AMACR was the first prostate cancer tissue biomarker identified. Currently, AMACR is more commonly applied to complement basal cell markers in antibody cocktail formats. The cocktails are now routinely utilized to resolve the diagnosis of “atypical glands” or to confirm the diagnosis of small volume cancer in needle biopsies (Fig. 9.1) [5]. Average sensitivity for the detection of limited prostate carcinoma in needle biopsies is in the range of 70–80 % with lower sensitivity reported in certain morphologic variants including foamy, pseudohyperplastic, and atrophic variants of usual acinar prostate adenocarcinoma [6, 7]. AMACR is expressed in ~ 90 % of irradiated prostate carcinomas; its expression is reduced in hormone-deprived cancers [8].

Positive AMACR staining supports a diagnosis of cancer in morphologically suspicious atypical glands. A diagnosis of cancer should not be reversed if AMACR and basal cell markers are negative and the morphology of the atypical glands is suspicious for carcinoma as a small proportion of prostate cancer lacks AMACR expression.

One of the challenges encountered during biopsy evaluation is the diagnosis of atypical glands suspicious for cancer (ATYP), which typically require immunohistochemistry (IHC) for further diagnostic work up. Two studies have demonstrated that in a subset of ATYP cases, positive AMACR may convert an ATYP to cancer diagnosis where morphology is suspicious for but not diagnostic of cancer and basal cell markers are negative [6, 9]. Caution should be exercised, however, before making a cancer diagnosis based on AMACR positivity alone as AMACR has significant limitations with prostate cancer specificity. The diagnosis of high-grade prostatic intraepithelial neoplasia (HGPIN), partial atrophy, adenosis (atypical adenomatous hyperplasia) and nephrogenic adenoma must be ruled out on morphological grounds before making a diagnosis of limited prostate cancer based on AMACR positivity.

Basal cell markers including high-molecular weight cytokeratin and p63, and prostate cancer marker AMACR (P504S), individually or as a part of PIN-4 cocktail, are currently the most commonly utilized IHC markers in clinical practice [32, 33]. AMACR is preferentially overexpressed in approximately 80 % of prostate cancer detected in prostate biopsies [5, 6, 32, 33]. However, its expression is also found in most cases of HGPIN, in a significant proportion of adenosis, nephrogenic adenoma and partial atrophy, and occasionally even in morphologically benign prostatic glands [6, 10–12]. Therefore, a tumor marker that demonstrates better specificity for prostate cancer and is not expressed in noncancerous lesions may complement basal cell markers and AMACR and will greatly facilitate the identification of limited cancer in prostate biopsies.

Several studies have analyzed the utility of ERG immunostain in the work up of limited prostate cancer and have consistently found high specificity of ERG for prostate cancer detection. The reported frequency of ERG expression in limited cancer ranges from 40 to 60 % [23–25, 27, 29, 34, 35]. ERG expression has been observed in a small proportion of HGPIN or benign glands, invariably associated with adjacent prostate cancer [23–25, 34]. Benign lesions distant from cancerous glands, including simple and partial atrophy, are typically negative for ERG. Overall, ERG has much higher specificity for prostate cancer than AMACR; hence, ERG staining in an atypical focus (where HGPIN or atypical glands adjacent to HGPIN can be excluded) supports a diagnosis of cancer, irrespective of AMACR staining. A representative example of limited prostate cancer stained with ERG antibody is represented in Fig. 9.3.

Only a few studies have examined the significance of ERG in the setting of ATYP [23, 24]. We studied 84 ATYP cases using multiplex ERG/AMACR/high molecular weight cytokeratin/p63 IHC to determine clinical utility of ERG in resolving an ATYP diagnosis [23]. A final diagnosis of benign , ATYP and cancer was rendered following review of morphology and all markers in 3, 30, and 51 cases, respectively. Of 51 cancer diagnoses, 45 and 94 % were positive for ERG and AMACR, respectively. Of 30 atypical diagnoses, 10 and 67 % were positive for ERG and AMACR, respectively. Of three benign diagnoses, none and 83 % were positive for ERG and AMACR, respectively. All three ERG-positive atypical cases were classified as “HGPIN with adjacent ATYP.” ERG was expressed in adjacent noncancer glands of 20 % of prostate cancers, while AMACR was expressed in noncancer glands in all diagnostic categories in 40 % of cases. In ERG-positive ATYP focus, the expression was predominantly uniform within the focus with minimal staining heterogeneity. Overall, ERG has a low sensitivity but high specificity for prostate cancer detection. Therefore, ERG positivity in small atypical glands where the diagnosis of HGPIN is excluded can be utilized to establish a definitive cancer diagnosis in the majority of ATYP cases.

