| Abstract|| |
Background: Cystic lymphoid hyperplasia (CLH) describes benign salivary lymphoepithelial cysts with a strong link to human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). The pathogenesis is related to ductal dilatation of entrapped salivary elements due to intranodal HIV-associated atypical lymphoid hyperplasia. Very little is known about the immunophenotypic profile of this entity. Aim: This study aims to describe the immunopathological features of a series of CLH cases in HIV-positive patients to clarify the etiopathogenesis. Materials and Methods: Paraffin-embedded tissue from 25 cases of parotid CLH in HIV seropositive patients was immunohistochemically analyzed with CD3, CD20, CD4, CD8, and p24 using standard procedures. Statistical Analysis: The data are mostly descriptive and were analyzed using EpiInfo (3.5.1) (CDC, Atlanta, USA); significant differences were analyzed using the Student's t-test and the Chi-square test with a statistical significance level of P < 0.05 being used. Results: Immunostaining showed a CD8:CD4 of ~1:1 except in selected cases with decreased CD4 and increased CD8 expression in the interfollicular (IF) areas. p24 staining revealed 100% specificity in HIV-associated CLH. Conclusion: The immunohistochemical description of CD20, CD3, CD4, and CD8 provides an understanding of CLH pathogenesis. CLH of parotid lymph nodes in confirmed HIV-positive patients with 100% specificity with HIV p24 antibody validates the strong association of CLH with HIV and AIDS. The CD4:CD8 ratio was ~1:1; however, increased CD8 expression within IF areas may indicate possible HIV-related CLH as compared to other cystic parotid lesions.
Keywords: Acquired immunodeficiency syndrome, cystic lymphoid hyperplasia, human immunodeficiency virus, lymphoepithelial cysts, salivary gland
|How to cite this article:|
Meer S, Dulabh S. Human immunodeficiency virus-associated cystic lymphoid hyperplasia: An immunohistochemical description. Indian J Pathol Microbiol 2017;60:336-40
|How to cite this URL:|
Meer S, Dulabh S. Human immunodeficiency virus-associated cystic lymphoid hyperplasia: An immunohistochemical description. Indian J Pathol Microbiol [serial online] 2017 [cited 2020 Feb 29];60:336-40. Available from: http://www.ijpmonline.org/text.asp?2017/60/3/336/215396
| Introduction|| |
Cystic lymphoid hyperplasia (CLH) refers to benign lymphoepithelial cysts (BLECs) of salivary glands, especially the parotid gland in human immunodeficiency virus (HIV)-seropositive individuals. The pathogenesis of CLH is related to ductal dilatation of entrapped salivary gland elements as a result of intranodal HIV-associated atypical lymphoid hyperplasia. In view of the atypical lymphoid proliferation surrounding the cystic spaces and its strong association with HIV and acquired immunodeficiency syndrome (AIDS), this study describes the immunopathological features of a series of CLH cases in HIV-positive patients with a view to elucidating the etiopathogenesis.
| Materials and Methods|| |
Formalin-fixed (10% neutral buffered formalin; 18–48 h) paraffin-embedded tissue from 25 cases of parotid CLH in HIV seropositive patients was retrieved from the archives of the Department of Oral Pathology, University of the Witwatersrand, Johannesburg, South Africa. The diagnosis was confirmed on review of hematoxylin and eosin (H and E) stained sections. Ethics clearance (M080927; M080850) for this study was granted by the Human Research Ethics Committee (Medical) of the University of the Witwatersrand, Johannesburg, South Africa.
Immunohistochemistry (IHC) was performed on deparaffinized 4 μ sections on each case with commercially available antibodies listed in [Table 1] using the Envision system (DakoCytomation, Glostrup, Denmark) on 3-aminopropyl-triethoxysilane (Sigma, St. Louis, Missouri, USA) glass slides. These were air-dried overnight, dewaxed, and hydrated through graded alcohols and water. These sections were immersed in citric acid (Sigma, St. Louis, Missouri, USA) buffer 0.01M, pH 6.0 and heated in a microwave oven (800W) on medium power for 10 min. For heat-induced epitope retrieval, the sections for the various antibodies were subjected to 1.0 mmol/L of ethylenediaminetetraacetic acid buffer (pH 9.0), heated in microwave (800W) on medium power for 10 min.
