Optimizing detection of RET and PPARg rearrangements in thyroid neoplastic cells using a home-brew tetracolor probe.

BACKGROUND: Fluorescence in situ hybridization (FISH) to identify specific DNA target sequences in the nuclei of nondividing cells of numerous solid neoplasms has contributed to the introduction of molecular cytogenetics as a useful adjunct to cytology, leading recently to the "marriage" of the 2 disciplines. Numerous cancer molecular markers can now be investigated using different technical approaches, at both the gene and expression levels, in biopsies of various suspected cancers, including differentiated thyroid carcinoma. The limited amount of bioptic material is often insufficient to carry out multiple tests, and optimizing handling of the biopsy is desirable. METHODS: We have developed a home-brew tetracolor break-apart probe able to simultaneously identify the 2 most common genetic alterations in differentiated thyroid carcinoma: RET/PTC variants in papillary thyroid carcinoma and PAX8/PPARg fusion and variants in follicular thyroid carcinoma. RESULTS: The probe had 100% specificity, 99.5% sensitivity, and ≥ 3% cutoff. The probe was tested on RET/PTC and PAX8/PPARg RT-PCR positive controls, and feasibility was assessed in 368 thyroid nodule fine-needle aspirations (FNA). In the latter analysis, 24 FNAs had split RET signal, and 9 had split PPARg signal. FISH analysis of available surgically removed nodules confirmed the sensitivity of FISH in detecting abnormal clones and oligoclones. CONCLUSIONS: The home-brew tetracolor probe showed high feasibility, optimizing the use of the biological material in relation to the available molecular tests and maximizing the FISH experimental and slide-scoring times. This probe may be considered an alternative to RT-PCR when recovery and quality of RNA amplification from FNA are insufficient.

Assessing RET/PTC in thyroid nodule fine-needle aspirates: the FISH point of view.

RET/PTC rearrangement and BRAF(V600E) mutation are the two prevalent molecular alterations associated with papillary thyroid carcinoma (PTC), and their identification is increasingly being used as an adjunct to cytology in diagnosing PTC. However, there are caveats associated with the use of the molecular approach in fine-needle aspiration (FNA), particularly for RET/PTC, that should be taken into consideration. It has been claimed that a clonal or sporadic presence of this abnormality in follicular cells can distinguish between malignant and benign nodules. Nevertheless, the most commonly used PCR-based techniques lack the capacity to quantify the number of abnormal cells. Because fluorescence in situ hybridization (FISH) is the most sensitive method for detecting gene rearrangement in a single cell, we compared results from FISH and conventional RT-PCR obtained in FNA of a large cohort of consecutive patients with suspicious nodules and investigated the feasibility of setting a FISH-FNA threshold capable of distinguishing non-clonal from clonal molecular events. For this purpose, a home brew break-apart probe, able to recognize the physical breakage of RET, was designed. While a ≥3% FISH signal for broken RET was sufficient to distinguish nodules with abnormal follicular cells, only samples with a ≥6.8% break-apart FISH signal also exhibited positive RT-PCR results. On histological analysis, all nodules meeting the ≥6.8% threshold proved to be malignant. These data corroborate the power of FISH when compared with RT-PCR in quantifying the presence of RET/PTC in FNA and validate the RT-PCR efficiency in detecting clonal RET/PTC alterations.

Trisomy 17 as a marker for a subset of noninvasive thyroid nodules with focal features of papillary carcinoma: cytogenetic and molecular analysis of 62 cases and correlation with histological findings.

CONTEXT: Differentiated carcinomas of the thyroid are divided into follicular thyroid carcinoma and papillary thyroid carcinoma (PTC), based on their propensity to invade and their cytological features [papillary carcinoma-type nuclear changes (PTC-NCs)]. PTC typically exhibits a diploid karyotype sometimes with inv10(q11.2q21.2), leading to rearranged RET gene. Follicular thyroid carcinomas are often aneuploid and may exhibit t(2;3)(q13;p25), resulting in PAX8-PPARgamma1 gene fusion. Isolated trisomy 17 has rarely been reported in thyroid lesions, and its significance is unknown. OBJECTIVE/DESIGN: Our objective was to determine whether isolated trisomy 17 corresponds to a specific histological or molecular thyroid tumor subset. Nine cases with isolated trisomy 17 were critically reviewed and investigated for RAS and BRAF mutations and for RET and PAX8-PPARgamma1 rearrangements. RESULTS: All nine cases were noninvasive, exhibited follicular growth pattern, and showed PTC-NCs focally defined within the nodule: four were PTCs follicular variant within larger tumors, and five were follicular-patterned nodules with incomplete cytological features of papillary carcinoma (variable proportion of cells with PTC-NCs scattered inside the lesion). RAS, BRAF V600E mutation, RET or PAX8-PPARgamma1 rearrangements were not identified. One case had BRAF K601E mutation. Only two of the 53 control cases showed focal PTC-NCs. CONCLUSIONS: Isolated trisomy 17 is associated with focal papillary carcinoma changes in follicular-patterned thyroid nodules and may be a marker for this subset of thyroid lesions that are often difficult to classify.

