False-Negative Histopathologic Diagnosis of Prostatic Adenocarcinoma

There are myriads of tissue artifacts that can generate difficulty in the interpretation of tissue sections from prostate needle core biopsies, potentially resulting in the inability to recognize and diagnose PAC. These artifacts can be the result of the biopsy procedure, the handling of the needle core tissue, and the fixation, processing, embedding, sectioning, and staining.⁷  Here we focus on crush and edge artifacts, thick sectioning, and overstaining. Frozen section and cautery artifacts are also addressed.

Crush artifact is the result of mechanical tissue compression and distortion by an outside force and most commonly occurs during processing of the tissue, especially if the tissue was manipulated with blunt instruments such as forceps. Microscopically, there is significant alteration of the architecture in crushed tissue, which makes evaluation of the glandular component challenging (Figure 1, A). In cases that should show uniformly shaped, small to medium-sized acinar adenocarcinoma glands, those glands may appear more complex in crushed tissue and often exhibit irregular luminal contours, mimicking those seen in benign prostatic glands. Cytologic and nuclear details may be obscured, and prominent nucleoli are rarely preserved in crushed areas of the tissue. Unfortunately, both tumor cells and inflammatory cells are susceptible to crush artifact, making it difficult, if not impossible, to distinguish between the two on H&E-stained sections with significant crush artifact. Procurement of additional sections (levels) may be attempted in an effort to sample noncrushed tissue, although this is in general a low-yield strategy. Another potential approach would be to apply 34βE12/p63/α-methylacyl coenzyme A racemase (AMACR) cocktail immunohistochemistry, which can highlight the absence of basal cells with the loss of 34βE12 and p63 antibody binding, along with overexpression of AMACR in PAC. This approach has been reported to be helpful in crushed margins on prostatectomy specimens.^8,9  One of these investigations⁸  found that the 34βE12/p63/AMACR cocktail immunostain was largely unaffected by crush artifact, whereas the second article⁹  reported diminished p63 immunoreactivity but retention of 34βE12 and AMACR immunoreactivity in the setting of crush/cautery artifact. Histomorphologic features in addition to confirmatory immunostaining are essential for the establishment of a diagnosis of malignancy,¹  and the results of the 34βE12/p63/AMACR cocktail immunostains should be interpreted within the histologic context of each case. In some cases with crush artifact, a diagnosis of atypia or atypical small acinar proliferation may need to be rendered. Adequate fixation, careful and gentle tissue handling and processing techniques, and optimized tissue-embedding protocols are required to eliminate crush artifact.

Crush artifact can also be found along the edge of needle core tissue because of shearing forces created during the biopsy procedure. Microscopically, a minute focus of PAC at the tissue edge is often fragmented (Figure 1, B) and reveals only a portion of its architecture, with incomplete gland outlines. Tumor cells can be elongated and exhibit indistinct cell borders and smudged chromatin. At higher magnification, nuclear enlargement and irregularity can be evident. A major challenge with PAC at the tissue edge is the missing of a small focus of atypical glands when screening at low magnification. If abnormal and distorted glands are observed at the edge, a search for continuity with better-preserved abnormal glands toward the center of the needle core tissue should be conducted as well as a study of additional sections (Figure 1, C).

Tissue sections that are too thick and/or overstained may cause difficulty in identification of PAC. Prostate needle core biopsy tissue should be cut at 3 to 5 μm for optimal histology. Sections may be too thick because of a uniform increase in thickness, an irregular thickness of the sections, and/or tissue folds. Evaluation for gland architectural disturbances, the presence of basal cells, and nuclear changes may be compromised in such cases (Figure 1, D). Overstaining of tissue sections can hinder the ability to judge nuclear atypia. Additional thin levels from the block should be ordered in these cases. Immunohistochemistry with 34βE12/p63/AMACR may also be helpful (Figure 1, E).

