Cowper Glands Identified in Prostate and Urethral Specimens: A Comprehensive Immunohistochemical Characterization and Potential Diagnostic Pitfall

Background

Named based on their periurethral location near the prostatic apex, bulbourethral glands are perhaps more familiarly known as Cowper glands,^1,2 and function to lubricate the urethra as a component of ejaculate fluid. In surgical pathology, recognition of Cowper glands plays an important role in prostate biopsy assessment as a benign mimicker of prostatic adenocarcinoma. ³ While morphology remains the key feature to accurately recognize Cowper glands from prostatic adenocarcinoma, in some instances the use of immunohistochemistry may serve as an adjunct diagnostic tool. ⁴ In this regard, although potential pitfalls of using immunohistochemistry for PSA and PSAP (which can be positive in Cowper glands^5,6) have been described, novel prostate-specific markers in Cowper glands have either only inspected in murine development models ⁷ or anecdotally alluded to, ⁸ without (to our knowledge) formal analysis in a series of patients. Herein we report our findings of traditional prostatic markers (PSA and PSAP), commonly used “cocktails” in prostate cancer diagnosis (PIN4), as well as novel purportedly prostatic-specific markers (NKX3.1 and PSMA) in the largest reported series of Cowper glands to date with the aim to educate readers on potential diagnostic pitfalls.

Methods and Materials

Five institutions were queried for prostate/urethral tissues (biopsies and/or resection) containing Cowper glands, with diagnoses from all patients confirmed by at least 2 genitourinary pathologists. Confirmatory diagnoses of Cowper glands from all specimens were made from hematoxylin and eosin (H&E) stained sections (dimorphic population of lobulated bland acini with foamy cytoplasm and duct[s], with skeletal muscle fibers ⁵ ). For those specimens that also had immunohistochemistry performed at the initial diagnostic assessment, the stains done (if any) were noted. Clinicopathological features for all patients were recorded.

Immunohistochemical stains were performed on formalin-fixed paraffin-embedded unstained slides cut from the original tissue blocks for PIN4 (clones XML26 + LL002 + BC4A4), NKX3.1 (clone EP356), PSAP (clone PASE/4LJ), PSA (clone ER-PR8), and PSMA (clone EP192) with appropriate controls for all slides. Of note, one paired H&E section was concurrently performed and reviewed at the time the unstained slides were cut to ensure lesional cells were present. Immunohistochemical expression was scored as negative, positive (weak), or positive (strong) for all slides with the following staining patterns considered positive: AMACR = cytoplasmic, p63 = nuclear, high molecular weight keratin = membranous/cytoplasmic, NKX3.1 = nuclear, PSAP = cytoplasmic, PSA = cytoplasmic, and PSMA = cytoplasmic.

Results

Twenty-one unique specimens containing Cowper glands were identified (all men), with an average age of 63 years (range 39-81 years). Four unique samples containing Cowper glands on the original prostate biopsies were excluded from the study, as the Cowper gland focus was no longer present on the attempted immunostaining (confirmed with paired additional H&E section performed). Table 1 depicts clinicopathological features for all 21 patients. Most specimens (15 of 21; 71%) were from prostate biopsies, typically with a clinical history of elevated PSA (11 of 15; 73%). However, 4 of 15 patients (27%) had a known prior history of prostate cancer at the time of biopsy (3 patients by prior prostate biopsy and 1 patient with a clinical history of metastatic prostate cancer but with a negative prior prostate biopsy). In 7 of the 15 (47%) prostate biopsies containing Cowper glands, prostate cancer was diagnosed in the same patient, with 2 containing concomitant prostate cancer (grade group 1 and grade group 3 prostate cancer, respectively) and Cowper glands in the same site (eg, same tissue core). Although all specimens contained a dimorphic population of lobules and ducts, the ducts were much more apparent in the larger resection specimens as compared to the prostate biopsies in which they were typically inconspicuous. For the 15 prostate biopsy samples, there was considerable variation in the specific prostate biopsy sites in which Cowper glands were identified (Table 1). Figures 1 to 3 depict prototypical histology and immunoprofile of study patients.

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Figure 1.

