CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Diagnostic value of fungal fluorescence in onychomycosis
Fluorescence of pathogenic fungi has been previously shown when hematoxylin-and-eosin–stained sections were examined under a fluorescence microscope. The authors hypothesized that this phenomenon could aid in evaluating nail specimens for onychomycosis. They conducted a study in which 48 routinely stained nail sections of periodic acid-Schiff (PAS)–positive onychomycosis, along with 23 PAS-negative control specimens with a clinical diagnosis of onychomycosis, were analyzed under a fluorescence microscope to determine the clinical usefulness of this technique. In most of the cases, fluorescence of fungal organisms was noted. Fungi were identified by their tubular or annular shapes with fluorescence surrounding them. The sensitivity and specificity of the method were 96 percent and 90 percent, respectively. In some cases, it was difficult to identify the fungi because of the relative paucity of organisms, weak fluorescence, and high background fluorescence of eosinophilic nail keratin. The authors concluded that fluorescence microscopy can be used as a rapid screening tool for identifying fungi in nail specimens.
Idriss MH, Khalil A, Elston D. The diagnostic value of fungal fluorescence in onychomycosis. J Cutan Pathol. 2013;40(4):385–390.
Correspondence: Dr. Dirk Elston at delston@ameripath.com
Distribution of microscopic melanoma metastases in sentinel lymph nodes: implications for pathology protocols
The authors investigated the utility of sectioning at multiple levels in the histopathologic analysis of sentinel lymph nodes for melanoma and the correlation of metastasis size with risk of subsequent metastasis. Metastatic melanoma was identified in sentinel lymph nodes (SLNs) from 91 of 475 (19 percent) melanoma patients who underwent SLN sampling at Massachusetts General Hospital between 2004 and 2008. All SLNs were evaluated by a nine-slide protocol: sets of MART-1, hematoxylin and eosin, and S100 stains at three distinct levels separated by 80 µm. The location and size of the tumor deposits were evaluated in the context of subsequent metastasis and overall survival. Of the 91 patients with positive sentinel nodes, all nine protocol slides were available for review in 61 (67 percent). Eleven of 61 patients had no tumor present in the first set of levels; two of these patients died of metastatic melanoma. Patients in whom 11 or more tumor cells were detected in the sentinel node had a greater chance of developing subsequent metastases when compared with patients in whom 10 or fewer tumor cells were detected (P=.05). Of those with either metastases greater than 2 mm in diameter or extracapsular extension, 50 percent developed metastases beyond the SLN basin. Eliminating one of the three levels in the SLN detection protocol would have led to a false-negative diagnosis in 18 percent of patients.
Lobo AZ, Tanabe KK, Luo S, et al. The distribution of microscopic melanoma metastases in sentinel lymph nodes: implications for pathology protocols. Am J Surg Pathol. 2012;36(12):1841–1848.
Correspondence: Dr. Lyn M. Duncan at duncan@helix.mgh.harvard.edu
Global mutational profiling of formalin-fixed colon cancers from a pathology archive
The advent of next-generation sequencing technologies, which significantly increase the throughput and reduce the cost of large-scale sequencing efforts, provides an unprecedented opportunity for discovery of novel gene mutations in human cancers. However, it remains a challenge to apply next-generation technologies to DNA extracted from formalin-fixed, paraffin-embedded cancer specimens. The authors described the development of a custom DNA capture method using next-generation for detecting 140 driver genes in five formalin-fixed, paraffin-embedded colon cancer samples using an improved extraction process to produce high-quality DNA. Isolated DNA was enriched for targeted exons and sequenced using the Illumina next-generation platform. An analytical pipeline using three software platforms to define single-nucleotide variants was used to evaluate the data output. Approximately 250 × average coverage was obtained, with more than 96 percent of target bases having at least 30 sequence reads. Results were then compared with previously performed high-throughput Sanger sequencing. Using an algorithm of needing a positive call from all three callers to give a positive result, 98 percent of the verified Sanger sequencing somatic driver gene mutations were identified by the authors’ method with a specificity of 90 percent. In all, 13 insertions and deletions identified by next-generation sequencing were confirmed by Sanger sequencing. The authors also applied this technology to two components of a biphasic colon cancer, which had strikingly different histology. Remarkably, no new driver gene mutation accumulation was identified in the more undifferentiated component. Applying this method to profiling of formalin-fixed, paraffin-embedded colon cancer tissue samples yields sensitivity and specificity for mutation detection equivalent to Sanger sequencing of matched cell lines derived from these cancers. This method enables high-throughput comprehensive mutational profiling of colon cancer samples and can easily be extended to allow targeted sequencing from formalin-fixed, paraffin-embedded material from other tumor types.
Adams MD, Veigl ML, Wang Z, et al. Global mutational profiling of formalin-fixed human colon cancers from a pathology archive. Mod Pathol. 2012;25(12):1599–1608.
Correspondence: Dr. Sanford D. Markowitz at joseph.willis@case.edun