Sections were then cooled for 20 min and immersed in 3% hydrogen peroxide in distilled water for 5 min and rinsed in tris-buffered saline (TBS) pH 7.6 with 0.1% Tween 20 (Sigma, St. Louis, Missouri, USA). The protein was blocked for 5–15 min to block endogenous peroxidase. Specimens were incubated with primary antibody for 20 min at room temperature using dilutions listed in [Table 1]. Sections were then incubated with the Envision detection kit (Dako, Denmark) for 20 min, rinsed with TBS Tween-20 and incubated for 3 min. Chromogen was applied for 10 min, and the color was developed with 3,3'-diaminobenzidine (Sigma) resulting in a brown reaction product. Thereafter, sections were lightly counterstained with H and E for 1 min. At the same time, the negative control was incubated for 1 h under the same conditions with the negative control reagent and buffer for each antibody. Appropriate positive controls were included along with negative controls.
Due to the nature of staining, immunopositivity for CD20, CD3, CD4, and CD8 was expressed semiquantitatively as a percentage as follows: <10%; 10%–50%; 50%–80%, and >80%. In view of the documented association of CLH with HIV and AIDS ,, and the HIV-induced histological changes, immunostaining was done with the monoclonal antibody HIV p24. Immunopositivity for p24 was expressed as either positive or negative and not according to intensity. In addition, p24 immunostaining was assessed in a control sample of 15 BLECs of the parotid gland in HIV-negative patients. The data in this study were mostly descriptive in nature.
| Results|| |
All 25 patients with CLH were HIV seropositive. There were 15 males and 10 females with a male:female ratio of 1.5:1. The age of patients ranged from 8 to 56 years (mean, 36.48; median, 37; range, 48).
The histologic features of all cases were classic for CLH as previously reported.,,,,, The BLECs were predominantly multicystic epithelial-lined cavities surrounded by dense atypical lymphoid hyperplasia with prominent germinal center (GC) formation. All cases occurred within lymph nodes approximating the parotid gland. The cyst lumens were filled with amorphous eosinophilic material with interspersed foamy macrophages and lymphocytes. The cyst lining ranged from stratified squamous to cuboidal to columnar and pseudostratified respiratory-type epithelium, with areas denuded of epithelium. There was moderately intense lymphocytic permeation of the cyst lining. Prominent subepithelial lymphoid condensation admixed with other acute and chronic inflammatory cells was noted within the reactive lymphoid proliferation adjoining the cyst lining.
The cystic spaces were bound by an atypical lymphoid proliferation dominated by large reactive follicles with serpiginous or hourglass shape GCs, and follicle lysis, similar to follicles seen in reactive lymph nodes in HIV-positive patients. In cases, there was conspicuous apoptosis with prominent tingible body macrophages. Follicles were devoid of a mantle zone of small lymphocytes (naked GCs). Perivascular clusters of monocytoid B-cells were evident. Occasional cases (8 of the 25 cases; 32%) showed scattered clusters (no more than 4 giant cells) of Warthin–Finkeldey type multinucleated giant cells. These giant cells were distinct from the Langhans-type multinucleated giant cells, with no associated necrosis or granulomatous inflammation. Infrequently, the lymphoid infiltrates showed a depletion of lymphocytes and an accumulation of macrophages and plasma cells. In a few cases, the follicles were atrophic with areas of hyalinization and diffuse fibrosis.
The CLH process in salivary gland parenchyma showed variable lymphoid proliferation around the intra- and inter-lobular ducts. Ductal dilatation produced early epimyoepithelial island formation.
