OUP user menu

Papillary Thyroid Carcinoma With Heterotopic Ossification Is a Special Subtype With Extensive Progression

Masahiko Takeda MD, Tetuo Mikami MD, PhD, Yoshiko Numata, Makito Okamoto MD, PhD, Isao Okayasu MD, PhD
DOI: http://dx.doi.org/10.1309/AJCPQZQN50HKIAHA 587-598 First published online: 1 May 2013

Abstract

An immunohistochemical study was conducted of 108 papillary carcinoma cases, including 48 cases with intratumoral heterotopic ossification (IHO). In 48 cases, papillary carcinoma with IHO was accompanied by nodular fibrosis. Cases of papillary carcinoma with IHO or nodular fibrosis showed higher incidences of lymph node metastasis, multifocal lesions, and extrathyroidal invasion than those without IHO and nodular fibrosis. A higher number of stromal myofibroblasts was observed in papillary carcinoma with IHO or nodular fibrosis than in that without fibrosis. Expression of both basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP)–2 was the highest in papillary carcinoma with IHO. Papillary carcinoma with IHO showed higher vascular invasion and higher numbers of capillaries expressing nestin, which is associated with high expression of vascular endothelial growth factor (VEGF). Papillary carcinoma with IHO is a unique subtype with extensive progression including frequent lymph node metastasis, multifocality, and invasive behavior. Papillary carcinoma with IHO was correlated with expression of bFGF, BMP-2, and VEGF in the carcinoma cells, leading to neovascularization.

Key Words:
  • bFGF
  • BMP-2
  • Heterotopic ossification
  • Nodular fibrosis
  • Myofibroblast
  • Papillary thyroid carcinoma
  • VEGF

The presence of calcification is one of the most significant findings in ultrasonography or computed tomography when evaluating thyroid nodules. Calcification is more frequently detected in papillary thyroid carcinoma than in other thyroid lesions.1 Papillary carcinoma represents the largest proportion of thyroid malignancies.

The stroma of papillary carcinoma consists of fibrous extracellular matrix and a community of stromal cells, including fibroblasts, endothelium, inflammatory cells, and calcification and intratumoral heterotopic ossification (IHO). IHO is an occasional histologic characteristic of papillary carcinoma. Bai et al2 reported that stromal calcification was associated with pT stage and lymph node metastasis. However, they failed to show the clinicopathologic significance of IHO in papillary carcinoma.

Tumor stroma interaction plays an important role in tumor invasion and metastasis of digestive organ cancers.3 Papillary carcinoma frequently contains nodular fibrosis (NF). This fibrosis is composed of a mixture of fibroblasts and various amounts of collagen fiber. α-Smooth muscle actin (αSMA)–positive fibroblasts (myofibroblasts), previously termed activated fibroblasts, are also present in areas of wound healing and chronic inflammation, and promote angiogenesis, stimulate proliferation of epithelial cells, and produce extracellular matrix, growth factors, and cytokines.4

Basic fibroblast growth factor (bFGF) is a potent chemotactic and mitogenetic factor for smooth muscle cells and myofibroblasts.5 bFGF makes up a complex family of signaling molecules involved in several physiologic processes, and has diverse biological functions, including involvement in cellular growth, differentiation, tumor invasion, and angiogenesis.6,7

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) superfamily, originally described as proteins that can induce ectopic bone formation when implanted into muscle tissue in vivo.8 Current evidence suggests that they also participate in various biological processes of cells, such as proliferation, differentiation, and apoptosis.9 BMP-2, which is a subtype of the BMP family, has been shown to play a crucial role in the occurrence and development of breast, lung, and colon cancers.1012 However, the relationship between BMP-2 and thyroid carcinoma has not been characterized.

In the present study, we histologically classified the intratumoral fibrosis and ossification status into 3 types: papillary carcinoma with IHO, papillary carcinoma with NF, and papillary carcinoma without NF. For comparison with papillary carcinomas, cases of follicular carcinoma were also examined. After the classification, clinicopathologic findings were examined, and the expressions of αSMA, bFGF, and BMP-2, as well as the state of vascular proliferation, were immunohistochemically analyzed, and roles of each protein in the formation of NF and IHO in thyroid papillary carcinoma were discussed.

