Microglandular hyperplasia: a model for the de novo emergence and evolution of endocervical reserve...

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Original contributions

Microglandular hyperplasia: a model for the de novoemergence and evolution of endocervical reserve cells

Agnieszka K. Witkiewicz MDa, Jonathan L. Hecht MD, PhDb, Aida Cviko MDa,Frank D. McKeon PhDc, Tan A. Ince MD, PhDa, Christopher P. Crum MDa,*

aDivision of Women’s and Perinatal Pathology, Department of Pathology, Brigham and Women’s Hospital, Boston,

MA 02115, USAbDepartment of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USAcDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

Received 13 April 2004; accepted 29 October 2004

0046-8177/$ – see front matter D 2005

doi:10.1016/j.humpath.2004.10.017

* Corresponding author.

E-mail address: [email protected]

Keywords:Microglandular

hyperplasia;

p63;

bcl-2;

Keratin;

Cervical neoplasms;

Reserve cells

SummaryBackground: Microglandular hyperplasia (MGH) of the cervix in human beings is associated early with

gland proliferation and terminates in mature squamous metaplasia. Using antibodies to basal cell

markers, we analyzed biopsies with MGH to profile the distribution and evolution of reserve cells and

their relationship to these epithelial components.

Design: Serial sections of 24 MGHs were subdivided into (1) early MGH with microacinar

proliferation, abundant subnuclear vacuoles, and a paucity of supporting stroma and (2) late MGH

with more prominent supporting stroma and/or squamous metaplasia. Serial sections were stained with

antibodies to p63, bcl-2, and keratin-5.

Results: Three patterns of p63 staining were observed corresponding to the age of the MGH: (1)

scattered staining of columnar cells, (2) focal subcolumnar staining in a reserve cell distribution, and (3)

linear subcolumnar arrays of p63-positive reserve cells that in some MGHs expanded into a squamous

metaplasia. Early acinar proliferations showed weak and focal columnar cell staining followed by focal

subcolumnar p63-positive cells. In late lesions, p63 staining was compartmentalized to the

extraglandular (or subcolumnar) areas. Stainings of p63, bcl-2, and keratin-5 were concordant. Staining

for keratin 14, which localizes to squamous cells, was variable.

Conclusions: The immunohistochemical profile in MGH indicates that reserve cells are created in

adulthood during specialized columnar proliferations. This columnar to reserve cell transition may

produce a stable population of reserve cells or a transition to squamous metaplasia. Similar patterns are

seen in cervical neoplasia, suggesting a link between benign and neoplastic cervical epithelial

differentiation.

D 2005 Elsevier Inc. All rights reserved.

Human Pathology (2005) 36, 154–161

Elsevier Inc. All rights reserved.

(C.P. Crum).

Microglandular hyperplasia: a model for the de novo emergence and evolution of endocervical reserve cells 155

1. Introduction

Reserve cells are an important component of the uterine

cervical transformation zone and are most conspicuous

beneath the columnar epithelium of the endocervix just

cephalad to the squamocolumnar junction [1]. This cell

population is considered important in the evolution of the

transformation zone during reproductive years, during

which reserve cells expand in number and undergo

squamous differentiation. Reserve cells presumably origi-

nate during development via the induction of basal cells

within preexisting mqllerian epithelium [2,3]. Cells analo-

gous to basal cells are also seen in the salivary gland, breast,

and prostate and can be distinguished from the overlying

columnar epithelium by expression of the p53 homolog, p63

[4,5]. Similarly, induction of basal cells in the urogenital

sinus epithelium may mediate the transition to urothelium,

which is also intensely p63-positive [3].

Precisely whether and how reserve cells are generated

during reproductive life has not been evaluated in detail.