CD20 and CD3
The hyperplastic lymphoid proliferation adjacent to cystic spaces showed strong CD20 expression, which was greatest in the GCs (>80%) compared to decreased CD3+ T-cell expression, which varied from <10% to areas showing 10%–50% staining. The CD20 and CD3 immunostaining recapitulated that of the normal distribution of CD20+ B-cells and CD3+ T-cells within the GCs and interfollicular (IF) areas, respectively. The abutment of GCs onto the overlying epithelium resulted in a strong CD20+ B-cell subepithelial presence. Most of the intra-epithelial lymphocytes were of a B-cell lineage (CD20+). B-cell permeation throughout the cyst lining varied greatly from <10% to areas showing >80% positivity [Figure 1]a. Where the lining was thin, i.e., 1–2 cell layers, very few B-cells infiltrated the epithelium as opposed to hyperplastic epithelial areas which showed greater B-cell representation (10%–50%). Up to 45% of these B-lymphocytes were seen free lying within the lumen.
|Figure 1: (a) CD20 expression of B-cells in germinal centers with lymphocyte permeation through cyst lining (L26, ×4); (b) CD3 positivity in interfollicular areas with minimal CD3+ T-cell epithelial permeation (polyclonal, ×4); (c) CD8 staining mimicked CD3 reactivity with a strong CD8+ presence in interfollicular areas (1A5, ×4); (d) CD4 reactivity was not as prominent and intense as CD8+ positivity within lymphoid hyperplasia (4B12, ×10); (e and f) most cases showed a similar CD8:CD4 ratio; CD8 positivity (1A5, ×40) overshadowing CD4 positivity (4B12, ×40); (g and h) p24 positivity in follicular dendritic cells in germinal centers of lymph node in cystic lymphoid hyperplasia (Kal-1, ×40)|
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CD3+ T-cell permeation within the cyst lining was noted in only about 10%, and this was especially in areas of hyperplastic epithelium. Prominent CD3 expression was noted in the IF areas (>80%) with CD20+ B-cell expression ranging between <10% and 50%–80%. The parotid gland parenchyma showed increased CD20+ B-cell periductal proliferation with scattered B-cells permeating the gland parenchyma interstitially. CD3+ T-cell periductal proliferation was low, and there was <10% T-cell permeation into adjacent salivary gland parenchyma [Figure 1]b.
CD8 and CD4
There was low to moderate CD8+ T-lymphocyte expression in the GCs (<10% to about 10%–50%); however, the staining intensity was more intense than the CD3+ and CD4+ T-lymphocyte expression (<10%) in the same area. In some cases, CD4 expression was greater within the GCs (50%–80%), with no clear demarcation between GC and IF areas. CD8 and CD4 reactivity was higher in the IF areas (>80%), with CD8 staining being more intense [Figure 1]c and [Figure 1]d.
CD8+ T-cell permeation of the cyst lining was <10%, with none of the free-lying cells within the lumen demonstrating CD8 positivity. The lining epithelium showed 10%–50% positivity with CD4 in some cases. CD8 and CD4 positivity in the subepithelial areas of the cyst wall was not as marked (50%–80%) as in the IF areas, and not as striking as the CD3 and CD20 positivity. CD8+ cell permeation into adjacent gland parenchyma was much higher than that of CD4+ cell permeation; however, CD4+ staining around entrapped salivary elements within lymph nodes was much stronger than that of CD8 positivity. CD4+ T-cells did not permeate the epithelium of smaller entrapped salivary gland ductules. CD8+ cells, however, permeated the ductules with a strong periductal band of CD8+ cells with infiltration into salivary gland interstitium. A similar CD8:CD4 ratio was noted in most cases, with CD8 positivity overshadowing CD4 positivity only isolated cases [Figure 1]e and [Figure 1]f.