View this table:
Table 1

Materials and Methods

Patients and Tumor Samples

The pathology files of Kitasato University Hospital (Sagamihara, Japan) included 207 surgically treated cases of papillary carcinoma seen between 2000 and 2007. On review of the histologic slides of these 207 cases, 48 cases with IHO and 26 cases with NF were included in this study. For comparison, 34 cases without NF were selected at random. For further comparison, 33 cases of follicular carcinoma seen during the same period were also included. Clinical information was obtained from the patients’ medical records. All of the patients were Japanese, ranging in age from 19 to 84 years (mean, 53.6 years).

The surgically resected specimens were routinely fixed in 10% buffered formalin, and the entire tumor and nontumorous thyroid tissues were cut into 0.3- to 0.5-cm thick slices. The size and gross appearance of the tumors were recorded, and the former was validated by comparison with tumor size on histologic slides. The sections were processed routinely and embedded in paraffin. One section from each of the removed lymph nodes was also processed routinely in the same way as the thyroid sections.

Histologic Examination

All sections were stained with H&E as well as elastica van Gieson, and histopathologically examined. Histologic types of thyroid carcinomas were determined according to the World Health Organization classification Table 1 and Table 2,8 and IHO in papillary thyroid carcinoma was defined as lamellar bone formation in the interstitial spaces. Band-like stromal calcifications of 1 mm or larger were recorded. It was clearly differentiated from psammoma bodies.

View this table:
Table 2

Nodular fibrosis was defined with the following criteria: (1) fibrosis composed of proliferating myofibroblasts and/or collagen fibers, with or without tumor cells, and (2) location within the tumor with a size of 5 mm or more in diameter. Fibrosis after preoperative fine-needle aspiration cytology could be excluded with these criteria.

Multifocality was defined as the presence of additional tumor foci noncontiguous to the primary tumors in the resected thyroid specimens. Extrathyroidal invasion was the extension of tumor beyond the thyroid capsule to the perithyroidal tissue. Extrathyroidal invasion was divided into the following 3 grades (extrathyroidal invasion 0–2) using the classification of the Japanese Society of Thyroid Surgery13 as follows: extrathyroidal invasion 0, invasion does not exceed thyroid capsule; extrathyroidal invasion 1, invasion exceeds thyroid capsule but remains in sternothyroid muscle or adipose tissue; extrathyroidal invasion 2, invasion reaches other organ (trachea, larynx, esophagus, recurrent nerve, carotid artery, internal jugular vein, and subcutaneous tissue).

Vascular invasion of cancer cells was estimated by observing 4-μm-thick histologic sections of the largest cut surface of formalin-fixed, paraffin-embedded blocks with double staining using H&E and elastica van Gieson. One focus of vascular invasion per histologic section was scored as V1, and 2 foci or more as V2.

In addition, inflammation status (chronic lymphocytic thyroiditis) of background thyroid tissue was examined in each case according to the criteria of Williams and Doniach14; grade 0, 0 to 1 focus per standard representative section (2 cm2); grade 1, 2 to 8 foci per standard representative section; grade 2, 9 to 40 foci per standard representative section; grade 3, more than 40 foci per standard representative section; and grade 4, more than half of the glandular parenchyma replaced in a standard representative section. A “focus” was defined as an aggregate of 50 or more lymphocytes. A nontumorous region was selected at least 5 mm from the borderline with the tumor to avoid reactive lymphoid cell infiltration around the tumor.14,15

Immunohistochemistry

Formalin-fixed and paraffin-embedded blocks of cancer lesions were selected. For the case of papillary carcinoma with IHO, the blocks adjacent to ossification but without ossification were used to avoid the influence of decalcification process on immunohistochemistry. The largest section of each tumor was immunostained. The primary antibodies used are listed in Table 3. The Envision + system (Dako, Glostrup, Denmark) was used for the primary antibodies against αSMA, bFGF, vascular endothelial growth factor (VEGF), CD34, and nestin, and the LSAB+ system-HRP (Dako) was applied for the antibody against BMP-2. Chromogenic fixation was carried out by immersing the sections in 3,3′-diaminobenzidine solution. Counterstaining was carried out with Mayer hematoxylin.

Immunohistochemical Examination

Immunohistochemical reactivity was evaluated blindly without knowledge of the clinicopathologic information.

