Impact of analytical variability on clinical interpretation of multiplex pneumococcal serology assays.

The response to pneumococcal vaccination can be used to assess a patient's humoral immune response to polysaccharide antigens. Multiplex assays measuring serotype-specific levels of pneumococcal antibodies are often used for this purpose, and clinical algorithms have been published to assist in the definition of an adequate immune response. We evaluated whether interlaboratory variability in multiplex pneumococcal serology assays would affect the clinical classification of the immune response. Specimens from 57 patients were analyzed at three reference laboratories with different multiplex assays to measure pneumococcal serology. Analytical correlation and clinical agreement in the classification of a patient's vaccination status by the three methods were compared. Although substantial variation in the quantitative antibody levels measured by different laboratories was seen, the qualitative classification of individual serologic results showed a high degree of agreement between labs and the ultimate classification of a patient as "protected" or "nonprotected" was the same for most patients. The majority of discordant classifications were driven by a systematic bias in results from one of the assays rather than by random error. These data suggest that the use of integrated assessments based on multiple serotypes can compensate for much of the analytical variability seen between laboratories. Knowledge of the analytical performance characteristics of a particular assay is most important when evaluating patients with results near clinical cut points.

ERG gene rearrangement status in prostate cancer detected by immunohistochemistry.

TMPRSS2-ERG, the most common gene fusion in prostate cancer, is associated with expression of a truncated protein product of the oncogene ERG. A novel anti-ERG monoclonal antibody has been recently characterized. We investigated the correlation between ERG rearrangement assessed by fluorescence in situ hybridization (FISH) and ERG expression detected by immunohistochemistry in a large cohort of patients treated with radical prostatectomy for clinically localized prostate cancer. Thirteen tissue microarrays comprising 305 tumors and a subset of 112 samples of nonneoplastic prostatic tissue were assessed for ERG rearrangement status by FISH and for ERG expression by immunohistochemistry. Accuracy of ERG detection by immunohistochemistry in predicting ERG status as assessed by FISH (criterion standard) was calculated in terms of sensitivity, specificity, positive and negative predictive values. Of 305 tumor foci, 103 (34%) showed ERG rearrangement by FISH. ERG was detected by immunohistochemistry in 100 (33%) cases, 99 of which were FISH positive. ERG detection by immunohistochemistry demonstrated a sensitivity and specificity of 96% and 99%, respectively, with positive and negative predictive values of 99% and 98%, respectively. None of the 112 samples of nonneoplastic prostatic tissue was rearranged by FISH or showed any ERG expression. In conclusion, ERG detection by immunohistochemistry in prostate cancer was highly predictive of ERG rearrangement as assessed by FISH in a large cohort of prostatectomy patients. Given the high yield and the easier task of performing immunohistochemistry vs. FISH, ERG assessment by immunohistochemistry may be useful for characterizing ERG status in prostate cancer.

The utility of ERG/P63 double immunohistochemical staining in the diagnosis of limited cancer in prostate needle biopsies.

Diagnosis of limited cancer can be challenging in prostate needle biopsies, and immunohistochemistry is commonly used in such settings. Recently, TMPRSS2:ERG gene rearrangement was found to be highly specific for and detected in approximately 50% of prostate cancer. Positive immunohistochemical staining with a novel anti-ERG antibody highly correlated with TMPRSS2:ERG gene rearrangement status. We developed a double immunohistochemical staining containing both erythroblastosis virus E26 oncogen (ERG) and basal cell marker P63 antibodies and evaluated its use in the diagnosis of limited cancer in prostate needle biopsies. A total of 77 prostate needle biopsies containing cancer occupying <1 mm of the length of only 1 core of the entire biopsy set were stained with the double stain containing ERG and P63 antibodies. ERG positivity and its staining intensity in cancerous and other noncancerous lesions were evaluated. ERG expression was detected in 42% (32 of 77) of cases, with strong, moderate, and weak staining intensity in 72%, 16%, and 12% of cases. The staining was uniform in 84% of cases and heterogeneous in 16% of cases with different staining intensities in >10% of cancerous cells. High-grade prostatic intraepithelial neoplasia was present in 17 cases, and in 5 (29%) cases ERG was positive in high-grade prostatic intraepithelial neoplasia glands, which were all immediately adjacent to or intermingled with ERG-positive cancerous glands. In 4 additional cases, positive ERG staining was found in morphologically benign glands, which were also immediately adjacent to or intermingled with ERG-positive cancerous glands. All other benign lesions distant from cancerous glands, including simple and partial atrophy, were negative for ERG. P63 was negative in all cancerous glands and positive in noncancerous lesions. The P63/ERG double immunostain combines the high sensitivity of P63 and the high specificity of ERG and may be potentially useful in the work-up of difficult prostate biopsies. The high specificity of ERG for the presence of cancer may have important implications for prostate biopsy interpretation and needs to be further validated in larger prospective studies.

