Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials.

PURPOSE: Knowledge 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. METHODS: The 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. RESULTS: Directing 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. CONCLUSION: We 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.

Identification of Helicobacter pylori in skin biopsy of prurigo pigmentosa.

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.

Parathyroid carcinoma arising from four-gland hyperplasia.

OBJECTIVE: To report the use of immunohistochemical staining for parafibromin, APC, and galectin-3 to evaluate the malignant potential of a resected parathyroid specimen in a patient initially presenting with primary hyperparathyroidism attributable to 4-gland hyperplasia, who subsequently developed metastatic parathyroid carcinoma. METHODS: We describe a patient with primary hyperparathyroidism who underwent a 3-gland resection of hypercellular parathyroid glands, with postoperative normalization of her serum calcium and parathyroid hormone levels. She returned 4 years later with recurrent hypercalcemia and underwent partial resection of her remaining hypercellular parathyroid gland, without improvement of her hypercalcemia. Selective venous sampling localized the source as draining into her azygos vein, and metastatic parathyroid carcinoma was ultimately diagnosed. 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 patients with parathyroid carcinoma. Selective venous sampling may help localize metastatic parathyroid carcinoma when the source is otherwise not apparent.

D2-40 lymphatic marker for detecting lymphatic invasion in thin to intermediate thickness melanomas: association with sentinel lymph node status and prognostic value-a retrospective case study.

BACKGROUND: Sentinel lymph node (SLN) status is the greatest prognostic factor of morbidity in melanoma. D2-40 antibody specifically marks lymphatic endothelium and has been used for identifying lymphatic invasion (LI) in multiple cancers. OBJECTIVE: We sought to determine the relationship between melanoma lymphatic invasion (as detected using D2-40 on primary melanoma biopsies/excisions) and the presence or absence of melanoma in subsequent SLN biopsy. METHODS: We retrospectively evaluated LI using D2-40 on primary biopsies/excisions from patients with thin to intermediate thickness (Breslow thickness: ≤2.0 mm) melanomas, who underwent lymphatic mapping and SLN biopsy, and whose SLN status was known. Sixty-four cases met the criteria and were available for analysis. We analyzed patient age, patient sex, mitotic rate, ulceration, tumor depth, and D2-40 detected LI as predictors of SLN status. RESULTS: Lymphatic invasion detection increased from 3.1% using hematoxylin and eosin only to 21.9% using D2-40. Twelve of 14 patients with D2-40 LI were SLN positive (positive predictive value, 85.7%). D2-40 LI was detected in the primary biopsy specimen of 12 of 18 patients with a positive SLN (sensitivity 66.7%). Of 50 patients without D2-40 LI, 44 were SLN negative (negative predictive value, 88.0%). Of 46 SLN-negative patients, 44 did not have D2-40 LI (specificity, 95.7%). LIMITATIONS: Results are retrospective and limited to SLN biopsy performed at one institution. CONCLUSIONS: On univariate and multivariate analysis, D2-40-detected LI was the most significant predictor of SLN status. D2-40 antibody staining to detect lymphatic invasion should be incorporated in routine melanoma biopsy evaluation.

Epithelial-mesenchymal transition markers in pancreatic ductal adenocarcinoma.

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.

Different molecular mechanisms underlie placental overgrowth phenotypes caused by interspecies hybridization, cloning, and Esx1 mutation.

To obtain a deeper insight into the genes and gene networks involved in the development of placentopathies, we have assessed global gene expression in three different models of placental hyperplasia caused by interspecies hybridization (IHPD), cloning by nuclear transfer, and mutation of the Esx1 gene, respectively. Comparison of gene expression profiles of approximately 13,000 expressed sequence tags (ESTs) identified specific subsets of genes with changed expression levels in IHPD, cloned, and Esx1 mutant placentas. Of interest, only one gene of known function and one EST of unknown function were found common to all three placentopathies; however, a significant number of ESTs were common to IHPD and cloned placentas. In contrast, only one gene was shared between IHPD and Esx1 mutant, and cloned and Esx1 mutant placentas, respectively. These genes common to different abnormal placental growth genotypes are likely to be important in the occurrence of placentopathy.