This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

Immunohistochemistry (IHC) is a powerful tool in the surgical pathologists' armamentarium. The requests for IHC and the list of monoclonal antibodies have increased tremendously in the past decade. Issues concerning technical reproducibility, uniformity of interpretation, inter-laboratory comparability, and quality assurance are assuming greater importance due to the increased availability of IHC and its impact on diagnosis and therapy. An attempt has been made to give a current perspective of this simple and yet, in some aspects, a complex tool.

Immunohistochemical techniques have become commonplace adjunctive aids in anatomic pathology. Although much has been written describing modifications of the basic techniques, sensitivity, and specificity of reagents, little has been published regarding the interlaboratory variability in immunostain results on a given test sample. The Cell Markers Survey of the College of American Pathologists was organized to address this question of interlaboratory variability and to disseminate information on the techniques and reagents currently available.

BACKGROUND Several studies have reported what seem to be false-positive results using the Food and Drug Administration (FDA)-approved HercepTest (Dako Corp, Carpinteria, CA) to profile Her-2/neu amplification and overproduction in breast carcinoma. False-positive status has been based on comparisons with gene copy enumeration by fluorescence in situ hybridization (FISH) and with comparisons to immunohistochemistry (IMH) results using a monoclonal antibody. However, simple overexpression by tumor cells that have normal gene copy has not been evaluated by profiling mRNA expression, ie, such cases could simply represent true-positive, transcriptionally upregulated overexpression. MATERIALS AND METHODS Four hundred infiltrating ductal carcinomas of breast were evaluated by IMH using monoclonal (CB11; Ventana Medical Systems, Inc, Tucson, AZ) and polyclonal (HercepTest; Dako) antibodies after antigen retrieval (AR). A polyclonal antibody sans AR (PCA/SAR) was also used. All IMH stains were evaluated and scored according to the guidelines for the FDA-approved HercepTest. A total of 145 of 400 carcinomas were subsequently evaluated by direct and digoxigenin-labeled (Dig) FISH, and 144 of 400 were evaluated by detection of mRNA overexpression via autoradiographic RNA:RNA in situ hybridization. RESULTS Overall HercepTest/CB11 IMH discordance was 12%. Expression of mRNA was highly concordant with FISH and DigFISH amplification and with CB11 and PCA/SAR immunohistology. IMH false-positive cases (no Her-2/neu gene amplification) occurred with both HercepTest (23%) and CB11 (17%), and the majority of false-positive results (34 of 44) were scored as 2+. All 2+ false-positive cases were mRNA-negative. Combined results of HercepTest and CB11 showed that 79%(38 of 48) of 3+ cases were Her-2/neu gene amplified, but only 17%(seven of 41) of 2+ cases had increased gene copy. CONCLUSION Discordant HercepTest/FISH results, and to a lesser extent discordance with CB11 IMH, are most commonly false-positive results with a score of 2+. The 2+ score as defined in the guidelines for the FDA-approved HercepTest should not be used as a criterion for trastuzumab therapy unless confirmed by FISH. Determination of Her-2 gene copy number by FISH may be a more accurate and reliable method for selecting patients eligible for trastuzumab therapy.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 21% of gastric and 33% of gastroesophageal junction (GEJ) adenocarcinomas. Trastuzumab has been approved for metastatic HER2-positive gastric/GEJ cancer in combination with chemotherapy. This retrospective analysis was undertaken to better define the clinicopathologic features, treatment outcomes, and prognosis in patients with HER2-positive adenocarcinoma of the esophagus/GEJ. Pathologic specimens from 156 patients with adenocarcinoma of the esophagus/GEJ treated on clinical trials with chemoradiation and surgery were tested for HER2. Seventy-six patients also received 2 years of gefitinib. Baseline characteristics and treatment outcomes of the HER2-positive and negative patients were compared both in aggregate and separately for each of the two trials. Of 156 patients, 135 had sufficient pathologic material available for HER2 assessment. HER2 positivity was found in 23%; 28% with GEJ primaries and 15% with esophageal primaries (P= 0.10). There was no statistical difference in clinicopathologic features between HER2-positive and negative patients except HER2-negative tumors were more likely to be poorly differentiated (P < 0.001). Locoregional recurrence, distant metastatic recurrence, any recurrence, and overall survival were also statistically similar between the HER2-positive and the HER2-negative groups, in both the entire cohort and in the gefitinib-treated subset. Except for tumor differentiation, HER2-positive and negative patients with adenocarcinoma of the esophagus and GEJ do not differ in clinicopathologic characteristics and treatment outcomes. Given the demonstrated benefit of trastuzumab in HER2-positive gastric cancer and the similar incidence of HER2 overexpression in esophageal/GEJ adenocarcinoma, further evaluation of HER2-directed therapy in this disease seems indicated.

