Standard indications for vulvar biopsy include diagnostic uncertainty, failed empiric treatment, and exclusion of neoplasia.1–3 Some authors advise a low threshold for biopsy to mitigate the problem of unsuspected dermatologic disease, which has been reported at 50% for mycosis and 60% for chronic vulvar pain.4–6 There is scant research on the reverse situation—nonconfirmatory histopathology in women suspected to have dermatologic disease.
In particular, the problem of a 30% nondiagnostic biopsy rate in clinically diagnosed lichen planus (LP) raises the possibilities of overidentification by clinicians and underreporting by pathologists.7–9 Vulvodynia, mycosis, and dermatitis present similarly to LP with painful erythema but have nonspecific histopathology. Thus, LP may not be the correct diagnosis in some women with a clinical suspicion unsupported by biopsy, a problem potentially compounded by the high placebo-response rate for vulvodynia.10,11 In contrast, some suspected LP cases with negative histopathology may represent true disease; biopsy may be nondiagnostic if performed at the wrong site or in the setting of waning or treated disease. A lack of consensus histopathologic criteria for vulvar LP may contribute to false-negative results.7,12–14 Clinicopathologic discordance presents a conundrum to the involved specialists and women who undergo an uncomfortable genital procedure without the benefit of a tissue diagnosis.
Several recent publications have highlighted site-specific challenges of histopathologic assessment of the inner vulva, including the presence of mucosa-associated lymphoid tissue (MALT), and epithelial regeneration as a manifestation of erosive LP.13–15 Using this information, we aim to review the histopathologic findings and clinical trajectory of women with a suspicion of dermatologic disease and a nondiagnostic vulvar biopsy.
MATERIALS AND METHODS
The local pathology database was searched for consecutive nondiagnostic biopsies of the inner vulva obtained between 2011 and 2015. Inner vulva was defined as medial labia minora, posterior fourchette, fossa navicularis, and vestibule. Reports considered that nondiagnostic included normal, nonspecific lymphocytic infiltrate, vestibular gland, maceration, old hemorrhage, and fibrosis. Clinical notes were reviewed to assess that both symptoms and an examination abnormality were present. Exclusion criteria were age less than 18 years, vestibulectomy and labiaplasty specimens, and biopsies containing hair-bearing skin.
Data were collected on age, clinician specialty, initial impression, examination findings, other vulvar dermatoses and biopsy results, duration of follow-up, treatment, and response. For women with at least one follow-up visit, clinicians provided a final diagnosis and these responses were categorized into LP, vulvodynia, or others. The vulvodynia category incorporated cases attributed to “associated factors” including neuropathic or referred pain, pelvic floor dysfunction or hypertonicity, and psychosexual problems.16 The Hunter New England Research Ethics and Governance unit approved this retrospective histopathologic case series (HREC 15/11/18/5.02), and signed written consent was obtained for use of the clinical photograph.
Histopathologic review was performed of slides stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). The pathologist (J.S.) was blinded to clinical impression and outcome. Biopsy site was recorded as hairless skin, mucocutaneous junction (MCJ), or squamous mucosa.15 The epithelium was evaluated for spongiosis, noted as present or absent. Epithelial thickness was measured at the thinnest site. Both the stromal lymphocytic infiltrate and exocytosis (presence of leukocytes in the epithelium) were assessed semiquantitatively as absent, sparse, moderate, or dense; other cell types were also recoded. Stromal sclerosis or fibrosis was reported as present or absent. The presence of spores or hyphae was sought on PAS-stained slides.
The epithelial basal layer was scrutinized for vacuolar change, apoptotic bodies, and squamatization. Perilymphocytic clearing sometimes seen with exocytosis was attempted to be distinguished from hydropic change within basal cells. Squamatization was defined as a change in morphology of normal basilar keratinocytes to horizontally disposed cells with a mature squamous appearance.17 Erosive LP required a diffuse change of absent rete ridges, epithelial thinning, a closely applied lymphocytic infiltrate, and either degenerative or regenerative basal layer change.13,14,18 A diagnosis of lichen sclerosus (LS) required basal layer changes, sclerosis in the upper dermis, and an underlying band-like lymphocytic infiltrate.18 Cases with a closely applied stromal lymphocytic infiltrate and basal layer damage that lacked the other diagnostic features of LP or LS were labeled nonspecific lichenoid reaction (see Figure 1).
