Papillary microcarcinoma with metastases showing faint (grade 1) p27 expression at ×20 magnification (A) and ×40 magnification (B).
Papillary microcarcinoma with metastases showing moderate (grade 2) p27 expression at ×20 magnification (A) and ×40 magnification (B).
Incidentally discovered papillary microcarcinoma with no evidence of metastasis showing strong (grade 3) p27 expression at ×20 magnification (A) and ×40 magnification (B).
Khoo MLC, Freeman JL, Witterick IJ, Irish JC, Rotstein LE, Gullane PJ, Asa SL. Underexpression of p27/Kip in Thyroid Papillary Microcarcinomas With Gross Metastatic Disease. Arch Otolaryngol Head Neck Surg. 2002;128(3):253–257. doi:10.1001/archotol.128.3.253
Papillary microcarcinomas (PMCs) of the thyroid (measuring less than 1 cm in maximum dimension) are extremely common incidental histologic findings, and most of these tumors are not considered clinically significant. However, rare PMCs behave aggressively and metastasize early, giving rise to clinically significant metastatic disease. We hypothesized that p27 and MIB-1/Ki-67 immunoreactivity would allow us to identify this small subgroup of PMCs that have the potential to behave aggressively.
We reviewed the histopathology reports of 2000 patients who underwent thyroid surgery at our institution between 1995 and 1999 and identified 22 patients who presented with gross regional metastases from a primary PMC. The primary and metastatic tumors were stained for ret, p53, p27, and MIB-1 using the avidin-biotin-peroxidase complex technique. A control group of 33 nonmetastasizing PMCs was also analyzed.
Immunoreactivity for ret, p53, and MIB-1 showed no difference between metastasizing and nonmetastasizing PMCs. In most tumors, ret was present, while p53 immunoreactivity was absent in all tumors. MIB-1 staining was present in a small number of cells in both groups of tumors. Immunoreactivity for p27 was quantitated by the intensity of expression as well as the distribution of positive cells within each tumor. All tumors showed lower p27 expression than normal thyroid tissue. However, metastasizing PMCs demonstrated a significantly lower expression of p27 than nonmetastasizing PMCs (P<.001).
Our results suggest that p27 immunohistochemical analysis may be a valuable diagnostic tool in predicting aggressive potential in PMCs.
PAPILLARY microcarcinomas (PMCs) of the thyroid are defined as small papillary carcinomas measuring less than 1 cm in maximum dimension. They are extremely common findings on histopathologic analysis following both thyroid surgery and autopsy, and their clinical significance is still debated. Several large studies have shown that the vast majority of PMCs behave in an indolent manner and remain dormant throughout a patient's lifetime. They almost never give rise to clinically overt disease, nor do they result in morbidity or mortality.1,2 Therefore, most clinicians regard these tumors as incidental findings of little clinical significance.
While papillary thyroid carcinomas have a strong propensity to metastasize to regional lymph nodes, clinically detectable lymph node metastases from PMCs are very uncommon. Nonetheless, on rare occasions, a PMC behaves aggressively and metastasizes early, presenting with clinically evident lymph node metastases. These tumors then seem to confer a definite morbidity and mortality.3 To date, traditional histopathologic assessment has not been able to distinguish between the typical PMC, which almost always remains quiescent, and the unusual PMC that has the potential to behave aggressively.
Several genetic alterations have been associated with the development of papillary thyroid carcinoma. Rearrangements of the ret gene on chromosome 10 have been well described. These ret/PTC rearrangements seem to be specific for papillary thyroid carcinoma and are thought to occur early in the carcinogenetic process.4 Inactivation of the p53 tumor suppressor gene does not seem to play a role in the development of well-differentiated carcinomas of the thyroid gland. However, when dedifferentiation to anaplastic carcinoma occurs, a p53 mutation is frequently present.5,6
Several other genes have also recently been implicated in thyroid carcinogenesis, including the tumor suppressor genes p27 and PTEN and the tumor oncogene cyclin D1. In an effort to identify PMCs that have the potential to behave aggressively, we studied 22 metastasizing PMCs and 33 nonmetastasizing PMCs using immunohistochemical analysis of ret, p53, MIB-1, and p27.