Chromosome 1p36.23 is frequently deleted in glioblastoma multiforme (GBM). miR-34a localizes in this region. Our experiments found that miR-34a was often deleted and epidermal growth factor receptor (EGFR) was frequently amplified in genomic DNA of 55 GBMs using single-nucleotide polymorphism DNA microarray. Notably, we found that the mean survival time was significantly shortened for patients whose GBMs had both EGFR amplification and miR-34a deletion. Expression of miR-34a was significantly lower in GBM samples compared with normal brain tissue. Forced expression of miR-34a in GBM cells decreased their ability to migrate and profoundly decreased their levels of cyclin-A1,-B1,-D1, and -D3, as well as cyclin-dependent kinase and increased expression of cyclin kinase inhibitor proteins (p21, p27). Also, human GBM cells (U251) stable overexpressing mir-34a formed smaller tumors when growing as xenografts in immunodeficient mice compared with wild-type U251 GBM cells. Furthermore, the protein expression of EGFR decreased in the cells with forced overexpression of miR-34a. Additional studies showed that mir-34a targeted Yin Yang-1 (YY1) and YY1 is a transcription factor that can stimulate the expression of EGFR. Thus, our data suggest that miR-34a acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell-cycle proteins and EGFR. Moreover, we discovered for the first time that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of GBM patients.Oncogene advance online publication, 14 May 2012; doi:10.1038/onc.2012.132.

The POISE Trial was a randomised, placebo-controlled, double-blind study of the effectiveness of perioperative beta-blockade in preventing cardiac events including death in 8351 patients. Our hypothesis was that knowledge of the results of the POISE Trial would either increase or decrease the use of effective perioperative beta-blockade, depending on the result. Patients presenting for non-cardiac surgery and at risk of perioperative cardiac events were recruited in two cohorts before and after the release of the POISE Trial results. Effective perioperative beta-blockade was defined as heart rate <65 beats per minute for at least 80% of the perioperative period in patients prescribed beta-blockers. Effective perioperative beta-blockade was achieved in 22 (11.5%) of 191 patients prescribed perioperative beta-blockade in the first cohort (n=392) and seven (6%) of 118 patients in the second cohort (n=241)(P=0.10). Effective heart rate control was achieved in 29 (9%) patients prescribed perioperative beta-blockers compared with 10 (3%) patients not prescribed perioperative beta-blockers (P=0.001). The rate of implementation of effective beta-blockade was low before POISE and this did not change significantly after publication. Our finding does not provide reliable evidence of a change in practice as a result of the POISE Trial.

The objective was to examine the potential benefits of using different combinations of multiple quarter milk samples compared with a single sample for diagnosing intramammary infections (IMI) in dairy cattle. Data used in the analyses were derived from 7,076 samples from 667 quarters in 176 cows in 8 herds in 4 locations (Minnesota/Wisconsin, n=4; Prince Edward Island, n=2; Ontario, n=1; New York, n=1). Duplicate quarter milk samples were collected at morning milking for 5 consecutive days. Cows were evenly distributed between early postparturient and mid- to late-lactation cows. All samples were frozen for shipping and storage, thawed once, and cultured in university laboratories using standardized procedures consistent with National Mastitis Council guidelines. The presence of specific pathogens was confirmed and identified using the API identification system (bioMerieux, Marcy l'Etoile, France) in each laboratory. A previously developed gold standard was applied to the first sample from d 1, 3, and 5 to classify infected quarters. The data were analyzed separately for coagulase-negative staphylococci (CNS) and Streptococcus spp. Various combinations of test results from d 2 and 4 were used in the test evaluation. These consisted of single samples (n=4), 2 sets of duplicate samples (2 samples collected on the same day), 2 sets of consecutive samples (2 samples collected 2 d apart), and 2 sets of triplicate samples (2 samples on the same day and a third sample 2 d apart). Series interpretation of duplicate or consecutive samples (i.e., positive=same pathogen isolated from both samples) resulted in the highest specificity (Sp; CNS Sp=92.1-98.1%; Streptococcus spp. Sp=98.7-99.6%), but lowest sensitivity (Se; CNS Se=41.9-53.3%; Streptococcus spp. Se=7.7-22.2%). Parallel interpretation of duplicate or consecutive samples (i.e., positive=pathogen isolated from either) resulted in the highest Se (CNS Se=70.8-80.6%; Streptococcus spp. Se=31.6-48.1%), but lowest Sp (CNS Sp=72.0-77.3%; Streptococcus spp. Sp=89.5-93.3%). The difference in estimates between single and duplicate samples was larger than between single and consecutive samples. Overall, triplicate samples provided the best combination of Se and Sp, but compared with a single sample, provided only a modest gain in Sp and little or no gain in Se.