In the setting of a normal basal layer, other dermatologic diagnoses were sought. Criteria for acute dermatitis included spongiosis and acanthosis, sometimes accompanied by epithelial and stromal eosinophils. Lichen simplex chronicus was diagnosed by orthokeratosis, hypergranulosis, acanthosis with broadening of the rete ridges, fibrosis of the papillary dermis, and absence of dermatophytes or other dermatosis.19 Plasma cell vulvitis required epithelial thinning, spongiosis, exocytosis, a moderate or dense infiltrate rich in plasma cells, and stromal hemosiderin.20–22 Stromal sclerosis with absent or sparse lymphocytes and a normal or squamatized epithelium yielded a diagnosis of vestibulovaginal sclerosis.17,23
Cases that did not meet dermatopathologic criteria for disease were categorized as vestibular gland, scar, and nondiagnostic with or without lymphocytic infiltrate. A minor vestibular gland appeared as a crypt of mucinous epithelium arising from squamous mucosa with periglandular mixed infiltrate (see Figure 2).24 Absent leukocytes, normal or squamatized epithelium, and stromal fibrosis were categorized as scar (see Figure 3). Cases categorized as “nondiagnostic with lymphocytic infiltrate” had moderate to dense stromal and/or epithelial lymphocytes (see Figure 4).
Cases were then stratified by biopsy site and by final clinical diagnoses of LP, vulvodynia, and others. Descriptive statistics were performed; clinical and histopathologic characteristics were compared with Fisher exact test.
There were 85 cases of nondiagnostic biopsy obtained from the inner vulva of symptomatic women; during this time, approximately 830 biopsies were submitted from this anatomic zone. Mean age was 53 years, with 58% of women older than 50 years. The pathology request form contained a provisional diagnosis of LP, LS, or plasma cell vulvitis in 49%; in the remainder, the clinician wished to exclude dermatologic disease. Clinicians requested exclusion of LP in 31 (72%), of LS in 8 (19%), and in 4 (9%) noted a symptom (see Table 1). Seventy women (82%) had at least one follow-up visit and a final clinical diagnosis: LP in 27 (38%), some other vulvovaginal condition in 28 (40%), and vulvodynia in 15 (21%). The “other” category included diagnoses of LS (8), plasma cell vulvitis (5), psoriasis (4), dermatitis (4), candidosis (3), estrogen deficiency (3), and aphthosis (1).
Erythema was the examination abnormality in 76%; other descriptors included white, orange, mottled, macerated, shiny, lacy, induration, and atrophy. At time of presentation to the specialist, 64% used a medication that affects vulvar assessment, most commonly topical corticosteroids, hormonal preparations, and neuromodulators, but also three each on antibiotics and antifungals. Topical corticosteroids preparations used at time of biopsy were high potency in 57%, medium in 21%, low in 7%, and unspecified in 14%. Two women were taking oral methotrexate, one for systemic lupus erythematosus and the other for presumed vulvovaginal LP; none were on other topical or systemic immunosuppressive medication. Women with a final diagnosis of vulvodynia were more likely to be on a neuromodulator at the intake visit [4/15 (27%) vs 2/70 (3%), p < .008] (see Figure 5).
Of 27 women who returned after an initial impression of LP, specialists maintained the diagnosis in 23 (85%). Four women with a low suspicion of LP were later assigned that diagnosis based on their clinical course. Lichen planus cases were more likely to be older than 50 years (p = .02), demonstrated vulvar architectural change (p = .001), and improved with steroid monotherapy (p = .003). Previous or subsequent biopsies demonstrated vulvar LP in 3 (11%) of 27, a lichenoid reaction on hairless skin in 2 (7%), LS on hair-bearing skin in 2, and vestibulovaginal sclerosis in 1 (4%).