All 25 cases (100%) stained positive with p24, the HIV antibody (Dako Glostrup, Denmark). p24 positivity was noted in follicular dendritic reticulum cells (FDRCs) in the GCs [Figure 1]g and [Figure 1]h. None of the Warthin–Finkeldey type multinucleated giant cells showed positive p24 immunostaining. All BLECs in the cohort of HIV-negative patients were negative for p24.
| Discussion|| |
Several studies have used IHC as an aid to define the reactive nature of CLH.,, Poletti et al. showed an abnormal presence of suppressor T-cells (Leu-2a-positive) and reduced numbers of helper T-cells (Leu-3a-positive) within Leu-14-positive follicular hyperplastic centers within the lymphoid proliferation. Anti-S-100 and DRC-1 antibodies highlighted FDRCs in these areas.
IHC findings in this study recapitulate that of previous reports and of that seen in HIV-induced lymphadenopathy, with intense CD20 positivity of B-cells within GCs and hyperplastic lymphoid follicles around cystic spaces and moderate numbers of CD3-positive T-cells within IF areas of the lymphoid proliferation.,, Like previous reports,, our study showed most intraepithelial lymphocytes within the cyst lining to be of B-cell lineage (CD20+) with only a few interspersed CD3+ T-lymphocytes, a finding corroborated by Kojima et al. in their series of three cases of HIV-unrelated parotid BLECs. The increased intraepithelial B-lymphocyte expression within the cyst lining was proportional to the degree of epithelial hyperplasia and is a feature consistent with both HIV ,,, and non-HIV associated CLH.
Although CD4+ expression was greater (50%–80%) in the GCs in some cases, CD8+ expression overshadowed that of CD4+, and our findings thus concur with that of Maiorano et al. who noted an increase in IF CD8+ suppressor cells over CD4+ T helper cells. Most instances in our series revealed a ratio of 1:1 for CD4:CD8 expression except in a few cases with decreased CD4 expression and increased CD8 expression within IF areas, a finding that was anticipated and consistent with previous findings.,, CD4+ cells showed increased proliferation around entrapped ducts without permeation, compared to CD8+ cells which readily permeated ducts with a strong band of CD8+ cells surrounding the duct.
Very few studies describe the presence of CD4+ T helper cells and CD8+ T suppressor cells in parotid lymphoepithelial lesions., Kreisel et al. showed depletion of CD4+ T-lymphocytes and increased numbers of CD8+ T-cells in HIV-associated lesions. The HIV-related cases in their series demonstrated a reduced CD4:CD8 ratio of <1, however, the ratio was normal in HIV-unrelated BLECs. In a case of long-standing HIV infection with asynchronous bilateral CLH 8 years apart, more pronounced depletion in IF CD4+ T-cells was noted in the later lesion, suggestive of disease progression.
The CD8+ T-cell infiltration of the salivary gland is very similar to that of diffuse infiltrative lymphocytosis syndrome (DILS). CD8+ T-cell salivary gland infiltration in DILS is related to persistent circulating CD8 lymphocytosis and diffuse CD8 lymphocytic tissue infiltration which causes generalized lymphadenopathy and parotid enlargement. This is an unusual host response to HIV, related to slower progression of HIV infection, probably due to a delay in depletion of CD4 T-cells and opportunistic infections.
Even though DILS is a late-stage manifestation of HIV infection, patients progress more slowly to clinical AIDS suggesting a favorable prognosis. Patients with DILS have a longer duration of disease compared to those without DILS and exhibit less advanced HIV disease stage. Blood studies typically reveal a CD4:CD8 ratio of 1:4. The favorable prognosis seen in DILS may be a result of transient expansion of the CD8+ T-cells in both tissues and peripheral blood in early phases of HIV infection. A similar development has not been consistently shown in CLH.,
Whether this applies to HIV-positive CLH is unclear, but the idea that CLH is a predecessor to DILS is questionable. CLH is an early manifestation of HIV infection, and even though isolated case reports reveal patients with elevated CD8+ T-cell counts, in the presence of depleting CD4+ T-cell counts, visceral CD8+ lymphocytic infiltration is not always present.,, It is currently not evident if CLH is a prerequisite for the development of DILS or whether all CLH patients will progress to DILS. As such, CLH and DILS are separate entities until further research proves otherwise.
