RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tumor material represents a valuable resource for the analysis of RNA-based biomarkers, both in research laboratories and in routine clinical testing. A robust and automated RNA-extraction method with a high sample throughput is required. METHODS: We evaluated extraction performance for 4 silica-based RNA-extraction protocols: (a) a fully automated, bead-based RNA-isolation procedure; (b) its manual counterpart; (c) a semiautomated bead-based extraction system; and (d) a manual column-based extraction kit. RNA from 360 sections (90 sections per extraction method) of 30 FFPE tumor blocks up to 20 years of age was purified and analyzed by quantitative reverse-transcription PCR for ESR1 (estrogen receptor 1), PGR (progesterone receptor), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)], and RPL37A (ribosomal protein L37a). RESULTS: The semiautomated protocol gave the best yield. The 3 bead-based methods showed good across-method correlations in both yield and relative mRNA amounts (r = 0.86-0.95 and 0.98, respectively). In contrast, correlations between any of the bead-based methods and the manual column-based method were worse (r = 0.77-0.95 and 0.96, respectively). The fully automated method showed the lowest variation from section to section (root mean square error, 0.32-0.35 Cq, where Cq is the quantification cycle) and required the least hands-on time (1 h). CONCLUSIONS: The fully automated RNA-purification method showed the best reproducibility in gene expression analyses of neighboring sections of tissue blocks between 3 and 20 years of age and required the least overall and hands-on times. This method appears well suited for high-throughput RNA analyses in both routine clinical testing and translational research studies with archived FFPE material.

The incidence of lymph node metastases in prostate carcinoma depends not only on tumor characteristics but also on surgical performance and extent of pelvic lymphadenectomy.

OBJECTIVES: The purpose of the present study was to determine whether predictions of the incidence of pelvic lymph node metastases in patients with similar prostate cancer characteristics are influenced by the extent of pelvic lymphadenectomy or surgical performance. MATERIAL AND METHODS: Data from a prostate cancer database were analyzed to investigate associations between incidence of lymph node metastasis and preoperative prostate-specific antigen level, clinical stage, biopsy Gleason score, extent of pelvic lymphadenectomy, and surgical performance. Subgroups of patients with the same characteristics were formed, and a multivariate analysis was performed. RESULTS: Data of 668 patients with cT1-T2c prostate cancer who underwent radical retropubic prostatectomy with pelvic lymphadenectomy were analyzed. Lymph node metastases were found in 8.7% of these patients. In the subgroup of patients undergoing limited pelvic lymphadenectomy, 6.3% were affected compared with 14.7% of patients undergoing extended pelvic lymphadenectomy (P<0.0005). In the subgroups of patients with the same tumor characteristics (with only two exceptions), the impact of the extent of lymphadenectomy on the incidence of lymph node metastases was evident. The results of the multivariate analysis corroborated the influence of the extent of pelvic lymphadenectomy (P<0.03) and surgical performance (P<0.04) on the incidence of lymph node metastases. CONCLUSIONS: The incidence of lymph node metastases was dependent not only on preoperative prostate-specific antigen level, clinical stage, and biopsy Gleason score but also to a large degree on surgical performance and the extent of pelvic lymphadenectomy. Our data suggest that a limited and/or not thoroughly performed pelvic lymphadenectomy results in failure to detect a relevant proportion of lymph node metastases.

Low frequency of molecular changes and tumor recurrence in inverted papillomas of the urinary tract.

AIM: Inverted papilloma (IP) of the urinary tract can be difficult to distinguish from noninvasive urothelial carcinoma with prominent inverted growth pattern (invNIUC). Ancillary markers may help to resolve such cases and clarify the reported malignant potential of some IPs. METHODS: Eighty-nine urothelial lesions initially diagnosed as IP were reviewed by 4 experienced urologic pathologists and studied immunohistochemically (Ki67, p53, CK20, MSH2, MLH1, and MSH6). Mutations of the FGFR3 gene, deletions (loss of heterozygosity) of 9p, 9q, and 17p, microsatellite instability, and elevated microsatellite instability at selected tetranucleotides were also analyzed. RESULTS: Considerable interobserver variability in histopathologic diagnoses was noticed. Only 62 (69.7%) initial diagnoses were confirmed by the review pathologists whereas 23 tumors (25.8%) were redefined as invNIUC. Molecular analyses revealed infrequent alterations in IPs, including microsatellite instability (1.8%), elevated microsatellite instability at selected tetranucleotides (13.2%), FGFR3 mutations (9.8%), 9p deletions (3.9%), 9q deletions (13.2%), 17p deletions (5.1%), nuclear p53 accumulation (18.9%), and aberrant immunostaining for MSH2 (5.8%), MLH1 (11.8%), and MSH6 (3.8%). IP and invNIUC differed in FGFR3 mutations and Ki-67 labeling index (P<0.001 each), and 9q loss of heterozygosity (P=0.03). There were fewer recurrences in IP (5.4%) compared with invNIUC (40.9%; P<0.0001). CONCLUSIONS: IP is a benign lesion that lacks specific genetic alterations found in exophytic noninvasive papillary urothelial tumors. These lesions could be reactive in nature, perhaps secondary to chronic inflammation or a neoplastic process that lack specific genetic alterations. Nevertheless given the clinical and molecular data of this study a conservative clinical approach is appropriate.