The aim of this study was to compare cardiac output and plasma propofol concentrations in the supine and prone positions in healthy adult patients presenting for lumbar spine surgery. Patients received propofol and remifentanil via effect-site steered target-controlled infusions. Cardiac output and plasma propofol concentration were compared during 20 minutes in the supine position and 20 minutes after positioning on a Wilson frame. Cardiac output did not change significantly over 20 minutes in either position (P = 0.37) and was similar at 20 minutes in the supine (6.1 [1.6] l/minute) and prone positions (6.1 [1.9] l/minute)(P = 0.87). Propofol concentrations were similar in the supine and prone positions at 20 minutes (2.55 [0.89] and 2.53 [0.90] microg/ml; P = 0.93). We conclude that prone positioning on the Wilson frame does not affect cardiac output or plasma propofol concentration.

The objective of this multi-state, multi-herd clinical trial was to report on the efficacy of using an on-farm culture system to guide strategic treatment decisions in cows with clinical mastitis. The study was conducted in 8 commercial dairy farms ranging in size from 144 to 1,795 cows from Minnesota, Wisconsin, and Ontario, Canada. A total of 422 cows affected with mild or moderate clinical mastitis in 449 quarters were randomly assigned to either (1) a positive-control treatment program or (2) an on-farm culture-based treatment program. Quarter cases assigned to the positive-control group received immediate on-label intramammary treatment with cephapirin sodium. Quarters assigned to the culture-based treatment program were not treated until the results of on-farm culture were determined after 18 to 24h of incubation. Quarters in the culture-based treatment program that had gram-positive growth or a mixed infection were treated according to label instruction using intramammary cephapirin sodium. Quarters assigned to the culture-based treatment program that had gram-negative or no-growth did not receive intramammary therapy. It was already reported in a companion paper that the selective treatment of clinical mastitis based on on-farm culture results decreases antibiotic use by half and tends to decrease milk withholding time without affecting short-term clinical and bacteriological outcomes. The present article reports on long-term outcomes of the aforementioned study. No statistically significant differences existed between cases assigned to the positive-control program and cases assigned to the culture-based treatment program in risk and days for recurrence of clinical mastitis in the same quarter (35% and 78 d vs. 43% and 82 d), linear somatic cell count (4.2 vs. 4.4), daily milk production (30.0 vs. 30.7 kg), and risk and days for culling or death events (28% and 160 d vs. 32% and 137 d) for the rest of the lactation after enrollment of the clinical mastitis case. In summary, the selective treatment of clinical mastitis based on on-farm culture resulted in no differences in long-term outcomes, such as recurrence of clinical mastitis in the same quarter, somatic cell count, milk production, and cow survival for the rest of the lactation after clinical mastitis.

