—  SHORT COURSE #13  —

Interpretation of Prostate Needle Biopsies

Case 2, 3A, 3B, 3C - An Approach to the Diagnosis of Limited Cancer with Prudent Utilization of Immunohistochemical Markers to Resolve the Diagnosis (continued)

Rajal Shah and Ming Zhou


Immunohistochemistry for Differential Diagnosis of Prostate Cancer
Immunohistochemistry has been widely used in work-up of difficult prostate biopsies (for review, see [1, 2, 3] ). It is used in two clinical settings. First, to distinguish prostate cancer from benign mimics. Basal cell markers (high molecular weight cytokeratin (HMWCK, CK903), p63) and alpha-metheyacyl-CoA-racemase (AMACR) are used for this purpose. Second, to differentiate poorly differentiated prostate cancer from transitional cell carcinoma, colonic adenocarcinoma and other metastatic carcinoma [30]. Prostate specific markers (prostate specific antigen (PSA) and prostate specific acid phosphatase (ASAP)), P501S (protein), cytokeratins (CK7, CK20, CK903) and p63 are used for this purpose [30].

Prostate basal cell markers
Antibodies to HMWCK are the first, and the most widely used, basal cell markers [2, 3]. HMWCKs constitute the cytoplasmic intermediate filaments, and are detected by several antibody preparations, including 34betaE12 (which includes antibodies against cytokeratins 1, 5, 10 &14), cytokeratins 5/6 and cytokeratin 14. These antibody preparations stain the cytoplasm of prostate basal cells.

P63, a p53 homolog, is a nuclear antigen that is expressed only in the prostate basal cells [4]. Because they target different antigens in basal cells, 34betaE12 and p63 can be used in the same immunostain reaction as basal cell cocktail to improve the detection of prostate basal cells [5, 6]. The use of antibody cocktail is also advantageous over either antibody used alone on prostate biopsies where the focus in question is often small and may be present only on one slide.

The diagnostic utility of basal cell markers rely on the fact that basal cells, therefore basal cell markers, are absent in prostate cancer. Prudent use of these markers has profound impact on the interpretation of prostate biopsies. Basal cell markers can be used to resolve atypical diagnoses and establish definitive cancer diagnosis in biopsies [7, 8]. They are particularly helpful in the diagnosis of pseudohyperplastic, foamy and atrophic prostate cancer, as these morphological variants often have deceptively bland H&E morphology and are difficult to diagnose in biopsy with limited material if basal cell markers are not used.

Several studies have compared the diagnostic utilities, including the sensitivity and specificity, of different basal cell marker preparations. In general, these basal cell markers have similar clinical utility, although the basal cocktail is slightly better than p63, and p63 is superior to HMWCK in diagnostic sensitivity, especially in TURP specimens with significant cautery artifact [5, 9]. The choice of basal cell marker is the matter of the preference and experience of individual laboratories. P63 and cocktail are routinely used in our laboratories.

There are several diagnostic pitfalls one needs to be aware of when using and interpreting the basal cell markers to aid the diagnosis of prostate cancer. Basal cell markers can occasionally be absent in noncancerous lesions for several reasons. HMWCK is susceptible to formalin fixation with progressive loss of immuno-reactivity with prolonged fixation time [10]. There is a centrifugal decrease of HMWCK staining in benign glands, with stronger staining in central and larger glands, and reduced, fragmented or even absent staining in smaller peripheral glands [11]. Benign conditions, including adenosis and partial atrophy, often have patchy and incomplete basal cell staining [12, 13]. Staining may even be absent in some glands. High-grade PIN often demonstrates fragmented basal cell staining [14]. Therefore, when evaluating basal cell immunostains, one has to study the benign glands on the same slide as internal positive control and accept a negative stain as truly "negative" only when the benign glands on the same slide are positive for basal cell markers.

Alpha-methyacyl-CoA-Racemase (AMACR)
AMACR is a tumor marker that is preferentially over-expressed in prostate cancer [15, 16, 17]. It is a key enzyme in the metabolism of branched chain fatty acids and bile acid intermediates. AMACR can be detected immunohistochemically with two different antibodies, a monoclonal antibody (P504S), and a polyclonal one [29]. Currently, the majority of the laboratories use P504S, because this is the antibody commercially available. Nevertheless, the two antibodies are quite comparable in terms of diagnostic sensitivity and specificity [18, 19, 29]. The over-expression of AMACR is not only limited in prostate cancer. It is also expressed in other tumors, including adenocarcinomas of colon, lung, breast, and papillary renal cell carcinoma [20].

A typical positive AMACR staining pattern in prostate cancer cells is apical granular. However, AMACR expression can also be detected by immunohistochemistry in morphologically benign glands, although it is not known whether these morphologically benign glands with AMACR expression have undergone pre-neoplastic transformation. However, it does raise the question as to what should be considered as positive AMACR staining diagnostically. Jiang et al [17] considered any strong and circumferential luminal AMACR staining as positive . We, on the other hand, define positive AMACR staining as the staining that is significantly stronger than that of adjacent benign glands [19]. For example, a prostate cancer with strong and circumferential AMACR staining will be considered negative if the adjacent benign glands have similar strong and circumferential staining. In contrast, a prostate cancer with weak apical staining will be considered positive for AMACR if the adjacent benign glands are completely negative.

The expression of AMACR in prostate cancer is often not uniform. Even within the same lesion, some glands have strong, some have weak, yet others have negative AMACR staining. Some of the morphological variants of prostate cancer, including foamy gland, pseudohyperplastic, and atrophic cancer, are less often to be positive and have weaker staining in cancer glands [19]. Overall AMACR is positive in approximately 80% of prostate cancer in needle biopsies [21]. Therefore, a positive staining supports a cancer diagnosis and a negative staining in suspicious focus may not exclude a cancer diagnosis.

Expression of AMACR is also found in a variety of other non-cancerous lesions, including up to 26% of benign glands, 15-27% of adenosis [18, 22], 3% of partial atrophy [28], and 35-58% of nephrogenic adenoma [23, 24], and majority of high-grade PIN [25]. The AMACR expression in these lesions is in general weak to moderate. However, it reinforces the notion that positive AMACR does not equate to prostate cancer.

Recently, several antibody cocktails have been formulated for the diagnosis of prostate cancer in biopsy. The diagnostic utility of basal cell cocktail has been discussed [5, 6]. AMACR and basal cell markers could be combined in single immunostaining reaction [26, 27]. This cocktail can simultaneously stain both benign and cancer glands. Benign glands are positive for basal cell marker and negative for AMACR, and cancer glands are positive for AMACR and negative for basal cell marker. This cocktail has several advantages over individual antibodies used alone. The biggest advantage is the conservation of tissue. For limited cancer in needle biopsies, the diagnostic material might be present only in one section; therefore, not enough for two separate immuno stains. In addition, it is more economical to combine two immunostaining reactions in one. The disadvantage is that the commercial antibody cocktail is pre-diluted, and the laboratories could not titrate the antibodies according to their own protocols.

It is important to know that immunohistochemical tests for prostate cancer should not be used as a screening test. Rather, it should be applied to selected cases whose differential diagnosis includes prostate cancer based on H&E examination. Interpretation of immunostaining should be in the context of H&E morphology. On H&E slide one should clearly define which glands are benign, and which glands are atypical. And for atypical glands, are they suspicious for cancer or are they more likely to represent benign mimickers of prostate cancer? Immunohistochemistry then can be performed to support or verify one's H&E impression. A diagnosis should never be established exclusively based on immunohistochemistry.

References:
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