Myofibroblast counts (MFCs) were collected immunohistochemically using αSMA-stained slides. αSMA-positive fibroblastic cells in intratumoral stroma were considered to be myofibroblasts. The immunostained sections were scanned using light microscopy at low magnification (×40), and the areas with the greatest number of distinctly highlighted αSMA-positive fibroblastic spindle cells were selected. Then the number of myofibroblasts was determined by counting all immunostained myofibroblasts in 5 areas, at a magnification of ×200 for each case using an ocular micrometer. The mean number of myofibroblasts in a field of 0.25 mm2 in each case was then calculated, and defined as the MFC in the tumor. For papillary carcinoma with IHO and papillary carcinoma with NF cases, both tumor and NF areas were evaluated independently. In follicular carcinoma, fibrosis was rarely observed. Therefore we examined only papillary carcinoma.

For bFGF, BMP-2, and VEGF, the stained slides were examined microscopically using the following parameters and semiquantitative criteria. Cytoplasmic immunostaining intensity in both tumor cells and stained area was evaluated. The intensity of the immunoreaction was classified into 4 levels: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. In addition, the percentage of immunoreactive cancer cells was recorded: 0, negative; 1, 1% to 25%; 2, 26% to 50%; 3, 51% to 75%; and 4, 76% to 100%. Both scores were multiplied to generate a total score of 0 to 12, according to the method of Sinicrope et al.16

View this table:
Table 3

CD34-expressing capillaries were counted to determine the microvascular density. Nestin-positive capillaries were considered as capillaries consisting of newly formed endothelial cells.17 In specimens using both immunostains, areas of highest density of capillaries were found by scanning tumor sections at low power (×100). Then the numbers of capillaries were counted in 5 different fields at high power (×400). The average number of capillaries per high-power field (×400) was defined as microvascular density with both immunostains.

Statistical Analysis

The Kruskal-Wallis test and Mann-Whitney U test were used to test among more than 3 groups. The χ2 test was carried out to analyze categorical data. The Stat View 5.0 computer program (Abacus Concepts, Berkeley, CA) was used for all statistical tests and management of the database. A P value less than .05 was considered to be significant.

Results

Clinicopathologic Data

The histopathologic variants18 with number of cases and clinicopathologic data for the examined cases are summarized in Tables 1 and 2. Most cases of papillary thyroid carcinoma were of the conventional type (Table 2). All cases of papillary carcinoma with IHO showed NF in the lesion, and the ossification was formed in the fibrotic area Image 1.

The mean ages of patients with papillary carcinoma with IHO, papillary carcinoma with NF, and papillary carcinoma without NF were 59.1 ± 14.8 years, 57.5 ± 16.9 years, and 51.7 ± 12.3 years, respectively. No difference was found between papillary carcinoma with IHO and papillary carcinoma with NF. Patients with papillary carcinoma with IHO and papillary carcinoma with NF were significantly older than those with papillary carcinoma without NF (P = .015 and P = .004, respectively). The mean size of each group of papillary thyroid carcinoma was not significantly different. The follicular carcinoma group was larger than any group of thyroid carcinoma (P < .0001).

Calcification was noted in 48 cases of papillary carcinoma with IHO (100%), 4 cases of papillary carcinoma with NF (15%), 4 cases of papillary carcinoma without NF (12%), and 2 cases of follicular carcinoma (6%). The incidence in papillary carcinoma with IHO was significantly greater than that in papillary carcinoma with NF or papillary carcinoma without NF (P < .001).

Multifocality was identified in 40 cases of papillary carcinoma with IHO (83%), 19 cases of papillary carcinoma with NF (73%), and 18 cases of papillary carcinoma without NF (53%). Papillary carcinoma with IHO showed a higher incidence of multifocality than papillary carcinoma without NF (P = .003).

Lymph node dissection was carried out in 113 patients. Cases of both papillary carcinoma with IHO and papillary carcinoma with NF showed significantly higher incidence of lymph node metastasis than cases of papillary carcinoma without NF (P = .0025 and P = .0165, respectively). Similarly, extrathyroidal invasion was more frequent in papillary carcinoma with IHO and papillary carcinoma with NF than in papillary carcinoma without NF (P < .0001 and P < .0001, respectively).

