Introduction 1 2 1 3 1 3 5 6 7 8 11 12 11 12 13 14 15 17 The aim of our study has been to analyze papillary thyroid carcinomas (PTC), the most frequent thyroid cancer, for the presence of MDM4 and its derivative forms to investigate alterations of these proteins in this tumor histotype and correlate them with histopathological features. In addition, we have analyzed MDM2 levels to assess the potential relationship between alterations of the two MDM family members. Our data have revealed the aberrant presence of variants MDM4-S and MDM4-211 messenger RNA (mRNA) in PTC as well as lack of correlation between MDM4 and MDM2 mRNA levels. Of note, levels of MDM4 mRNA were significantly downregulated in tumor samples in comparison to normal counterparts, and such downregulation appears significantly associated with tumor stage. Materials and methods Tissue samples and patients Fifty-seven papillary thyroid carcinomas and 57 matched normal thyroid tissue samples from the contra-lateral lobe (CTRL) of 57 patients were studied. In addition, to confirm statistical analyses, 26 papillary thyroid carcinomas and three normal thyroid tissues from independent individuals were analyzed. All specimens were obtained from patients undergoing surgery at the University of Perugia from 1997 to 2007. Before the surgical procedure, all patients signed informed consent forms for collection of fresh thyroid samples for genetic studies. All specimens were sampled from the primary tumor at the time of surgery, snap frozen, and stored at −80°C until use. Tumors containing at least 70% of tumor cells based on the hematoxylin–eosin staining were selected. All normal thyroid tissue from the contra-lateral lobe were histopathologically analyzed for the presence of tumor. Available medical records of the patients were consulted to gain information about the clinical features of the disease and, when possible, the tumor stage was defined according to the sixth edition of the “American Joint Committee on Cancer” Cancer Staging Manual (American Joint Committee on Cancer 2002) based on pathological tumor–node–metastasis parameters and distinguishing patients in two groups: (1) patient age < 45 years (stage I and II), (2) patient age ≥ 45 years (stage I, II, III, IV). The total of 83 papillary thyroid carcinomas included 55 of the classic variant, 20 of the follicular variant, five of other variants (tall cell, solid, diffuse sclerosing), and three showing the coexistence of PTC histology with areas of dedifferentiation. The mean period of follow-up was 45.8 ± 30.4 months. Genetic analysis of TP53 mutation, BRAF mutations and ret/PTC rearrangements TP53 p53 18 18 Quantitative real-time PCR 13 n 11 Western blot analysis 13 Statistical analysis U p Results Analysis of MDM4 and MDM2 levels in papillary thyroid tumors 1 . Table 1 Histopathological features of the 57 Papillary Thyroid Tumors (PTC)   n n n BRAF mutation 35 (61) 20 (35) 2 (3) Ret/PTC rearrangement 5 (9) 50 (88) 2 (3) TP53 mutation 0 (0) 57 (100) 0 Extra-thyroidal invasion 15 (26) 41 (72) 1 (2) Multifocality 24 (42) 32 (56) 1 (2) Nodal metastasis 24 (42) 33 (58) 0 Distant metastasis 6 (10) 35 (62) 16 (28) n I 15 (75) 0 5 (25) II 0 (0) 15 (75) 5 (25) n ) I 8 (22) 18 (48) 11 (30) II 2 (5) 24 (65) 11 (30) III 3 (8) 23 (62) 11 (30) IV 13 (35) 13 (35) 11 (30) Histology Classic 38 (67) 19 (33) 0 Follicular 13 (23) 44 (77) 0 Other 4 (7) 53 (93) 0 Dedifferentiated 2 (3) 55 (97) 0 ND 2 19 20 21 Table 2 Tumor sample data Sample M4 M4 T/N MDM4-S/flM4 MDM4-211 MDM4-211 to flM4 M2 T/N M4 to M2 TP53 status BRAF mut Ret/PTC rearr. a Tumor stage Multifocality N1 M1 b 1 9.42 0.9 1.8 1.36 0.14 1 1.3 wt + − 1 3 − − − 2 2 18.96 1.3 5.2   1.4 2.5 wt − − 4 4 + + + 4 3 29.34 1.6 0.4 9.03 0.31 1.5 3.5 wt − − 1 1 − − − 2 4 