Role of endobronchial ultrasound in diagnosis and molecular assessment of metastatic melanoma.

BACKGROUND AND OBJECTIVE: Vemurafenib is a new inhibitor of the mutated BRAF oncogene. In the presence of mutated BRAF in metastatic melanoma, treatment with vemurafenib leads to a reduction in mortality and in tumour progression when compared with chemotherapy. This study describes nine cases in which endobronchial ultrasound (EBUS) guided transbronchial needle aspiration (TBNA) was used to assess mediastinal and hilar lymph nodes for the presence of metastatic melanoma and demonstrates the ability to detect mutations in BRAF on the tissue obtained. METHODS: A retrospective review was performed of all patients who had a history of melanoma and underwent EBUS TBNA to investigate hilar or mediastinal lymphadenopathy for the presence of metastatic melanoma. RESULTS: In seven of the nine cases, metastatic melanoma was confirmed on cytology. The two negative cases were proven to be true negatives by follow up or by demonstrating an alternate diagnosis. In five cases, analysis for BRAF mutation was performed. Four cases were positive for mutation and one demonstrated wild-type BRAF. CONCLUSIONS: Tissue samples obtained from EBUS TBNA are adequate to diagnose metastatic melanoma in hilar and mediastinal lymph nodes and to detect the presence or absence of mutations in the BRAF gene. Our findings suggest that close collaboration between bronchoscopists and pathologists will be needed to implement BRAF testing in routine practice in the era of targeted therapy.

Primary mediastinal seminoma: a comprehensive assessment integrated with histology, immunohistochemistry, and fluorescence in situ hybridization for chromosome 12p abnormalities in 23 cases.

Accurate diagnosis of mediastinal seminoma is critical because of its favorable response to radiation therapy and/or cisplatin-based chemotherapy. Immunohistochemical staining for OCT4 has recently been validated as a powerful tool for detecting gonadal seminoma. However, discrepancies between the genetic alterations and immunoprofiles of mediastinal and testicular seminomas have been reported, raising the question of whether techniques that are useful in the diagnosis of gonadal seminoma are applicable to its mediastinal counterpart. The present study was conducted to evaluate the morphologic and immunohistochemical characteristics and chromosomal abnormalities of 12p in 23 primary mediastinal seminomas and to compare their applicability as diagnostic tools. Dual-color fluorescence in situ hybridization (FISH) analyses for chromosome 12p and immunostains for OCT4, c-kit, placental-like alkaline phosphatase, CD30, and a panel of cytokeratins, including cytokeratin AE1/AE3 (AE1/3), high molecular weight cytokeratin (34betaE12, HMWCK), CAM5.2, cytokeratin 7 (CK7), cytokeratin 20 (CK20), and epithelial membrane antigen were performed. Lymphocytic infiltration was found in all 23 cases (100%). The incidence of other histologic characteristics were as follows: fibrous septa/stroma (21 cases, 91%), prominent tumor cell nucleoli (21 cases, 91%), clear tumor cell cytoplasm (20 cases, 87%), distinct tumor cell borders (20 cases, 87%), granulomatous inflammation (17 cases, 74%), cellular pleomorphism (10 cases, 43%), necrosis (8 cases, 35%), prominent cystic change (2 cases, 8%), intercellular edema (1 case, 4%), and syncytiotrophoblasts (1 case, 4%). The mean mitotic count was 4.4 (range 0 to 16) per 10 high-power fields. Moderate to strong nuclear OCT4 staining was identified in all 23 cases (100%). Seventeen tumors (74%) showed membranous expression of c-kit, with variable staining intensity and percentages. Weakly to moderately intense immunostaining for placental-like alkaline phosphatase was identified in 10 cases (43%) with occasional background staining artifact. The incidences of positive staining were 43% for AE1/3, 39% for HMWCK, 48% for CAM5.2, 39% for CK7, and 9% for epithelial membrane antigen, respectively. In most cases, these epithelial markers highlighted only a small proportion of tumor cells with variable intensities. Immunostaining for CD30 and CK20 was completely negative in all seminomas. Twenty-two seminomas (96%) revealed chromosome 12p abnormalities, including 12p amplification in 20 cases (87%) or i(12p) in 15 cases (65%). Lymphocytic infiltration is the most common histologic feature observed in primary mediastinal seminoma and both OCT4 immunostain and FISH for 12p abnormalities can be very helpful in diagnosing mediastinal seminoma. The intense staining pattern of OCT4 and the high sensitivity of FISH make them superior to other auxiliary diagnostic utilities for detecting seminoma. In addition, the incidences of cytokeratin expression of primary mediastinal seminoma are similar to those of its gonadal counterpart and pathologists must exercise caution in the interpretation of epithelial markers in mediastinal neoplasms.

Expression of hMLH1 and hMSH2 and assessment of microsatellite instability in testicular and mediastinal germ cell tumours.

The aim of this study was to investigate DNA mismatch repair deficiency in male germ cell tumours. We analysed the expression of two mismatch repair proteins, human mutL homologue 1 (hMLH1) and human mutS homologue 2 (hMSH2), and evaluated the frequency of microsatellite instability with 10 mononucleotide and two dinucleotide repeat sequences, in 39 paired tumour/normal DNA samples obtained from 17 testicular and two mediastinal germ cell tumours. In all 19 cases, hMLH1 and hMSH2 both showed nuclear immunolocalization in invasive and testicular in-situ tumours. In non-neoplastic seminiferous tubules, hMLH1 was expressed only in premeiotic germ cells, while hMSH2 was seen in all stages of spermatogenesis. Genetic analysis of dinucleotide markers revealed loss of heterozygosity in one of two testicular yolk sac tumours at D18S58 and an allelic shift at D2S123 in two of three testicular embryonal carcinomas, while none of the 12 seminomas exhibited a genetic abnormality at these loci. No abnormalities were demonstrated with the 10 mononucleotide markers. The two mediastinal germ cell tumours showed no genetic instability or allelic loss with all 12 markers. We suggest that genetic alterations as assessed by microsatellite analysis in germ cell tumours may reflect tissue maturation and phenotypic differentiation rather than tumour progression. In addition, we suggest that hMLH1 and hMSH2 genes may not be implicated in the genesis of germ cell tumours.