We reviewed our database on thyroid cancer and the histopathology reports of 2000 patients who had undergone thyroid surgery at our institution between 1995 and 1999 and identified 30 patients who had pathologically proven lymph node metastases from a primary PMC in the thyroid gland (T1 N1 disease). Among these, 22 patients had gross (≥2 cm) nodal metastases from an occult PMC of the thyroid gland. These patients initially presented with clinically evident lymph node metastases from an unknown primary tumor. Only after fine needle aspiration cytology, and in some cases lymph node excision biopsy, were these metastases shown to be of thyroid origin. Subsequent thyroidectomy then revealed the primary lesion to be a PMC.
The other 8 patients had undergone thyroidectomy for various nonmalignant indications and had been incidentally found to have a PMC as well as microscopic metastatic deposits in a perithyroidal lymph node. Because the significance of these lesions with micrometastatic foci remains unknown,3 we elected to exclude these from further analysis.
We immunohistochemically analyzed archival paraffin-embedded tissue from the 22 tumors with gross metastatic disease for ret, p53, MIB-1, and p27 staining. The control group, 33 randomly selected nonmetastasizing PMCs, underwent similar analysis. These PMCs were selected from thyroid glands that had been excised for nodular hyperplasia. Papillary microcarcinomas from glands containing malignancy were not included. To extend our analysis on p27, we studied 2 groups of larger papillary carcinomas as well, 14 with gross nodal metastases and 20 without nodal involvement. We hypothesized that p27 expression might predict the metastatic potential of papillary carcinomas in general, and we included these larger tumors as an additional control. In selecting these papillary carcinomas, we selected typical papillary cancers between 2 and 4 cm in size without evidence of extrathyroidal extension. We also excluded tumors with poor histologic features such as tall-cell or columnar-cell differentiation and tumors with poorly differentiated or anaplastic foci.
The metastasizing and nonmetastasizing PMCs were analyzed by immunohistochemical analysis using antibodies for ret, p53, MIB-1, and p27. The 2 groups of larger papillary carcinomas were analyzed for MIB-1 and p27.
Formalin-fixed, paraffin-embedded sections 3 µm thick were dewaxed in toluene and rehydrated through graded alcohols to water. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide. Antigen retrieval was performed in 10mM citrate buffer (pH, 6.0) inside a microwave pressure cooker for p53, MIB-1, and p27, and by formic acid pretreatment for ret. Endogenous biotin detection was blocked with the Avidin-Biotin Blocking Kit (Vector Lab Inc, Burlingame, Calif). Primary antibody incubations were carried out at room temperature as follows: ret, rabbit polyclonal (C-19) (Santa Cruz Biotechnology Inc, Santa Cruz, Calif), 1:1000 dilution, overnight incubation; p53, mouse monoclonal (D0-7) (Novocastra Ltd, Newcastle upon Tyne, England), 1:100 dilution, 1-hour incubation; MIB-1, mouse monoclonal (MIB-1) (Immunotech, Marseille, France), 1:200 dilution, 1-hour incubation; and p27/Kip1, mouse monoclonal (57) (BD PharMingen, Mississauga, Ontario), 1:1000 dilution, 1-hour incubation. Following washing in phosphate-buffered saline, secondary incubations were carried out with biotin antimouse/antirabbit IgG followed by streptavidin-HRP (horseradish peroxidase) (Signet Pathology System, Dedham, Mass) for 30 minutes. Immunoreactivity was revealed by incubation in 3-amino-9-ethylcarbazol. Slides were counterstained in hematoxylin and mounted with Crystal Mount (Biomeda Corp, Foster City, Calif).
