RET/PTC rearrangement occurring in primary peritoneal carcinoma.

RET/PTC rearrangements are initiating events in the development of a significant proportion of papillary thyroid carcinomas. Activated RET/PTC mutations are thought to be restricted to thyroid disease, but this study proposes that these events may also occur in nonthyroid tumors. A total of 57 nonthyroid papillary tumors were examined for RET/PTC rearrangements using interphase fluorescence in situ hybridization, Taqman reverse transcriptase polymerase chain reaction, and immunohistochemistry. Taqman single nucleotide polymorphism detection was used to analyze for expression of mutated BRAF T1799A. In all, 20% (3/15) of primary peritoneal carcinoma had detectable RET/PTC1 rearrangements by all 3 methodologies. A further case of similar histotype had an alternate RET/ PTC rearrangement. No RET/PTC1 rearrangements were detected in the remaining tumor cohort. All 57 tumors were homozygous for wild-type BRAF. The results indicate that RET/PTC rearrangements occur in a small subset of nonthyroid papillary tumors. These rearrangements may not be directly implicated in tumor growth; rather representing "passenger" mutations reflecting RET instability in secondary tumor subclones.

ret/PTC-1 expression alters the immunoprofile of thyroid follicular cells.

BACKGROUND: Hashimoto Thyroiditis (H.T.) is a destructive autoimmune thyroid condition whose precise molecular pathogenesis remains unclear. ret/PTC-1 is a chimeric transcript which has been described in autoimmune thyroid disease (AITD) and thyroid neoplasia. The purpose of this study was to observe the immunogenic effect exposure to H.T. and control lymphocyte supernatant would have on normal (Nthy-ori) and ret/PTC-1 (TPC-1) expressing thyroid cell line models. RESULTS: A 2 x 2 matrix comprising Nthy-ori and TPC-1 cell lines and H.T. and control lymphocyte supernatant was designed and utilised as follows; activated lymphocytic supernatant from a H.T. and normal control were co-cultured with a cell line derived from normal thyroid (Nthy-ori) and also a cell line derived from a papillary thyroid carcinoma that endogenously expresses ret/PTC-1 (TPC-1). The co-cultures were harvested at 0, 6 and 18 hour time points. Gene expression analysis was performed on RNA extracted from thyrocytes using TaqMan Immune profiling Low-Density Arrays (Applied Biosystems, CA, USA) comprising gene expression markers for 93 immune related targets plus 3 endogenous controls. Stimulation of the normal thyroid cell line model with activated T cell supernatant from the H.T. donor yielded global up-regulation of immune targets when compared with control supernatant stimulation. In particular, a cohort of targets (granzyme B, CD3, CD25, CD152, CD45) associated with cytotoxic cell death; T cell receptor (TCR) and T cell signaling were up-regulated in the normal cell line model. When the ret/PTC-1 expressing thyroid cell line was co-cultured with H.T. lymphocyte supernatant, in comparison to control supernatant stimulation, down-regulation of the same subset of immune targets was seen. CONCLUSION: Co-culturing H.T. lymphocyte supernatant with a normal thyroid cell line model leads to over-expression of a subset of targets which could contribute to the pathogenesis of H.T. via cytotoxic cell death and TCR signalling. Stimulation of the ret/PTC-1 positive cell line with the same stimulus led to a down-regulated shift in the gene expression pattern of the cohort of immune targets. We hypothesize that ret/PTC-1 activation may dampen immunogenic responses in the thyroid, which could possibly facilitate papillary thyroid carcinoma development.

Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials.

BACKGROUND: Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62-164 bp). RESULTS: Results from the Bioanalyzer and TaqMan data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70 degrees C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification. CONCLUSION: Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.

p16(INK4A) genetic and epigenetic profiles differ in relation to age and site in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) typically affects male smokers older than 55 years. Recently, an increase in the incidence of HNSCC in young adults has been recognized, many of them nonsmokers and females. Functional inactivation of p16 is known to be a common event in HNSCC, mainly by either deletion or methylation. A previous study by this group has shown that p16 deletions in HNSCC are significantly associated with age. The primary objective of this study was to evaluate additional molecular alterations of p16 in HNSCC, specifically in relation to age, site, and human papillomavirus (HPV) status. Patients ranging in age from 22 to 76 years with HNSCC were prospectively identified (n = 24). Methylation-specific polymerase chain reaction and immunohistochemistry were used to evaluate p16 gene inactivation and p16 protein expression, respectively. HPV 16 status was determined for each case. Overall, p16 inactivation was a frequent event detected in 46% of cases. Methylation of p16 was more often detected in females than males (P = .05). All cases showing p16 methylation were from the anterior tongue, and 75% of them were young patients. The results indicate that p16 methylation is a more common event in those younger than 40 years in contrast to p16 deletions, which are more common in those older than 40 years. Consequently, it appears that specific modes of inactivation of p16 in HNSCC are related to specific patient risk profiles. Interestingly, HPV 16 messenger RNA was detected exclusively in HNSCC from the base of tongue lesions and was only found in males. This differs from the patient profile of HNSCC in the young, which affects the anterior tongue and commonly females, thus, making it highly unlikely that this virus is a primary causative agent of HNSCC in these young adults.