The objective of this multi-state, multi-herd clinical trial was to evaluate the efficacy of using an on-farm culture system to guide strategic treatment decisions in cows with clinical mastitis. The study was conducted in 8 commercial dairy farms ranging in size from 144 to 1,795 cows from Minnesota, Wisconsin, and Ontario, Canada. A total of 422 cows affected with mild or moderate clinical mastitis in 449 quarters were randomly assigned to either (1) a positive-control treatment program or (2) an on-farm, culture-based treatment program. Quarter cases assigned to the positive-control group received immediate on-label intramammary treatment with cephapirin sodium. Quarters assigned to the culture-based treatment program were cultured on-farm and treated with cephapirin sodium after 18 to 24h of incubation if they had gram-positive growth or a mixed infection. Quarters with gram-negative or no growth did not receive intramammary therapy. The proportion of quarter cases assigned to positive-control and culture-based treatments that received intramammary antibiotic therapy because of study assignment was 100 and 44%, respectively; the proportion of cases that received secondary antibiotic therapy was 36 and 19%, respectively; and the proportion of cases that received intramammary antibiotic therapy because of study assignment or secondary therapy was 100 and 51%, respectively. A tendency existed for a decrease in the number of days in which milk was discarded from cows assigned to the culture-based treatment program versus cows assigned to the positive-control group (5.9 vs. 5.2 d). No statistically significant differences existed between cases assigned to the positive-control and cases assigned to the culture-based treatment program in days to clinical cure (2.7 vs. 3.2 d), bacteriological cure risk within 21 d of enrollment (71 vs. 60%), new intramammary infection risk within 21 d of enrollment (50 vs. 50%), and treatment failure risk (presence of infection, secondary treatment, clinical mastitis recurrence, or removal from herd within 21 d after enrollment; 81 vs. 78%). In summary, the use of an on-farm culture system to guide the strategic treatment of clinical mastitis reduced intramammary antibiotic use by half and tended to decrease milk withholding time by 1 d, without significant differences in days to clinical cure, bacteriological cure risk, new intramammary infection risk, and treatment failure risk within 21 d after the clinical mastitis event.

The implementation and enforcement of the College of American Pathologists Survey Checklist ANP 22432 has renewed attention on the issue of outdating of antibodies used for immunohistochemical analysis. The current study examined the staining patterns of 26 recently acquired primary antibodies and their expired counterparts. Two reviewers examined sequential sections of formalin-fixed, paraffin-embedded tissue samples for staining intensity and percentage of positivity. Appropriate positive and negative control studies were performed. Of the 26 antibodies, 20 exhibited no difference in percentage of positivity or staining intensity. Of the remaining 6, 3 showed better performance with the expired cohort and 3 with nonexpired antibodies. However, no antibody staining characteristics varied by more than 1 step, and in no case was positive staining lost after antibody expiration. Negligible differences exist in immunostaining between outdated and current antibodies. Thus, exemption for primary antibodies from existing regulations would conserve resources without adversely impacting patient care.

A 48-year-old woman attended a physician because of a solitary cutaneous nodule on the left lower leg. Microscopic examination of the excisional specimen revealed a dermal tumor composed of nests of epithelioid cells exhibiting clear cytoplasm. They had centrally located vesicular nuclei with distinct nucleoli. A rich network of capillaries was present throughout. The tumor showed an infiltrative border. There was no epidermal involvement. Periodic acid-Shif (PAS) and PAS-Diastase stains demonstrated glycogen deposition within the cytoplasm of the clear cells. Immunohistochemical evaluation revealed that the tumor cells were positive for HMB-45 and microftalmia associated transcription factor (MITF). Focal desmin positivity was also seen. The tumor cells were negative for S-100 protein, alfa smooth muscle actin, HHF-35, and various cytokeratins. The case is one of a primary cutaneous pecoma. Pecomas are rare, recently described mesenchymal tumors composed of perivascular epithelioid cells. They constitute a spectrum of lesions in different organs including angiomyolipoma of the kidney and liver, sugar tumor of the lung, lymphangiomatosis, and lymphangiomyoma. Primary cutaneous PEComas are exceptionally rare and have only recently been recognized. To date, these are approximately 22 cases in the English literature. Follow-up data is limited but they appear to behave in a benign fashion. We report an additional case with the goal of alerting dermatopathologists to this distinctive unusual neoplasm.

Epithelioid angiomyolipoma (AML) is an uncommon renal mesenchymal tumor with malignant potential and is frequently associated with tuberous sclerosis. Extrarenal AMLs are rare, and to the best of our knowledge, this is the first reported case of a primary monotypic epithelioid AML of adrenal gland in a patient without evidence of tuberous sclerosis. The patient is a 42-year old man who presented with retroperitoneal hemorrhage resulting from spontaneous rupture of adrenal mass. Histologically, the tumor showed a prominent component of epithelioid smooth muscle cells with slightly pleomorphic nuclei, sometimes with prominent nucleoli and eosinophilic cytoplasm resembling oncocytic tumors. Epithelioid cells were positive for melanoma (HMB45 and positive MelanA) and smooth muscle markers (alpha-smooth muscle-specific actin), but not for epithelial markers (cytokeratin, EMA). Differential diagnosis from renal cell carcinoma, adrenal gland carcinoma, and metastatic carcinoma is often challenging because of its epithelioid morphology. Because primary and secondary malignant tumors are much more common and aggressive neoplasms, establishing the correct diagnosis has important therapeutic and prognostic implications.