Frozen section artifact is most commonly encountered during intraoperative consultation, when frozen section evaluation is requested by urologists to assess for margin status during a radical prostatectomy procedure.¹⁰  These sections can be associated with significant freezing, crushing, and/or cautery artifact (Figure 1, F and G). Microscopically, there is distortion of both the benign and malignant glands, with shrunken cells and artifactual stromal spaces. On higher magnification, adenocarcinoma glands exhibit disordered architecture and nucleomegaly, but it may not be possible to appreciate nuclear detail, intranuclear detail, and nucleomegaly.

Cautery (thermal injury) artifacts are often generated during transurethral resection of the prostate for benign prostatic hyperplasia. Significant thermal injury, resulting in smudged nuclei with elongation/streaming and complete obliteration of nuclear features (Figure 1, H), may preclude definitive evaluation of the prostate glands on H&E sections. Additional sections (levels) may be obtained to attempt to sample noncauterized tissue. Application of 34βE12/p63/AMACR immunohistochemistry may also be a diagnostic aid (Figure 1, I).⁹ 

Minimal or limited adenocarcinoma of the prostate in needle core tissue is defined as a minute focus of adenocarcinoma less than 1 mm long¹¹  or involving less than 5% of the tissue (Figure 2, A through D).^12,13  A false-negative diagnosis of PAC is most often due to minimal size of the prostate cancer in needle core tissue.^3–5 

One study of 196 needle biopsy cases demonstrated a false-negative rate of 1.1% for the diagnosis of PAC, based on central rereview by urologic pathologists of prostate needle cores initially diagnosed as benign from patients enrolled in a prostate cancer screening program.³  The median number of glands of adenocarcinoma in these false-negative cases was 9, with a range of 1 to 56.³  More than one-half (51%) of the missed cases of adenocarcinoma had few atypical glands (with <10 glands).³  In 4 cases location of the small cancer at the biopsy edge or in a small biopsy fragment were thought to be contributing factors to the failure to diagnose adenocarcinoma.³  Additional reasons for missing small adenocarcinomas included noninvasive architecture (in 49% of cases), adenocarcinoma gland size similar to surrounding benign glands (in 37% of cases), and spacing of adenocarcinoma glands that was similar to surrounding benign glands (in 14% of cases).³  Absence of patterns indicative of invasion,¹³  including crowded glands, small glands intercalating among larger benign glands, and rows of glands extending across the width of the core, can create problems in diagnostic recognition of minimal or limited adenocarcinoma. A few scattered small pale acinar adenocarcinoma glands can be particularly difficult to diagnose as malignant (Figure 2, C and D). Finally, another pitfall is that one-third of the small cancers were deceptively benign-appearing variants, including pseudohyperplastic and foamy gland variants (see section below on deceptively benign histologic variants of prostatic adenocarcinoma).

A false-negative rate of 1.1% for the diagnosis of PAC was also reported in a second study in which 793 prostate needle cores initially diagnosed as benign by standard examination of H&E-stained sections were subsequently stained for AMACR in immunohistochemistry.⁴  All cases that were initially missed had only small foci of PAC (defined as ≤1 mm or <5% of a core) present on routine H&E-stained sections. Most (67%) of these small carcinomas showed foamy/pseudohyperplastic or atrophic features (see section below on deceptively benign histologic variants of prostatic adenocarcinoma). The authors concluded that the use of AMACR immunohistochemistry has the potential to decrease the false-negative rates of malignancy.⁴  There should be a low threshold for use of immunohistochemistry for basal cells and AMACR in supporting the diagnosis of minimal adenocarcinoma, especially when atrophic, foamy, microcystic, and pseudohyperplastic features are present.

A third study also employed AMACR immunohistochemistry to establish a false-negative rate of 3.3% for the diagnosis of PAC for 1003 cases classified as benign on routine H&E slides.⁵  The average length of adenocarcinoma was 0.43 mm (range, 0.2–1.5 mm). The most common reason for missing adenocarcinoma was few malignant glands (<10) combined with the lack of histoarchitectural features because of the presence of the carcinoma at the edge of the core or marked intermingling of the carcinoma glands with benign glands. Additional confounding factors included crush artifact, atrophic or pseudohyperplastic adenocarcinoma, and inflammatory infiltrate in the stroma. Inflammation in prostate needle core tissue is more often associated with benign prostatic glands than it is with glands of adenocarcinoma,¹⁴  and only rarely does inflammation obscure prostatic adenocarcinoma glands leading to a false-negative diagnosis.

