Analysis of Immunohistochemical Stain Usage in Different Pathology Practice Settings

Anatomic Pathology / Immunohistochemistry Use by Practice Type Analysis of Immunohistochemical Stain Usage in Different Pathology Practice Settings Akeesha A. Shah, MD, Henry F. Frierson, Jr, MD, and Helen P. Cathro, MBChB Key Words: Immunohistochemistry; Academic; Private practice; Commercial Abstract This study compares the use of immunohistochemistry (IHC) for diagnosing carcinoma in private practice and commercial settings with use in a single academic center. H&E-stained slides and IHC stains, when present, of recently diagnosed carcinomas (n = 200) from patients referred to our institution for treatment were reviewed by a resident and midand senior-level pathologists. Diagnostic agreement between academic and referral pathologists was 98%; the former group used IHC stains in 11% and the latter in 26% of cases (P <.0001). Pathologists from commercial laboratories (12% of referrals) used IHC in 38% of cases, whereas private/hospital-based community laboratories (86% of referrals) used them in 24%. The average number of stains ordered per case was similar among all groups. We suggest that the use of IHC may reflect both the degree of experience of the pathologist and the pathology practice setting. For more than 25 years, immunohistochemistry (IHC) has greatly assisted surgical pathologists in the diagnosis of neoplastic diseases. Currently, IHC has a wide variety of uses, including, but not limited to, confirmation of diagnosis, determination of the primary site of origin for metastatic tumors, classification of neoplasms, use as a prognostic and predictive biomarker, and identification of infectious organisms. Residents at all levels of training in surgical pathology are exposed to IHC and must become proficient in its application, interpretation, and limitations. With its proliferation in pathology practice settings of all types, the temptation to overuse it continues. IHC should, of course, complement and not supersede information gleaned from the clinical context and the H&E-stained morphologic appearance. The pathology literature is inundated with articles that address the usefulness of diagnostic IHC algorithms and panels for tumor classification. However, there is a paucity of studies examining the patterns of usage of IHC among pathologists, possibly because of the inherent bias likely to be present in the study design. Despite this limitation, our anecdotal experience is that IHC usage varies among practice types as well as according to the pathologist s level of experience. Hence, we examined the application of IHC in a group of patients with carcinomas referred to the University of Virginia (UVA) Health System (Charlottesville, VA) for treatment and compared its frequency of use among different practice settings and experience level among the authors of this study. Materials and Methods Two hundred nearly consecutive cases from October 2010 to October 2011 were selected for review from the files Downloaded from https://academic.oup.com/ajcp/article-abstract/138/6/831/1766291 Am J Clin Pathol 2012;138:831-836 831 831 831

Shah et al / Immunohistochemistry Use by Practice Type of the Department of Pathology at UVA. The cases comprised carcinomas from patients chiefly referred to our institution for definitive treatment and consisted mainly of biopsy specimens that yielded the initial diagnosis of invasive cancer. Patients with a prior diagnosis of cancer were excluded from this study. Hematopathology, neuropathology, renal pathology, and cytopathology cases were also excluded. The cases were blindly and individually reviewed by each of the 3 authors. These study pathologists represented a midlevel resident and midlevel and senior-level faculty pathologists. In addition to the H&E stains, the study pathologists were provided with the age and sex of each patient, the location of the biopsy specimen, and a brief clinical history taken from the referring institution s surgical pathology report. Study pathologists recorded their diagnosis based solely on review of the H&E-stained sections. They had no prior knowledge of any previously performed IHC stains. After a review of the H&E slides, study pathologists recorded the IHC stains they would have ordered to aid their diagnosis as if they were signing out the case as the primary pathologist. Only after recording their diagnosis were they given access to the IHC stains or results of the IHC stains that had been previously performed, either by the referring institution or by the UVA pathologist who had initially reviewed and signed out the