At the time of histopathologic review, 9 biopsies (11%) were interpreted as nonspecific lichenoid reaction and 2 (2%) as LS; 7 (64%) of these had basilar apoptotic bodies. Although none met criteria for LP, there was concordance with a clinical diagnosis of LP in 6 (22%) of 27 (see Table 2). An assessment of nonspecific lichenoid reaction or LS was more common in women with clinical LP than in the other diagnostic categories [7/27 (26%) vs 4/58 (7%), p = .03]. One case of clinically diagnosed plasma cell vulvitis showed typical stromal features of hemosiderin and plasma cells but lacked spongiosis. No case showed spores or hyphae on PAS.
Histopathologic review yielded several findings of unknown significance. Four cases showed focal basal layer vacuolar change or squamatization occurring for an area of 30 or fewer cells (see Figure 6). Eighteen cases showed marked perilymphocytic haloes; 3 of these had a neutrophil predominance (see Figure 7). In some of these, it was difficult to know whether the basilar clearing was entirely related to exocytosis and spongiosis or whether there also were hydropic keratinocytes. Suprabasilar apoptotic bodies were seen in two cases with a normal basal layer (see Figure 7).
Table 3 displays clinical and histopathologic characteristics grouped by type of epithelium. Biopsy of hairless skin was more likely from a circumferential abnormality [18/23 (78%) vs 16/62 (26%), p < .001], whereas the lesion was more likely to be focal when squamous mucosa was sampled [20/32 (62.5%) vs 16/53 (30%), p = .025]. Biopsies from MCJ were more likely to be associated with Candida albicans on culture [8/30 (27%) vs 3/55 (5%), p = .014] and to be nondiagnostic with lymphocytic infiltrate [14/30 (47%) vs 9/55 (16%), p = .005]. On histopathologic review, biopsies of hairless skin were more likely to be categorized as nondiagnostic without lymphocytic infiltrate [17/23 (74%) vs 23/62 (37%), p = .003], whereas squamous mucosa specimens were more likely to have some other diagnosis—vestibular gland in 3, vestibulovaginal sclerosis in 2, and 1 each with dermatitis, plasma cell vulvitis, and scar [8/32 (25%) vs 3/53 (6%), p = .02]. These three cases of vestibular gland were all described as multifocal or localized erythema with an initial impression of plasma cell vulvitis.
In this study of nondiagnostic biopsies of inner vulva from symptomatic women, initial clinicopathologic discordance was only partially corrected by careful clinical and histopathologic review. Although clinicians modified the diagnosis to vulvodynia in a few cases after negative biopsy and follow-up, usually they retained a diagnosis of chronic dermatosis, particularly LP, which was inconsistent with the pathology. It is possible that some women thought to have LP instead to have vulvodynia; clinical improvement due to placebo effect removes an early imperative for clinicians to modify their diagnoses. During the course of follow-up, additional treatments may be provided for presumed comorbid pain conditions, or symptoms may remit in keeping with the natural history of vulvodynia.25
However, 18% of clinical LP cases had previous or subsequent supportive histopathology, so clinicians should not reflexively dismiss a dermatologic diagnosis after a single negative biopsy. A potential explanation for nondiagnostic results is suboptimal site or timing of biopsy. There is scant literature regarding when and where to biopsy, beyond the traditional guidance to choose the lesion periphery or worst area. This study suggests several low-yield situations. Multifocal erythema near the hymenal base likely represents vestibular gland openings or vulvodynia.11,26 Diffuse erythema around the MCJ is associated with candidosis; women with risk factors for mycosis may benefit from microbiologic assessment and treatment before biopsy.4,27 The MCJ is also where lymphocytic infiltrates potentially representing MALT are commonly found; when the abnormality is diffuse, hairless skin may yield a specimen that is easier to interpret. When vulvar architecture is normal, biopsy of bland poorly demarcated erythema is unlikely to yield a diagnosis, particularly if the woman has comorbid musculoskeletal or neuropathic pain conditions.