Quantitation of immunoreactivity was done jointly by 2 of the authors (M.L.C.K. and S.L.A.); ret and p53 immunoreactivity was assessed by the presence or absence of staining in some or all of the tumor cells. MIB-1 expression was quantified as a labeling index based on the number of positive tumor cells per high-power field. Only nuclear staining in thyroid follicular cells was considered positive staining for p53 and MIB-1. Immunoreactivity displayed by fibrovascular stromal and lymphoid cells within the tumors was not considered. Small tumors were assessed en toto. For larger tumors, 3 separate high-power fields per tumor were studied and counted, and the final index was an average of the 3 counts.
Expression of p27 was assessed based on the intensity of nuclear staining within the tumor cells. Again, immunoreactivity displayed by fibrovascular stromal and lymphoid cells was not considered. The intensity of staining was graded as 0 to 4 as follows: grade 0 for total absence of staining; grade 1 for faint nuclear staining (requiring high-power assessment); grade 2 for moderate nuclear staining (easily seen); grade 3 for strong nuclear staining (but noticeably less than normal); and grade 4 for staining as strong as adjacent normal thyroid tissue.
Quantitation of p27 immunoreactivity was based on the prevalent intensity of staining within the tumor. Most specimens displayed a uniform intensity of staining throughout the tumor, and in such cases the quantitation was straightforward. For the occasional tumor with a more heterogeneous expression, we graded the p27 immunoreactivity according to the intensity displayed by the majority of tumor cells.
For each group of metastasizing PMCs and carcinomas, we used a corresponding group of nonmetastasizing tumors as external controls. Internal control within each slide was provided by adjacent normal thyroid or lymphoid tissue, which invariably showed strong nuclear staining for p27 and served as the benchmark for assessment of intensity.
Statistical analysis was performed using the t test for MIB-1 expression and the χ2 test for p27 expression. For the latter calculation, as very few tumor samples showed either grade 0 or grade 4 p27 expression, we merged grades 0 and 1, and grades 3 and 4. This resulted in 3 grades per tumor group: grade 0-1, grade 2, and grade 3-4. Statistical significance was ascribed at P<.05.
There was no difference in the immunoreactivity for ret and p53 between the metastasizing and nonmetastasizing PMCs. In most of the tumors ret was present in both groups, while p53 immunoreactivity was absent in all tumors in both groups.
MIB-1 immunoreactivity was present in a small number of cells in both metastasizing and nonmetastasizing PMCs. In the nonmetastasizing microcarcinomas, the MIB-1 labeling index ranged from 0 to 30, with a mean of 6.89 and a median of 4.5. In the metastasizing microcarcinomas, the MIB-1 labeling index ranged from 2 to 37, with a mean of 10.17 and a median of 6.5. As is apparent, there was considerable overlap in the labeling indices between the 2 groups, and while the mean and median labeling indices were slightly higher in the group with metastases, these differences were not statistically significant.
Nuclear p27 immunoreactivity was very strong in normal thyroid follicular cells, fibrovascular stromal cells, and lymphocytes. Almost all the tumors, including both larger carcinomas and microcarcinomas, showed lower than normal p27 expression, though the degree of underexpression varied. Of 89 tumors, only 4 showed normal (grade 4) p27 expression. Two of these were nonmetastasizing PMCs, while the other 2 were nonmetastasizing papillary carcinomas.
For the PMCs as well as the larger papillary cancers, the tumors with metastases showed significantly less p27 expression than the tumors without metastases (P<.001 and P<.005, respectively). Of the 22 metastasizing PMCs, 15 tumors (68.2%) showed very faint (grade 0 and 1) p27 expression (Figure 1) compared with only 6 tumors with moderate (grade 2) staining (Figure 2) and 1 tumor with strong (grade 3) staining. Conversely, of the 33 nonmetastasizing PMCs, only 6 tumors (18.2%) showed faint (grade 1) p27 expression compared with 15 tumors with moderate (grade 2) staining, 10 tumors with strong (grade 3) staining (Figure 3) and 2 tumors with very strong (grade 4) staining.
