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Immunohistochemical Evaluation of Prostate Needle Biopsies Using Saved Interval Sections vs New Recut Sections From the Block
A Prospective Comparison

Omar Hameed MBChB, Peter A. Humphrey MD, PhD
DOI: http://dx.doi.org/10.1309/AJCPNYKDOVQLSNR8 683-688 First published online: 1 May 2009

Abstract

The best method of tissue sampling for performance of immunohistochemistry on prostate needle biopsy tissue sections has not been established. The aim of this study was to prospectively compare the usefulness of using saved interval sections vs new recut sections from the paraffin block in the immunohistochemical evaluation of prostate needle biopsy (PNB) material. Seventy (10.3%) of 682 PNB specimens consecutively examined over a 1-year period were evaluated immunohistochemically. Of these cases, 38 were concurrently evaluated using saved interval sections (2 levels) and recut sections. Identical staining results were achieved in 21 cases (55%). Loss of the atypical/ malignant focus of interest was seen in 3 (8%) of the interval sections vs 19 (50%) of the recut sections (P < .0001). The mean linear extent of the foci that were lost in the recut sections (0.75 mm) was significantly less than that of the foci that were not lost in the recuts (1.32 mm; P = .025). Fifteen (79%) of the 19 foci that were lost in the recut sections were 1 mm or less in length vs 7 (37%) of the 19 not lost in the recuts (P = .021). These findings support the practice of cutting and saving 2 interval levels of unstained sections of PNB specimens for potential immunohistochemical evaluation.

Key Words:
  • Prostate
  • Carcinoma
  • Immunohistochemistry
  • α-Methylacyl-CoA racemase
  • AMACR
  • p63
  • Interval sections

Most prostate needle biopsy specimens are diagnosed as benign or malignant based solely on examination of routine H&E-stained sections. Not infrequently though, foci of minimal (limited; <1 mm)1 and/or unusual variants of prostatic adenocarcinoma (PC), as well as benign mimickers of carcinoma, are encountered for which immunohistochemical analysis is often used to help determine the diagnosis.2,3 Because of their small size, the problem occasionally encountered is that such lesions may be present on only one or a few sections and can be cut through with subsequent leveling of the paraffin block to obtain additional sections for immunohistochemical analysis. A previous study found this to occur in 9% of cases that were evaluated during a 4-year period,4 after which the practice at the authors’ institutions was changed so that unstained interval sections were cut from all blocks of prostate needle biopsy specimens and saved for potential immunohistochemical evaluation. Although a subsequent study has since validated this practice,5 it was retrospective, and, to our knowledge, there are no prospective studies that compare the usefulness of immunohistochemical analysis performed on saved interval sections vs new recut sections obtained from the paraffin block. The present study was specifically performed to address this issue.

Materials and Methods

A prospective study was designed to evaluate the use of immunohistochemical analysis in the evaluation of routine prostate needle biopsy specimens as used by a group of surgical pathologists in a large academic medical center (Lauren V. Ackerman Laboratory of Surgical Pathology, Washington University School of Medicine, St Louis, MO) during a 1-year period (July 15, 2005, to July 14, 2006). After institutional review board approval, routine prostate biopsy specimens were subjected to a specific sectioning and evaluation protocol Figure 1, and immunohistochemical analysis was performed using antibodies directed against high-molecular-weight cytokeratin (HMWK; 34βE12), p63, α-methylacyl Co-A racemase (AMACR), and/or a p63/AMACR antibody cocktail,6 as selected by the primary attending pathologist signing out the case. The study protocol did not specify which saved interval level (level 2 and/or 4) to use for staining with these antibodies, but the negative control slides were always recut from the block.

At the end of the prospective study period, all immunohistochemically stained sections were collected and reevaluated by the study pathologists to compare the results of immunohistochemical analysis performed on saved unstained interval sections with those performed on newly recut sections from the block. Specifically, the frequency at which atypical or malignant foci were no longer visible on the immunohistochemically stained sections was evaluated and compared between the 2 groups. In addition, the linear extent of such foci (measured by using an ocular micrometer) and the number of atypical or malignant glands present were also measured and compared between the 2 groups.