Aneuploidy in oncocytic lesions of the thyroid gland: diffuse accumulation of mitochondria within the cell is associated with trisomy 7 and progressive numerical chromosomal alterations.

Oncocytic cells are characterized by a greatly increased number of mitochondria that distend the cell cytoplasm and result in a distinctive granular appearance of the cell on conventional histology sections. Oncocytes are frequently found in metabolically active human tissues including the thyroid gland, and, as a general rule, when their proportion in a thyroid tumor is greater than 75% the tumor is referred to as oncocytic (Hürthle cell) adenoma or carcinoma. Such tumors represent a subset of thyroid lesions, and recently, both interphase fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) studies reported that they may show aneuploidy, with widespread numerical chromosomal alterations. In contrast, very few cases have been studied by conventional cytogenetic analysis. Whether the cells with chromosomal changes are the same as those with mitochondrial accumulation or whether lesions only partially composed of oncocytic cells also have cytogenetic alterations is unclear. To investigate the relationship between acquisition of the oncocytic phenotype and numerical chromosomal changes, we analyzed a random selection of thyroid lesions with (18 cases) and without (11 cases) morphological evidence of oncocytic differentiation. Lesions with oncocytes included hyperplastic nodules, adenomas, Hürthle cell tumors, and papillary carcinomas with lymphocytic stroma (Whartin-like tumors of the thyroid). Karyotypic changes were analyzed by cytogenetic analysis, FISH, or CGH, and the results were compared with in situ analysis of mitochondrial accumulation by immunofluorescence. A striking correlation between the presence of oncocytes and the presence of aneuploid katyotypes was seen in the oncocytic follicular thyroid nodules, but not in the oncocytic papillary tumors. Structural chromosome changes or normal karyotypes were observed in the lesions lacking oncocytic features. Extending the FICTION technique to the evaluation of a cytoplasmic antigen (mitochondrial membrane antigen), we pursued the simultaneous visualization of both mitochondrial increase and numerical chromosomal alterations, and showed that oncocytes of follicular lesions are prone to become aneuploid. Our data support the contention that follicular tumors composed of oncocytes should be regarded as a distinct subset.

Topographical localization of glial cell line-derived neurotrophic factor in the human brain stem: an immunohistochemical study of prenatal, neonatal and adult brains.

As a step towards the identification of the neuronal populations responsive to glial cell line-derived neurotrophic factor (GDNF) in the human nervous system and their changes with age, this study reports on the immunohistochemical localization of the protein GDNF in the autoptic normal human brain stem of pre- and full-term newborns and adult subjects. Two different anti-GDNF polyclonal antibodies were used. Western blot analysis on homogenates of human and rat brain and recombinant human GDNF resulted in differential detection of monomeric and dimeric forms of the proteins. The ABC immunohistochemical technique on cryostat tissue sections showed an uneven distribution of GDNF-like immunoreactive nerve fibers and terminals and neuronal cell bodies. Immunoreactive elements were mainly localized to the spinal trigeminal, cuneate, solitary, vestibular, and cochlear sensory nuclei, dorsal motor nucleus of the vagus nerve, ventral grey column, hypoglossal nucleus, dorsal and ventrolateral medullary reticular formation, pontine subventricular grey and locus coeruleus, lateral regions of the rostral pontine tegmentum, tectal plate, trochlear nucleus, dorsal and median raphe nuclei, caudal and rostral linear nuclei, cuneiform nucleus, and substantia nigra. Comparison between pre- and full-term newborns and adult subjects revealed changes with age in density of positive innervation and frequency of immunoreactive perikarya. The results obtained provide detailed information on the occurrence of GDNF-like immunoreactive neurons in the human brain stem and suggest that the protein is present in a variety of neuronal systems, which subserve different functional activities, at developmental ages and in adult brains.