When the prostate biopsy is well-oriented and contains ample lobulated dimorphic acini with numerous admixed ducts, Cowper's gland recognition remains a morphological diagnosis (A: 20×, B: 100×, C: 200×; H&E). However, in some instances where there are Cowper's glands (boxed) and concomitant prostatic adenocarcinoma in the same patient, the admixed ducts may be overlooked by crowded acini with frothy cytoplasm and luminal secretion (reminiscent of mucinous fibroplasia), mimicking foamy gland pattern prostatic adenocarcinoma (D: 20×; E: 200×; H&E). Strong staining for NKX3.1 in Cowper's glands (F: 200×, NKX3.1) is a further potential pitfall for misdiagnosis as prostatic adenocarcinoma. Abbreviation: H&E, hematoxylin and eosin.

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Figure 2.

When Cowper's glands elements in a prostate biopsy are smaller and distorted, the characteristic dimorphic population and admixed ducts may not always be present in a particular level section (boxed duct only present in subset of Cowper clusters; A, C: 100×, H&E). Strong staining for NKX3.1 in Cowper's gland (B, D: 100×; NKX3.1) was observed in 100% of study patients, and in this regard is an important pitfall to recognize to avoid misclassification as foamy gland pattern, atrophic pattern, or post-treatment prostatic adenocarcinoma. Instead, relying on basal markers (high molecular weight keratin/p63) is most useful in distinguishing Cowper glands (retains basal cell markers) from prostatic adenocarcinoma. Abbreviation: H&E, hematoxylin and eosin.

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Figure 3.

Although some (25%) Cowper glands from our study showed weak PSMA positivity, all (100%) showed retained basal cell reactivity by PIN4 stain (A: 200×, H&E; B: 200×, PSMA; C: 200×, PIN4). Similarly, some (57%) Cowper glands from our study showed weak PSAP positivity compared to all (100%) showing diffuse strong positivity for NKX3.1 (D: 200×, H&E; E: 200×, PSAP; F: 200×, NKX3.1). Abbreviation: H&E, hematoxylin and eosin.

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Table 1.

Clinicopathological Features of Incidental Cowper Glands Identified in Prostate/Urethral Specimens.

Patient	Procedure	Age (years)	Clinical presentation	Serum PSA (ng/mL)	Prior PCA/ grade group	Prostate site	Diagnosis for patient	Diagnosis for site	Initial IHC done at diagnosis
1	Prostate biopsy	72	Elevated PSA	80.3	N/A	Left posterior transition zone	GG4	Benign	NKX3.1, PSA, PIN4
2	Prostate biopsy	63	h/o PCA on active surveillance	3.2	Yes (GG1)	Right midline apex	GG2	HGPIN	NKX3.1, PSA, PIN4
3	Prostate biopsy	62	Elevated PSA	5.0	N/A	Right midline apex	GG2	Benign	NKX3.1, PSA, PIN4
4	Prostate biopsy	80	Elevated PSA	4.0	N/A	Right anterior medial	ASAP	Benign	NKX3.1, PSA, PSAP, PIN4
5	Prostate biopsy	76	Nodular prostate	3.0	N/A	Left medial mid	Benign	Benign	NKX3.1, PSA, PSAP, PIN4
6	Prostate biopsy	64	Nodular prostate	3.5	N/A	Right anterior lateral	Benign	Benign	NKX3.1, PSA, PSAP, PIN4
7	Prostate biopsy	64	Elevated PSA	6.0	N/A	Right medial	Benign	Benign	NKX3.1, PSA, PSAP, PIN4
8	Prostate biopsy	81	h/o PCA on active surveillance	7.6	N/A	Left anterior	GG1 (1/14 cores), GG2 (2/14 cores), GG3 (2/14 cores)	GG3	None
9	Prostate biopsy	66	Elevated PSA	2.8	N/A	Left apex/mid anterior	ASAP	ASAP	None
10	Prostate biopsy	65	Elevated PSA	8.3	No (ASAP on one core)	Left anterior apex	GG5 (1/12 cores), GG1 (1/12 cores)	Benign	None
11	Prostate biopsy	68	Elevated PSA	11.6	N/A	Right anterior	GG5 (1/12 cores), GG3 (1/12 cores), GG2 (1/12 cores)	Benign	None
12	Prostate biopsy	60	Elevated PSA	4.4	N/A	Right anterior	GG1 (right sided 5/12 cores)	Benign	None
13	Prostate biopsy	71	Elevated PSA	294.3	No (clinical evidence of metastasis)	Left	Benign	Benign	None
14	Prostate biopsy	60	Elevated PSA	8.5	N/A	Left	Benign	Benign	None
15	Prostate biopsy	67	Elevated PSA, history of PCA	6.3	Yes (per report; grade unknown)	Left medial base/mid	Benign	Benign	None
16	Radical prostatectomy	63	Prior PCA on prostate biopsy	4.2	Yes (GG2)	Apex	GG2, pT3a	N/A	None
17	Urethroplasty, partial prostatectomy	48	Posterior urethral stricture	N/A	N/A	Anterior	Benign	Benign	None
18	Urethral stricture excision	21	Urethral stricture	N/A	N/A	Apex	Benign	Benign	None
19	Cystoprostatectomy	62	Urothelial carcinoma	N/A	N/A	Apex	Urothelial carcinoma	N/A	None
20	Colon resection	39	Rectal adenocarcinoma	N/A	N/A	Apex	Colonic adenocarcinoma	N/A	None
21	Urethra resection	80	Melanoma of urethra	N/A	N/A	Apex	Focal residual melanoma	N/A	None