An important clinical question remains: is a positive ERG staining used merely to confirm a malignant diagnosis that could otherwise be established based on routine hematoxylin and eosin (H&E) histology and traditionally utilized PIN-4 cocktail antibodies composed of AMACR and basal cell markers? Alternatively, could a positive ERG staining be used to convert an atypical diagnosis to cancer in cases that otherwise would not be diagnostic of prostate cancer based on histology and traditionally utilized AMACR and basal cell markers? In our experience addressed in the earlier section of utility of ERG in resolving an “ATYP” diagnosis, traditionally utilized AMACR and basal markers were adequate to resolve ATYP diagnosis in the vast majority of cases [23]. However, owing to the high specificity of ERG for prostate cancer, ERG positivity in small atypical glands where the diagnosis of HGPIN was excluded helped establish a definitive cancer diagnosis in small proportion of additional ATYP cases where either morphology and/or traditional markers were not deemed adequate enough to offer a definitive cancer diagnosis. In this series, 12/48 (28 %) atypical diagnoses based on morphology, AMACR, and basal cell markers were changed to cancer after incorporating a positive ERG staining. These cases were morphologically suspicious for cancer and in all cases AMACR was expressed but a definitive diagnosis of cancer could not be rendered due to either quantitatively or qualitatively less than optimal morphology or inconclusive basal cell IHC. A positive AMACR staining was not sufficient in these cases to make a definitive cancer diagnosis as AMACR is also known to be expressed in a significant proportion of benign prostate cancer mimics. In this study, 67 % of lesions which were classified as atypical and 40 % of noncancer glands in all diagnostic categories demonstrated AMACR expression, indicating that AMACR expression is not cancer specific and by itself is not sufficient to convert an atypical diagnosis to cancer. On the contrary, ERG expression in small proportion of benign or HGPIN glands has been predominantly reported in the setting of adjacent cancer, therefore ERG positivity in small atypical glands, where the diagnosis of PINATYP or HGPIN is excluded, is virtually diagnostic of cancer [23, 24]. An example of “ATYP” (diagnosis based on morphology, AMACR, and basal cell markers) due to qualitatively less than optimal morphological features converted to cancer after incorporating positive ERG staining is represented in Fig. 9.4a–c.

Previous FISH-based evaluations of the genomic rearrangements associated with prostate cancer development have consistently revealed that about 20 % of HGPIN lesions in proximity to cancer are also positive for ERG rearrangement with identical ERG gene fusions. Evaluation of ERG oncoprotein expression in whole mount sections using clone 9FY has revealed a strong concordance between focally ERG-positive HGPIN and homogenously ERG-positive prostate cancer in 96.5 % of cases [31]. These findings indicate a clonal relationship between gene fusion-positive HGPIN and cancer and thus potential implications for utilization of ERG as a marker for prostate cancer risk stratification in patients with HGPIN diagnosis. Two studies examined the significance of ERG-positive HGPIN for future cancer risk stratification and came to contrasting conclusions. Gao et al. found that the presence of ERG rearrangement in HGPIN lesions detected on initial biopsy warrants repeat biopsy [36]. He et al., however, did not find the utility of ERG to stratify cancer risk associated with HGPIN [37]. Patients with initial HGPIN in biopsies and at least one follow-up prostate biopsy were included and were immunostained for ERG. The cancer detection rate was not significantly different between ERG-positive and ERG-negative HGPIN cases. The authors concluded that ERG expression is distinctly uncommon in isolated HGPIN (5.3 %) and positive ERG expression is not associated with increased cancer detection in subsequent repeat biopsies [37].