14.93 1.7 9.7   1.1 2 wt − + 1 1 + − − 1 5 4.39 0.3 0.9   0.4 1.1 wt − − 4 4 − + + 3 6 11.16 0.4 1.3   0.5 1.9 wt − − 4 4 − + + 3 7 12.60 0.8 0.7 8.94 0.71 0.7 3.9 wt − − 1 1 + + − 1 8 4.44 0.4 0.8   2.1 0.7 wt + − n.d. n.d. − − n.d. 1 9 15.51 1.4 0.6   1.4 1.6 wt + − 1 4 + + − 1 10 8.63 0.3 0.6   0.9 1.3 wt − − n.d. n.d. + − n.d. 1 11 5.96 0.2 1.2   0.9 0.7 wt − − 2 4 − + − 1 12 8.11 0.5 1.1   1 0.9 wt + − 3 1 + + − 1 13 6.25 0.4 1.3 1 0.16 1.8 0.8 wt + − n.d. n.d. − + n.d. 2 14 6.13 0.4 1.5   0.8 0.9 wt + − n.d. n.d. − − n.d. 1 15 8.34 0.3 1.6   0.6 1.2 wt − − 1 1 − + − 1 16 3.82 0.2 1.4 1.23 0.32 0.8 0.5 wt + − 3 1 − − − 1 17 18.83 1 0.8   0.3 8.8 wt + − 2 4 + + − 2 18 6.32 1.2 0.7   1.1 1.2 wt + − 1 1 − − − 1 19 3.61 0.2 1.8   2.6 0.1 wt − − 1 1 − − − 2 20 5.12 0.3 3   0.9 0.8 wt + − n.d. 1 + − − 1 21 8.4 0.5 4.2   0.6 1.1 wt − + 2 n.d. + − n.d. 1 22 5.1 0.3 n.d.*   1 0.6 wt − + 1 3 − − − 1 23 7.06 0.9 0.9   2.6 0.7 wt + − n.d. n.d. − − n.d. 1 24 8.85 0.6 0.1   1 1.4 wt + − 1 1 + − − 1 25 6.21 0.6 0.1   2.3 0.6 wt − − n.d. n.d. + − n.d. 1 26 9.61 1 0.1   1.2 1.1 wt + − 1 1 − − − 2 27 7.11 0.3 0.6   0.4 1.6 wt + − n.d. n.d. + + n.d. 1 28 3.53 0.2 n.d.   0.2 1.4 wt + − 1 2 − − − 2 29 5.76 0.4 2.2   0.7 1.2 wt + − n.d. n.d. + − n.d. 1 30 10.7 1 1   1.9 1.2 wt + − 1 1 − − − 1 31 11.47 0.3 n.d.   0.8 0.9 wt − + 3 1 + + − 1 32 14.42 1.5 n.d.   2.4 1.4 wt + − 1 1 − − − 1 33 3.92 0.3 0.2   0.4 1.7 wt + − 3 4 − − + 1 34 11.96 0.7 1   0.5 2.8 wt − − 1 1 − − − 2 35 3.69 0.1 1.8 1.17 0.32 0.4 0.7 wt + − 1 4 + + − 2 36 8.03 0.4 2.7   0.7 1.4 wt + − 1 1 − − − 1 37 21.33 2 0.4   0.8 5.6 wt − − 1 1 + + − 4 38 5.56 0.4 4.7   1.6 0.6 wt + − 3 4 + + − 2 39 1.12 0.1 1.1   2.4 0 wt − − 1 4 − + − 2 40 13.98 1.4 1.4   3.5 0.9 wt + − 1 1 + − − 2 41 5.84 0.3 3.6   1 0.6 wt + − 3 4 − + + 1 42 7.36 0.3 1.3 1.83 0.25 0.9 0.8 wt + − 1 1 − − − 1 43 4.77 0.3 1.2   0.8 0.8 wt − + 3 n.d. − + n.d. 4 44 2.45 0.1 2.2   0.7 0.4 wt + − 1 3 − + − 1 45 5.12 0.3 0.7   0.8 0.7 wt + − 3 4 − + + 1 46 3.1 0.3 2.1   1.6 0.2 wt + − 1 1 − − − 1 47 10.27 1.1 2.4   19.6 0.1 wt + − 1 1 + + − 2 48 12.13 1.1 2.4   1.1 2.2 wt + − n.d. n.d. + − n.d. 1 49 7.94 0.8 1.6   0.8 2.2 wt − − 3 1 + + − 1 50 14.72 0.8 2.8   0.8 2.2 wt + − 3 4 n.d. + − 1 51 15.67 1 1   1 2.2 wt + − 1 2 − − − 1 52 66.49 3.6 0.6   3.6 2.2 wt + − 1 n.d. + − n.d. 1 53 28.94 1.4 1.7   1.4 2.2 wt + − 1 n.d. − − n.d. 1 54 3.72 0.4 0.3   2.8 0.5 wt − − 1 n.d. + + n.d. 4 55 5.3 0.4 0.7   1.8 0.5 wt + − 1 1 − − − 1 56 2.04 0.6 0.9   2.3 0.3 wt n.d. n.d. 1 n.d. + − n.d. 1 57 6.87 0.7 n.d.   1.4 0.8 wt n.d. n.d. 1 n.d. − − n.d. 1 n d N1 M1 a b 1 p 1 W = p 1 p = 1 Fig. 1 a–d superimposed line M SD e f line series notched box and whiskers Crosses circles p  U = p p 2 Fig. 2 p  3 = p = 3 Fig. 3 Asterisks section sign Arrows  3 Analysis of intraindividual variation of MDM4 and MDM2 levels 4 2 4 2 3 Fig. 4 a b superimposed line  Correlation of MDM4 and MDM2 with tumor histopathological features 1 2 V600E V599Ins V600E 22 = p = 5 3 p = Fig. 5 a line series notched box and whiskers Crosses circles p b line series notched box and whiskers Crosses circles p  Table 3 Summary of significant correlations of MDM4 and MDM4 T–N values with histopathological features   Multifocality Tumor Stage Presence Absence I II–IV n n n = 23 n = 18 MDM4 T–N Mean ± SD 0.9 ± 0.76 0.56 ± 0.43 0.81 ± 0.54 0.51 ± 0.43 Median ± IQR 0.63 ± 0.83 0.38 ± 0.6 0.65 ± 0.81 0.3 ± 0.58 MDM4 Mean ±SD 12.4 ± 12.6 8.1 ± 6.4 10.28 ± 5.9 8.38 ± 5.9 Median ± IQR 8.74 ± 8.7 6.19 ± 5.1 8.85 ± 5.44 5.7 ± 8.0 p 3 n p 5 3 On the contrary, MDM2 levels as well as MDM2 T-to-N ratio did not show correlation with any histopathological parameter. Characterization of MDM4 variants in thyroid tumors 2 11 1 13 2 13 3 1 11 p Discussion 3 23 24 25 11 25 6 7 26 1 27 11 2 28 1 3