The missed small adenocarcinomas in these 3 series were in most cases Gleason grade 3 + 3 for a score of 6 of 10 (grade group 1).^3–5  Of the 174 total false-negative cases 164 (94%) were grade group 1. Missed high-grade cases included 5 cases of 3 + 4 for a score of 7 of 10 (grade group 2), 4 Gleason grade cases of 4 + 4 for a score of 8 of 10 (grade group 4), and one case of 5 + 5 for a score of 10 of 10 (grade group 5). Missing minimal low-grade (grade group 1, Gleason score 6 of 10) adenocarcinoma of the prostate may not necessarily affect clinical outcome, although clinical follow-up data for these patients are limited. In the one study performed in a screening setting, the men with a false-negative diagnosis appeared to be in a curable stage at the time of eventual PAC diagnosis.³  However, establishment of the diagnosis of minimal grade group 1 adenocarcinoma may be a determinant for enrollment into an active surveillance program. Additionally, in the few men with missed high-grade (grade groups 2–5), PAC active therapy may be indicated.

The 2016 World Health Organization classification recognizes 4 deceptively benign-appearing histologic variants of acinar adenocarcinoma of the prostate: atrophic, foamy gland, microcystic, and pseudohyperplastic.¹⁵  These prostatic adenocarcinoma variants can be misdiagnosed as benign conditions, particularly when the foci of adenocarcinoma are small. In one series of false-negative prostate cancer diagnoses in needle biopsy tissue, two-thirds of the missed cancers were deceptively benign variants (foamy, pseudohyperplastic, and atrophic variants).⁴ 

Atrophic pattern adenocarcinoma can be seen in a sporadic setting or after hormonal or radiation therapy. This variant is characterized by cytoplasmic volume loss, similar to benign atrophy.^16–18  Microscopically, an infiltrative or crowded arrangement of glands displaying cytoplasmic volume loss, nucleomegaly, and/or macronucleoli are characteristic of this variant. The glands are typically single, separate, and small, but occasionally cystically dilated glands may also be lined by carcinoma cells that show cytoplasmic volume loss.¹⁹  Nuclear atypia is variable. In some glands of atrophic-pattern adenocarcinoma the nuclei are compressed and without nucleoli, which can lead to failure of diagnostic recognition (Figure 3, A). In most cases there is intermingled usual acinar adenocarcinoma with a moderate amount of cytoplasm, a finding that is an important diagnostic clue. Atrophic-pattern adenocarcinoma glands demonstrate diffuse and complete absence of basal cells (using 34βE12 and p63 antibodies) and overexpression of AMACR by immunohistochemistry (Figure 3, B). However, AMACR is overexpressed in a reduced percentage of cases of atrophic-pattern adenocarcinoma, being positive in about 70% of 23 cases,²⁰  compared with 80% to 100% of usual acinar adenocarcinomas reported in the literature.²¹  So, a negative AMACR immunostain is noninformative in the differential distinction versus benign atrophy.

Atrophic-pattern adenocarcinoma can occasionally be missed, especially when focal, and in cases in which the background benign prostate shows significant atrophic changes. Another problem is the underestimation of the extent of cancer in needle core tissue when atrophic-pattern adenocarcinoma glands are mistaken for benign atrophic glands. At the same time, pure atrophic-pattern adenocarcinoma should be rarely, if ever, diagnosed because the distinction between pure atrophic-pattern adenocarcinoma and benign atrophic glands can be extremely challenging. Benign atrophy can simulate atrophic-pattern adenocarcinoma in needle biopsy tissue with disordered or crowded gland growth, some degree of nuclear atypia, and a cancerlike immunophenotype in some glands.²¹  The basal cell layer can be fragmented in benign atrophy, with some glands lacking basal cells or possessing a partial basal cell lining, compared with complete and diffuse loss in all adenocarcinoma glands. In addition, AMACR overexpression may be observed in benign atrophy, particularly partial atrophy, and so immunoreactivity for that marker should be considered within the context of the histopathologic and basal cell marker findings.