BACKGROUND The heterogeneous histological features of melanoma may often overlap with melanocytic nevi. For this reason, pathologists have sought after immunohistochemistry to assist with difficult cases. Recently, Wilms' tumor 1 protein (WT1) has been suggested to differentiate between melanoma and melanocytic nevi. OBJECTIVE Our objective was to determine whether immunohistochemistry analysis of WT1 expression is a reliable tool in differentiating cutaneous melanoma from melanocytic nevi. METHODS Forty-five melanoma and 43 melanocytic nevi were immunostained with anti-WT1 monoclonal antibody (clone 6F-H2). RESULTS Forty of the 45 cutaneous melanoma (89%) and 22 of the 43 melanocytic nevi (51%) stained (> 10% cells) for WT1. The highest sensitivity for WT1 was expressed by nodular melanoma (19/20), superficial spreading melanoma (8/10) and Spitz nevi (9/11). At the threshold of above 75% WT1-stained cells, the specificity for melanoma was 95% but the sensitivity was only 31%. At the threshold of 10%, the sensitivity increased to 89% but the specificity decreased to only 49%. Finally, at the threshold of 25% and 50%, the sensitivity and specificity were 71%, 61% and 64%, 77%, respectively. CONCLUSIONS Our data suggest that melanoma is associated with increased WT1 expression. However, as a single immunostaining marker, WT1 is not sufficient for distinguishing melanoma from melanocytic nevi.

Tumors that originate from neural crest-derived cells represent a heterogeneous group of neoplasms including benign and malignant tumors with melanocytic and schwannian differentiation. The immunophenotype of these tumors is well known but little is known about the expression of smooth muscle/myofibroblastic markers in these tumors. A total of 590 neural crest-derived tumors (50 benign schwannomas, five malignant peripheral nerve sheath tumors, 80 neurofibromas, 240 nevocytic nevi, 115 primary melanomas, and 100 melanoma metastases) were studied with respect to alpha-smooth muscle actin and muscle-specific actin expression. alpha-Smooth muscle actin and muscle-specific actin-positive tumor cells with a co-expression of S-100 protein were found in one benign schwannoma, one primary cutaneous melanoma, and four melanoma metastases. Four of these cases were examined ultrastructurally, but typical actin filaments with focal densities were not found in any of the four. Other immunohistochemical markers examined including desmin, h-caldesmon and smooth muscle myosin heavy chain were negative in the tumor cells. The present results suggest that neural crest-derived tumors could show expression of alpha-smooth muscle actin on rare occasion.

Epithelioid angiomyolipoma (eAMLoma) is an uncommon renal mesenchymal tumor with malignant potential and is frequently associated with tuberous sclerosis (TSC). It is composed of polygonal large-sized tumor cells arranged in an epithelioid manner. Differential diagnosis from renal cell carcinoma (RCC) is often challenging because of its epithelioid morphology. Herein is reported three cases of eAMLoma, involving one in a 28-year-old man with TSC and two in women without TSC (34 and 62 years of age, respectively). The male TSC patient had microscopic conventional AMLomas in the same kidney. All patients were positive for melanoma (reactive with HMB45 antibody, and positive for melan A, tyrosinase and microphthalmia transcription factor) and smooth muscle markers (positive for alpha-smooth muscle-specific actin), but not for epithelial markers (cytokeratin, epithelial membrane antigen). In particular, the translocation RCC is an important differential diagnostic candidate, in terms of the positive reaction with HMB45 and morphological similarity. The present tumor samples did not show any reactivity for transcription factor binding to IGHM enhancer 3 or transcription factor EB, which excluded the possibility of translocation RCC. The possibility of eAMLoma should be evaluated as a diagnostic candidate, especially in cases of renal tumors (i) in young patients;(ii) associated with TSC; or (iii) with an epithelioid morphology and a high nuclear grade.

