In this study, we sought to determine if parathyroid neoplasm had distinct miRNA signature. We found that the miRNA expression profile by unsupervised cluster analysis showed clustering of most parathyroid carcinomas together. As expected, in the supervised cluster analysis there was complete segregation of parathyroid samples by tumor type (parathyroid adenoma versus parathyroid carcinoma). We further validated our results using RT-PCR. Approximately two-thirds (9 of 13) of the miRNA expression levels showed good correlation with the microarray data. All the validated miRNA had the same pattern of downregulation in carcinoma compared to adenoma. Furthermore, miR-126*, miR-26b and miR-30b were significantly differentially expressed between parathyroid adenoma and parathyroid carcinoma by quantitative RT-PCR. MiR-126* levels was the most accurate for differentiating between parathyroid carcinoma and parathyroid adenoma (71% sensitivity, 82% specificity).
Parathyroid carcinoma is rare and little is known about the genetic basis for it. Moreover, distinguishing between benign and malignant parathyroid tumors can be extremely difficult3, 5, 6, 18
. It is not rare for a patient with presumed benign disease to be diagnosed with parathyroid carcinoma after developing local recurrence or distant metastases19
. Some histologic criteria for identifying parathyroid carcinoma (thick capsule, fibrous bands, nuclear pleomorphism, increased mitosis, capsular invasion and vascular invasion) are subjective and leave room for misdiagnosis5
. In fact, most parathyroid carcinomas are diagnosed after recurrent disease develops. Some markers (galectin-3 and Rb overexpression, loss of CDC73/HRPT2) may be helpful for diagnosing parathyroid carcinoma. The absence of parafibromin (CDC73/HRPT2) staining has been reported to have 31% to 100% sensitivity and 41% to 100% specificity for distinguishing benign from malignant parathyroid tumors3, 20, 21
. The identification and use of adjunct markers to improve the accuracy of parathyroid carcinoma diagnosis may have several clinical implications. For example, those patients found to have histologic equivocal criteria for parathyroid carcinoma but suggestive of parathyroid carcinoma on marker analysis, for carcinoma, may have more close follow up or reoperation with more aggressive surgical resection if tumor margins are positive.
In recent years, there has been a great deal of advancement in our knowledge regarding the role of miRNA in tumorigenesis10
. However, in the current literature there are no studies that have attempted to show the miRNA signature for parathyroid carcinoma, adenoma and hyperplasia. There has been one study which has evaluated miRNA expression profiling in a limited number of samples and parathyroid tumor types. Corbetta and colleagues found 17 miRNA differential expressed between 4 parathyroid carcinoma samples compared to 2 normal parathyroid glands out of 362 miRNAs profiled22
. In contrast to our findings, unsupervised hierarchical cluster analysis did not show separate clustering of parathyroid tumor types comparing the 6 samples. The investigators validated 4 miRNAs (miR-139, miR-296, miR-222 and miR-503) to be differentially expressed in 4 parathyroid carcinoma samples compared to 2 normal parathyroid gland samples. None of the miRNAs identified to be differentially expressed in their study were differentially expressed in our study. Our study design and array platform used was different than the study by Corbetta et al. as was the goal of our study22
. We profiled a greater number of miRNAs among a greater number of parathyroid tumor types using a larger number of samples per histologic group, which were compared to reference pooled normal parathyroid glands. The strength of such an approach is it allowed us to determine if the miRNA profile was different among the different tumor types providing at least some clues to the miRNA signature of parathyroid tumor types, for what often can be a clinically and histologically heterogeneous group of tumor types. Thus, the clustering of most parathyroid carcinoma samples together by unsupervised analysis lends some support to a miRNA signature being present which is emphasized with the complete separation between parathyroid carcinoma and adenoma by supervised cluster analysis.
A formal diagnostic accuracy analysis of the significantly differentially expressed miRNAs between parathyroid adenoma and carcinoma showed one miRNA with good accuracy (miR-126*) by quantitative RT PCR. For these reasons, we believe are study results are unique and provide some insight into miRNA expression in parathyroid tumors and identify possible candidate miRNAs which may have a role in parathyroid tumorigenesis.
Again, by unsupervised clustering all but one parathyroid carcinoma grouped in two distinct clusters. For parathyroid hyperplasia and parathyroid adenoma there was no distinct pattern observed in the unsupervised clustering. One parathyroid adenoma and one parathyroid hyperplasia clustered with parathyroid carcinoma. Given the current limitations in the clinical and histologic classification of some parathyroid tumors, it is unclear if these samples were misclassified as parathyroid adenoma and hyperplasia but only long term clinical follow up will be able to help clarify this issue.
We also conducted a target search for the 13 differentially expressed miRNA between carcinoma and adenoma (FDR<0.01) looking at genes implicated in parathyroid dysregulation as potential targets. The genes of interest were CDC73/HRPT2, Rb1, Galectin 3 (Lgals 3), Ki-67 (MKI67), CCND 2, CASR or VDR3, 4, 8, 9
. Our search identified CDC73 as a potential target for miR-28, but we did not find a relationship between miR-28 and CDC73 levels using RT PCR and immunohistochemistry. We also examined the chromosomal location of the 13 miRNAs and none of the target genes and miRNAs shared a common chromosomal location. The most common reported chromosomal imbalance in parathyroid carcinoma is loss of the 1q21-q32 region which is the coding region for CDC 737
. None of our miRNAs were located in that region. Another common site for chromosomal imbalance is at 9q33-qter, this imbalance is reported in up to 24% of tumor samples23
. This region is the same chromosomal region for miR-126* (9q 34.3). Kytola and colleagues reported this region to be a region of genomic gain by comparative genomic hybridization but miR-126* (the minor strand) and miR-126 (the major strand) were both downregulated in our carcinoma samples 23
The 3 miRNAs (miR-126*, miR-30b, miR-26b) that were significantly dysregulated in carcinoma versus adenoma have been implicated in several malignancies24–29
. For example, the major strand of miR-126* has been implicated in non-small cell carcinoma30, 31
, progression of adenoma to carcinoma in colon cancer32
and some leukemia33
. Mir-126 is believed to regulate the epidermal growth factor-like domain 7 (EGFL7), which is involved in cellular migration and angiogenesis31
. Also, an upregulation in miR-126 has been shown in vitro to decrease growth rate of cell lines from lung cancer origin30
In summary, miRNAs are differentially expressed in parathyroid neoplasms. Most miRNA are downregulated in parathyroid carcinoma while in parathyroid hyperplasia most miRNA are upregulated. MiRNA profiling shows distinct differentially expressed miRNAs by tumor type which may serve as helpful adjunct to distinguish parathyroid adenoma from carcinoma.