Atrophic-pattern adenocarcinoma is considered Gleason pattern 3, with a score of 6 of 10, and grade group 1. The admixed usual acinar adenocarcinoma is most often also pattern 3, although in the whole gland 13% of cases harbor high-grade pattern 4.¹⁷ 

Foamy gland adenocarcinomas are deceptively benign because of a frequent lack of nuclear atypia.^22–26  This variant can be sporadic but can also be secondary to androgen-deprivation and radiation therapy changes (see below). Microscopically, foamy gland adenocarcinoma is characterized by abundant xanthomatous or foamy cytoplasm with pyknotic nuclei (Figure 3, C).^22–26  Not all foamy gland carcinoma cells have pyknotic nuclei and foamy gland carcinoma is usually combined with nonfoamy usual-acinar adenocarcinoma with nuclear atypia. Architectural growth patterns span the spectrum from single and separate well-formed glands of Gleason pattern 3 (grade group 1) through high-grade patterns 4 and 5, including single cell arrangements. Rare cases of foamy ductal adenocarcinoma have been described.²⁷  Most cases (80% of 81 total) in needle biopsy tissue are Gleason score 6 of 10 (grade group 1), with Gleason grade 3 + 4 for 7 of 10 (grade group 2) being the next most common grade.²⁵  There should be a low threshold for application of basal cell (34βE12/p63) and AMACR immunohistochemistry. Moreover, AMACR is an excellent marker for foamy gland carcinoma, with 95% sensitivity (Figure 3, D).²⁵ 

Foamy gland carcinoma may be missed when the malignant glands are misinterpreted as benign glands with foamy change or foamy macrophages. Underdiagnosis of foamy gland carcinoma can occur when glands with foamy cytoplasm are thought be benign mimickers of carcinoma, such as partial atrophy with foamy changes.²⁸  Foamy gland carcinoma can also be mistaken for histiocytes and prostatic xanthoma.^29,30  To address this differential diagnosis a search for coexisting foamy adenocarcinoma without nuclear pyknosis and coexisting nonfoamy gland adenocarcinoma should be undertaken. An immunohistochemical marker panel can be employed: pan-cytokeratin, prostate-specific antigen, prostatic specific acid phosphatase, and NK3 homeobox 1 (NKX3.1) immunostains are positive in foamy gland adenocarcinoma whereas the macrophage markers CD68 and lysozyme should produce negative results. These markers can aid in confirmation of foamy gland carcinoma when foamy histiocytes are being considered.^31,32  Finally, a diagnosis of minimal foamy gland carcinoma of less than 1 mm is particularly challenging and an alternative diagnosis of atypia (atypical foamy glands) has been given in a few cases.³³ 

Foamy gland carcinoma has a similar prognosis as that of nonfoamy gland adenocarcinoma of the prostate.²⁶ 

Microcystic adenocarcinomas display intermediate-sized dilated glands, which on average are 5 to 10 times the size of usual small acinar adenocarcinoma glands.¹⁹  Microscopically, the tumor cells show nuclear atypia as observed in usual acinar adenocarcinoma, with enlarged nuclei and prominent nucleoli. Occasionally there can be cytoplasmic volume loss with compressed nuclei and dense chromatin. The expansion of the luminal spaces generates a rounded profile, and the luminal cell-lining layer is typically flat (Figure 3, E). Admixed usual small acinar adenocarcinoma is often seen. Identification at lower scanning magnifications of intraluminal crystalloids and blue mucin in dilated glands should raise the possibility of microcystic adenocarcinoma and instigate further evaluation. Immunohistochemical stains show the dilated glands to lack basal cells (34βE12⁻ and p63⁻) and almost all cases (96%; 51 of 53) express AMACR (Figure 3, F).¹⁹ 

The dilated glands of microcystic adenocarcinoma challenge the conventional concept that usual acinar adenocarcinoma with a single open lumen is a small gland malignancy. Although most dilated prostatic glands represent dilated benign glands, such as cystic atrophy, diagnostic awareness of the existence of cystically dilated acinar adenocarcinoma is important. Assessment for adjacent usual small acinar adenocarcinoma and immunohistochemistry for basal cells and AMACR are important diagnostic aids. Microcystic adenocarcinoma is Gleason pattern 3, Gleason score 6 of 10, and grade group 1.