case. It should be noted that in those instances in which the study pathologist would have required additional IHC stains, they were not actually performed because the study was carried out retrospectively and tissue specimens were not available. IHC stains used for prognostication or in guiding treatment, such as estrogen and progesterone receptors in breast carcinoma, were not recorded. Referring institutions were categorized as commercial or private, including hospital-based, the former being defined as those whose pathologists had no hospital affiliation. All commercial pathology laboratories included in this report had operations in multiple states. Using the referring institution s surgical pathology report, the diagnoses and number of IHC stains performed in each case, as well as the type of IHC stain performed by the primary pathologists, were recorded after the study pathologists had made their interpretations. To help overcome the inherent bias in the study, namely, that all the included cases were known by the study pathologists to likely represent carcinomas, 200 consecutive sitematched biopsy specimens with a first-time carcinoma diagnosis made at our institution from May 2009 to November 2011 were selected as control cases. All were of a similar level of complexity as the study cases. These cases were not signed out by either of the study pathologists. The pathologists signing out these cases were all surgical pathology faculty members at UVA and are referred to as control pathologists throughout this article. It should be mentioned that UVA does not have a specialty sign out system, but rather all surgical pathologists sign out all types of cases except for complex hematopathology, dermatopathology, neuropathology, and renal pathology cases. All the faculty members have areas of special interest in tumor pathology, and interdepartmental consultations are frequent. The level of experience of the other faculty members is summarized as follows: senior (n = 5) and midlevel (n = 4), with senior pathologists defined as those in practice for more than 10 years. The diagnosis of the control pathologists as well as the number of IHC stains used were recorded. Diagnostic agreement, the average number of cases for which IHC stains were used, the average number of IHC stains per case, as well as the average number of IHC stains used with respect to organ system, were compared among study pathologists, referring pathologists, and control pathologists. P values were obtained using the Fisher exact test, with P <.05 considered significant (Graphpad Software, La Jolla, CA). Results Anatomic sites for the 200 referral cases included prostate (41), endometrium (33), other genitourinary (29), gastrointestinal and hepatobiliary tracts (27), lung (21), head and neck, (19), breast (16), other gynecologic (11), and bone/soft tissue (3) Figure 1. Of the 200 cases, 172 were from private 19 (9.5%) 21 (10.5%) 16 (8.0%) 11 (5.5%) 27 (13.5%) 3 (1.5%) 41 (20.5%) 29 (14.5%) 33 (16.5%) Prostate Endometrium Other genitourinary Gastrointestinal and hepatobiliary Lung Head and neck Breast Other gynecologic Bone and soft tissue Figure 1 Distribution of organ systems among the 200 referral/control cases. 832 Am J Clin Pathol 2012;138:831-836 Downloaded 832 from https://academic.oup.com/ajcp/article-abstract/138/6/831/1766291

Anatomic Pathology / Original Article practice/hospital-based pathology groups (n = 55), 24 were from commercial groups (n = 12), and 4 were from academic medical centers (n = 4). Overall diagnostic agreement between study and referring pathologists was 98%. Discrepant diagnoses were found in 4 cases, each of which was of genitourinary origin. One case was interpreted as a poorly differentiated carcinoma by the referring pathologist and as clear cell renal cell carcinoma by the study pathologists. The second case was diagnosed as a condyloma by the referring pathologists and interpreted as squamous cell carcinoma by study pathologists. The third case was diagnosed as an adenocarcinoma by referring pathologists and interpreted as urothelial carcinoma by study pathologists. The fourth case was diagnosed as chromophobe carcinoma by referring pathologists and interpreted as an oncocytoma by study pathologists. Where there was disagreement among study pathologists, a consensus was reached in all cases when 2 of the 3 study pathologists concurred. IHC stains were/would have been used by both referring and study pathologists in 3 of the 4 cases. Referring and study pathologists used IHC stains in 26% and 11% of total cases, respectively (P <.0001) Table 1. Pathologists from commercial