Figure 1

Protocol for the evaluation of routine prostate needle biopsy specimens during the prospective study period. AMACR, α-methylacyl coenzyme A racemase; IHC, immunohistochemical analysis.

The χ2 test, the Fisher exact test, or the McNemar test was used to compare categorical data as statistically appropriate, and the Student t test was used to compare continuous data. A P value of .05 or less was considered statistically significant.

Results

During the 1-year study period, 682 consecutive prostate needle biopsy cases were evaluated. Of these, 376 (55.1%) were diagnosed as benign, and 232 (34.0%), 46 (6.7%), and 28 (4.1%) were diagnosed as PC, focal glandular atypia/atypical small acinar proliferation, and high-grade prostatic intraepithelial neoplasia, respectively. Immunohistochemical analysis was performed on 70 (10.3%) specimens, including 3 (0.8%), 45 (19.4%), 18 (39%), and 4 (14%) cases eventually diagnosed as benign, PC, focal glandular atypia, and high-grade prostatic intra-epithelial neoplasia, respectively.

Of the 70 cases, 38 (54%) were evaluated per study protocol and had immunostains performed concurrently on interval and recut sections with identical staining results seen in 21 (55%) of them Image 1. Loss of the atypical or malignant foci of interest was seen in 3 (8%) of the interval sections vs 19 (50%) of the recut sections (P < .0001; McNemar test) Image 2. In 2 of the 3 cases in which the focus of interest was lost in the interval sections, it was also lost in the recut sections. The linear extent of the foci that were lost in the recut sections (mean, 0.75 mm; range, 0.15–2.7 mm) was significantly less than the linear extent of foci not lost in the recuts (mean, 1.32 mm; range, 0.25–2.8 mm) (P = .025; Student t test). Moreover, 15 (79%) of the 19 foci lost in the recut sections were 1 mm or shorter vs 7 (37%) of 19 not lost in the recuts (P = .021; χ2). Although the number of glands in the foci that were lost in the recut sections (mean, 19.2; range, 4–59) was also less than the number of glands that were not (mean, 34; range, 3–110), the difference approached but did not reach statistical significance (P = .069; Student t test).

Discussion

In this prospective study, we have clearly shown that use of saved unstained interval sections for the immunohistochemical evaluation of prostate needle biopsy specimens is more useful diagnostically than using new recut sections from the paraffin block because it is significantly less likely to be associated with loss of representative tissue for immunohistochemical analysis. Limited data exist on the diagnostic usefulness and yields of the use of interval sections vs recut sections in immunohistochemical staining of prostate needle core tissues, and such data were from a study that was retrospective.5 Our study is the first prospective study specifically designed to directly address this issue. Prospective research studies reduce the capacity for selection bias that is a prominent feature of retrospective studies.

Image 1

A, B, and C, A prostate needle biopsy specimen displaying a focus of foamy gland prostatic adenocarcinoma that measured 2.0 mm in length (H&E, A, x40; B, x 100; and C, x400). D and E, Immunohistochemical analysis using a p63/α-methylacyl coenzyme A racemase antibody cocktail was concurrently performed on a saved interval section (D, x 100) and a newly recut section from the block (E x 100). The focus of malignancy was present in both of these sections, and the pattern of staining was identical for confirming the diagnosis of malignancy.

Image 2

A, B, and C, A prostate needle biopsy specimen displaying an area of focal glandular atypia suggestive of carcinoma that measured 0.8 mm in length (H&E, Ax 40; Bx 100; and Cx 400). D and E, Immunohistochemical analysis using a p63/α-methylacyl coenzyme A racemase antibody cocktail was concurrently performed on a saved interval section (Dx 100) and a newly recut section from the block (Ex 40). The pattern of staining seen on the interval section confirmed the diagnosis of carcinoma, while the malignant focus (and adjacent benign tissue) was no longer present in the newly recut section.