Effect of BRAFV600E mutation on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model.

BACKGROUND: microRNAs (miRNAs) are a group of non-coding single stranded RNAs measuring approximately 22 nucleotides in length that have been found to control cell growth, differentiation and apoptosis. They negatively regulate target genes and have recently been implicated in tumourigenesis. Furthermore, miRNA expression profiling correlates with various cancers, with these genes thought to act as both tumour suppressors and oncogenes. Recently, a point mutation in the BRAF gene leading to a V600E substitution has been identified as the most common genetic change in papillary thyroid carcinoma (PTC) occurring in 29-69% of cases. This mutation leads to aberrant MAPK activation that is implicated in tumourigenesis. AIM: The aim of this study was to identify the effect that BRAF oncogene has on post-transcriptional regulation in PTC by using microRNA analysis. RESULTS: A unique miRNA expression signature differentiated between PTC cell lines with BRAF mutations and a normal thyroid cell line. 15 miRNAs were found to be upregulated and 23 miRNAs were downregulated. Several of these up/down regulated miRNAs may be involved in PTC pathogenesis. miRNA profiling will assist in the elucidation of disease pathogenesis and identification biomarkers and targets.

Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.

BACKGROUND: Archival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens. RESULTS: Our results show that miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR. The levels of expression of miRNA detected in total RNA extracted from FFPE were higher than that extracted from snap frozen cells when the quantity of total RNA was identical. This phenomenon is most likely explained by the fact that larger numbers of FFPE cells were required to generate equivalent quantities of total RNA than their snap frozen counterparts. CONCLUSION: We hypothesise that methylol cross-links between RNA and protein which occur during tissue processing inhibit the yield of total RNA. However, small RNA molecules appear to be less affected by this process and are recovered more easily in the extraction process. In general miRNAs demonstrated reliable expression levels in FFPE compared with snap frozen paired samples, suggesting these molecules might prove to be robust targets amenable to detection in archival material in the molecular pathology setting.

BRAF T1799A mutation occurring in a case of malignant struma ovarii.

Struma ovarii is an extremely rare tumor that occasionally undergoes malignant transformation. Because struma ovarii is composed of thyroid tissue, it is conceivable that the pathogenetic events involved in thyroid follicular transformation may take place also in struma ovarii. The authors describe a case of a classical variant of papillary thyroid carcinoma arising in a struma ovarii of a 22-year-old female. The tumor was heterozygous for BRAF T1799A mutation. No ret/ PTC-1 or ret/PTC-3 rearrangements were detected. This finding would suggest that malignant struma ovarii is similar histologically and genetically to primary papillary thyroid carcinoma.

Effect of ret/PTC 1 rearrangement on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model.

BACKGROUND: microRNAs (miRNAs) are a group of non-coding single stranded RNAs measuring approximately 22 nt in length that have been found to control cell growth, differentiation and apoptosis. miRNAs negatively regulate their target genes and recently have been implicated in tumourigenesis. Furthermore, miRNA expression profiling correlates with various cancers, with these genes thought to act as both tumour suppressors and oncogenes. ret/PTC 1 is an oncogene with constitutive kinase activity implicated in the development of papillary thyroid carcinoma (PTC). This rearrangement leads to aberrant MAPK activation that is implicated in PTC tumourigenesis. AIM: The aim of this study was to identify the effect that ret/PTC 1 has on transcription and post-transcriptional regulation in PTC by using DNA microarray and microRNA analysis. RESULTS: DNA microarray analysis revealed a group of genes differentially expressed between normal thyroid cell lines and those harbouring a ret/PTC 1 rearrangement.Furthermore, a unique miRNA expression signature differentiated between PTC cell lines with ret/PTC 1 and a normal thyroid cell line. 21 miRNAs showed significant overexpression and 14 miRNAs showed underexpression in these cell lines when compared to normal thyroid. Several of these up/down regulated miRNAs may be involved in PTC pathogenesis.

Array comparative genomic hybridisation analysis of gamma-irradiated human thyrocytes.