laboratories (12% of referral cases) used IHC in 38% of cases, whereas those from private or hospital-based community laboratories (86% of referral cases) used IHC in 24% of cases. The total number of cases in which IHC was used varied among study pathologists to a smaller degree; the senior pathologist used stains in 6% of cases, the midlevel pathologist in 14% of cases, and the resident in 10% of cases. The greatest differences between referring and study pathologists in IHC usage were seen for the following sites: lung (71% vs 38%; P =.062), prostate (27% vs 5%; P =.013), breast (31% vs 13%; P = not significant [NS]), and other gynecologic (27% vs 9%; P = NS). When IHC stains were performed, the average number of stains used per case by pathologists from private groups Table 1 Comparison of the Number of Immunohistochemistry Cases Between Referring and Study Pathologists * No. of Cases With Immunohistochemistry Referring Study Organ System (n = 200) Pathologists Pathologists P Value Prostate (n = 41) 11 (27) 2 (5).013 Endometrium (n = 33) 2 (6) 0.492 Other genitourinary (n = 29) 6 (21) 3 (10).47 Gastrointestinal and 5 (19) 1 (4).192 hepatobiliary (n = 27) Lung (n = 21) 15 (71) 8 (38).062 Head and neck (n = 19) 4 (21) 3 (16) 1.00 Breast (n = 16) 5 (31) 2 (13).394 Other gynecologic (n = 11) 3 (27) 1 (9).587 Bone and soft tissue (n = 3) 1 (33) 1 (33) 1.00 Total cases 52 (26) 21 (11) <.0001 * Data are given as number (percentage) unless otherwise indicated. was 4, whereas those in commercial laboratories as well as the study pathologists used an average of 3 stains per case. The average number of stains used per case did not vary with experience level among study pathologists; all used an average of 3 stains per case. Excluding bone/soft tissue, which had too few cases for analysis, the organ system with the highest average number of stains per referral case was head and neck, with an average of 6 IHC stains per case; study pathologists used an average of 3 stains per case. For 4 other organ systems, private pathologists used more stains on average than study pathologists: 5 vs 2 IHC stains used per lung case; 4 vs 2 stains per gastrointestinal/hepatobiliary case; 3 vs 2 stains per gynecologic case; and 3 vs 1 stain per breast case. The majority of commercial cases were prostate biopsies, and these pathologists used a broader range of stains per case than either private or study pathologists Table 2. For the site-matched control group, the overall IHC rate of usage was 13%, which is not statistically different from Table 2 Immunohistochemistry Ordering Practices Among Commercial, Private, and Study Pathologists Distributed by Organ System Commercial Pathologists Private Pathologists Study Pathologists No. of Cases Mean No. of No. of Cases Mean No. of No. of Cases Mean No. of Organ System With IHC Stains (Range) With IHC Stains (Range) With IHC Stains (Range) Prostate 6 3 (1-7) 5 2 (1-4) 2 2 (2-5) Endometrium 1 1 (NA) 1 5 (NA) 0 NA Other genitourinary 0 NA 6 3 (1-4) 3 3 (3-4) Gastrointestinal and 0 NA 5 4 (1-7) 1 2 (2-3) hepatobiliary Lung 0 NA 15 5 (1-9) 8 2 (2-4) Head and neck 0 NA 3 6 (3-13) 3 3 (1-7) Breast 1 1 (NA) 4 3 (1-7) 2 1 (NA) Other gynecologic 1 6 (NA) 2 3 (1-4) 1 2 (NA) Bone and soft tissue 0 NA 0 0 1 2 (2-5) IHC, immunohistochemistry; NA, not available. Downloaded from https://academic.oup.com/ajcp/article-abstract/138/6/831/1766291 Am J Clin Pathol 2012;138:831-836 833 833 833

Shah et al / Immunohistochemistry Use by Practice Type study pathologists at 11% (P =.64). With the exclusion of bone/soft tissue, the lung was the organ with the greatest average usage of IHC for control pathologists, as well as for referral and study pathologists. In contrast to referring and study pathologists, the control pathologists used more IHC stains for endometrial cases (6% and 0% vs 18%, respectively) Table 3. The top 10 IHC stains used by referral pathologists were thyroid transcription factor 1 (TTF-1), prostatic intraepithelial neoplasia 4 (PIN4) cocktail, and cytokeratin 7 (CK7) (each used at the same frequency), CK5/6, and cytokeratin cocktail, and then CK20, p63, synaptophysin, chromogranin, and highmolecular-weight kininogen (HMWK) Table 4. Discussion Before IHC, which has been performed in diagnostic pathology for nearly 3 decades, a number of other techniques were used to augment basic H&E histology. The Society for Histochemistry was founded in Germany in 1952, and American pathologists readily embraced new histochemical stains in the practice of diagnostic pathology, as documented by the leading pathology textbooks of the time. 1,2 Transmission Table 3 Control Pathologists Use of Immunohistochemistry Organ System (n = 200) Prostate (n = 41) 7 (17) Endometrium (n = 33) 6 (18) Other genitourinary (n = 29) 0 Gastrointestinal and hepatobiliary (n = 27) 5 (19) Lung (n = 21) 5 (24) Head and neck (n = 19) 0 Breast (n = 16) 0 Other gynecologic (n = 11) 1 (9) Bone and soft tissue (n = 3) 1 (33) Total cases 25 (13) No. (%) of Cases With Immunohistochemistry electron microscopy (EM) was the next tool used to aid the histopathologist, but its widespread dissemination was limited by cost; it was primarily used in academic medical centers and Veterans Affairs hospitals. 