For the larger papillary carcinomas, the results were similar. Of the 14 papillary carcinomas with metastases, 7 (50%) showed faint (grade 1) p27 expression while 7 (50%) showed moderate (grade 2) p27 expression. However, of the 20 papillary carcinomas without metastases, only 2 (10%) showed faint (grade 0-1) p27 expression compared with 8 with moderate (grade 2) staining, 8 with strong (grade 3) staining, and 2 with very strong (grade 4) staining. The results of the immunostaining for p27 are summarized in Table 1 and Table 2.
Papillary thyroid microcarcinomas with gross metastatic disease are extremely uncommon. However, a small subgroup of PMCs have the potential to behave aggressively and metastasize early, resulting in increased morbidity and mortality.3
Routine histopathologic analysis is unable to distinguish between the typical PMC, which tends to remain quiescent, and the rare PMC with the potential for aggressive behavior. We undertook this study to try to identify immunohistochemical markers that might allow us to recognize the potentially aggressive lesions. We hypothesized that one or several tumor genes might influence the behavior and metastatic potential of PMCs.
A small number of PMCs are considered incidental findings yet are found to be associated with micrometastatic disease in lymph nodes on careful histologic examination. We identified 8 such cases in our series. The true incidence of this phenomenon is not known, and the clinical significance of these lesions remains unclear.3 Indeed, the clinical significance of micrometastatic spread of larger papillary thyroid carcinomas remains controversial. We therefore elected to omit these cases from our analysis. However, it will be interesting to apply the results of our study to larger groups of thyroid carcinomas with micrometastases.
The ret/PTC gene rearrangement, which may take one of several forms, has been shown to occur only in papillary carcinomas of the thyroid gland7 and is not seen in other tumors of thyroid follicular cell origin. These rearrangements result in fusion of the intracellular domain of the ret protooncogene with N-terminal portions of other genes that are expressed in follicular epithelium. Rearrangements of ret in papillary thyroid carcinoma have been shown to occur early in the carcinogenetic process.4 Interestingly, the incidence of ret/PTC rearrangement seems to be higher in PMCs than in larger papillary carcinomas. Members of our group7 have reported that the incidence of ret/PTC rearrangement in PMCs was almost 80% vs 40% for larger papillary carcinomas.
The significance of ret rearrangements in papillary thyroid carcinoma is still unclear. Several reports have suggested that ret rearrangement may predict aggressive behavior, including local invasion and the potential for metastases.8 However, expression of ret/PTC gene products seems to be reduced in papillary carcinomas with aggressive histologic features such as columnar and tall-cell papillary variants and papillary tumors showing dedifferentiation toward poorly differentiated and anaplastic carcinoma. Other reports have suggested that ret/PTC positivity predicts an indolent phenotype.9,10
We hypothesized that immunoreactivity for ret might help distinguish between metastasizing and nonmetastasizing PMCs. However, we found that most of the microcarcinomas stained for ret under immunohistochemical analysis, and there was no difference between the 2 groups. These results are similar to those previously reported.7 This indicates that while ret rearrangement plays a role in the development of papillary carcinoma, it does not influence the metastatic potential of early papillary thyroid carcinomas.
p53 Mutation resulting in protein overexpression is a common feature of poorly differentiated and anaplastic thyroid carcinomas but is uncommon in well-differentiated thyroid carcinomas. However, p53 overexpression is seen with increasing frequency in papillary carcinomas with poor histologic features such as tall-cell or columnar-cell morphologic characteristics11 and in tumors with evidence of dedifferentiation.6 Overexpression of p53 has also been linked to the potential for papillary carcinomas to metastasize to regional lymph nodes.5,12
We studied p53 immunoreactivity in both metastasizing and nonmetastasizing PMCs. None of the tumors showed nuclear accumulation of p53 protein. Therefore, while p53 inactivation may play a role in the dedifferentiation of well-differentiated thyroid carcinoma, it does not seem to play a role in the potential for PMCs to metastasize.
