 Persistent or recurring
PTGC can occur, particularly in pediatric cases (up to 50%),
and usually is located in the same lymph node region.4,5
PTGC also may occur as a predominant pattern in lymph
node hyperplasia, designated as florid PTGC. This manifestation is described classically in children or in young males in
their second to third decade of life who have an asymptomatic, solitary, enlarged lymph node (cervical most frequent
followed by inguinal and then axillary).5-7
PTGC also has been described in association with
Hodgkin disease. It has been reported that 16% to 20% of
cases of PTGC are associated with Hodgkin disease,
mostly lymphocyte predominant Hodgkin disease
(LPHD).5-7 Poppema8 and Poppema et al9 suggested an
association between PTGC and LPHD based on similar
morphologic features and the observation that both entities
may occur together in the same lymph node. PTGC may
coexist or precede LPHD or be present in subsequent
lymph node biopsy specimens following a diagnosis of
lymphoma.10,11 However, in prospective studies, the identification of a significant component of PTGC in a lymph
node was not associated definitively with an increased risk
of developing LPHD.4,5,10
PTGC usually is identified at low magnification as welldefined nodules that are larger than adjacent germinal
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
centers. High power reveals the nodules as composed of a
polymorphous mixture of predominantly small lymphocytes
with morphologic features suggestive of mantle B cells and
T lymphocytes and scattered residual follicle center cells.10,12
The mantle B cells are small with round to slightly irregular
nuclear contours, and they express surface IgM and surface
IgD, characteristics of mantle B cells.12 However, tingible
body macrophages and mitoses typically are absent.
Although PTGC has long been believed to be part of
the spectrum of lymphoid hyperplasia, the potential relationships between hyperplastic follicles (HFs), follicular
lysis (FL), and PTGC has not been well illustrated. Only a
few studies have suggested that PTGC is caused by the
ingression of T cells and mantle B cells.10,12 These studies
are limited further by the small numbers of cases studied
and by the use of immunohistochemical analysis on frozen
tissue sections, which do not provide optimal morphologic
features for evaluation.
We sought to thoroughly examine the morphologic
ingression of mantle B cells and T cells into germinal centers
to delineate the relationships of HF, FL, and PTGC by using
a panel of immunophenotypic markers including CD20 (to
highlight the different lesions), bcl-2, and CD3 on paraffinembedded tissue sections. By defining mantle cells by the
immunophenotype of bcl-2+/CD3– and T cells as CD3+, we
attempted to define and quantitate the small lymphoid
component in HF, FL, and PTGC to gain insight into these
processes. Our results document the ingression of T cells and
mantle B cells in FL and PTGC, although the mantle B-cell
component predominates in PTGC, suggesting that HF, FL,
and PTGC constitute a spectrum of evolution in resolution of
lymphoid hyperplasia with sequential ingression of T cells
followed by mantle B cells. Furthermore, the expression of
bcl-2 in mantle B cells is maintained in PTGC. This persistence of bcl-2 expression may cause pitfalls in the differential diagnosis of florid PTGC vs follicular lymphoma, particularly the so-called floral variant.
All 19 cases were evaluated for the distribution of
mantle B cells and T-cell lymphocytes by immunohistochemical techniques using commercially available monoclonal antibodies (DAKO, Carpinteria, CA) to bcl-2 protein
(clone 124; dilution 1:80), CD3 (clone T3-4B5; dilution
1:50), and CD20 (clone L26, pan–B-cell marker; dilution
1:2,000) in sequential sections. All slides were stained using
an automated immunostainer (Ventana, Tucson, AZ). The
slides underwent heat-induced epitope retrieval using a pressure cooker (CD3 and bcl-2) or microwave irradiation
(CD20) in citrate buffer before staining. Staining was
detected using a diaminobenzidine chromogen. Appropriate
positive and negative controls were used in all cases.
To assess the expression of bcl-2, CD3, and CD20 in
HF, FL, and PTGC, evaluation of staining in sequential
sections was analyzed. CD20 staining was used to highlight
the different lesions. In each lymph node, the number of
CD3+ cells (T cells) and bcl-2+ cells (T cells and mantle B
cells) per 100 cells in 5 high-power fields (×400) of HF,
FL, and PTGC were enumerated by 2 of us (C-C.C. and
V.O.). The central areas of HF, FL, and PTGC were
selected for the enumerating to avoid counting the mantle
zone B cells. This led to enumeration of, in general, 1 highpower field per HF lesion, 1 high-power field per FL
lesion, and 1 to 2 high-power fields per PTGC lesion. For
each high-power field, CD3+ or bcl-2+ cells were divided
by the total number of cells and then multiplied by 100 to
obtain the quantity of CD3+ or bcl-2+ cells per 100 cells.