3 Although few general surgical pathologists currently use EM on a regular basis for tumor examination, it has been pointed out that depending on the tumor type this method may be more cost-effective than a panel of IHC stains. 4 Similarly, immunofluorescence microscopy is used by few pathologists, mainly renal and dermatopathologists, in part because of the requirement for an immunofluorescence microscope. In situ hybridization microscopy is another spinoff from histochemistry that combines molecular analysis with slide-based techniques. Beginning in the 1980s, IHC quickly supplanted histochemistry, being particularly attractive because of its ability to more accurately suggest the specific type or the anatomic site of origin for various cancers. 5-9 IHC can be performed rapidly without expensive specialized equipment and is increasingly being automated. Once the pattern of staining in control tissue is ascertained, IHC is relatively straightforward to interpret. Unfortunately, it can easily be abused, both by over- and misutilization. Except in selected circumstances, the use of a single IHC stain is not optimal, with logical IHC algorithms for particular differential diagnoses being far more discriminating and accurate. Clinical demand for rapid turnaround of cases has no doubt contributed to the problem of overutilization, with pathologists unwilling to hold a case for a second round of IHC. The use of IHC has become so prevalent that some pathologists may spend less time examining the H&E stain in favor of evaluating the IHC results. Other problems with IHC relate to quality of staining, antibody sensitivity and specificity, interpretation, and their regulation. 10-12 Potential conflict of interest with regard to ordering IHC stains is likely to receive increasing scrutiny in this age of Medicare cost rationalization. The type of IHC stain as well as the number used may also affect costs, eg, IHC cocktails processed using separate vials for each antibody can be charged as multiple IHC stains, despite being performed on a single Table 4 The 10 Most Used Immunohistochemical Stains by Organ Site Organ System TTF-1 CK7 PIN4 CK5/6 CK CK20 p63 SYN CHR HMWK Prostate 15 2 7 Endometrium 1 1 Other Genitourinary 1 3 1 3 Gastrointestinal and hepatobiliary 1 3 1 2 3 1 Lung 13 7 8 6 5 7 5 7 Head and neck 1 3 1 1 1 Breast 1 Other Gynecologic 1 2 1 1 1 1 Bone/soft tissue 1 Total 15 15 15 14 13 12 11 9 8 7 CHR, chromogranin; CK, cytokeratin; HMWK, high-molecular-weight kininogen; PIN4, prostatic intraepithelial neoplasia 4; SYN, synaptophysin; TTF-1, thyroid transcription factor 1. 834 Am J Clin Pathol 2012;138:831-836 Downloaded 834 from https://academic.oup.com/ajcp/article-abstract/138/6/831/1766291

Anatomic Pathology / Original Article slide, depending on the payor. IHC cocktails processed using multiple antibodies per single vial can only be charged once. Very few studies have addressed the overall patterns of IHC usage, in part because of the difficulty of performing such studies without bias. One study addressing second opinion surgical pathology review found that repeating IHC stains and adding new ones to the panel resulted in a change in diagnosis in 18.3% of cases for which IHC was performed. 13 These authors concluded that repeating or performing IHC in cancer cases referred to a tertiary care center for treatment was important for patient care. An analysis of the costeffectiveness of IHC in 1998 found a usage rate of 6% at a general hospital, which contrasts with the 26% rate by referral pathologists in our study. 