Applying Standard Clinical Chemistry Assay Validation to Droplet Digital PCR Quantitative Liquid Biopsy Testing.

BACKGROUND: Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS: We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS: For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS: Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes.

CONTEXT: Measurement of thyroglobulin (Tg) by mass spectrometry (Tg-MS) is emerging as a tool for accurate Tg quantification in patients with anti-Tg autoantibodies (TgAbs). OBJECTIVE: The objective of the study was to perform analytical and clinical evaluations of two Tg-MS assays in comparison with immunometric Tg assays (Tg-IAs) and Tg RIAs (Tg-RIAs) in a cohort of thyroid cancer patients. METHODS: A total of 589 samples from 495 patients, 243 TgAb-/252 TgAb+, were tested by Beckman, Roche, Siemens-Immulite, and Thermo-Brahms Tg and TgAb assays, two Tg-RIAs, and two Tg-MS assays. RESULTS: The frequency of TgAb+ was 58%, 41%, 27%, and 39% for Roche, Beckman, Siemens-Immulite, and Thermo-Brahms, respectively. In TgAb- samples, clinical sensitivities and specificities of 100% and 74%-100%, respectively, were observed across all assays. In TgAb+ samples, all Tg-IAs demonstrated assay-dependent Tg underestimation, ranging from 41% to 86%. In TgAb+ samples, the use of a common cutoff (0.5 ng/mL) for the Tg-MS, three Tg-IAs, and the USC-RIA improved the sensitivity for the Tg-MSs and Tg-RIAs when compared with the Tg-IAs. In up to 20% of TgAb+ cases, Tg-IAs failed to detect Tg that was detectable by Tg-MS. In Tg-RIAs false-high biases were observed in TgAb+ samples containing low Tg concentrations. CONCLUSIONS: Tg-IAs remain the method of choice for Tg quantitation in TgAb- patients. In TgAb+ patients with undetectable Tg by immunometric assay, the Tg-MS will detect Tg in up to 20% additional cases. The Tg-RIA will detect Tg in approximately 35% cases, but a significant proportion of these will be clinical false-positive results. The undetectable Tg-MS seen in approximately 40% of TgAb+ cases in patients with disease need further evaluation.

Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline.

OBJECTIVE: The aim was to formulate clinical practice guidelines for pheochromocytoma and paraganglioma (PPGL). PARTICIPANTS: The Task Force included a chair selected by the Endocrine Society Clinical Guidelines Subcommittee (CGS), seven experts in the field, and a methodologist. The authors received no corporate funding or remuneration. EVIDENCE: This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe both the strength of recommendations and the quality of evidence. The Task Force reviewed primary evidence and commissioned two additional systematic reviews. CONSENSUS PROCESS: One group meeting, several conference calls, and e-mail communications enabled consensus. Committees and members of the Endocrine Society, European Society of Endocrinology, and Americal Association for Clinical Chemistry reviewed drafts of the guidelines. CONCLUSIONS: The Task Force recommends that initial biochemical testing for PPGLs should include measurements of plasma free or urinary fractionated metanephrines. Consideration should be given to preanalytical factors leading to false-positive or false-negative results. All positive results require follow-up. Computed tomography is suggested for initial imaging, but magnetic resonance is a better option in patients with metastatic disease or when radiation exposure must be limited. (123)I-metaiodobenzylguanidine scintigraphy is a useful imaging modality for metastatic PPGLs. We recommend consideration of genetic testing in all patients, with testing by accredited laboratories. Patients with paraganglioma should be tested for SDHx mutations, and those with metastatic disease for SDHB mutations. All patients with functional PPGLs should undergo preoperative blockade to prevent perioperative complications. Preparation should include a high-sodium diet and fluid intake to prevent postoperative hypotension. We recommend minimally invasive adrenalectomy for most pheochromocytomas with open resection for most paragangliomas. Partial adrenalectomy is an option for selected patients. Lifelong follow-up is suggested to detect recurrent or metastatic disease. We suggest personalized management with evaluation and treatment by multidisciplinary teams with appropriate expertise to ensure favorable outcomes.

Companion-diagnostic testing limited to KRAS codons 12 and 13 misses 17% of potentially relevant RAS mutations in colorectal cancer.

