PMC9478947_01
(A) CT scan performed on November 2008, before Sorafenib is started. Large lymph node metastases (between 2 and 5.5 cm in diameter) localized in the right hilar region across the pulmonary artery and the main bronchus. (B) Thyroglobulin (Tg) biomarker trend during Sorafenib treatment and after surgical removal of metastases performed after Sorafenib treatment (anti-Tg antibodies persistently negative): Tg values were significantly reduced in the first few weeks of Sorafenib therapy and remained low throughout treatment on different doses. In July 2009, after surgical removal of lymph node and lung metastases, Tg values rose suddenly. (C, D) CT scan performed on July 2009, after following Sorafenib withdrawal and surgical removal of lymph node and lung metastases. Ten liver metastases of 1-2.5 cm size and adrenal metastases (5 cm on the left and 4 cm on the right) were observed. (E) CT scan performed on January 2009, during Sorafenib treatment, showing significant reduction in lymph node metastases (maximum diameter 1 cm). (F) Chronological description of serum suppressed thyroglobulin levels and treatments carried out for thyroid carcinoma.
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In addition, Ki-67 immunostaining showed a proliferation index >5% in both the primary tumor and the distant metastasis ( Supplementary Figure 1 ). Thus, in November 2008, due to the presence of progressive, symptomatic, iodine-refractory, unresectable distant metastases ( Figure 1A ), after informed consent of the patient, we started treatment with Sorafenib, the only TKI available at the time. During Sorafenib treatment, lymph node and lung metastases showed a significant volume reduction, and serum thyroglobulin (Tg) decreased from 1,700 to 55 ng/ml ( Figure 1B ). Tg values, without a substantial reduction in side effects. The dose was then increased to 600 mg/day with a new decrease in Tg levels ( Figure 1B ). In June 2009, given the significant reduction of metastases burden ( Figure 1C ), the patient underwent right pneumonectomy and mediastinal lymphadenectomy. Tg levels increased progressively, and a total body CT scan, negative for neck and chest metastases, showed multiple liver and adrenal metastases ( Figures 1D-F ). The patient's general conditions deteriorated rapidly, so no further treatments were possible, and he died in September 2009.
Sequencing of the PCR amplicons corresponding to exon 7 of PTEN and to exons 5 and 9 of TP53 genes and of RT-PCR amplicon corresponding to exon 5 of TP53 in FFPE and frozen samples. (A) The PTEN p.P248Tfs*5 mutation was found in heterozygosity in the DNA extracted from FFPE primary TC and lung and lymph node metastases samples obtained before and after Sorafenib treatment. (B) Different mutational patterns observed for TP53 in four sections of primary TC. The first sample showed the presence of TP53 p.C135Y mutation in exon 5 and c.920-2A>G splicing variant in intron 8, the second harbored two TP53 mutations (p.M133R and p.C135Y), the third had only the p.M133R mutant, while the last had no mutations. (C) The c.920-2A>G splicing variant was found in all metastases analyzed with the exception of the lymph node metastasis obtained after Sorafenib treatment. (D) The analysis of TP53 transcript encompassing exon 5 showed the presence of the C135Y mutation in homozygous state in the cDNA obtained from frozen primary TC. TC, thyroid cancer; MTS, metastasis.