Fractional polynomials in a new metastatic renal carcinoma continuous prognostic index involving histology, laboratory, and clinical predictors.

BACKGROUND: We analyzed the effect of histology and various clinical and laboratory predictors in a new continuous prognostic index for metastatic renal-cell carcinoma based on fractional polynomials. PATIENTS AND METHODS: We evaluated 322 metastatic renal-cell carcinoma patients treated with subcutaneous recombinant cytokine-based home therapies in consecutive trials. We evaluated papillary histology, along with age, disease localizations, C-reactive protein, and neutrophil count in a new prognostic index, which was based on the multivariable fractional polynomial (MFP) algorithm. RESULTS: The MFP model allowed us to divide patients into three risk groups, using seven selected significant prognostic factors: histology, neutrophils, C-reactive protein, bone metastases, liver metastases, lymph node metastases, and age. Using the multivariable fractional polynomial model, median overall survival for high-, intermediate-, and low-risk patients was 10 months (n=80), 23 months (n=162), and 41 months (n=80) (scheme A; p

Differential diagnosis of bladder versus colorectal adenocarcinoma: keratin 7 and GATA3 positivity in nuclear ß-catenin-negative glandular tumours defines adenocarcinoma of the bladder.

AIMS: This study evaluates immunohistochemical markers for the differential diagnosis of primary bladder adenocarcinoma (BAC) from secondarily involving colorectal adenocarcinoma (CAC). Additional staining of putative precursor lesions (cystitis cystica et glandularis (CC) and intestinal metaplasia (IM)) supports insights into metaplastic cell development and aberrant differentiation in tumours. METHODS: Tissue microarray sections of formalin-fixed, paraffin-embedded tissues from clinically verified 11 BAC, 11 CAC, 18 invasive urothelial carcinomas (UCs), 22 normal urothelium samples, 25 CC and 15 IM were stained for keratin 7, 5/6, 5/14 and 20, ß-catenin, e-cadherin, cadherin 17, cdx2, uroplakin II and III, CD10, androgen receptor (AR), S100P, MUC2, MUC5AC and GATA3 expression. Data were analysed using Kruskal-Wallis/Dunn's multiple comparison test and Fisher's exact test. RESULTS: Significant difference (p<0.05) between all three tumour groups was observed for keratin 7 only. Further significant difference between BAC and CAC was found for GATA3 and nuclear ß-catenin staining. BAC-positive/CAC-negative markers without significance were: p63, keratin 5/6, 5/14, uroplakins II/III and AR. CC showed a urothelial phenotype (p63+, GATA3+, S100P+, uroplakin+ in single cells) with initial signs of intestinal differentiation (single cells cdx2+ or cadherin 17+). IM displayed a full intestinal phenotype (p63-, all urothelial markers-, cdx2/MUC2/MUC5AC+, cadherin17+). CONCLUSIONS: Differential diagnosis of BAC and CAC remains difficult, but positive staining for keratin 7 in nuclear ß-catenin-negative tumours argues for BAC. Additional markers like GATA3 and p63 may be added, as positivity in some cases may be helpful. However, for reliable histological diagnosis, knowledge of comprehensive clinical data is still essential.

Diagnostic Accuracy of Renal Mass Biopsy: An Ex Vivo Study of 100 Nephrectomy Specimens.

We investigated the diagnostic accuracy of renal mass biopsy in an ex vivo model, as well as compared the agreement of the preoperative radiological diagnosis with the final pathologic diagnosis. Two 18-gauge needle-core and 2 vacuum-needle biopsies were performed ex vivo from the tumors of 100 consecutive patients undergoing radical nephrectomy between 2006 and 2010. The median tumor size was 5.5 cm. There was no significant difference with regard to cylinder length or tissue quality between the sampling methods. At least 1 of 4 needle cores contained diagnostic tissue in 88% of patients. Biopsy specimens identified clear cell (54%), papillary (13%), or chromophobe (5%) renal cell carcinoma; urothelial carcinoma (6%); oncocytoma (5%); liposarcoma (1%); metastatic colorectal carcinoma (1%); squamous cell carcinoma (1%); unclassified renal cell neoplasm (1%); and no tumor sampled (12%). The sensitivity of the biopsy for accurately determining the diagnosis was 88% (95% CI: 79% to 93%). The specificity was 100% (95% CI: 17% to 100%). Biopsy grade correlated strongly with final pathology (83.5% agreement). There was no difference in average tumor size in cases with the same versus higher grade on final pathology (5.87 vs 5.97; P = .87). Appraisal of tumor histology by radiology agreed with the pathologic diagnosis in 68% of cases. Provided that the biopsy samples the tumor tissue in a renal mass, pathologic analysis is of great diagnostic value in respect of grade and tumor type and correlates well with excisional pathology. This constitutes strong ground for increasingly used renal mass biopsy in patients considering active surveillance or ablation therapy.