However, studies have established the following scenario

which includes the following steps: (1) induction of p63

occurs in the columnar epithelium lining the vagina and

cervix during reproductive life, giving rise to subcolumnar

cells identical to reserve cells in the adult cervix [Fig. 1(A)],

(2) these cells do not emerge in the p63-null mouse, and (3)

administration of estrogens to mice at 1-month age—or the

onset of estrus—results in the conversion of these cells to

squamous epithelium [Fig. 1(B)] (C.P. Crum and F.D.

McKeon, unpublished data). The implication from these

observations is that reserve cells can be induced from

mqllerian epithelium and convert to squamous cells. The

importance of these observations lies in the fact that such

cells may be integral to changes in cell differentiation in

both benign and neoplastic states [5,6].

Fig. 1 (A) Vaginal epithelium in the newborn mouse consists of mqllerthe reserve cells convert to mature squamous mucosa with loss of the c

Although experimental studies of cervical transformation

zone in human beings are not feasible, evidence exists for the

coexistence of glandular proliferation and squamous differ-

entiation. A common morphological entity in the cervix of

reproductive-age women is microglandular hyperplasia

(MGH) [7]. Microglandular hyperplasia may present as a

predominately microacinar proliferation in its early phase

and ultimately undergoes conversion into squamous meta-

plasia. Presumably, the transition from a purely columnar to

a squamous mucosa entails the presence of reserve cells.

The purpose of this study was to explore the relationship

between glandular proliferation in MGH, reserve cells, and

the onset of squamous metaplasia using p63, keratin, and bcl-

2 staining to localize reserve cells in these conditions. The

implications of this process for the varied cell phenotypes

seen in normal and abnormal cervix are discussed.

2. Methods

2.1. Case selection and histological classification

With the approval of the Brigham and Women’s Hospital

institutional review board, cervical samples, including cone

biopsies containing foci of MGH (24 cases), were identified

during the routine surgical pathology practice in our

departments.

2.2. Immunohistochemistry

2.2.1. Antibodies2.2.1.1. p63. Preparation of a monoclonal antibody to p63

was described in detail previously [8]. Briefly, a cDNA

fragment containing the N-terminal portion (amino acids

1-205) of DNp63 was overexpressed as a glutathione

S-transferase fusion protein and used for subsequent

ian epithelium with subcolumnar cells. (B) After estrogenic stimuli,

olumnar cells.

Fig. 3 Localization of p63 in the transformation zone from a

cervical conization specimen. In the upper part of the field, p63-

positive cells are distributed at the base of small acini and between

acini of immature columnar epithelium. Below, p63-positive cells

are discretely arranged beneath mature columnar epithelial cells.

Fig. 2 (A) Type 1 MGH with microacinar architecture and prominent subnuclear vacuoles, (B) type 2 MGH with supporting stroma and

conspicuous reserve cells, and (C) type 2 MGH with developing squamous metaplasia.

A.K. Witkiewicz et al.156

monoclonal antibody production. The antibody generated

by this construct reacted with all p63 proteins, including the

TA-p53 targets (F. McKeon, unpublished data). However,

the dominant p63 protein detected in epithelium is DNp63

(C.P. Crum and F.D. McKeon, unpublished data).

Immunostaining for p63 was performed on 4-lm depar-

affinized sections using horseradish peroxidase–conjugated

goat antimouse IgG antibody as previously described [8]. In

each case, stromal cells were used as an internal negative

control. Previously described nuclear staining in basal and

parabasal cells of the ectocervix and maturing transforma-

tion zone served as an internal positive control [5].

2.2.1.2. Keratin 5/6. Keratin 5/6 is a previously described

marker of reserve cells [9] and was detected using a mouse

monoclonal antibody to a conjugated synthetic peptide

derived from the carboxy terminus (clone D5/16B4, dilution

1:10, Zymed Laboratories, San Francisco, Calif). Stains

were performed on 4-lm deparaffinized sections using

diaminobenzidine detection. Antigen retrieval involved

microwave treatment for 30 minutes at 1998F in 1mmol/L

EDTA buffer pH 8. The section of normal skin was used as

a positive control.