Cardiac ankyrin repeat protein (CARP) is highly expressed in cardiac muscles and detectable in normal skeletal muscles. Arpp, a close homolog of CARP, has been demonstrated to be useful for distinguishing rhabdomyosarcoma from other malignant tumors. However, the CARP distributions among malignant tumors have been poorly investigated. Here, we analyzed the comprehensive expression of CARP in malignant tumors and evaluated its potential use for rhabdomyosarcoma diagnosis. A total of 159 malignant tumors, including 34 rhabdomyosarcomas, 85 non-rhabdomyosarcomas, and 40 carcinomas, were immunohistochemically analyzed for CARP expression. Cytoplasmic expression of CARP was detected in 29 (85%) of 34 rhabdomyosarcomas. The immunoreactivity was observed in both small cells with little differentiation and differentiated tumor cells with abundant eosinophilic cytoplasm. In contrast, focal immunoreactivity for CARP was only observed in 5 (4%) of 125 non-rhabdomyosarcomas, comprising 2 malignant fibrous histiocytomas, 1 angiosarcoma, 1 epithelioid sarcoma, and 1 squamous cell carcinoma of the lung. Comparative analysis of the CARP expression profiles with those of myogenic markers in rhabdomyosarcomas revealed that myogenin (88%) and desmin (88%) exhibited the best sensitivity, followed by CARP (85%), MyoD (82%), muscle-specific actin (79%), and myoglobin (65%). MyoD (96%) and myoglobin (96%) had the best specificity, followed by CARP (95%), myogenin (95%), desmin (89%), and muscle-specific actin (86%). Our results indicate that CARP is a sensitive and specific marker for rhabdomyosarcoma and that it will be useful for the differential diagnosis of rhabdomyosarcoma.

A case of hepatic clear cell myomelanocytic tumor in a 31-year-old woman presenting clinically with abdominal pain is reported. Histopathologic examination showed a lesion characterized by a population of large epithelioid cells with clear or eosinophilic granular cytoplasm, rich in glycogen. Immunohistochemically, the tumor cells were positive for HMB-45, Melan-A and muscle-specific actin, but negative for epithelial markers, desmin, S-100 protein, and neuroendocrine markers. Ultrastructurally, the tumor cells had abundant glycogen, well-developed rough endoplasmic reticulum, microtubules and aberrant melanosomes. Clinical and pathologic features with a brief review of the relevant literature for hepatic CCMMT as a variant of perivascular epithelioid cell tumor (PEComa) are discussed.

Undifferentiated malignant neoplasms are a daunting diagnostic problem for anatomical pathologists, calling for a tour de force in morphological skill, clinicopathologic correlation, and application of adjunctive laboratory studies. The most useful approach to these lesions begins with generic classification into 1 of 4 histologic categories: small round cell; spindle cell; large polygonal cell (epithelioid); and pleomorphic neoplasms. Once that step has been accomplished, one can systemically apply corresponding groups of antibody reagents in immunohistologic studies and interpret the results in an algorithmic fashion. This review presents the tumor markers that are the most useful in this contextual approach, as well as the specific algorithmic structures that can be applied to the 4 specified tumor groups. Other selected problems in the diagnosis of morphologically ambiguous tumors are considered as well.

CONTEXT We have developed tissue microarray-based surveys to allow laboratories to compare their performance in staining predictive immunohistochemical markers, including proto-oncogene CD117 (c-kit), which is characteristically expressed in gastrointestinal stromal tumors (GISTs). GISTs exhibit activating mutations in the c-kit proto-oncogene, which render them amenable to treatment with imatinib mesylate. Consequently, correct identification of c-Kit expression is important for the diagnosis and treatment of GISTs. OBJECTIVE To analyze CD117 immunohistochemical staining performance by a large number of clinical laboratories. DESIGN A mechanical device was used to construct tissue microarrays consisting of 3 x 1-mm cores of 10 tumor samples, which can be used to generate hundreds of tissue sections from the arrayed cases, suitable for large-scale interlaboratory comparison of immunohistochemical staining. RESULTS An initial survey of 63 laboratories and a second survey of 90 laboratories, performed in 2004 and 2005, exhibited >81% concordance for 7 of 10 cores, including all 4 GIST cases, which were immunoreactive for CD117 with >95% staining concordance. Three of the cores achieved less than 81% concordance of results, possibly due to the presence of foci of necrosis in one core and CD117-positive mast cells in 2 cores of CD117-negative neoplasms. CONCLUSIONS There was good performance among a large number of laboratories performing CD117 immunohistochemical staining, with consistently higher concordance of results for CD117-positive GIST cases than for nonimmunoreactive cases. Tissue microarrays for CD117 and other predictive markers should be useful for interlaboratory comparisons, quality assurance, and education of participants regarding staining nuances such as the expression of CKIT by nonneoplastic mast cells.