A repeat prostate biopsy after 3–6 months is recommended for patients with ATYP diagnosis due to its high predictive value for cancer detection in repeat biopsy. In a study of follow-up biopsies from 103 patients with a preliminary diagnosis of ATYP, ERG expression was detected in 16 cases (15.5 %). Of these 16 ERG-positive cases, the atypical glands were positive for ERG in nine [34]. Five of these patients (55.6 %) had cancer on repeat biopsies, compared with 42 of the 87 (48.3 %) with ERG-negative preliminary biopsies. The authors concluded that ERG expression is unlikely to help identify patients suitable for subsequent biopsies. Of note, in the study the repeat biopsies were not directed to the ERG-positive ATYP sites. Overall, additional biopsy studies assessing ERG-positive HGPIN and ATYP lesions are needed to evaluate more thoroughly the utility of measuring ERG expression as prostate cancer risk stratification in subsequent biopsies in patients with HGPIN or ATYP.

Atypical cribriform lesions of the prostate gland are defined as cribriform or rarely solid prostate glands populated by cytologically malignant cells with preservation of basal cells. It may represent cribriform HGPIN or intraductal carcinoma of the prostate (IDC-P) [38]. IDC-P is almost always associated with high-grade and high-volume invasive carcinoma. On the other hand, cribriform HGPIN is a putative neoplastic precursor lesion; recent studies have shown that the significance of HGPIN as a predictive marker of cancer has reduced significantly in the range of 25 % [39, 40]. The diagnosis of focal HGPIN, defined by < 2 cores involvement, currently does not mandate a repeat biopsy within the first year of diagnosis [39]. In contrast, prostate cancer with associated IDC-P component has a significantly worse prognosis than cancer without IDC-P [40]. Therefore, the distinction of cribriform PIN from IDC-P is of paramount importance due to its widely differing clinical significance. In a study evaluating ERG gene fusions in a subset of cribriform HGPIN (noncancer-associated atypical cribriform lesions) and IDC-P lesions (cancer-associated atypical cribriform lesions) in totally embedded radical prostatectomy specimens, isolated cribriform HGPIN lesions consistently lacked ERG gene rearrangements, while IDC-P lesions regardless of their morphologic spectrum were highly enriched in these gene fusions [38]. ERG gene rearrangements were observed in up to 75 % of IDC-P. Therefore, all cancer-associated atypical cribriform lesions essentially represent an intraductal spread of prostate cancer, and ERG IHC has potential utility in stratification of an atypical cribriform lesion encountered in prostate biopsy. Essentially all ERG expressing atypical cribriform lesions, especially when associated with adjacent prostate cancer represent examples of IDC-P.

ERG is known to be expressed in endothelial cells, and oncogenic ERG gene fusions occur in subsets of prostatic carcinoma, acute myeloid leukemia, and Ewing sarcoma [41, 42]. In vascular tumors, ERG oncoprotein is expressed in a broad range of tumors including hemangiomas, lymphangiomas, angiosarcomas, epithelioid hemangioendotheliomas, and Kaposi sarcomas. Among nonvascular mesenchymal tumors, Ewing sarcoma and blastic extramedullary myeloid tumors express ERG. Among epithelial tumors, diffuse ERG expression is largely restricted to prostatic adenocarcinomas. Rare other carcinomas and epithelial tumors that may demonstrate focal ERG expression include large cell undifferentiated pulmonary carcinomas, mesotheliomas, thymomas, squamous cell carcinomas of the skin and lung, carcinosarcomas of the uterus, gastrointestinal stromal tumors, hepatocellular carcinomas, teratomas of the testis, anaplastic carcinomas of the thyroid, giant cell tumors of the tendon sheath, and benign fibrous histiocytomas of the skin [43]. Overall, ERG has a very narrow biological role in highly selected tissues and among epithelial tumors in appropriate clinical setting, strong and diffuse ERG expression would essentially support the diagnosis of prostate carcinoma [25]. Similarly, in the setting of small cell carcinoma of unknown origin, ERG positivity would support the prostatic origin of small cell carcinoma [44, 45].