The susceptibility of thyroid epithelium to radiation-induced carcinogenesis is well recognised. In this context, thyroid carcinogenesis is associated with specific somatic ret/papillary thyroid carcinoma (PTC) rearrangements and morphologically with the papillary phenotype. Previous studies have demonstrated the possibility of inducing ret rearrangements in vitro using X-rays. The purpose of our study was to assess whether gamma (gamma) radiation using a Caesium 137 source can induce specific ret rearrangements in a human thyroid epithelial cell culture model. We further hypothesised that if radiation-induced thyroid carcinogenesis is associated with non-random rearrangement events, then DNA copy gain and loss induced by irradiation may also occur in a non-random manner. We irradiated SV40-immortalised human thyroid epithelial cells with incremental doses of gamma-radiation and, using TaqMan reverse-transcription polymerase chain reaction, looked for the presence of the common ret rearrangements. Cohorts showing evidence of ret/PTC chimeric transcripts were further analysed using microarray comparative genomic hybridisation (CGH) to detect copy gain and loss associated with radiation. Four Grays of gamma-radiation was sufficient to induce ret/PTC-3. In this model, transcripts of ret/PTC-1 were not detected, and we suggest that the type of radiation may influence the resulting rearrangement that occurs. Using array CGH, we have demonstrated a predominant pattern of subtelomeric deletions occurring in association with this radiation cohort and raise the possibility that chromosome 10 may be a hotspot for radiation-induced damage for as yet unknown reasons.

A molecular expression signature distinguishing follicular lesions in thyroid carcinoma using preamplification RT-PCR in archival samples.

Follicular variant of papillary thyroid carcinoma is a lesion that frequently causes difficulties from a diagnostic perspective in the laboratory. The purpose of this study was to interrogate a cohort of archival thyroid lesions using gene expression analysis of a panel of markers proposed to have utility as adjunctive markers in the diagnosis of thyroid neoplasia and follicular variant of papillary thyroid carcinoma in particular. Laser Capture Microdissection was used to procure pure cell populations for extraction. In addition a novel, multiplex preamplification technique was used to facilitate analysis of multiple targets. The panel comprised: HLA-DMA, HLA-DBQ1, CD74, CSNK1G2, IRF3, KRAS2, LYN, MT1K, MT1X, RAB23, TGFB1 and TOP2A, with CDKN1B as an endogenous control. Expression profiles for each target were generated using TaqMan Real-Time PCR. HLA-DMA, HLA-DQB1, MT1X, CSNK1G2 and RAB23 were found to be differentially expressed (P<0.05) when comparing follicular adenoma and follicular variant of papillary thyroid carcinoma. Comparison of follicular adenoma and follicular thyroid carcinoma groups showed significant differential expression for MT1K, MT1X and RAB23 (P<0.05). Comparison of the papillary thyroid carcinoma group (classic and follicular variants) and the follicular adenoma group showed differential expression for CSNK1G2, HLA-DQB1, MT1X and RAB23 (P<0.05). Finally, KRAS2 was found to be differentially expressed (P<0.05) when comparing the papillary thyroid carcinoma and follicular thyroid carcinoma groups. This panel of molecular targets discriminates between follicular adenoma, papillary thyroid carcinoma, follicular variant of papillary thyroid carcinoma and follicular thyroid carcinoma by their expression repertoires. It may have utility for broader use in the setting of fine-needle aspiration cytology and could improve the definitive diagnosis of certain categories of thyroid malignancy.

Distinct array comparative genomic hybridization profiles in oral squamous cell carcinoma occurring in young patients.

BACKGROUND: Oral cancer typically affects smokers older than 50 years of age. Recently, however, a marked increase in the number of patients 40 years old and younger, many with no history of tobacco smoking, has been noted. Studies in this age group have so far been restricted to genomic areas well recognized as abnormal in typical patients with oral cancer. The aim of this study was to assess genomic aberrations in oral cancer, using comparative genomic hybridization (CGH) microarray technology, and to compare the genomic aberration profile of patients older than 40 years old with those 40 years old and younger. METHODS: Tumor samples from 20 patients with oral cancer (age range, 21-78; 10 smokers and 10 nonsmokers) were laser microdissected, and array CGH was used to identify genomic imbalances in these two cohorts. RESULTS: The older cohort showed high numbers of gains and losses in contrast to very few copy number changes in the younger nonsmoker cohort. In concurrence with the literature, tumors from the older cohort manifested deletions involving 3p and 9p21 and gains involving 3q, 5q, 7p, 8q, 11q, and 20q. The younger group, particularly the nonsmokers, showed very few changes overall, and the aberrations were not in the sites classically associated with oral cancer. Deletion of CDKN2A (p16) was completely absent in the younger group but was present in 50% of the older cohort. CONCLUSIONS: We have demonstrated that there is far less genomic instability in young nonsmokers with oral cancer than found in typical patients with oral cancer. These observations indicate that oral cancer presenting at a younger age, particularly in nonsmokers, has a genomic profile different from the classically described oral cancer.