Detection of BRAF mutation in the cytocentrifugation supernatant fluid from fine-needle aspiration of thyroid lesions may enhance the diagnostic yield.

OBJECTIVE: BRAF mutations using cellular DNA from fine-needle aspiration (FNA) specimens are commonly used to support the diagnosis of papillary thyroid carcinoma (PTC). The goal of this study was to preliminarily evaluate the diagnostic utility of detecting BRAF mutations in the routinely discarded FNA specimen supernatant fluid. MATERIALS AND METHODS: Seventy-eight FNAs of thyroid lesions were evaluated for BRAF mutations using both cellular and supernatant DNA. BRAF mutation data were correlated with cytology and surgical pathology. RESULTS: Of the 78 samples evaluated, 68 (87%) had amplifiable DNA in the supernatant with 2 (3%) positive for BRAF mutations. These two samples showed no mutations in the cellular counterpart. Among the 11 samples showing morphologic findings (FNA/surgical pathology) suspicious/diagnostic of PTC, 6 (55%) samples (one supernatant and five cellulars) were positive for BRAF mutations. This suggests that testing supernatant DNA in FNA specimens may increase the diagnostic yield by 1/11 (9%) in this setting. CONCLUSIONS: The vast majority of routinely discarded FNA supernatants contain amplifiable DNA. In addition, profiling the mutations of BRAF and other genes using supernatant DNA may provide valuable diagnostic information to assist the diagnosis of PTC in patients with clinical/morphologic findings suspicious for malignancies and cellular DNA showing no mutations.

Portal Venous Blood Circulation Supports Immunosuppressive Environment and Pancreatic Cancer Circulating Tumor Cell Activation.

OBJECTIVES: Aggressive spread and liver metastases are predominant features of pancreatic ductal adenocarcinoma (PDAC). This study investigates activation of PDAC circulating tumor cells (CTC) and immunosuppression in the portal venous system. METHODS: Portal venous and peripheral blood were collected during pancreaticoduodenectomy from patients with PDAC (n = 21) or other non-PDAC pancreatic conditions (n = 20). Circulating tumor cells were isolated by fluorescence-activated cell sorting and characterized for messenger RNA (mRNA) expression and acetylated chromatin encoding K-RAS exon 12 mutation (K-RASmut). Myeloid-derived suppressor cells (MDSC) were identified using flow cytometry. RESULTS: Pancreatic ductal adenocarcinoma K-RASmut mRNA expression in portal venous blood CTC was significantly elevated compared with preoperative and postoperative peripheral blood (P = 0.0123 and P = 0.0246, respectively). There was no significant variation in total CTC numbers between portal and peripheral blood.Portal venous M-MDSC were elevated compared with peripheral blood in PDAC patients (P = 0.0065). M-MDSC increases correlated with K-RASmut mRNA-expressing CTC present in PDAC portal blood (P < 0.0001). CONCLUSIONS: Association of MDSC with active CTC in portal venous blood may support immunosuppression within the portal venous circulation to promote PDAC CTC survival.

Get in the "groove" with new molecular technology.

The advantages of MGB real-time PCR technology are: use of short, or highly conserved, specific sequence; specific primers and non-cleavable probes; post-PCR melt-curve analysis; universal cycling conditions; open platform; compatible with most real-time PCR instruments; unsurpassed sensitivity; multiplexing with a rich proprietary dye set; and the ability to tailor designs to detect most mutations.

Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer.

Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection System (PE Applied Biosystems). Losses in the sequences D22S274 (22q13.31) and in the DIA1 (22q13.2-13.31) gene were detected in 10 out of 40 cases (25%) each. Statistically significant correlations were observed for patients with relative copy number loss of the marker D22S274 and stages T3-T4 of disease (P=0.025), family history of cancer (P=0.001), and death (P=0.021). Relative copy number loss involving the DIA1 gene was correlated to family history of cancer (P<0.001), death (P=0.002), and consumption of alcohol (P=0.026). Log-rank test revealed a significant decrease in survival (P=0.0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer.

Early hematopoietic reconstitution after clinical stem cell transplantation: evidence for stochastic stem cell behavior and limited acceleration in telomere loss.

Our inability to purify hematopoietic stem cells (HSCs) precludes direct study of many aspects of their behavior in the clinical hematopoietic stem cell transplantation (HSCT) setting. We indirectly assessed stem/progenitor cell behavior in the first year after HSCT by examining changes in neutrophil telomere length, X-inactivation ratios, and cycling of marrow progenitors in 25 fully engrafted allogeneic HSCT recipients. Donors were sampled once and recipients at engraftment and 2 to 6 months and 12 months after HSCT. Telomere length was measured by an in-gel hybridization technique, X-inactivation ratios were measured by the human androgen receptor assay, and cell cycle status was determined by flow cytometric analysis of pyronin Y- and Hoechst 33342-stained CD34(+)CD90(+) and CD34(+)CD90(-) marrow cells. Compared with their donors, recipients' telomeres were shortened at engraftment (-424 base pairs [bp]; P <.0001), 6 months (-495 bp; P =.0001) after HSCT, and 12 months after HSCT (-565 bp; P <.0001). There was no consistent pattern of change in telomere length from 1 to 12 months after HSCT; marked, seemingly random, fluctuations were common. In 11 of 11 informative recipients, donor X-inactivation ratios were faithfully reproduced and maintained. The proportion of CD34(+)CD90(+) progenitors in S/G(2)/M was 4.3% in donors, 15.7% at 2 to 6 months (P <.0001) after HSCT, and 11.5% at 12 months after HSCT (P <.0001, versus donors; P =.04, versus 2-6 months). Cycling of CD34(+) CD90(-) progenitors was largely unchanged. We infer that (1) HSCT-induced accelerated telomere loss is temporary and unlikely to promote graft failure or clonal hematopoietic disorders and (2) the striking fluctuations in telomere length and variation in pattern of telomere loss reflect stochastic determination of HSC fate after HSCT.