BACKGROUND: KRAS codons 12 and 13 mutations are commonly used to identify colorectal carcinoma (CRC) patients who are unlikely to benefit from anti-EGFR therapy. However, humans have four different homologous RAS proteins and no routine screening is performed for the other mutation sites. Non-screened mutations may still be present in a significant subset of patients without KRAS codon 12 and 13 mutations. METHODS: We developed a LightCycler screening assay that encompasses codons 12, 13 and 61 of all RAS genes. Screen-positive specimens were characterized by Sanger sequencing. 130 CRC specimens were screened for all RAS genes. The results for KRAS codons 12 and 13 were compared with an FDA approved method (Qiagen). RESULTS: Twenty-nine of 130 specimens (22.3%) were positive for KRAS codons 12 and 13, with 100% congruence with the Qiagen method. Six additional specimens were identified to have mutations. One mutation in HRAS codon 61, two in KRAS codon 61, and three in NRAS codon 61. CONCLUSION: Limiting RAS testing to only KRAS codons 12 and 13 in companion diagnostic testing of CRC results in nearly 1/5 of patients with RAS mutations not being excluded from costly EGFR antagonist treatment, despite likely futility. Inclusion of all RAS genes in companion diagnostic screening is warranted.

The Putative PAX8/PPARγ Fusion Oncoprotein Exhibits Partial Tumor Suppressor Activity through Up-Regulation of Micro-RNA-122 and Dominant-Negative PPARγ Activity.

In vitro studies have demonstrated that the PAX8/PPARγ fusion protein (PPFP), which occurs frequently in follicular thyroid carcinomas (FTC), exhibits oncogenic activity. However, paradoxically, a meta-analysis of extant tumor outcome studies indicates that 68% of FTC-expressing PPFP are minimally invasive compared to only 32% of those lacking PPFP (χ(2) = 6.86, P = 0.008), suggesting that PPFP favorably impacts FTC outcomes. In studies designed to distinguish benign thyroid neoplasms from thyroid carcinomas, the previously identified tumor suppressor miR-122, a major liver micro-RNA (miR) that is decreased in hepatocellular carcinoma, was increased 8.9-fold (P < 0.05) in all FTC versus normal, 9.2-fold in FTC versus FA (P < 0.05), and 16.8-fold (P < 0.001) in FTC + PPFP versus FTC - PPFP. Constitutive expression of PPFP in the FTC-derived cell line WRO (WRO-PPFP) caused a 5-fold increase of miR-122 expression (P < 0.05) and a striking 5.1-fold reduction (P < 0.0001) in tumor progression compared to WRO-vector cells in a mouse xenograft model. Constitutive expression of either miR-122 or a dominant-negative PPARγ mutant in WRO cells was less effective than PPFP at inhibiting xenograft tumor progression (1.8-fold [P < 0.001] and 1.7-fold [P < 0.03], respectively). PPFP-induced up-regulation of miR-122 expression was independent of its known dominant-negative PPARγ activity. Up-regulation of miR-122 negatively regulates ADAM-17, a known downstream target, in thyroid cells, suggesting an antiangiogenic mechanism in thyroid carcinoma. This latter inference is directly supported by reduced CD-31 expression in WRO xenografts expressing PPFP, miR-122, and DN-PPARγ. We conclude that, in addition to its apparent oncogenic potential in vitro, PPFP exhibits paradoxical tumor suppressor activity in vivo, mediated by multiple mechanisms including up-regulation of miR-122 and dominant-negative inhibition of PPARγ activity.

Distinct genetic changes characterise multifocality and diverse histological subtypes in papillary thyroid carcinoma.

AIMS: This study was undertaken to investigate the genetic factors underlying the development of multifocality and phenotypic diversity in multifocal papillary thyroid carcinoma (mPTC). METHODS: Loss of heterozygosity (LOH) and BRAF(V600E) mutation status were analysed in a total of 55 individual tumour foci from 18 cases of mPTC. The genetic findings and morphology of tumour foci were then compared. RESULTS: Multifocal PTC LOH rates were higher than observed previously in solitary PTC. Different patterns of LOH and BRAF(V600E) positivity separated follicular variant tumours and tumour foci from other PTC histological subtypes. In five cases, genetic alterations were detected in morphologically normal thyroid epithelium. CONCLUSIONS: These findings support the concept that multifocal PTCs develop through clonal selection from a field of pre-neoplastic cells, with morphotype differentiation correlating with specific tumour-genetic alterations. The relatively high genetic disarray in multifocal PTC may underlie their ability to spread throughout the thyroid gland.