From a pathological perspective, this study highlights a major problem in vulvar histopathology—the definition of normal. Fundamental knowledge gaps are exacerbated by the use of imprecise terminology. “Inflammation” is commonly used to describe the presence of stromal lymphocytes but actually refers to the pathologic process of leukocytes causing tissue damage. Stromal lymphocytes without evidence of epithelial injury may represent MALT or may be the infiltrate associated with dermatosis or mycosis.26,28 Several studies have documented a moderate infiltrate in absence of dermatologic disease, at rates that vary by specimen type: 15% of perilesional specimens of hairless skin and squamous mucosa, 71% of vestibulectomies, and 73% of biopsies from the Bartholin gland opening of asymptomatic controls.15,24,26 Features of local immune activation, such as germinal centers, antigen-presenting dendritic cells, macrophages, and mast cells, have been documented both in controls and vulvodynia cases.29,30 Currently, there is no reliable assay for lymphocytic activity that distinguishes normal immune function from a disease process. Thus, without histopathologic evidence of tissue damage, pathologists cannot reliably distinguish between inflammation and MALT.
Specimens with moderate lymphocytic infiltrate pose substantial challenges for pathologists. Exocytosis with perilymphocytic clearing can mimic or mask vacuolar change, especially when combined with spongiosis. A nearby vestibular gland may be the source of an infiltrate, but the crypt may not be contained within the specimen. Stromal capillaritis and hemosiderin accompanied by plentiful plasma cells likely represent plasma cell vulvitis, but pathologists may not assign this diagnosis in biopsies lacking epithelial changes. As a result of this complexity, histopathologic review sometimes yields a different interpretation of findings, especially when combined with the clinical photograph and context. We constructed an algorithm to assist with clinicopathologic assessment of these challenging cases (see Figure 8).
The limitations of this study are those inherent to the retrospective design, including incomplete clinical data and practice differences among clinicians. Some clinicians may be more likely than others to modify their initial impression after a nondiagnostic biopsy, but we were unable to ascertain any statistical differences because of the large number of referring providers. A comprehensive examination of the mouth and extragenital skin was not always documented. Forty-four percent of cases had no microbiological assessment, yet when performed, 31% had a pertinent positive result. Wet mounts are not routinely performed by many Australian dermatologists and gynecologists. There was substantial variation in the information written on pathology requisition forms. To facilitate clinicopathologic correlation, notes should provide a provisional diagnosis and competing differential diagnoses. Location should be described unambiguously, for example, “right inferior inner labium minus,” rather than with a clock-face position or general terms such as introitus and vulva. Clinical photographs were not obtained or available in all cases and would have permitted a more nuanced description of anatomic location and clinical findings. Finally, our results and algorithm are most applicable to clinicians with access to a skilled vulvar pathologist.
There is broad scope for research in this area. Study of the variation in clinical appearance and histology of the inner vulva is urgently required to improve our understanding of the relationship between pain, erythema, and MALT. This may assist clinicians in distinguishing the erythema of chronic dermatoses from that due to neurogenic inflammation or from the physiologic erythema of some fair-skinned women and perhaps help them avoid unnecessary biopsy. It is possible that the histopathologic phenomenon of exocytosis with focal basal layer degeneration is a manifestation of MALT. There is scant literature to validate the description of plasma cell vulvitis in dermatopathology textbooks.20–22 More information about the clinical course of women with presumed LP and nondiagnostic biopsy would be best obtained in prospective trials that require histopathology for inclusion and use this information to stratify response to interventions.7,13
In summary, nondiagnostic biopsies from the inner vulva should prompt thoughtful multidisciplinary review, but more research is required to resolve the problem of clinicopathologic discordance through better understanding of vulvar histology and pathophysiology.
The authors thank Dr Jenny Bradford and Dr Judith Fleming for their assistance with access to clinical records.
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Keywords:Copyright © 2018 by the American Society for Colposcopy and Cervical Pathology
vulva; nondiagnostic biopsy; lichen planus; vulvodynia; candidosis