2.2.1.3. Keratin 14. Keratin 14 (CK14) is a type I

intermediate filament distinguishing stratifying epithelial

cells from simple epithelium and is strongly associated with

squamous differentiation [9]. This protein was detected

using a monoclonal antibody (clone, dilution 1:50, Research

Diagnostics, Flanders, NJ) to a conjugated synthetic peptide

derived from the carboxy terminus. Stains were performed

on 4-lm deparaffinized sections using diaminobenzidine

detection after 30-minute microwave treatment for antigen

retrieval.

2.2.1.4. Bcl-2. Bcl-2 plays a central role in the inhibition of

apoptosis, has been localized to basal squamous and

subcolumnar reserve cells, was detected using an antibody

to a synthetic peptide composed of amino acids 41 to 54 of

the BCL-2 protein (clone 124, dilution 1:25 Dako Corp,

Carpinteria, Calif) [5]. Stains were performed on 4-lmdeparaffinized sections using mouse secondary antibodies

and diaminobenzidine detection after antigen retrival (mi-

crowave, 30 minutes).

3. Results

3.1. Cases

Twenty-four cases of MGH were obtained via a search of

pathology diagnostic records with the approval of the

institutional review board at Brigham and Women’s

Fig. 4 Distribution of p63 in type 1 MGH. (A) Low cuboidal (immature) columnar cells line small acini, with weak or negative nuclear

staining for p63. (B) Focal and linear staining of microacini at variable intensity. Small groups or single nuclei are present beneath or between

acini. (C) Another field from this biopsy depicts areas with minimal or no staining (lower left), staining confined principally to basal

columnar epithelial cells (lower right) and subcolumnar cells (middle and middle right).


Hospital. The mean age of 24 patients was 33 (range, 27-57)

years. The primary histological diagnosis was high-grade

squamous intraepithelial lesion in 16 cases, low-grade

squamous intraepithelial lesion in 3 cases, endocervical

polyp in 2, and endometrial polyp in 3 cases. Twelve

patients underwent cone procedure, 8 biopsies, and 4

polypectomy. The cases were not selected based on the

primary diagnosis and were incidental to and spatially

separated from the squamous intraepithelial lesions for

which the procedures were performed.

Cases of MGH were identified using established criteria

and were subdivided into 2 categories: (1) early MGH with

microacinar architecture, abundant subnuclear vacuoles, and

a paucity of supporting stroma [Fig. 2(A)] and (2) late or

Fig. 5 Distribution of p63 in type 2 MGH. (A) Linear arrays of p63-p

(B) Immature (above) and mature (below) columnar cells maturation wi

normal cervix.

evolving hyperplasias with more prominent supporting

stroma, with or without squamous metaplasia [Fig. 2(B)

and (C)]. Early MGHs were present in 12 cases and were

characterized by proliferation of closely packed glands of

varying size often with cystic dilatation with little interven-

ing stroma [Fig. 2(A)]. Cuboidal or columnar cells

commonly exhibiting subnuclear and supranuclear vacuoles

lined the glands. The nuclei of the lesional cells were

uniform. The intraglandular mucinous secretions often

contained inflammatory cells, predominantly neutrophils.

Of 12 cases of late MGH, 3 exhibited transition from early

to late changes, and 9 had more prominent supporting

stroma, with various degree of squamous metaplasia, with

stromal hyalinization present in 1 case [Fig. 2(B) and (C)].

ositive reserve cells are now extensively distributed beneath acini.

th prominent subnuclear p63-positive cells, similar to that seen in

Fig. 6 (A) Early squamous metaplasia in an expanding population of subcolumnar cells. (B) Squamous differentiation is now more

conspicuous, obscuring some acini.

Fig. 7 Endocervical columnar epithelium with reserve cells (A) showing coexpression of p63, (B) keratin 5 (C), and bcl-2 (D).