Development and validation of a comprehensive mutation and deletion detection assay for SDHB, SDHC, and SDHD.

BACKGROUND: Lack of sequencing validation and complexity of deletion testing hinder genetic diagnosis of SDH-associated paraganglioma/pheochromocytoma. METHODS: We developed sequencing assays and multiplex ligation-dependent probe amplification (MLPA) deletion detection for SDHB, SDHC and SDHD. Clinical performance was validated on 141 blinded samples, previously tested at NIH. RESULTS: Sequencing and deletion detection were highly reproducible and agreed with previous NIH results in 99.3% and 100%, respectively. CONCLUSIONS: DNA sequencing combined with MLPA allows reliable and simplified genotyping of SDHB, SDHC and SDHD.

Evaluation of the PAX8/PPARG translocation in follicular thyroid cancer with a 4-color reverse-transcription PCR assay and automated high-resolution fragment analysis.

BACKGROUND: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. METHODS: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. RESULTS: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100-10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell-derived neoplasms. CONCLUSIONS: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

The role of the PAX8/PPARgamma fusion oncogene in the pathogenesis of follicular thyroid cancer.

When identified at early stages, most well-differentiated thyroid cancers are readily treated and yield excellent outcomes. Follicular thyroid cancer (FTC) however, when diagnosed at a late stage, may be very resistant to treatment, and exhibits 10-year survival rates less than 40%. Despite substantial progress in recent years, we still have limited understanding of the molecular and biological interrelationships between the various subtypes of benign and malignant follicular thyroid neoplasms. In contrast to the wealth of information available regarding papillary thyroid carcinoma (PTC), the triggering mechanisms of FTC development and the major underlying genetic alterations leading to follicular thyroid carcinogenesis remain obscure. Recent studies have focused on a chromosomal translocation, t(2;3) (q13;p25), fusing PAX8, a transcription factor that is essential for normal thyroid gland development, with the peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the steroid/thyroid nuclear receptor family. This chromatin rearrangement results in the expression of a PAX8/PPARgamma fusion protein, designated PPFP, whose incidence is relatively common in FTC and may represent an initiating event in the genesis of FTC. Here we review progress on the studies of PPFP that assess its involvement in FTC tumorigenesis.

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.

Procalcitonin: a marker for the diagnosis and follow-up of patients with medullary thyroid carcinoma.

CONTEXT: Calcitonin (CT) is the main medullary thyroid carcinoma (MTC) tumor marker. However, it has several limitations, including a concentration-dependent biphasic half-life, sensitivity to rapid in vitro degradation, and the presence of different isoforms/fragments. Procalcitonin (PCT), the prohormone of calcitonin, is free of these limitations but is currently used only as a sepsis marker. OBJECTIVES: The objective of the study was to determine whether PCT is suited as a MTC tumor marker by comparing the diagnostic performance of PCT with that of CT in MTC. DESIGN: PCT and CT were measured in a total of 835 subjects, including normal volunteers (n = 197) and patients with active-MTC (n = 91), cured-MTC (n = 42), neuroendocrine tumors (n = 225), mastocytosis (n = 48), follicular cell-derived thyroid carcinoma (cured = 120, persistent/recurrent = 55), and benign thyroid disease (n = 57). RESULTS: PCT levels were significantly higher in the active-MTC patients (mean 126.4 ng/ml) than the cured-MTC patients (mean <0.1 ng/ml). The overall concordance between the two markers was 95.7% (kappa = 0.81). Receiver-operating characteristic curve analysis showed no significant difference in diagnostic performance between CT and PCT. PCT's diagnostic sensitivity and specificity were 91 and 96%, respectively. The corresponding values for CT were 99 and 98%. Analyte stability studies showed that CT is very unstable in vitro with a decrease of 35-50% from the original value 24 h after the blood draw, whereas PCT levels did not significantly change during this time. CONCLUSIONS: A strong correlation was observed between PCT and CT levels in patients with MCT. Given PCT's greater analytical stability, we conclude that it represents a promising complementary MTC tumor marker.

The Role of the PAX8/PPARgamma Fusion Oncogene in Thyroid Cancer.