Abbreviations: ASAP, atypical small acinar proliferation; GG, grade group; HGPIN, high-grade prostatic intraepithelial neoplasm; IHC, immunohistochemistry; N/A, not applicable; PCA, prostatic adenocarcinoma.

At the time of original diagnosis, immunohistochemistry was performed for Cowper gland assessment in 7 of 21 (33%) patients overall; among the prostate biopsies, immunohistochemical staining was performed in 7 of 15 (47%) patients. Table 2 depicts the immunoprofile for Cowper glands from the 21 patients (for PSAP: 7 patient samples either failed/were unavailable for staining; for PSMA: 5 patient samples were unavailable for staining). In all 21 patients (100%), Cowper glands showed strong positive staining for NKX3.1 as well as retained basal cell markers staining (strong positive staining for high molecular weight keratin and p63 immediately surrounding Cowper glands) (Figure 1). Only 1 Cowper gland sample (1 of 21; 5%) showed positive staining (weak) for AMACR. Although PSA stain was negative in all 21 samples containing Cowper gland samples, PSAP was positive in 57% of samples (8 of 14; mostly weak staining) while PSMA was positive in 25% of samples (4 of 16; mostly weak staining).

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Table 2.

Immunoprofile of Incidental Cowper Glands Identified in Prostate/Urethral Specimens.

Discussion

Cowper glands recognition remains one of the key histoanatomic “small gland pattern” benign mimics of prostatic adenocarcinoma, ³ namely Gleason pattern 3 prostatic adenocarcinoma (including foamy gland pattern), atrophic pattern adenocarcinoma, and post-treatment prostatic adenocarcinoma.^4,9 Although in many instances the characteristic dimorphic population of lobulated acini and ducts is sufficient for accurate morphological recognition of Cowper glands,^5,6 often at prostate biopsy core ends where the specimens containing Cowper glands can be distorted, the duct component may not be immediately apparent or require additional level sections for accentuation. In this regard, immunohistochemistry may be incorporated to distinguish Cowper glands from prostatic adenocarcinoma. While pitfalls of positivity with some of the traditional prostate immunostains have been previously reported or done so in an isolated setting (eg, not as the commonly used “cocktail” marker), to our knowledge a comprehensive evaluation of both traditional and purportedly prostate-specific novel markers in Cowper glands has not been previously performed, prompting the current study (the largest Cowper gland collection to date).

Previous studies have explored PSA immunoreactivity in Cowper glands, with an incidence ranging from 0% to 80%.^5,6,10 Similarly, PSAP staining in Cowper gland has been described, ranging from 0% to 27%.^5,10 In our series, although PSA stain was negative in all patients, PSAP was positive in 57% of our Cowper glands study (Table 2), which, in general, supports that traditional prostate marker positivity in Cowper glands is an important pitfall to recognize. Whereas one prior study also investigated PAP stain in Cowper gland (negative in all their tested samples ⁶ ), this marker was not available in our laboratories for testing.

In regard to allegedly prostate-specific novel markers, although NKX3.1 is alluded to as positive in Cowper glands ⁸ (which we entirely concur based on our anecdotal experience), this study is the first to our knowledge to formally describe NKX3.1 positivity (typically strong/diffuse) in a large series of Cowper glands. Cowper glands are known to develop from the membranous section of the pelvic urethra, where they (similar to prostatic glands) are thought to arise from the urogenital sinus. The NKX3.1 gene encodes a homeobox-containing transcription factor and is considered to play an important role in prostatic gland and duct development and proliferation. ¹¹ Based on murine model expression of NKX3.1 stain in both prostatic tissue and Cowper gland and the aforementioned positivity of “traditional” prostate markers PSA/PSAP in Cowper glands, it may not be surprising to see NKX3.1 positivity (typical of most prostate cancers ¹¹ ) in all (100%) our series of clinically performed prostate samples containing Cowper glands. Nonetheless, it remains an important diagnostic pitfall for pathologists to be aware of if using this stain in isolation to distinguish Cowper glands from prostatic adenocarcinoma (notably, employing a PIN4 stain or related “cocktail” marker would demonstrate retained basal cell staining and obviate this “pitfall”). Similarly, although perhaps less “novel” than NKX3.1, PSMA stain is a fairly sensitive and specific marker for prostatic adenocarcinoma ¹² but can be positive in some Cowper glands (25% in our series, the first reported incidence). Interestingly, Bartholin glands in female patients, which are considered to be homologous to the bulbourethral (Cowper) glands in males, have also been shown to express NKX3.1. ¹³