Image 1

Subtypes of thyroid carcinomas. A, Papillary carcinoma with intratumoral heterotopic ossification. Arrow indicates papillary carcinoma cells between bone trabeculae within nodular fibrosis (NF) (H&E, ×100). B, Higher magnification of A (H&E, ×200). C, Papillary carcinoma with NF. Note prominent stromal fibrosis. Arrow indicates carcinoma cell nests (H&E, ×100). D, Carcinoma cell nests in C in higher magnification. Arrow indicates a nucleus with pseudoinclusion (H&E, ×400). E, Papillary carcinoma without NF (H&E, ×100). F, Follicular carcinoma. Note capsular invasion of tumor cells (elastica-H&E, ×200).

Chronic lymphocytic thyroiditis in background thyroid tissue was assessed, as summarized in Table 4. No significant differences were seen among the 3 groups of patients with papillary carcinoma (P = .067).

Myofibroblast Count

Immunoreactive αSMA was positive in stromal myofibroblasts Image 2. In tumor areas, papillary carcinoma with IHO and papillary carcinoma with NF possessed significantly greater MFCs than did papillary carcinoma without NF (P = .017 and P = .004, respectively) Figure 1A. In NF, papillary carcinoma with NF had greater MFCs than did papillary carcinoma with IHO (P = .002) Figure 1B.

View this table:
Table 4

bFGF and BMP-2 Protein Expression

bFGF was expressed in the cytoplasm and nucleus of cancer cells Image 3A and Image 3D. BMP-2 was localized in the cytoplasm of cancer cells Image 3B and Image 3E. Both bFGF and BMP-2 showed strong positivity in cancer cells around fibroblastic foci (Image 3A and Image 3B). Both bFGF and BMP-2 expressions were higher in papillary carcinoma than in follicular carcinoma. bFGF score of papillary carcinoma with IHO was the highest in papillary carcinoma, with papillary carcinoma with NF showing intermediate levels and papillary carcinoma without NF having the lowest immunopositivity Figure 2A. BMP-2 score had a similar pattern to bFGF score Figure 2B.

Image 2

Representative α smooth muscle actin (αSMA)–positive myofibroblasts. A, Cancer cells and αSMA+ myofibroblast-rich stroma in a case of papillary carcinoma with intratumoral heterotopic ossification (×100). B, Cancer cells and αSMA+ myofibroblast-rich stroma in a case of papillary carcinoma with nodular fibrosis (NF) (×100). C, Cancer cells and αSMA+ myofibroblast-poor stroma in a case of papillary carcinoma without NF (×100). D, Hyalinized fibrotic (αSMA+ myofibroblast-poor) stroma of the same case as A (×400). E, Fibrotic (αSMA+ myofibroblast-rich) stroma of the same case as B (×400).

VEGF Expression

VEGF expression in cancer cells was significantly higher in papillary carcinoma with IHO and papillary carcinoma with NF than in papillary carcinoma without NF. No significant difference was observed between papillary carcinoma with IHO and papillary carcinoma with NF Image 3C, Image 3F, and Figure 2C.

Vascular Invasion of Cancer Cells

Vascular invasion of cancer cells was identified by means of elastica van Gieson staining. Vascular permeation by cancer cells was frequently found in the order of papillary carcinoma with IHO, papillary carcinoma with NF, and papillary carcinoma without NF, the difference between each group being significant (Table 1).

Microvessel Density Identified Using CD34 and Nestin

Microvascular density identified with CD34 showed no significant difference among the 3 groups of papillary carcinoma. On the other hand, microvascular density examined with nestin staining, which can identify newly formed capillaries, increased in the order of papillary carcinoma without NF, papillary carcinoma with NF, and papillary carcinoma with IHO, the difference being significant Image 3G, Image 3H, Image 3I, Image 3J, and Figure 3.

Figure 1

Comparison of myofibroblast count (MFC) among papillary carcinomas (PAP) with intratumoral heterotopic ossification (IHO), with nodular fibrosis (NF), and without NF. A, MFC in tumor area (T); B, MFC in NF area (*P < .05, P < .01).