There were no significant differences in the counts obtained
by the 2 pathologists. The number of mantle B cells per
100 cells was defined as the number of bcl-2+ cells per 100
cells minus the number of CD3+ cells per 100 cells. The
numbers (means and SDs of all high-power fields enumerated by the 2 pathologists) of T cells and mantle B cells in
each morphologic component then were compared by using
the Student t test.
Results
Materials and Methods
We retrieved 19 paraffin-embedded lymph nodes
containing follicular hyperplasia, FL, and PTGC from the
pathology files at the University of Utah Health Sciences
Center, Salt Lake City, and from our hematopathology consultation service. All cases were reviewed to confirm the diagnosis and the presence of the aforementioned morphologic
features. None of the patients had a known history of HIV
infection or lymphoma, in particular Hodgkin lymphoma. Of
19 cases, 13 had no known evidence of developing lymphoid
malignant neoplasms (range of follow-up period, 2.5 to 9.2
years). The remaining 6 cases were lost to follow-up.
All 19 lymph node sections had a background of reactive follicular hyperplasia showing HFs and contained 1 or
more PTGC (range, 1-6). None of the cases had clinical or
morphologic evidence of LPHD or features of florid PTGC.
FL also was present in 13 of 19 lymph nodes.
Hyperplastic Follicles
H&E examination revealed that HFs were composed of
germinal centers surrounded by intact and well-defined
mantle zones ❚Image 1A❚. As shown in ❚Table 1❚ and ❚Image
1B❚, CD3 identified a small number of T cells within the
germinal centers. Staining for bcl-2 was strongly positive in
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A
B
C
D
E
F
G
H
I
❚Image 1❚ A-C, The histologic features and
immunophenotype of hyperplastic follicle (HF). A, HF is
composed of germinal centers surrounded by intact and welldefined mantle zones (H&E, ×100). B, CD3 identifies a small
number of T cells within the germinal centers (CD3, ×100). C,
Quantities of bcl-2+ cells in the germinal centers are similar
to CD3+ cells identified, indicating a minimal ingression of
mantle B cells, defined as bcl-2+/CD3– (bcl-2, ×100). D-F,
The histology and immunophenotype of follicular lysis (FL). D,
In FL, the germinal center is distorted by tongues and
clusters of dark blue mantle cells impinging on and invading
the reactive germinal center (H&E, ×100). E, T cells are
present in greater numbers in FL than in HF (CD3, ×100). F,
The bcl-2+ cells comprise a significantly larger population in
the follicle center than did the CD3+ cells, indicating the
ingression of mantle B cells (bcl-2, ×100). G-I, The histology
and immunophenotype of progressive transformation of
germinal centers (PTGC). G, PTGC is identified as large, welldelineated nodules without evident mantle zones or tingible
body macrophages (H&E, ×100). H, The density of CD3+ T
cells in PTGC is similar to that of FL but significantly higher
than that of HFs (CD3, ×100). There is a marked ingression of
mantle B cells, highlighted by the differential between CD3
staining (H) and bcl-2 staining (I, bcl-2, ×100). I, The large
numbers of mantle B cells and T cells in PTGC led to the
characteristic appearance of diffuse bcl-2 staining in PTGC.
mantle B cells and T cells within the paracortical area, but
the quantities of bcl-2+ cells enumerated in the germinal
centers (28.58 ± 7.23 cells per 100 cells) were similar to
those of the CD3+ cells identified (Image 1B) ❚Image 1C❚.
Thus, the numbers of mantle B cells (defined as bcl2+/CD3–) ingressing into the follicle center was relatively
small in HFs (Table 1).
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Follicular Lysis
In FL, the germinal center was distorted by tongues and
clusters of dark blue mantle cells impinging on and invading
the reactive germinal center ❚Image 1D❚. T cells were present
in greater numbers in FL than in HF (P < .0001; Table 1)
❚Image 1E❚. In FL, bcl-2+ cells constituted a significantly
larger population in the follicle center than did the CD3+
cells, indicating the ingression of mantle B cells ❚Image 1F❚.
This resulted in a relative increase of the mantle cell component in FL compared with HF (P < .001; Table 1).
Progressive Transformation of Germinal Centers
PTGC was identified as large well-delineated nodules
without evident mantle zones or tingible body macrophages
❚Image 1G❚. The density of CD3+ T cells in PTGC was
similar to that of FL but significantly higher than that of HF
(P < .0001; Table 1) ❚Image 1H❚. There was a marked ingression of mantle B cells, highlighted by the differential
between CD3 staining and bcl-2 staining (Image 1H) ❚Image
1I❚, compared with FL (P < .0001; Table 1). The large
numbers of mantle B cells and T cells in PTGC led to the
characteristic appearance of diffuse bcl-2 staining in PTGC
(Image 1I).