There is significant reported variability in the rate of basal cell positivity around Cowper gland acini for high molecular weight keratin, ranging from 10% to 100%.^5,6 Although we observed 100% basal cell positivity for high molecular weight keratin in our Cowper glands series (Table 2), it is notable that our study is the first (to our knowledge) to employ Cowper glands assessment in the form of a “cocktail” immunostain (PIN4), performed as such to simulate the practice commonly implemented in routine surgical pathology assessment of prostate biopsies.^14–16 Moreover, ours is the first to report that p63 staining mirrors that of high molecular weight keratin, both being uniformly positive (strong) in the basal cells around Cowper glands acini. We note that although typically negative in Cowper glands, AMACR can rarely be positive (weak staining noted in 1 of 21 patients).

The most notable benign mimicker of Cowper glands to be aware of includes mucinous metaplasia. ¹⁷ Although there can be significant morphological overlap of mucinous metaplasia and Cowper glands, appreciating adjacent prostatic acini with only partial mucinous change can be helpful in the former, whereas recognition of an intimately admixed duct can be helpful in the latter. In some instances, the anterolateral or apical location of the prostate biopsy site may be useful in Cowper gland recognition, although as shown in our study, relying on location may not be reliable (Table 1). Similarly, while Cowper glands characteristically contain admixed skeletal muscle fibers, this is not a specific feature and can be seen in both mucinous metaplasia and prostatic adenocarcinoma. As mucinous metaplasia and Cowper glands may share a similar immunoprofile, it remains a morphological and likely academic distinction, with the only important clinical distinction being separation from prostatic adenocarcinoma.

As this was a retrospective study, one limitation of our study is the inability to estimate the incidence of encountering Cowper gland sampling on routine prostate biopsy. Although Cowper glands encountered during prostate needle biopsy have been previously reported in smaller series,^5,6 in one of these studies the incidence of identifying Cowper glands was estimated at 0.006% ⁵ (based on our anecdotal experiences, it may not be all that uncommon, particularly in the modern era of multisite magnetic resonance imaging-guided prostate biopsies). Another limitation of our study is that although some prior reports included SMA staining and/or special stains for mucin in the assessment of Cowper glands,^5,6 we did not evaluate these, focusing our assessment on more routinely employed stains. Moreover, while P501S (prostein) is another historical “prostate-specific” marker generally on par in sensitivity to PSA and showing characteristic clumped granular perinuclear (Golgi pattern) immunoreactivity,^18,19 this marker was not available in our laboratories for testing (notably also recognized as uncommonly utilized by many pathologists ²⁰ ). Nonetheless, given recently observed reactivity in many extra-prostatic tumor types, ²¹ we suspect that Cowper glands would also show P501S positivity, although formal confirmatory assessment would be of interest. Finally, while it could be argued that given the deeper locations of some of our study specimens, these could instead represent mucinous metaplasia and not Cowper glands, it should be mentioned that specimens lacking intimately admixed ducts were explicitly excluded from our study to maintain purity.

In summary, in most instances recognition of the dimorphic population of lobulated bland acini and admixed duct(s) by H&E assessment is sufficient to recognize Cowper glands. However, in some instances, there is a considerable overlap with prostatic adenocarcinoma (namely foamy gland pattern, atrophic pattern, and post-treatment adenocarcinoma), and immunohistochemistry can be helpful. In this regard, caution should be rendered when using prostate-specific markers (PSA, PSAP, PSMA, and NKX3.1), as these can show considerable staining in Cowper glands and be a diagnostic pitfall. Instead, findings from the current study support basal markers (high molecular weight keratin/p63; either individually or in a “cocktail” approach) and PSA to be useful in distinguishing Cowper glands (retains basal cell markers) from prostatic adenocarcinoma.