Discussion

No significant clinicopathologic characteristics were previously reported on the ossification of thyroid carcinoma. IHO was found in 23.1% of papillary carcinoma cases (48 of 207) in the current study. Previous reports on the incidence of IHO are limited. Bai et al2 reported an incidence of 16.0% (29 of 181 papillary carcinomas) in China, and Yamashita et al19 reported an incidence of 17.6% (6/34) in Japan. These incidence rates were not significantly different from ours (P = .101 and P = .619, respectively). No reports of IHO in Caucasian patients have been published, to our knowledge. Since the 2 aforementioned articles were from East Asia, some racial differences or influences of regional and dietary factors might exist in the difference between IHO incidence rates. Further studies are needed to clarify this point. IHO was not found in any cases of follicular carcinoma, indicating that IHO was a feature specific to papillary thyroid carcinomas.

In the current study, the clinicopathologic examination revealed, for the first time, that papillary carcinoma with IHO had high incidences of multifocality, lymph node metastasis, and extrathyroidal invasion. Although conventional thyroid papillary carcinoma generally has a good prognosis, pTNM classification, including lateral lymph node metastasis and extrathyroidal invasion, still reflects different prognostic outcomes.2022 Accordingly, IHO can be a valuable factor for predicting the prognosis of papillary carcinoma. IHO was revealed to be tightly associated with intratumoral NF, but not associated with chronic lymphocytic thyroiditis. Therefore, we focused our attention on the mechanism of how the stromal fibrosis is induced in papillary carcinoma lesion, and how IHO starts in association with fibrosis.

Image 3

Basic fibroblast growth factor (bFGF), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) expression in papillary carcinoma. A case of papillary carcinoma with intratumoral heterotopic ossification (A, B, and C), and a case of papillary carcinoma without nodular fibrosis (NF) (D, E, and F). Expression of bFGF (A); BMP-2 (B); and VEGF (C) is remarkable in the tumor cells of papillary carcinoma with IHO, whereas papillary carcinoma without NF shows no expression of the 3 proteins (D, E, and F). Representative vascular channels immunostained with CD34 and nestin in a case with papillary carcinoma with IHO (G, H) and a case of papillary carcinoma without NF (I, J). Vascular channels immunostained with anti-CD34 are shown in both cases (G, I). However, neovascularization immunostained with antinestin antibody is much more extensive in papillary carcinoma with IHO (H) than in papillary carcinoma without NF (J). (A, B, F, G, H, I, and J, ×100; C, D, and E, ×200.)

The presence of stromal myofibroblasts in papillary carcinoma with IHO and papillary carcinoma with NF was detected on immunohistochemical examination as αSMA-positive spindle cells in the stroma, particularly in NF. MFCs in cases with IHO and with NF were significantly higher than in cases without NF. In addition, papillary carcinoma with NF had more myofibroblasts than did papillary carcinoma with IHO. We consider that the nodular fibrotic area hyalinizes over time, and heterotopic ossification occurs in the old fibrotic region.

Tissue integrity is generally maintained by the stroma. In cancer, however, tumor cell invasion is driven by the stroma.4 Myofibroblasts and cancer-associated fibroblasts are important components of the tumor stroma.23 Myofibroblastic stroma has been reported to correlate with poor prognosis in several tumor types, including prostate,24 breast,25 colorectal,26 and oral cancers.27 A high level of myofibroblastic stroma has been reported to be associated with local recurrence.25 Although αSMA-positive myofibroblastic stroma may serve as a general marker of carcinoma aggressiveness, it is not yet clear why certain tumors develop this stroma. The origin of myofibroblasts in tumor stroma remains controversial, but local fibroblasts and bone marrow–derived precursors are considered to be the main progenitor cells.23

Figure 2

Comparison of immunoreactive scores of basic fibroblast growth factor (bFGF; A), bone morphogenetic protein-2 (BMP-2; B), and vascular endothelial growth factor (VEGF; C) among papillary carcinoma (PAP) with intratumoral heterotopic ossification (IHO), with nodular fibrosis (NF), and without NF (*P <.05, P < .01).

Figure 3

Comparison of microvascular density (MVD) and neovascularization examined with CD34 (A) and nestin (B) immunostaining among papillary carcinoma (PAP) with intratumoral heterotopic ossification (IHO), with nodular fibrosis (NF), and without NF (*P < .05, P < .01). NS, not significant.