Immunohistochemical analysis is increasingly being used in the evaluation of prostate needle biopsy specimens: a recent study found that it was being performed on up to 44% of cases submitted for second opinion in recent years, compared with fewer than 3% a decade earlier,7 and another reported that immunohistochemical analysis was used in the evaluation of 22% of routine prostate needle biopsy specimens.8 The most frequent uses of immunohistochemical analysis in this arena are to aid in the diagnosis or exclusion of minimal and unusual variants of PC and to help distinguish between PC and its numerous benign mimickers.2,3

Antibodies directed against HMWK, p63, and AMACR, as well as various antibody cocktails directed against these molecules, are all being used for these purposes. One of the advantages of using antibody cocktails is the potential for simultaneous assessment of more than one marker in minute, diagnostically difficult foci on a single section, especially when these are limited in extent and present on only a single or a few sections. A significant problem with small atypical foci in needle biopsy samples is that, even when antibody cocktails are used, they are not infrequently cut through with subsequent leveling of the paraffin block to obtain additional sections for performance of immunohistochemical analysis. In these cases, immunohistochemical analysis can be performed on one of the H&E-stained sections directly,9 after “protecting” part of the section,10 or after transfer to charged slides.6 In contrast with direct immunostaining, which can be associated with significant technical problems,9 use of the other 2 methods can be quite advantageous in certain situations.3,6,10

An alternative approach is to cut and save unstained interval sections at the outset for potential immunohistochemical analysis. Green and Epstein5 found in their institution that in 31 of 74 needle biopsy specimens that originally required HMWK immunostaining of the saved sections for diagnosis, the lesion was no longer present on additional sections taken from the paraffin block. Thus, immunostaining of saved interval unstained sections was integral to the diagnosis of 2.8% of the prostate needle biopsy cases submitted during the 2-year period of their study.5 This is certainly a compelling reason to consider saving interval sections on prostate needle biopsy samples; however, it not without cost. In 1999 when the aforementioned study was published, the estimated additional cost of this practice was calculated to be $13.20 per case. Considering that they were integral to the diagnosis in only up to 3% of cases, a presumed low cost-benefit ratio might be one possible explanation why, in a recent international survey of more than 90 genitourinary pathologists,11 only 48% reported cutting and saving interval sections in their institutions. In addition, it is unclear whether the availability of economic resources is also related to the regional variation in this practice, which was twice as frequently reported by North American pathologists than pathologists elsewhere (58% vs 29%).11

It is interesting that the present study found that the proportion of cases in which immunohistochemical analysis on interval sections was integral to the diagnosis was identical to that reported by Green and Epstein.5 Although we did not perform a formal cost analysis, this suggests that cutting and saving only 2 additional interval levels on 2 slides from each paraffin block, as we did, is at least as useful diagnostically as cutting and saving 4 additional interval levels (the method reported by Green and Epstein5) and, as such, more cost-effective. Additional studies are obviously still needed to determine the optimal number of interval sections to cut and save for potential immunohistochemical analysis.

In summary, we have shown that, compared with new recut sections from the block, the use of 2 saved interval levels for the immunohistochemical evaluation of prostate needle biopsy specimens was significantly less likely to be associated with loss of the atypical focus of concern. As expected, loss of such foci was more frequently seen in smaller foci (≤1 mm long), but was also seen in foci nearly 3 mm long, suggesting that the benefit of this practice is not necessarily limited to the diagnosis or exclusion of minimal PC. The data support the routine cutting of unstained interval sections for prostate needle core cases, and, indeed, based on these data, this approach has been adopted as routine practice at both of our hospitals.

Notes

Upon completion of this activity you will be able to:

  • define the proportion of prostate needle biopsy specimens in which immunohistochemistry is currently being used in their evaluation, and the proportion of urological pathologists who routinely cut and save interval sections for potential immunohistochemistry.

  • list the different prostate needle biopsy sections on which immunohistochemistry can be performed.

  • compare the utility of immunohistochemistry performed on saved interval sections of prostate needle biopsy material with that performed on new recut sections from the block.

  • identify the most cost-effective prostate needle biopsy sectioning method that can reduce the incidence of noncontributory immunohistochemical stains due to loss of the focus of concern.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 745. Exam is located at www.ascp.org/ajcpcme.

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