MIB-1/Ki-67 expression is a marker of proliferative activity and has been shown to be significantly higher in poorly differentiated and anaplastic carcinomas than in well-differentiated carcinomas.13- 15 The relationship between MIB-1 labeling index and the risk of metastases is, however, less clear. In a study of follicular thyroid carcinoma, Erickson et al13 found significantly higher MIB-1 expression in patients with distant metastases. Similarly, Sugitani et al3 found a significantly higher MIB-1 labeling index in patients with PMCs who died of distant metastases. However, Tallini et al14 found no association between MIB-1 expression and metastases in their patients with well-differentiated thyroid carcinomas.
In our study, the mean and median MIB-1 labeling indices were higher in the metastasizing PMCs than in the nonmetastasizing microcarcinomas. However, the differences were not statistically significant.
The p27/Kip-1 gene is a tumor suppressor gene that encodes a 27-kd protein, a cyclin-dependent kinase inhibitor. This protein acts as a negative regulator of the cell cycle, controlling G1 to S phase transition. Reduced p27 function has been implicated in the pathogenesis of several malignancies. While rearrangements and mutations of the p27 gene are uncommon, underexpression of nuclear p27 protein has been shown to occur in cancers of several organs.16- 25 Furthermore, p27 underexpression seems to predict a more aggressive tumor phenotype as well as poorer survival. Underexpression of p27 has also been associated with an increased risk of lymph node metastases in adenocarcinomas of the colon,16 stomach,17,18 breast,19 prostate,20 and salivary gland.21 In the thyroid gland, p27 is strongly expressed in the nuclei of normal follicular thyroid cells but is underexpressed in hyperplastic and neoplastic thyroid disease.22 Thyroid tumors have been shown to express significantly less p27 than hyperplastic nodules,23 and malignant tumors express significantly less p27 than do benign tumors.13,24 Poorly differentiated thyroid carcinomas also express less p27 than well-differentiated thyroid carcinomas.25 Expression of p27 has also been reported to show an inverse correlation with MIB-1 expression.22
In this study, we hypothesized that PMCs that demonstrated aggressive potential and early metastases might have lower p27 expression than typical PMCs. We quantified the expression of p27 by assessing the intensity of nuclear staining in thyroid tumor cells. Previous studies have quantified p27 expression based on a labeling index of positive cells per 1000 cells counted. This strategy was effective when comparing groups with different thyroid diseases (eg, hyperplasia vs neoplasia or benign tumors vs malignant tumors). However, in our study we were comparing groups with similar pathologic characteristics, and we found that the percentage of cells expressing p27 was very similar between the 2 groups of microcarcinomas and the 2 groups of papillary carcinomas. It was the intensity of p27 expression that varied between tumors. We therefore graded the tumors according to the intensity of p27 expression.
Our results show that p27 is significantly underexpressed in metastasizing PMCs compared with nonmetastasizing PMCs. Thus, p27 immunohistochemical analysis seems useful to distinguish the rare PMCs that have the potential to behave aggressively from the typical PMCs that tend to remain quiescent. Our results also show that p27 underexpression predicts an increased risk of lymph node metastases in larger papillary thyroid carcinomas. This further illustrates the value of p27 immunohistochemical analysis in predicting behavior in papillary thyroid cancer. Our data indicate that PMCs that underexpress p27 should no longer be regarded as incidental findings of little significance, but as true papillary carcinomas with the potential for aggressive behavior.
In conclusion, p27 seems to be significantly underexpressed in PMCs as well as in larger papillary carcinomas of the thyroid that metastasize to regional lymph nodes. Immunohistochemical analysis for p27 may be a valuable diagnostic tool in distinguishing PMCs of the thyroid with aggressive potential from the more typical indolent lesions and may also prove useful for predicting the metastatic potential of larger papillary cancers of the thyroid.
Accepted for publication November 2, 2001.
This study was presented at the annual meeting of the American Head and Neck Society, Palm Desert, Calif, May 16, 2001.
We would like to thank James Ho and Kelvin So for excellent technical assistance in the completion of this work.
Corresponding author: Sylvia L. Asa, MD, PhD, Department of Pathology, University Health Network, 610 University Ave, Suite 4-302, Toronto, Ontario, Canada M5G 2M9 (e-mail: firstname.lastname@example.org).