The mutual interaction between cancer cells and myofibroblasts depends on many invasive growth-promoting factors, through direct cell-cell contacts and paracrine signals.3 We found here a trend of the highest expression of bFGF and BMP-2 in papillary carcinoma with IHO.

It has been suggested that bFGF participates in tumor invasion, fibroblastic stromal reaction, and lymph node metastasis. bFGF can stimulate proliferation and differentiation of fibroblasts and vascular endothelial cells.6,7 Recent studies have shown that bFGF is overexpressed in various cancers, such as lung cancer,28 breast cancer,29 and colon cancer,30 demonstrating a close association with angiogenesis, as well as with the progress of cancer. Previous studies of thyroid carcinoma reported a trend of higher expression of bFGF in carcinomas than in benign proliferative lesions, and in benign lesions than in normal tissue.31 However, there were no reports of the relationship among bFGF expression, stromal fibrosis, and IHO.

TGF-β is the most potent cytokine driving myofibroblast transdifferentiation.4 BMP-2 is a member of the TGF-β superfamily.9 BMP-2 was originally described as a protein that can induce ectopic bone formation.8 Immunohistochemical studies using anti–BMP-2 antibody have shown heterotopic ossification to be associated with neoplasms in the lung.32 IHO is seen more frequently in papillary thyroid carcinoma than in other organ carcinomas. Thus, BMP-2 expression in carcinoma cells was considered to be important for the IHO.

Papillary carcinoma with IHO was found to have a higher level of neovascularization, as seen with nestin-positive microvascular density, than other groups. Recent reports indicated that intratumoral lymphatics in papillary thyroid carcinomas were associated with lymph node metastasis,33 and that VEGF expression in papillary carcinoma cells correlated with lymph node metastasis.34,35 Because the anti-VEGF antibody used in our immunohistochemical study was produced by immunization with the N-terminus of VEGF-A, simple comparison of our results with those of previous reports34,35 was difficult. However, frequent lymph node metastasis in papillary carcinoma with IHO can be explained by our results.

Our study included only 1 case of the follicular variant of papillary carcinoma. In the literature from Western countries, the incidence of follicular variant is much higher. The follicular variant comprised 20% to 38% of papillary carcinoma cases.36,37 However, much lower figures were reported from East Asia. In a report by Zhu et al38 from China, 7 (4.5%) of 155 papillary carcinoma cases were of the follicular variant. There was no significant difference between their figures and ours (P = .192). Although the reason for these differences between the West and Asia is unknown, racial differences or dietary factors might influence the incidence of the follicular variant.

Follicular carcinomas were also immunohistochemically examined in this study. From the results of bFGF, BMP-2, and VEGF, follicular carcinoma was considered to be clearly different from papillary carcinoma.

Figure 4

Schematic outline of our hypothesis about papillary carcinoma with intratumoral heterotopic ossification.

In conclusion, papillary carcinoma with IHO showed frequent lymph node metastasis, multifocality, and extrathyroidal invasion, suggesting different prognostic outcomes from those seen in other conventional papillary carcinomas. It is considered that bFGF produced by carcinoma cells stimulates myofibroblast proliferation, which results in NF, and that old NF shows ossification induced by BMP-2 from carcinoma cells. Such carcinoma cells tend to produce VEGF, which leads to neovascularization and lymphatic invasion Figure 4. Thus, papillary carcinoma with IHO should be regarded as a unique subtype of thyroid carcinoma.

CME/SAM

Upon completion of this activity you will be able to:

  • list histologic characteristics of papillary thyroid carcinomas with heterotopic ossification.

  • describe the biological potential of papillary thyroid carcinomas with heterotopic ossification.

  • compare patterns of extrathyroidal spread between papillary carcinomas with heterotopic ossification and classical papillary carcinomas of thyroid.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 691. Exam is located at www.ascp.org/ajcpcme.

Footnotes

  • This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (21791641).

References

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
  22. 22.
  23. 23.
  24. 24.
  25. 25.
  26. 26.
  27. 27.
  28. 28.
  29. 29.
  30. 30.
  31. 31.
  32. 32.
  33. 33.
  34. 34.
  35. 35.
  36. 36.
  37. 37.
  38. 38.
View Abstract