Pseudohyperplastic adenocarcinoma can be mistaken for usual epithelial hyperplasia, complex normal glands, or high-grade prostatic intraepithelial neoplasia.^34–37  Pseudohyperplastic adenocarcinoma is among the most common causes for a false-negative diagnosis on needle core biopsies.^3,4  Microscopically, pseudohyperplastic adenocarcinoma reveals architectural complexity (Figure 3, G) including papillary infoldings, luminal undulations, branching, and in some cases, cystic dilatation. In radical prostatectomy tissue, infiltrative growth pattern can be seen in a few cases,³⁴  but this can be difficult to appreciate in prostate needle core biopsy tissue. Rather, crowding of complex glands should lead to consideration of pseudohyperplastic adenocarcinoma. Usually there is significant nuclear atypia, with rounded nuclei and prominent nucleoli, although there is a report of a few cases with foamy cytoplasmic features associated with the loss of nuclear atypia.³⁸  Features associated with usual acinar adenocarcinoma, such as intraluminal crystalloids, pink amorphous secretions, and blue mucin, can also be seen in a few cases.^34,37  Although these findings are neither sensitive nor specific, their presence should still prompt the pathologist to study the complex glands at high magnification. The mean length of pseudohyperplastic carcinoma in needle biopsy tissue is around 4 mm (range, 1–10 mm),³⁷  although a few cases less than 1 mm have been reported. Three of these cases of minimal pseudohyperplastic adenocarcinoma had been mistaken for hyperplasia, adenosis, and prostatic intraepithelial neoplasia.³⁹ 

The standard diagnostic approach for all deceptively benign-appearing prostatic adenocarcinomas should be used for pseudohyperplastic adenocarcinoma: a search for adjacent usual small acinar adenocarcinoma should be made and immunohistochemistry for basal cells and AMACR should be performed (Figure 3, H). Note that 77% of cases (13 of 17) are AMACR positive.⁴⁰  Although supportive and extremely useful in establishing the diagnosis, AMACR positivity is not required for the diagnosis. Like most deceptively benign-looking prostatic adenocarcinomas (with the exception of some foamy gland carcinomas), the Gleason pattern is 3, Gleason score is 6 of 10, and grade group is 1.

Single cell adenocarcinoma is, by definition, a Gleason pattern 5 and grade group 4 or 5 disease,²  and thus missing these cells on prostate needle core biopsies has potentially severe consequences. Single cell prostatic adenocarcinoma is often admixed with other high-grade patterns.⁴¹  These high-grade PACs are typically extensive in needle core tissue and involve multiple cores,⁴¹  such that Gleason scores 8 to 10 of 10 adenocarcinomas are readily detected in most cases. However, in 5% of 268 cases of Gleason score 8 to 10 of 10 the cancer is less than 1 mm long; it is these cases in particular that may go unnoticed. Microscopically, tumor cells are singly dispersed and show nuclear abnormalities such as enlargement, hyperchromasia, and prominent nuclei (Figure 4, A and B). Some cells can be signet ring-like. When foamy gland carcinoma comprises single cells, diagnostic appreciation of malignancy may be very difficult. Single cell malignancy can be a result of androgen-deprivation therapy or radiotherapy (see below).

Recognition of single cell adenocarcinoma requires careful scrutiny of areas in which there is hypercellularity, with examination at higher magnification. Evaluation for other patterns of high-grade prostatic adenocarcinoma admixed should be initiated. Single cell, linear array, and small sheet arrangements of Gleason pattern 5 (grade group 5) adenocarcinoma should not be misdiagnosed as lymphocytic infiltrates,⁴²  which are very common in prostatic needle core tissue. In cases in which the nuclear features are not diagnostic, immunohistochemistry for pancytokeratin, NKX3.1, prostate-specific antigen, and CD45 can be helpful.

