Validation of the Cut-points Recommended for ROMA Using the Roche Elecsys CA125 and HE4 Assays.

Assessing the risk of malignancy in patients with a pelvic mass helps triage women suspected of ovarian cancer to specialized gynecologic oncologists to improve treatment outcomes. To this end, several algorithms have been proposed; most notably, the Risk of Ovarian Malignancy Algorithm (ROMA) based on CA125, HE4, and menopausal status. However, appropriate decision cut-points for the ROMA score depends on the choice of analytical assays used. This study validates the use of the Roche Elecsys CA125 and HE4 assays for ROMA calculation in a cohort of 207 women who presented to Mayo Clinic with a pelvic mass. Results were compared to a definitive histologic diagnosis in each case. Clinical performance of ROMA scores derived using these assays was similar to stated claims and indicates that recommended cut-points are acceptable for clinical use.

Development and characterization of a differentiated thyroid cancer cell line resistant to VEGFR-targeted kinase inhibitors.

BACKGROUND: Vascular endothelial growth factor-targeted kinase inhibitors have emerged as highly promising therapies for radioiodine-refractory metastatic differentiated thyroid cancer. Unfortunately, drug resistance uniformly develops, limiting their therapeutic efficacies and thereby constituting a major clinical problem. APPROACH AND METHODS: To study acquired drug resistance and elucidate underlying mechanisms in this setting, BHP2-7 human differentiated thyroid cancer cells were subjected to prolonged continuous in vitro selection with 18 µM pazopanib, a clinically relevant concentration; acquisition of pazopanib resistance was serially assessed, with the resulting resistant cells thereafter subcloned and characterized to assess potential mechanisms of acquired pazopanib resistance. RESULTS: Stable 2- to 4-fold in vitro pazopanib resistance emerged in response to pazopanib selection associated with similar in vitro growth characteristics but with markedly more aggressive in vivo xenograft growth. Selected cells were cross-resistant to sunitinib and to a lesser extent sorafenib but not to MAPK kinase (MEK1/2) inhibition by GSK1120212. Genotyping demonstrated acquisition of a novel activating KRAS codon 13 GGC to GTT (glycine to valine) mutation, consistent with the observed resistance to upstream vascular endothelial growth factor receptor inhibition yet sensitivity to downstream MAPK kinase (MEK1/2) inhibition. CONCLUSIONS: Selection of thyroid cancer cells with clinically utilized therapeutics can lead to acquired drug resistance and altered in vivo xenograft behavior that can recapitulate analogous drug resistance observed in patients. This approach has the potential to lead to insights into acquired treatment-related drug resistance in thyroid cancers that can be subjected to subsequent validation in serially collected patient samples and that has the potential to yield preemptive and responsive approaches to dealing with this important clinical problem.

RNA sequencing identifies multiple fusion transcripts, differentially expressed genes, and reduced expression of immune function genes in BRAF (V600E) mutant vs BRAF wild-type papillary thyroid carcinoma.

CONTEXT: The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. OBJECTIVE: RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. DESIGN: BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. PATIENTS: BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. RESULTS: RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. CONCLUSION: BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.

Succinate dehydrogenase gene mutations are strongly associated with paraganglioma of the organ of Zuckerkandl.

Organ of Zuckerkandl paragangliomas (PGLs) are rare neuroendocrine tumors that are derived from chromaffin cells located around the origin of the inferior mesenteric artery extending to the level of the aortic bifurcation. Mutations in the genes encoding succinate dehydrogenase subunits (SDH) B, C, and D (SDHx) have been associated with PGLs, but their contribution to PGLs of the organ of Zuckerkandl PGLs is not known. We aimed to describe the clinical presentation of patients with PGLs of the organ of Zuckerkandl and investigate the prevalence of SDHx mutations and other genetic defects among them. The clinical characteristics of 14 patients with PGL of the organ of Zuckerkandl were analyzed retrospectively; their DNA was tested for SDHx mutations and deletions. Eleven out of 14 (79%) patients with PGLs of the organ of Zuckerkandl were found to have mutations in the SDHB (9) or SDHD (2) genes; one patient was found to have the Carney-Stratakis syndrome (CSS), and his PGL was discovered during surgery for gastrointestinal stromal tumor. Our results show that SDHx mutations are prevalent in pediatric and adult PGLs of the organ of Zuckerkandl. Patients with PGLs of the organ of Zuckerkandl should be screened for SDHx mutations and the CSS; in addition, asymptomatic carriers of an SDHx mutation among the relatives of affected patients may benefit from tumor screening for early PGL detection.

Mutant BRAF(T1799A) can be detected in the blood of papillary thyroid carcinoma patients and correlates with disease status.

CONTEXT: The BRAF(T1799A) transversion is the most frequent morphotype-specific somatic mutation in papillary thyroid carcinoma (PTC). The ability to detect this mutation in the circulation could aid in diagnosis and follow-up of PTC patients. OBJECTIVE: Our objective was to develop and clinically validate a sensitive and specific assay for the detection of BRAF(T1799A) in blood samples from PTC patients. DESIGN: We developed an allele-specific real-time PCR method for the detection of BRAF(T1799A) in blood samples and studied prospectively blood samples from 193 patients with thyroid cancer (173 PTC, 20 non-PTC) attending for routine follow-up. The results of molecular testing were correlated with disease status and thyroglobulin measurements. BRAF(T1799A) status of the original tumor samples was also confirmed, where available. RESULTS: The assay had a detection sensitivity of fewer than one heterozygote BRAF(T1799A)-carrying cell per 100,000 diploid cells, without detectable cross-reactivity between wild-type BRAF and BRAF(T1799A). Circulating BRAF(T1799A) was detected in 20 of 173 PTC patients and in none of the 20 non-PTC patients. BRAF(T1799A)-positive samples contained between one in 326 and fewer than one in 100,000 copies of BRAF(T1799A). Tissue BRAF status correlated with blood BRAF status, whereas BRAF(T1799A) positivity in blood correlated with the presence of active disease at the time of the blood draw, with eight of the 38 PTC patients with persistent/recurrent disease being positive for circulating BRAF(T1799A) (relative risk vs. circulating BRAF(T1799A)-negative, 2.55; P < 0.04). CONCLUSIONS: BRAF(T1799A) can be detected in the blood of PTC patients with residual or metastatic disease and may provide diagnostic information.