Fig. 8 Schematic of the evolution of MGH, eventuating in either squamous metaplasia (right) or quiescent linear rows of reserve cells

beneath mature columnar mucosa (left).


In these, the stromal infiltration by acute and chronic

inflammatory cells varied from mild (3 cases) to moderate

(6 cases). The basement membrane was not obscured by the

inflammation and was easy to evaluate in early and late

MGH cases. In addition to prominent stroma, late MGH

cases were characterized by glands with more cuboidal than

columnar cells with eosinophilic cytoplasm largely devoted

of vacuoles. Squamous metaplasia was identified in all cases

and was prominent in 5 cases.

3.2. Immunohistochemistry

3.2.1. p63As reported previously, p63 strongly stains mature squa-

mous epithelium and scattered reserve cells in the transfor-

mationzone (Fig.3) [5].Maturecolumnarcellsdidnot stain. In

MGH, immunostaining with p63 correlated closely with the

pattern ofMGH. In type 1MGHswith prominent microacinar

architecture, p63 localized with variable but mostly weak

intensity in individual columnarcell nuclei [Fig. 4(A)and (B)].

Less commonly, p63 was localized in a linear continuous

pattern in the microacini [Fig. 4(C)].

In type 2 lesions, p63 localized to basal and subcolumnar

regions with a linear arrangement surrounding the epithe-

lium [Fig. 5(A) and (B)]. In addition to extra-acinar staining,

a second pattern emerged in some foci, with expansion of

the acinar lining cells and frank conversion to a squamous

phenotype with variable stratification. This pattern was

associated with progressive ablation of the columnar

architecture, with maintenance of the microglandular growth

pattern [Fig. 6(A) and (B)].

3.2.2. Cytokeratins and bcl-2Staining for bcl-2 and keratin 5/6 followed a similar

pattern to p63 [Fig. 7(A-D)]. In contrast, keratin 14 was

uniformly negative in the early and evolving phases of

MGH, emerging only when stratified squamous cells were

present.

4. Discussion

Microglandular hyperplasia is well recognized as a

preexisting condition for squamous metaplasia in the cervix.

The lesions consist of numerous small glandular spaces

lined by mucinous or eosinophilic columnar cells. The

ramifying pattern of glands, often arising from the normal

columnar cervical epithelium, increases in complexity,

acquires subnuclear vacuoles, and undergoes squamous

differentiation. For descriptive purposes, we view this as a

sequential process and designate simpler cuboidal to

columnar gland patterns as bearly Q and those undergoing

squamous metaplasia as blate,Q but the process of squamous

metaplasia is not well understood.

Studies using electron microscopy and immunostains for

keratin subtypes have identified a population of breservecellsQ in the stroma underlying the columnar epithelium of

MGH that are thought to be the basis of squamous

metaplasia at that site. These cells are not present in normal

cervical columnar epithelium or stroma [10]. Hypotheses

concerning reserve cell origins at the transition zone of the

cervix include (1) an ingrowth of basal cells from the

stratum germinativum of adjacent normal stratified squa-

mous epithelium, (2) origin from undifferentiated fetal rests

within the stroma, (3) origin from endocervical stromal

cells, and (4) origin from cervical columnar cells (reviewed

in Ref. [11]). The reserve cells giving rise to metaplasia in

MGH may be analogous to those at the transition zone.

Given the irregular architecture and location of MGH above


the transition zone, however, an origin from basal cells of

stratified squamous epithelium is excluded.