Discussion
In the present study, we revisited the morphologic and
immunophenotypic features of PTGC and documented the
ingression of mantle B cells and T cells into germinal
centers as originally suggested by Lennert and MüllerHermelink.1 Most important, by enumerating the T cells and
mantle B cells by the combination of bcl-2 and CD3
staining, we illustrated that HF, FL, and PTGC seem to be a
sequential morphologic evolution of lymphoid follicular
hyperplasia characterized by the ingression of T cells
followed by mantle B cells.
The bcl-2 protein is a regulator of lymphocyte apoptosis
and has been shown to be expressed normally in mantle B
cells and T cells, but it is absent in nonneoplastic follicle
center cells.13 Because of the strong expression of bcl-2 within
benign mantle B cells, we used bcl-2+/CD3– to identify and
enumerate the mantle B cells and to demonstrate sequentially
increased degrees of mantle cell ingression throughout the
morphologic spectrum of HF, FL, and PTGC. We further
demonstrated that as the mantle B cells began infiltrating the
germinal centers, as in HL and PTGC, they retained their bcl2 expression and, ultimately, together with infiltrating T cells,
formed the large bcl-2+ nodule characteristic of PTGC.
The pathogenesis of PTGC remains unclear. Our observation that the amounts of mantle B cells and T cells were
increased sequentially from HF to FL to PTGC supports the
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
❚Table 1❚
The Quantity of T Cells and Mantle B Cells in Hyperplastic
Follicles, Follicular Lysis, and PTGC*
T cells (CD3+)
Mantle B cells
(bcl-2+/CD3–)
Hyperplastic
Follicles
Follicular Lysis
PTGC
25.8 ± 7.8
2.1 ± 1.6
39.8 ± 10.0
12.3 ± 11.4
36.5 ± 12.1
41.0 ± 22.5
PTGC, progressive transformation of germinal centers.
* Data are given as mean ± SD per 100 cells.
concept that PTGC is part of the spectrum of reactive follicular hyperplasia and, possibly, the ultimate fate of a follicular
center in response to antigen simulation.4,5,14 This concept
would envision a stimulated follicle to undergo sequential
steps of follicular hyperplasia, FL, and PTGC as a
continuum. The concept is sustained further by a recent
study demonstrating that somatic hypermutation, clonal
expansion, and selection also can occur in the disorganized
PTGC microenvironment, as well as in classic stimulated
germinal centers.15
The observation that areas of PTGC are bcl-2+ owing
to the marked ingression of mantle B cells and T cells in the
present study may have important diagnostic significance.
Cases of florid PTGC have been reported,2 as have studies
describing follicular lymphoma “mimicking” PTGC, particularly the floral variant of follicular lymphoma.16,17 In the
floral variant, more than half of the neoplastic follicles were
surrounded by prominent mantle zones that irregularly
invaginated the follicle centers, giving the appearance of
PTGC.16 An initial diagnosis of atypical hyperplasia or
PTGC was made in 21% of these cases.16 It must be emphasized that bcl-2 can be positive in the nodules of both entities, as described in our study, and should not be automatically interpreted as the bcl-2 positivity of follicular
lymphoma.
An accurate diagnosis requires a detailed morphologic
evaluation and adequate ancillary studies. The diagnosis of
the floral variant of follicular lymphoma is supported by
the identification of the densely packed nodules of atypical
follicular center cells. Extranodal extension may be present.
By contrast, PTGC usually is associated with a background
of reactive follicular hyperplasia. A distinctive wreath of
epithelioid histiocytes occasionally surrounds the PTGC.
Immunohistochemical staining for CD10 can be helpful.
Only a few CD10+ follicle center B cells are present in
PTGC, while abundant CD10+ cells are observed in the
floral variant of follicular lymphoma (unpublished observation). Furthermore, interfollicular CD10+ lymphocytic
infiltrates strongly support the diagnosis of follicular
lymphoma.18 In difficult cases, molecular studies to detect
t(14;18) and clonal immunoglobulin gene rearrangement
can be used to confirm the diagnosis of follicular
lymphoma.
Our results document the sequential ingression of T
cells and mantle B cells in FL and PTGC, with the mantle B
cells predominating in PTGC. These findings suggest that
HF, FL, and PTGC are a sequential spectrum of lymphoid
follicular hyperplasia. We also demonstrated that mantle B
cells retain their bcl-2 positivity after ingression into FL and
PTGC. Recognizing bcl-2 positivity in PTGC may avoid the
potential pitfall of misinterpreting PTGC as follicular
lymphoma, particularly the floral variant.
From the 1Department of Pathology, Medical College of
Wisconsin, Milwaukee; 2Department of Pathology, Mercy
Hospital, Coon Rapids, MN; and 3ARUP Institute and
Department of Pathology, University of Utah, Salt Lake City.
Address reprint requests to Dr Perkins: University of Utah
Health Science Center, 50 N Medical Dr, Salt Lake City, UT 84132.
Acknowledgment: We thank the ARUP Institute for financial
and technical support of this study.
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