MicroSEC filters sequence errors for formalin-fixed and paraffin-embedded samples.

The clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded samples and results in many sequencing errors. We identified that most of these errors are detected in chimeric reads caused by single-strand DNA molecules with microhomology. During the end-repair step of library preparation, mutations are introduced by the mis-annealing of two single-strand DNA molecules comprising homologous sequences. The mutated bases are distributed unevenly near the ends in the individual reads. Our filtering pipeline, MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in formalin-fixed, paraffin-embedded samples without over-eliminating the mutations detected also in fresh frozen samples. Amplicon-based sequencing using 97 mutations confirmed that the sensitivity and specificity of MicroSEC were 97% (95% confidence interval: 82-100%) and 96% (95% confidence interval: 88-99%), respectively. Our pipeline will increase the reliability of the clinical sequencing and advance the cancer research using formalin-fixed, paraffin-embedded samples.

Impact of examining additional deeper sections on the pathological diagnosis of endoscopically resected early gastric cancer.

OBJECTIVES: The pathological diagnosis of endoscopically resected early gastric cancer (EGC) is performed by evaluating a few representative sections from the specimen. We aimed to determine whether evaluating twice as many sections as usual by essentially cutting the original sections in half could improve the pathological diagnosis of EGC. METHODS: We retrospectively investigated 85 EGC in 82 patients who had undergone endoscopic resection at our hospital from August 2008 to October 2012. EGC without indications of curative resection were excluded. We re-examined the original paraffin blocks after shaving away approximately half their original thickness, and evaluated whether the pathological diagnoses were affected. This technique essentially doubled the number of sections examined. RESULTS: Ten pathological diagnoses of 68 EGC (14.7%) were changed from curative resection to non-curative resection when we evaluated twice as many sections as in the standard method. The median tumor size was 25 mm in the changed diagnosis group versus 14.5 mm in the no change group (P = 0.03). The univariate analysis also showed that tumor size was a significant predictor of changed diagnosis (P = 0.015). Both the changed diagnosis group and no change group had no recurrence during follow up. CONCLUSIONS: Histological evaluation of twice as many sections as usual changed the initial pathological diagnosis of EGC, although the clinical implication of an additional deeper section was controversial because there was no recurrence. Our analysis also emphasized the importance of detailed histological evaluation to confirm a radical cure in endoscopic resection, especially in the case of larger EGC.

Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues.

The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.

Availability and quality of paraffin blocks identified in pathology archives: a multi-institutional study by the Shared Pathology Informatics Network (SPIN).

BACKGROUND: Shared Pathology Informatics Network (SPIN) is a tissue resource initiative that utilizes clinical reports of the vast amount of paraffin-embedded tissues routinely stored by medical centers. SPIN has an informatics component (sending tissue-related queries to multiple institutions via the internet) and a service component (providing histopathologically annotated tissue specimens for medical research). This paper examines if tissue blocks, identified by localized computer searches at participating institutions, can be retrieved in adequate quantity and quality to support medical researchers. METHODS: Four centers evaluated pathology reports (1990-2005) for common and rare tumors to determine the percentage of cases where suitable tissue blocks with tumor were available. Each site generated a list of 100 common tumor cases (25 cases each of breast adenocarcinoma, colonic adenocarcinoma, lung squamous carcinoma, and prostate adenocarcinoma) and 100 rare tumor cases (25 cases each of adrenal cortical carcinoma, gastro-intestinal stromal tumor [GIST], adenoid cystic carcinoma, and mycosis fungoides) using a combination of Tumor Registry, laboratory information system (LIS) and/or SPIN-related tools. Pathologists identified the slides/blocks with tumor and noted first 3 slides with largest tumor and availability of the corresponding block. RESULTS: Common tumors cases (n = 400), the institutional retrieval rates (all blocks) were 83% (A), 95% (B), 80% (C), and 98% (D). Retrieval rate (tumor blocks) from all centers for common tumors was 73% with mean largest tumor size of 1.49 cm; retrieval (tumor blocks) was highest-lung (84%) and lowest-prostate (54%). Rare tumors cases (n = 400), each institution's retrieval rates (all blocks) were 78% (A), 73% (B), 67% (C), and 84% (D). Retrieval rate (tumor blocks) from all centers for rare tumors was 66% with mean largest tumor size of 1.56 cm; retrieval (tumor blocks) was highest for GIST (72%) and lowest for adenoid cystic carcinoma (58%). CONCLUSION: Assessment shows availability and quality of archival tissue blocks that are retrievable and associated electronic data that can be of value for researchers. This study serves to compliment the data from which uniform use of the SPIN query tools by all four centers will be measured to assure and highlight the usefulness of archival material for obtaining tumor tissues for research.

Intraoperative cytologic diagnosis of sentinel node metastases in breast cancer.

OBJECTIVE: To clarify the usefulness of imprint cytology for intraoperative investigations of sentinel lymph nodes in breast cancer, comparing the results with those of examinations using frozen and permanent sections. STUDY DESIGN: The material consisted of 303 sentinel lymph nodes from 124 cases of clinically node negative breast cancer. Touch imprint cytologic slides and frozen sections were obtained from the same cut surface of the sentinel nodes. Correlations with the final histopathologic results in paraffin sections were evaluated. RESULTS: The sensitivity, specificity and accuracy of imprint cytology were 70.3%, 99.6% and 96.0%, and those of frozen sections were 83.8%, 100%, 98.0%, respectively. The values were improved when the 2 methods were combined (89.2%, 99.6%, 98.3%), though the concordance between imprint cytology and frozen section was 91.9%. CONCLUSION: Both imprint cytology and frozen section are useful for evaluating sentinel lymph node status in breast cancer. However, the 2 techniques should be combined to improve the diagnostic sensitivity.

Accuracy of frozen section diagnoses of breast lesions after introduction of a national programme in mammographic screening.

AIMS: By introducing mammography screening programmes, the size of the detected breast lesions became smaller and the histopathological interpretation problems greater. The study's aim was to analyse the risks and possible limitations of the frozen section method. METHODS AND RESULTS: Frozen section consultations of breast lesions (n=559) 2 years before and 6 years after launching a national mammographic screening programme in 1992 were evaluated in regard of the benign/malignant ratio, tumour size, preoperative frozen section results and final permanent section diagnoses. The breast frozen section examinations of 1990 compared with those from 1998 declined from 70.7% (299/423) to 62.2% (260/418) (P < 0.01), the benign/malignant ratio from 1.09 to 0.54 (P < 0.0001), the rate of the conclusive, correct frozen section diagnoses from 96.3% to 91.9% (P < 0.03). The sensitivity dropped from 92.3% to 87.6%, the negative predictive value from 95.7% to 88.3%, whereas the negative likelihood ratio rose from 0.08 to 0.12. The 'small' (< or = 10 mm) invasive breast carcinomas increased from 14.2% to 22.3% (P < 0.01) and the 'in situ' carcinomas from 2.1% to 6.6% (P < 0.05). CONCLUSIONS: The declining sizes of breast tumours (< or = 10 mm), especially from radiologically detected lesions and sometimes without a macroscopic correlate, create new limitations and changing indications in the histopathological interpretation. Considering the performance of new diagnostic methods (i.e. large core needle biopsies), frozen sections of surgical specimens should not be the primary diagnostic procedure for breast lesions and should be performed only after other preoperative methods have failed.