Patterns of cytokeratin expression described in the

transition zone support a columnar cell origin. Reserve

cells express keratins CK5, 8, 17, 18, and 19. Expression of

CK14 is variable. Columnar epithelium away from the

transition zone expresses CK7, 8, 18, and 19. However,

columnar cells overlying reserve cells in the transition zone

also express CK5 and 17, similar to reserve cells, indicating

that these cells may undergo a low level of squamous

differentiation, a concept supported by the distribution of

p63 immunostaining in this study [9]. Mature ectocervical

squamous epithelia express CK4, 13, and 14, but lack CK8,

18, and 19. Thus, reserve cells are taken either to have a

tendency to bidirectional differentiation or to be an

intermediate step in the transition from columnar to

squamous cells [12].

The keratin content of reserve cells is complex and

argues against their stromal origin because endocervical

stroma contains no cytoskeletal keratin proteins. It is

unlikely that stromal cells migrating through the basal

membrane would initiate expression of multiple keratin

types. Also, vimentin, so characteristic of stromal cells, is

not expressed in reserve cells [9,13]. The stroma of MGH is

morphologically distinct from cervical stroma with a more

epithelioid phenotype and the presence of acute and chronic

inflammation.

The p63 staining patterns we observed are consistent

with the emergence of reserve cells during the evolution of

MGH and before squamous metaplasia. The relative paucity

of reserve cells in type 1 microacinar lesions and their

abundance in lesions with multilayered epithelium and

squamous differentiation are consistent with an evolution

from microacinar/columnar MGH to more complex MGH

with metaplasia. We have observed similar phenomena in

the endometrium, where tubal and mucinous metaplasias

may harbor prominent p63-positive basal or subcolumnar

cells [14]. The endometrium normally does not contain

reserve cells, and their emergence suggests their origin from

preexisting components of the epithelium [2,3].

Our data also support a columnar cell origin of the reserve

cells. In early lesions, weak immunoreactivity for p63

localized to single cells and progressed to a single layer

pattern of stronger staining columnar cells. In more mature

lesions, more intense staining was seen, localizing to basal

and subcolumnar regions. With squamous metaplasia,

staining was present in the multilayered metaplastic cells

filling the microacini. Staining for other reserve cell markers

including keratin 5/6 and bcl-2 [5,9] showed a similar pattern

to p63, whereas CK14, a mature squamous cell marker, was

only present in epithelia exhibiting squamous differentiation.

A columnar cell origin for reserve cells is also consistent

with previous observations at the transition zone where there

is a correlation between p63 and CK14 staining in immature

squamous epithelium; p63 tended to extend beyond the

CK14 zone into the adjacent columnar cells [5].

Why MGH is not more frequently associated with human

papillomavirus (HPV)–related lesions remains unclear. Pos-

sible explanations include lack of receptors in MGH for HPV

virion attachment, inability to sustain HPV infection in a

reserve cell population not undergoing squamous differenti-

ation (and therefore not promoting viral replication), and

protection of acini from infection by the overlying epitheli-

um. Nonetheless, the ability of the cervical epithelium to

generate reserve cells is mirrored in the range of intra-

epithelial neoplasms seen in the cervix. They include the

following: (1) poorly differentiated adenocarcinomas in situ

(stratified mucin-producing intraepithelial lesions) in which

p63-positive cells are focally present in the basal cell layers

[5] [Fig. 3(O)], (2) rare p63-positive subcolumnar mucin-

producing precursor lesions that correspond to the transition

from columnar to reserve cell [6] [Fig. 3(B)], (3) immature

bmetaplasticQ squamous intraepithelial lesions (SILs) that

follow the growth pattern of MGH-associated squamous

metaplasia, and (4) a range of metaplastic growth patterns in

SIL variously termed atypical immature metaplasia, meta-

plastic SIL, and recently, beosinophilic dysplasiaQ[5,6,15,16]. A simplified explanation for these disparate

phenotypes is that epithelial differentiation is governed by 2

switch points in endocervical epithelial cell fate, including the

transition from columnar to reserve cell, and reserve cell to

squamous differentiation (Fig. 8). The same molecular

pathways governing these switches in MGH might explain

the multiplicity of neoplastic phenotypes observed.

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