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The molecular analysis of the DNA obtained from the formalin-fixed paraffin-embedded (FFPE) primary TC revealed the presence of a frameshift variant in exon 7 of PTEN (c.741dupA, p.P248Tfs*5) ( Figure 2A ) and a missense mutation in exon 5 of TP53 gene (c.404G>A, p.C135Y) in heterozygosis, both absent in the corresponding contralateral normal thyroid tissue (data not shown). On the other hand, a heterogeneous TP53 molecular profile was observed in these samples: one harbored the TP53 p.C135Y mutant together with a splicing mutation in intron 8 (c.920-2A>G), the second harbored the TP53 p.C135Y variant together with another missense variant (c.398T>G, p.M133R), the third sample had only the p.M133R mutation, and the last had no TP53 mutations ( Figure 2B ). Evaluating the presence of these variants in patient's FFPE lung and lymph node metastatic samples, the PTEN frameshift was detected in all tissues obtained pre- and post-Sorafenib treatment but, in the latter, at very low allelic frequency ( Figure 2A ). On the contrary, the two TP53 missense mutations in exon 5 were absent in all metastatic samples analyzed, while the splicing variant c.920-2A>G was present in lung metastases obtained both pre- and post-treatment and in the lymph node metastasis before starting Sorafenib ( Figure 2C ). TP53 p.C135Y mutation was found in homozygosis in the primary TC tissue ( Figure 2D ). The DNA recovered from the sample harbored the p.C135Y mutation in heterozygosis. It is interesting to note that there is an almost total loss of MMR proteins in the tumor area corresponding to PDTC ( Supplementary Figure S2 ).
Immunohistochemistry for p53 protein in FFPE primary TC, contralateral normal thyroid tissue, and lung and lymph node metastases obtained before and after Sorafenib treatment. (A) The immunostaining for p53 was wild type in the contralateral normal thyroid tissue (inset a*) and abnormal/over-expressed with high nuclear expression in primary TC, both FTC/PDCT (inset b*) and PDTC areas (inset c*). (B-D) Lung metastases obtained before (B) and after Sorafenib treatment (D) and lymph node metastasis before TKI (C) showed an abnormal/cytoplasmatic p53 staining with low nuclear expression (for each is shown a selected inset area). (E) The lymph node metastasis obtained after the TKI treatment showed an abnormal/over-expressed p53 pattern with high nuclear expression. FTC, follicular thyroid cancer; PDTC, poorly differentiated thyroid cancer; NT, normal thyroid; TKI, tyrosine kinase inhibitor. Scale bar are shown for each images.
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A peculiar pattern was observed in tissues analyzed: p53 was abnormal/over-expressed with high nuclear expression in primary TC (both FTC and PDTC areas) and normal in the contralateral normal thyroid tissue ( Figure 3A ). As far as metastases are concerned, all metastatic tissues had an abnormal/cytoplasmatic p53 staining with low nuclear expression ( Figures 3B-D ) with the exception of the lymph node obtained after Sorafenib treatment showing an abnormal/over-expressed p53 pattern with high nuclear expression ( Figure 3E ).
Immunohistochemistry for p53 and DNA mismatch repair (MMR) proteins and microsatellite instability (MSI) detection in available samples before and after Sorafenib treatment (A, C, D) A positive nuclear staining was observed for MHL1, MSH2, MSH6, and PMS2 proteins in all analyzed tissues samples. However, primary TC shows light staining and subclonal loss of expression in all MMR proteins. (B) CNV analysis shows the presence of a LOH for both BAT-25 and BAT-26 loci in the primary TC of our patient. (E) Peak analysis of D2S123 and D5S346 loci, performed using Genemapper 5 software, is clearly differently shaped in the lymph node obtained after Sorafenib treatment with respect to those of and contralateral normal thyroid tissue DNA, indicating a high microsatellite instability. TC, thyroid cancer; MTS, metastasis; NT, normal thyroid; CNV, copy number variation.
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Finally, evaluating the expression of mismatch repair proteins (MMRs), a positive nuclear staining was observed for all these markers (MHL1, MSH2, MSH6, and PMS2) in the tissues analyzed ( Figures 4A-C ), although not all nuclei in the primary tumor showed the expression of MMR antigens ( Figure 4A ). Copy number variation (CNV) analysis showed the presence of a loss of heterozygosity (LOH) for both loci in the primary TC ( Figure 4D ). On the other hand, a high MSI for D2S123 and D5S346 loci was found only in the lymph node metastasis obtained after Sorafenib treatment, as shown in  Figure 4E .