TMPRSS2-ERG gene fusion prevalence and class are significantly different in prostate cancer of Caucasian, African-American and Japanese patients.

BACKGROUND: Prostate cancer (PCa) exhibits significant differences in prevalence and mortality among different ethnic groups. The underlying genetics is not well understood. TMPRSS2-ERG fusion is a common recurrent chromosomal aberration in PCa and is however not studied among different ethnic groups. We examined the prevalence and class of TMPRSS2-ERG gene fusion in PCa from Caucasian, African-American, and Japanese patients. MATERIALS AND METHODS: A tissue microarray of PCa from 42 Caucasians, 64 African-Americans, and 44 Japanese patients who underwent radical prostatectomies (RP) was studied for TMPRSS2-ERG fusion using a multicolor interphase fluorescence in situ hybridization assay for ERG gene break-apart. RESULTS: TMPRSS2-ERG gene fusion was present in 50% (21/42) of Caucasians, 31.3% (20/64) of African-Americans, and 15.9% (7/44) of Japanese (P=0.003). The gene fusion through translocation, deletion, or both occurred in 61.9% (13/21), 38.1% (8/21), and 0% (0/21) in Caucasians, 20% (4/20), 60% (12/20), and 20% (4/20) in African-Americans, and 71.4% (5/7), 28.6% (2/7), and 0% (0/7) in Japanese patients (P=0.02). A multivariate analysis demonstrated that TMPRSS2-ERG gene fusion correlated with the ethnicity (P=0.03), marginally correlated with the pathologic stage (P=0.06), but not other clinicopathologic parameters, including age, preoperative PSA levels, and Gleason score. CONCLUSIONS: The prevalence and class of TMPRSS2-ERG are significantly different in PCa of Caucasian, African-American, and Japanese patients. Future studies of the molecular pathways implicated in TMPRSS2-ERG gene fusion may shed light on the disparity in prevalence and mortality of PCa among different ethnic groups and help design better prevention and treatment strategies.

ERG rearrangement is present in a subset of transition zone prostatic tumors.

A majority of prostate cancers exhibit a recurrent gene rearrangement involving chromosome 21. In approximately 90% of cases, the rearrangement is characterized by fusion of the androgen-regulated gene TMPRSS2 with the oncogene ERG. A recent study suggested that TMPRSS2-ERG gene fusion is lacking in cancers arising from the transition zone of the prostate. A dominant transition zone cancer was detected in 62/397 (16%) patients who underwent radical prostatectomy at our institution and were reviewed and mapped by a single pathologist. In 46/62 specimens, a secondary tumor was identified in the peripheral zone of the prostate. A tissue microarray containing both transition and peripheral zone tumors was constructed and evaluated for gene fusion analysis. TMPRSS2-ERG fusion status was determined using a multicolor interphase fluorescence in situ hybridization assay for ERG break-apart. The median age of the patients was 59 years. Prostatectomy Gleason score was 6 in 21, 7 in 34, and ≥8 in 7 cases. Median tumor volume was 200 mm(2). TMPRSS2-ERG gene fusion was present in 7/59 (12%) transition zone, and in 12/35 (34%) peripheral zone tumors. Transition zone fusion-positive cases were larger than their negative counterparts. No significant correlation was found between fusion status and Gleason score or pathologic stage. Gene fusion through deletion occurred in 4/7 transition zone and 7/12 peripheral zone tumors. Transition zone prostate cancers are considered biologically and genetically different from peripheral zone tumors. Although ERG rearrangement is more common in peripheral zone tumors, we have detected TMPRSS2-ERG fusion in a subset of transition zone cancers (12%). The lower frequency of gene fusion in transition zone prostate cancer may suggest distinct molecular alterations from peripheral zone tumors and the association with a high tumor volume may indicate a growth advantage for transition zone tumors harboring the gene fusion. Further studies are necessary to confirm this hypothesis.