Androgen-deprivation therapy is generally used in the treatment of locally advanced or metastatic prostate cancer, and repeat needle biopsy of the prostate after this type of treatment is uncommon. Ideally, the clinical history of the previous diagnosis of prostatic adenocarcinoma and therapy should be provided with the specimen. However, this information may not be available at the time of histologic examination of prostate needle core tissue. Hormonal treatment should be suspected when needle core tissue has androgen-deprivation therapy changes in benign tissue such as diffuse benign gland atrophy, with a paucity of benign glands and stromal dominance, basal cell prominence, luminal cells with cleared cytoplasm and pyknotic nuclei, immature squamous metaplasia, and urothelial metaplasia.^42,43 

The architectural histopathologic changes in PAC after androgen-deprivation therapy include glands with compressed lumina, single cells, and cords, clusters, chains, and solid sheets.⁴⁴  There can be a decrease in size and density of glands of adenocarcinoma and cytoplasmic volume loss, similar to benign prostatic atrophy.^45,46  At low-power magnification there may be an apparent absence of glands but increased cellularity (Figure 5, A). Cytologically, the malignant cells can have pyknotic nuclei without nucleoli, and vacuolated/foamy cytoplasm (Figure 5, B). These features can be variable and some carcinoma cells that are resistant to androgen deprivation may not show architectural and/or cytologic changes related to the therapy.

Carcinoma cells with androgen-deprivation therapy can simulate macrophages, so immunohistochemical stains for pancytokeratin, NKX3.1, prostate-specific antigen, AMACR, and basal cell markers should be applied to aid in identification of histologically inconspicuous carcinoma cells with treatment effect. One caveat is that AMACR is down-regulated by androgen-deprivation therapy, with only 45% (9 of 20 localized cancers) to 71% (35 of 49) of treated PACs having positive results.^47,48  When treatment effect is present, a Gleason grade is usually not assigned.

The histopathologic appearance of prostatic adenocarcinoma after radiation therapy is variable, from no effect to marked effect (Figure 5, C).^42,44,49  Radiation therapy can cause a decrease in the number of adenocarcinoma glands and can produce changes similar to those seen in androgen-deprivation therapy with haphazardly distributed single cells and carcinoma cells having foamy cytoplasm and pyknotic nuclei. These single cells with marked radiotherapy effect can be deceptively benign appearing and confused with macrophages (Figure 5, C and D). Features that are most helpful in the diagnosis of PAC after radiation therapy include the following: infiltrative growth pattern, perineural invasion, blue mucin secretions, and complex architecture. The single malignant cells with marked radiotherapy effect can be difficult to detect, especially when they are widely scattered, can be deceptively benign appearing, and can be confused with macrophages. Basal cell and AMACR immunostains are of value in this setting, not only in confirmation of the malignant nature of cells and glands with marked radiotherapy effect but also in highlighting p63⁺/high–molecular-weight cytokeratin⁺/AMACR⁻ benign glands with radiotherapy-induced nuclear atypia. Moreover, AMACR expression is not affected by radiotherapy. Marked radiotherapy effect on prostatic adenocarcinomas should be reported because these patients have the same prognosis as patients without carcinoma.⁵⁰  Gleason grade and grade group should be assigned only to those postradiation prostatic adenocarcinomas in which there is no treatment effect.

Failure to diagnose adenocarcinoma of the prostate in sections of prostate tissue may have profound clinical consequences. It is therefore critical to recognize and understand those factors that most commonly constitute the basis for false-negative diagnosis of prostate cancer. These factors include tissue processing and sectioning artifacts; minimal adenocarcinoma of limited size; deceptively benign-appearing variants of acinar adenocarcinoma including atrophic, foamy gland, microcystic, and pseudohyperplastic variants; single cell carcinoma; and treatment effects, particularly hormonal therapy and radiotherapy effects, on prostatic carcinoma. Before rendering a diagnosis of benign prostatic tissue, the diagnostic histopathologist should generate a differential diagnostic checklist with these factors and consider whether the observed histopathologic findings may be due to malignancy simulating benign prostatic tissue.