14 This study reached the conclusion that IHC is extremely cost-effective; however, the analytic algorithm was based on the premise that the correct diagnosis could not have been reached without IHC. To our knowledge, the current study is the first of its kind to examine IHC on the basis of pathology practice setting as well as the level of experience of the pathologist. We found a significant difference between referring and study pathologists in the frequency with which IHC was performed, with referring pathologists using IHC in more cases than study pathologists (26% vs 11%; P <.0001). Pathologists from commercial laboratories used IHC more often than pathologists from private laboratories (38% vs 24%). Most of the cases sent from commercial laboratories consisted of prostate specimens, with the PIN4 cocktail being the most commonly ordered IHC stain. In private practice laboratories, the lung was the organ site with the highest number of IHC stains; TTF-1 was the most commonly used stain. The increased use of TTF-1, p63, and CK5/6 is likely caused by the perceived need to more accurately classify lung cancers, as targeted therapies for specific histologic types are now available. Despite the differences among commercial, private, and study pathologists in total number of cases in which IHC was performed, no significant difference was found in the average number of IHC stains used per case. Private groups used an average of 4 stains per case, whereas commercial and study pathologists used 3 per case. Among the study pathologists, no statistically significant difference was seen in the average number of IHC stains used per case based on level of experience. However, overall, the senior pathologist used IHC in fewer cases (6%) than both the midlevel pathologist (14%) and the midlevel resident (10%). Senior pathologists were trained during an era in which H&E histology was emphasized, whereas midlevel pathologists are likely to be more dependent on IHC. The resident s lower rate of IHC usage compared with the midlevel pathologist may be explained partly by uncertainty about the correct use of IHC. Other pathologists in our department used IHC no more than study pathologists except for endometrial cases; this finding is most likely because one of the faculty pathologists recently published a study on using IHC in gynecologic pathology, thus stimulating an awareness of its potential usefulness in this setting. The top 10 IHC stains used by referring pathologists, in descending order, were as follows: TTF-1, CK7, and PIN4 (each used at the same frequency), followed by CK5/6 and pankeratin, and then CK20, p63, synaptophysin, chromogranin, and HMWK. In our opinion, CK7 and CK20 are among the most commonly overused IHC stains, especially as there are more tumor-specific reagents that yield fruitful information. Our study has several biases and limitations. An important bias was that the cases analyzed were all referral cases of known first-time diagnoses of carcinoma. Despite this knowledge, study pathologists attempted to approach the cases as though they were being diagnosed for the first time and as though they were not necessarily malignant. Cases were primarily biopsies that were not particularly diagnostically complex but represented routine cancer diagnoses that would be made on a daily basis. Additional limitations were that the cases selected did not represent all organ sites; also all of the sites present in this study were not equally represented. Our study was relatively small (200 referral cases), and a larger study, especially one in which the case mix differed from ours, might show additional or contradictory findings. In conclusion, although diagnostic agreement among pathologists in several practice settings was high, referral pathologists used IHC stains in a higher proportion of cases than those at our academic medical center. Interestingly, the average number of stains used per case was very similar among pathologists of the 3 practice types. An analysis of the particular stains suggests that the increase in targeted therapy for lung carcinoma, in particular, may be driving some usage patterns. The pattern for IHC usage may also reflect the degree of experience of the individual pathologist. From the Department of Pathology, University of Virginia Health System, Charlottesville, VA. Support for this study was provided by the Department of Pathology at the University of Virginia Health System. This study was presented in part at the 2012 Annual Meeting of the United States and Canadian Academy of Pathology Meeting; March 2012; Vancouver, Canada. Address reprint requests to Dr. Cathro: Department of Pathology, University of Virginia Health System, PO Box 800214, Charlottesville, VA 22908; hpc4f@virginia.edu. References 1. Gossner W. A brief history of the Society for Histochemistry; its founders, its mission and the first 50 years. Histochem Cell Biol. 2002;118:91-94. Downloaded from https://academic.oup.com/ajcp/article-abstract/138/6/831/1766291 Am J Clin Pathol 2012;138:831-836 835 835 835

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