PMC8807656_01
(A) Ultrasound examination revealed a heterogeneous nodule in the left lobe of the thyroid. (B) CT examination revealed 4-cm low-density lesions in the left lobe of the thyroid gland. (C, D, E) Direct cell smears show abundant cells with diffuse distribution of small- to medium-sized atypical monolayer protocells, and the foci of sudden keratosis could be seen from low magnification in (C) and high magnification in (E). (E) Tumor cells have round to oval nuclei with bare and scanty cytoplasm and vague to prominent nucleoli. The chromatin of the nucleus varies from mostly light open chromatin to hyperchromatic. (F) Positive immunoreaction with NUT protein in the nucleus of the carcinoma cells.
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University (Shandong, China) for a hard and painless neck nodule that had persisted for approximately 3 months. Ultrasound examination revealed a 3.8 x 2.7-cm irregular heterogeneous nodule in the left lobe of the thyroid gland with multiple point-like strong echoes ( Figure 1A ), which was further confirmed by CT ( Figure 1B ). Direct cell smears revealed an abundance of diffuse, small- to medium-sized, atypical dispersed monolayers of primitive cells with a few lymphocytes in the background ( Figures 1C-E ). The chromatin of the nucleus varied from mostly light, open chromatin to hyperchromatin ( Figure 1E ). Sudden keratosis was observed ( Figures 1C, E  ). A positive immunoreaction with the NUT antibody was found in the nuclei of the carcinoma cells ( Figure 1F ).
(A) Nests and sheets of primitive cells with high nuclear/cytoplasmic ratios infiltrate surrounding normal thyroid tissue. (B) Sudden keratosis foci could be seen. (C) Coagulative necrosis. (D) Lymphoid vessel involvement. (E) Immunoreaction with EMA in the cell membrane of the NUT carcinoma cells. (F) Immunoreaction with NUT protein in the nucleus of the NUT carcinoma cells. (G) Positive immunoreaction with TTF-1 in the NUT midline carcinoma cells; internal positive control of follicular thyroid epithelium is seen in the right part of the picture. (H) Positive immunoreaction with c-myc was found in approximately 80% of the carcinoma cells. (I, J) Immunoreaction with CK19 in sudden keratoses and a few NUT midline carcinoma cells. (J) The Ki-67 labeling index is high. (K) Positive immunoreaction with PD-L1 in carcinoma cells and a few inflammatory cells. (L) FISH shows splitting of the translocated NUTM1.
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NC. Nests and sheets of primitive cells with a high nuclear/cytoplasmic ratio infiltrate surrounding normal thyroid tissue were present ( Figure 2A ). There was squamous differentiation with prominent red medium-sized nuclei, suggestive of NC ( Figure 2B ). Coagulative necrosis and lymphatic vascular involvement were frequently observed, with mitotic rates as high as 3-6/5 high visual fields ( Figures 2C, D  ). Positive immunoreaction with EMA and the NUT protein was observed in the carcinoma cells but not in the sudden keratoses ( Figures 2E, F  ). A positive TTF-1 immune response was observed in approximately 60% of the carcinoma cells, suggesting that the tumor originated from follicular thyroid cells ( Figure 2G ), whereas only a few carcinoma cells were PAX-8 positive. A positive c-myc immunoreaction was found in approximately 80% of the carcinoma cells ( Figure 2H ). The CK19-positive immune response in the cytoplasm was mainly observed in cells with squamous differentiation ( Figure 2I ). The Ki-67 labeling index was approximately 60% ( Figure 2J ). The PD-L1 combined proportion score was 30% ( Figure 2K ). Dual-color FISH was further applied to confirm the split-apart translocated NUTM1, as shown in  Figure 2L .
Genomic rearrangements of BRD-NUT gene fusion and detailed introduction. (A) Circos plot showing the genetic variants identified in a thyroid NUT carcinoma genome. The corresponding genes are labeled inside the circus. The arc connecting the chromosomes signifies translocation detected in sequencing data. (B) Schematic ideograms show gene fusions between BRD4 (chr.19p13.1) and NUTM1 (hr. 15q14). The arrowheads indicate the breakpoints of BRD4 (exon 11) and NUTM1 (exon 2/3). Each base in the fusion plot is drawn 5' to 3'. (C) RT-qPCR showed that the expression level of BRD4-NUT fusion in cancer tissue was 3.58 higher than that in normal tissue, further confirming the results of WGS.
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Six SVs were located in the intron region of UTR3 and ncRNA, and the last SV was located in UTR5. Structural variation of NUTM1 occurred in the exon region at 15q14, while BRD4 SV occurred in the UTR3 region at 19p13.12 ( Figures 3A, B  ). According to the fusion information analysis, the BRD4-NUT fusion gene is formed by the fusion of exons 2 and 3 of the NUT gene and exon 11 of the BRD4 gene ( Figure 3A ). qRT-qPCR showed that the expression level of BRD4-NUT fusion in the cancer tissue was 3.58 higher than that in the normal tissue, further confirming the results of WGS ( Figure 3C ).
Time diagram from June 2019 to March 2020 of the patient: on June 18, 2019, thyroid nodule was identified by ultrasound and CT examination; on June 29, 2019, thyroid fine-needle aspiration was performed, and poorly differentiated carcinoma was suggested by cytology; on July 18, 2019, total thyroidectomy and lateral cervical lymph node dissection were performed. Thyroid NUT carcinoma was confirmed by morphology, immunohistochemistry, and genetic alterations. The combined proportion score of PD-L1 revealed to be 30%. On October 30, 2019, the patient received combined chemotherapy and PD-1 inhibition therapy (epirubicin 140 mg/dl, paclitaxel liposomes 240 mg/d2, and carelizumab 200 mg/dl). On November 19, 2019, CT examination revealed lateral cervical lymph node metastasis and bone metastasis on the 8th rib and L2 vertebra. Zoledronic for anti-bone metastasis was added to the previous treatment plan. On December 14, 2019, physical examination revealed a 5-cm mass on the right subscapular corner of the patient. Core needle biopsy revealed metastatic NUT carcinoma with focal necrosis (<10%), suggesting a poor response to the treatment. PET-CT showed multiple bone metastases. Due to the rapid progression, the chemotherapy regimen adjusted to cisplatin 50 mg/dl, 40 mg/d2,3+, and etoposide 0.1 dl-5. The patient died on March 20, 2020.
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However, distant metastases were confirmed gradually, as shown in  Figure 4 .