Thyroid cancer is uncommon and exhibits relatively low mortality rates. However, a subset of patients experience inexorable growth, metastatic spread, and mortality. Unfortunately, for these patients, there have been few significant advances in treatment during the last 50 years. While substantial advances have been made in recent years about the molecular genetic events underlying papillary thyroid cancer, the more aggressive follicular thyroid cancer remains poorly understood. The recent discovery of the PAX8/PPARgamma translocation in follicular thyroid carcinoma has promoted progress in the role of PPARgamma as a tumor suppressor and potential therapeutic target. The PAX8/PPARgamma fusion gene appears to be an oncogene. It is most often expressed in follicular carcinomas and exerts a dominant-negative effect on wild-type PPARgamma, and stimulates transcription of PAX8-responsive promoters. PPARgamma agonists have shown promising results in vitro, although very few studies have been conducted to assess the clinical impact of these agents.

Plasma chromogranin A or urine fractionated metanephrines follow-up testing improves the diagnostic accuracy of plasma fractionated metanephrines for pheochromocytoma.

CONTEXT: The initial diagnosis of pheochromocytoma relies on plasma fractionated metanephrines levels. Normal levels exclude pheochromocytoma, but positive tests have a low positive predictive value due to the disease's rarity. OBJECTIVES: The objective of the study was to evaluate three approaches to distinguish between true-positive and false-positive tests: 1) increased cutoff for plasma fractionated metanephrines, 2) measurement of serum/plasma chromogranin A (CGA), and 3) urine fractionated metanephrine testing. DESIGN: We studied retrospectively all Mayo Clinic patients with positive plasma fractionated metanephrine tests over a 15-month period and determined their final diagnosis based on histology, imaging, additional biochemical tests, and more than 1 yr follow-up. For a subgroup, urine fractionated metanephrine results were available. All original plasma samples were retested for CGA. RESULTS: Of 140 patients, 40 had a chromaffin tumor confirmed and 100 excluded, indicating a positive predictive value of plasma fractionated metanephrines of 28.6%. Increasing the threshold for a positive test improved specificity to 98% but missed eight cases (20%). Incorporation of urine fractionated metanephrine testing as follow-up test achieved 80% specificity and 91% sensitivity. The corresponding figures for CGA were 71 and 87% for all patients and 89 and 87% when patients taking proton pump inhibitors were excluded. CONCLUSIONS: Unless plasma fractionated metanephrines levels are elevated more than 4-fold above the upper limit of normal, patients with a positive plasma fractionated metanephrines test should be evaluated with urine fractionated metanephrines and serum/plasma CGA assays before being subjected to imaging or invasive diagnostic tests.

Serum thyroglobulin, high-resolution ultrasound, and lymph node thyroglobulin in diagnosis of differentiated thyroid carcinoma nodal metastases.

CONTEXT: Clinically enlarged cervical lymph nodes in patients with a history of thyroid cancer are usually assessed by fine-needle aspiration biopsy (FNAB) followed by cytology with or without tissue core. Thyroglobulin (Tg) is frequently elevated in malignant FNAB needle-wash specimens and may possibly augment or replace cytology. Furthermore, the combination of undetectable serum Tg and an innocuous ultrasound might altogether obviate the need for biopsy. OBJECTIVES: The objectives of the study were to: 1) determine an appropriate diagnostic cutoff for Tg levels in FNAB; 2) assess the diagnostic performance at this cutoff; and 3) compare serum Tg and FNAB needle-wash Tg levels to determine whether serum Tg levels predict positive Tg FNAB. DESIGN: This was a retrospective study of 122 FNAB samples in 88 athyrotic thyroid cancer patients. RESULTS: Fifty of 52 nonmalignant FNAB samples (96.2%) had Tg 1 ng/ml or less. All 70 malignant FNAB had Tg greater than 1 ng/ml. Of 103 specimens with diagnostic cytology, five (4.9%) had discordant Tg results; in four of these FNAB Tg was concordant with the final diagnosis. Eighteen of 19 (94.7%) FNAB with nondiagnostic (n = 16) or absent (n = 3) cytology were correctly classified by FNAB needle-wash Tg. Undetectable (<0.1 ng/ml) serum Tg was associated with a negative diagnosis in 21 of 23 biopsies (91.7%); the two cancer-positive samples were both serum Tg autoantibody positive and classified as suspicious by ultrasonography. CONCLUSIONS: Nodal FNAB needle-wash Tg measurements complement cytology in thyroid cancer follow-up and might substitute for it. The combination of unremarkable ultrasonography and an undetectable serum Tg in Tg autoantibody-negative patients might obviate the need for FNAB.

