PurposeKnowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.MethodsThe test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.ResultsDirecting this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.ConclusionWe present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.

Objectives-Expression of transcription factors that mediate epithelial-mesenchymal transition (EMT), such as Twist and Slug, is correlated with poor prognosis in many tumor types. Selected EMT markers were studied in a series of pancreatic ductal adenocarcinomas (PDAs) and benign pancreatic tissues to determine whether expression levels correlated with diagnosis, histologic grade, or patient outcome.Methods-Immunohistochemical stains for Twist, Slug, and N-cadherin were performed using a tissue microarray containing 68 PDAs and 38 samples of normal pancreas or chronic pancreatitis tissues.Results-Twist and Slug were identified in both the nucleus and cytoplasm of benign pancreatic ductal epithelium, chronic pancreatitis, and PDA. Compared with normal ductal epithelium, nuclear levels of Twist are decreased in PDA. None of the other EMT markers showed significant differences in staining indices among the diagnostic groups. There were no correlations between EMT marker expression and histologic grade. Epithelial-mesenchymal transition marker expression was not associated with N-cadherin expression, patient outcome, or duration of survival.Conclusions-Decreased expression of nuclear Twist is observed in malignant pancreatic epithelium. However, use of Twist as a diagnostic marker is precluded because decreased expression is also seen in chronic pancreatitis. None of the markers studied were predictive of patient outcome. Keywords epithelial-mesenchymal transition; pancreatic ductal adenocarcinomaDespite advances in radiologic imaging procedures, the diagnosis of pancreatic ductal adenocarcinoma (PDA) remains difficult, particularly for tumors of low stage. Invasive diagnostic procedures such as endoscopic ultrasonography or computed tomography-guided fine-needle aspiration or core biopsy often yield ambiguous diagnoses such as "atypical" or "suspicious for malignancy" when the tumor is of low histologic grade (well-differentiated) or when the amount of diagnostic material is limited. Tumor markers that assist in the pathologic diagnosis of PDA would be clinically useful in this frequent and challenging clinical distinction. MATERIALS AND METHODS Case RetrievalThe surgical pathology files at the Vanderbilt University Medical Center were searched for pancreatic resection specimens performed for either PDA or nonneoplastic disease with existing tissue blocks suitable for construction of a tissue microarray (TMA). A total of 72 cases were identified, 4 of which were resected for benign disease (2 secondary to trauma, 1 for a benign pancreatic neoplasm, and 1 for a benign stricture of the common bile duct). Patient charts were reviewed to record pathologic data (histologic grade, tumor location, tumor size, and pTNM status), demographic data (patient age and sex), and follow-up information (outcome and interval from date of surgical resection to death). The study protocol was approved by the institutional review boards at both Vanderbilt University and Dartmouth College. TMA ConstructionAll tissue samples were ...

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A 23-year-old Chinese man presented with a 3-year history of a pruritic eruption. On examination, pink urticarial papules associated with hyperpigmented reticulated patches were noted on his neck, back, and upper chest. Histopathology revealed vacuolar interface dermatitis and numerous gram-negative rods within a dilated hair follicle. The organisms were reactive with anti-Helicobacter pylori immunohistochemisty. The histologic findings and clinical presentation support the diagnosis of prurigo pigmentosa. Additional testing demonstrated a positive urease breath test and serum H. pylori IgG antibodies. The patient was referred to gastroenterology and treated with appropriate antibiotics. After treatment, esophagogastroduodenoscopy revealed chronic gastritis without evidence of H. pylori infection and his skin showed reticulated hyperpigmented patches without evidence of active inflammatory papules. Although previous reports have associated prurigo pigmentosa to H. Pylori gastritis, this is the first report of H. pylori organisms identified in a skin biopsy of prurigo pigmentosa.

Objective-To report the use of immunohistochemical staining for parafibromin, APC and galectin-3 to evaluate the malignant potential of the resected parathyroid specimen in a patient initially presenting with primary hyperparathyroidism secondary to four-gland hyperplasia who subsequently developed metastatic parathyroid carcinoma.Methods-We describe a patient with primary hyperparathyroidism who underwent a 3 gland resection of hypercellular parathyroids with post-operative normalization of her serum calcium and parathyroid hormone levels. She returned four years later with recurrent hypercalcemia and underwent partial resection of her remaining hypercellular gland without improvement of her hypercalcemia. Selective venous sampling localized the source as draining into her azygous vein, and she was ultimately diagnosed with metastatic parathyroid carcinoma.Results-Immunohistochemical staining for parafibromin, APC and galectin-3 suggested the malignant potential of the atypical adenoma removed during the patient's original operation, which is believed to be the source of her metastatic disease. Access to this information by the treating surgeon may have prompted a more extensive en bloc resection or more vigilant follow-up that could have altered the patient's clinical course.Conclusion-Immunohistochemical staining for parafibromin, APC and galectin-3 can be used to help distinguish the source of metastatic disease in parathyroid carcinoma. Selective venous sampling may help localize metastatic parathyroid carcinoma when it is otherwise not apparent.