HIV salivary enlargement raises concern as to whether this is an early indicator of HIV infection or late-stage manifestation of AIDS-related complex. Sperling et al. showed CLH in early HIV infection when CD4+ T-lymphocyte counts are high. In contrast, later studies showed a higher incidence with depleting CD4+ counts.,, Clinicians believe that CLH occurs in states of altered immunity with diminished CD4+ T-cell counts and elevated CD8+ T-cell counts. Schiødt et al. showed CD4:CD8 counts of 280:1138 and 225:900 cells/mm 3 at initial and follow-up examinations, respectively, a ratio of roughly 1:4. Whether parotid enlargement is an early manifestation of HIV infection or a prodrome to AIDS, occurring with later widespread dissemination and depleted CD4+ T-cell counts is unclear.
Our study did not IHC analyze the cyst lining. To enhance the definition of epithelial and lymphoid components seen in AIDS-related CLH, Poletti et al. confirmed positivity with cytokeratin and epithelial membrane antigen (EMA) and negativity with carcinoembryonic antigen within the tonsil-like squamous cyst epithelium. The squamous cyst epithelium was predominantly keratin-positive, and while normal squamous epithelium does not usually show EMA immunoreactivity, stronger EMA reactivity was noted within the cyst wall when compared to salivary gland epithelium. Furthermore, the squamous epithelium contained a proper set of accessory cells of cell-mediated immune response lineage. Leukocyte common antigen positivity revealed intraepithelial lymphocytes and intraepithelial macrophages which were also positive for α1-antichymotrypsin and vimentin. OKT6 and anti-S100 protein antibodies disclosed scattered intraepithelial Langhans cells.
The HIV p24 antibody (Dako, Glostrup, Denmark) used in this study identifies part of the gag protein, a core component of the HIV-1 viral particle. Several attempts have tried to elucidate the role of HIV infection in the development of parotid BLECs with this antibody.,, Uccini et al. showed the cystic fluid component of LECs in HIV-positive patients to be a viral reservoir for HIV-1. Even though all our cases showed positivity of follicular dendritic cells for HIV-1 p24 antigen, our previous report showed 93.9% positivity in 164 cases of CLH. Although not absolute, in most cases, the positive p24 is indicative of a strong association between HIV and CLH and probable progression of the disease process.
IHC as an accessory tool has failed to clarify the pathogenesis of CLH as to whether it originates within intraparotid lymph nodes or occurs as secondary lymphocytic infiltration of the parotid gland following generalized lymphadenopathy. d'Agay et al. claimed that human mucosal lymphocyte (HML)-positive lymphocytes indicated the primary involvement of epithelial structures in CLH with lymphoid hyperplasia occurring secondarily. HML antibody is specific for a membrane molecule on HMLs and stains most intraepithelial and lamina propria lymphocytes in different mucosae but seldom stains lymphoid organs. However, Maiorano et al. showed that LECs have similar IHC in both HIV-positive and HIV-negative patients with simultaneous involvement of intrasalivary lymph nodes and salivary gland parenchyma. There is as yet no clear consensus; however, use of IHC provides a mechanism of assessing tissue involvement in these lesions.
| Conclusion|| |
This descriptive immunohistochemical study elucidates an understanding of the pathogenesis of CLH. CLH cases occurred within peri- and intra-parotid lymph nodes, in confirmed HIV-positive patients with 100% specificity with the HIV p24 antibody corroborating the strong association of CLH with HIV and AIDS. The CD4:CD8 ratio was ~1:1; however, the increased CD8 expression within IF areas may indicate possible HIV-related CLH as compared to other cystic parotid lesions.
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Department of Oral Pathology, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, WITS 2050
Source of Support: None, Conflict of Interest: None