The paired box-8/peroxisome proliferator-activated receptor-gamma oncogene in thyroid tumorigenesis.

The American Cancer Society estimates 30,180 new cases of thyroid cancer in the United States in 2006. Of all thyroid cancers, 15-20% are follicular thyroid carcinoma (FTC), making this the second most common thyroid malignancy (after papillary carcinoma). A proportion of FTC has been found to be associated with a chromosomal translocation, t (2, 3)(q13;p25), which fuses the thyroid-specific transcription factor paired box-8 with the peroxisome proliferator-activated receptor-gamma nuclear receptor, a ubiquitously expressed transcription factor. This fusion event causes expression of a paired box-8/peroxisome proliferator-activated receptor-gamma fusion protein (PPFP). PPFP is detected in approximately 30% of FTC. In this report we review data on the role of PPFP in FTC, its mechanism of oncogenesis, and PPFP targeting as a strategy in thyroid cancer treatment.

PPARgamma staining as a surrogate for PAX8/PPARgamma fusion oncogene expression in follicular neoplasms: clinicopathological correlation and histopathological diagnostic value.

The PAX8/PPARgamma (PPFP) fusion-oncogene is moderately specific for follicular thyroid carcinomas (FTC). It remains unknown whether this can be translated into improved diagnosis, classification, or outcome prediction. We studied a cohort of well-characterized follicular adenomas (FA), FTC, and Hurthle cell carcinomas (HCC) from patients with complete clinical follow-up, to determine whether PPARgamma immunohistochemistry (as a surrogate of PAX8/PPARgamma expression) helps to distinguish FA from FTC and to assess its diagnostic accuracy as an adjunct to frozen section. We also correlated PPARgamma staining with clinical outcomes to assess its role as a prognostic marker.PPARgamma staining was more common in FTC (31 of 54; 57%) than in HCC (one of 23; 4%) or FA (four of 31; 13%) (P < 0.000001). Adjunctive use of PPARgamma immunohistochemistry improved diagnostic sensitivity of intraoperative frozen section from 84% to 96% (P < 0.05) but reduced specificity from 100% to 90% (P < 0.05). PPARgamma staining was associated with favorable prognostic indicators (female gender, better tumor differentiation, and lesser risk of metastases).PPARgamma staining may be helpful in the differential diagnosis of FA, FTC, and HCC, particularly when diagnostic sensitivity of histomorphology is reduced (e.g. during intraoperative frozen section). PPARgamma staining also shows an association with favorable prognosis and may have a role in risk stratification.

The PAX8/PPAR gamma fusion oncogene as a potential therapeutic target in follicular thyroid carcinoma.

Follicular thyroid carcinoma (FTC) accounts for approximately 20% of all thyroid cancers, and up to 40% of the deaths associated with this disease. Current treatment approaches include surgery, followed by radioactive iodine therapy. However, a significant proportion of locally advanced and metastatic FTC fails to concentrate iodine. Because traditional chemotherapeutic agents have not been shown to alter outcomes in this disease, novel therapeutic strategies are needed for advanced disease. Recently, a genomic rearrangement has been identified in up to 50% of FTC, involving a translocation event between chromosome regions 3p25 and 2q13. This translocation fuses the thyroid-specific transcription factor PAX8 gene with the PPARgamma gene, a ubiquitously expressed transcription factor. We have confirmed that this Pax8/PPARgamma fusion gene (designated PPFP) is an oncogene, which accelerates cell growth, reduces rates of apoptosis and permits anchorage independent and contact uninhibited growth of a thyroid cell line. The action of PPFP arises, at least in part, through its activity as a dominant-negative inhibitor of the wild-type PPARgamma transcription factor. Although the mechanism by which PPFP impairs PPARgamma activity remains unknown at this time, it is likely to be mediated by competition for the genomic PPARgamma response elements, the endogenous ligand, or various cofactors, including the Retinoid X Receptor (RXR). Consequently, modulation of PPFP activity might be possible through the use of PPARgamma agonists, RXR-agonists, or specific modulators of PPFP itself. Alternatively, modulation of several down-stream regulatory pathways may become possible, as the consequences of PPARgamma inhibition become better known. PPFP represents a potential novel target for the management of advanced FTC.