Lateral lymph node metastasis in papillary thyroid microcarcinoma: a study of 5241 follow-up patients.

PURPOSE: To investigate the impact of lateral lymph node metastasis in papillary thyroid microcarcinoma (PTMC). METHODS: 5241 PTMC patients with follow-up information were enrolled in the current study. These patients underwent primary surgery in our situation from January 1997 to December 2016. Additionally, a validation cohort consisting of 274 PTMC patients who underwent primary surgery between January 2020 and December 2021 was also included. Univariable and multivariate logistic analyses were conducted to identify the association between clinicopathologic features and lateral lymph node metastasis (LLNM). Kaplan-Meier survival curve analysis was used to calculate the disease-free survival (DFS) rate. The fitting curve was generated to identify the quantitative relationship between central lymph node metastases (CLNM) and LLNM. RESULTS: Of 5241 PTMC patients, cervical lymph node metastasis was detected in 1494 (28.5%) cases, including 1364 (26.0%) with CLNM only and 130 (2.5%) with LLNM. With a median follow-up time of 60 months (interquartile range [IQR], 44-81), recurrence was detected in 114 patients (2.2%). Multivariate Cox regression analyses showed that LNM was the only independent risk factor for recurrence, with HR values of 3.03 in CLNM and 11.14 in LLNM, respectively. Tumor diameter >0.5 cm (hazard ratio [HR]:1.80), multifocality (HR:2.59), bilaterality (HR:2.13), extrathyroidal invasion (HR:2.13), and CLNM (HR:5.11) were independent risk factors for LLNM. The prevalence of LLNM escalated significantly with increasing number of lymph node involvement in CLNM when stratified by the number of metastatic lymph nodes and trend was observed similarly in the validation cohort. The fitting curve showed that the incidence of LLNM could be as high as 20.7% when the number of CLNM ≥ 5. CONCLUSIONS: By analyzing a large database with follow-up information, our study provides evidence that LLNM is significantly correlated with tumor recurrence in patients with PTMC. Tumor size (>0.5 cm), multifocality, bilaterality, extrathyroidal extension (ETE) and CLNM are independent risk factors for LLNM.

Long-term impact of prophylactic central neck dissection in non-invasive classic papillary thyroid carcinoma.

BACKGROUND: The utilization of prophylactic central neck dissection (pCND) in cases of non-invasive clinical node-negative (cN0) papillary thyroid carcinoma (PTC) remains a topic of debate, with a dearth of long-term evidence. MATERIALS AND METHODS: We retrospectively reviewed 1181 cN0 PTC patients from 1997 to 2011. Of these, 641 underwent pCND (pCND + group) and 540 did not (pCND-group). Propensity score matching (PSM) was used to identify similar patients. Event-free survival and long-term complications including permanent hyperparathyroidism and permanent recurrent laryngeal nerve (RLN) paralysis were analyzed after PSM. RESULTS: The pCND + group had more aggressive characteristics. In the matched cohort after PSM, the 5-year, 10-year, and 15-year EFS rates were 98.9 %, 98.2 %, and 97.1 % for the pCND + group, and 97.7 %, 97.1 %, and 97.1 % for the pCND-group, respectively. There was no statistically significant difference in EFS rates between the two groups (Log Rank P = 0.38). There was no statistically significant difference in the incidence of permanent hyperparathyroidism (3.3 % vs. 1.5 %, P = 0.08) and permanent RLN paralysis (1.7 % vs. 0.9 %, P = 0.13) between the pCND+ and pCND- groups. CONCLUSION: Our study, with a median follow-up duration of 107 months, indicates that pCND does not lead to a significant reduction in nodal recurrence among non-invasive cN0 PTC patients.

Combined fine-needle aspiration and selective intraoperative frozen section to optimize prediction of malignant thyroid nodules: A retrospective cohort study of more than 3000 patients.

Background: Preoperative fine-needle aspiration (FNA) is widely used to differentiate malignant from benign thyroid nodules, while intraoperative frozen sections (FS) are suggested as a systematic supplement for intraoperative decision-making, but limitations still remain for both procedures. Methods: Medical records of 3807 patients with thyroid nodules who underwent both pathological diagnoses (FS and FNA) at our hospital were reviewed. The diagnostic accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FNA and FS were also evaluated. We further designed an optimal integration scheme (FNA+selective FS) to predict thyroid nodule malignancy. Finally, the efficiency of the proposed integrated diagnostic model was validated using an independent external cohort. Results: For distinguishing malignant nodules, FNA had an accuracy of 90.3%, sensitivity of 90.7%, specificity of 85.2%, PPV of 98.8% and NPV of 40.4%. In contrast, the FS represented higher discriminative power (Accuracy, 94.5%; Sensitivity, 94.1%; Specificity, 100%; PPV, 100%; and NPV, 55.6%). we proposed the selective usage of FS (removed nodules with Bethesda category VI from routine FS, ~1/3 of total). The integrated new diagnostic model of FNA plus selective FS (FNA+sFS) achieved accuracy of 96.9%, sensitivity of 97.3%, specificity of 92%, PPV of 99.4%, and NPV of 71.6% (NRI=0.135, 95% CI 0.103-0.167, P <0.001) and was successfully applied to an external cohort (N=554). Conclusion: Compared with the FNA diagnostic system, FS has an increased ability to distinguish benign and malignant thyroid nodules. The newly proposed integrated diagnostic model of FNA + selective FS can optimize the accuracy of diagnosis.

Preoperative Thyroid Peroxidase Antibody Predicts Recurrence in Papillary Thyroid Carcinoma: A Consecutive Study With 5,770 Cases.

Background: Thyroid autoimmunity is common in papillary thyroid carcinoma (PTC) and was believed to confer a better prognosis; however, controversy still remains. This study aimed to investigate the prognostic value of chronic lymphocytic thyroiditis (CLT) and preoperative thyroid peroxidase antibody (TPOAb) in PTC patients. Methods: A retrospective analysis was performed on 5,770 PTC patients who underwent surgical treatment with pathologically confirmed PTC in our institution between 2012 to 2016. The patients were divided into groups with respect to the coexistence of CLT or preoperative TPOAb levels. The clinicopathological characteristics and disease-free survival (DFS) rates were compared between the groups. Results: The coexistence of CLT was likely to have bilateral, multifocal tumors. Particularly, PTC patients with TPOAb++ (>1,000 IU/L) had a larger tumor size (p = 0.007) and higher rates of bilaterality and multifocality than those with TPOAb- (TPOAb< 100 IU/L), while for lymph node metastasis and extrathyroidal extension, there is no statistical difference. Tumor recurrence was found in 15 of 425 (3.5%), 9 of 436 (2.1%), and 56 of 3,519 (1.6%) patients with TPOAb++, TPOAb+, and TPOAb-, respectively (p = 0.017). On univariate analysis, TPOAb++ was correlated with tumor recurrence, with a hazard ratio of 2.20 [95% confidence interval (CI), 1.25-3.89], which remained as an independent risk factor at 1.98 (95% CI, 1.10-3.55) on multivariate analysis. PTC patients with TPOAb++ had the lowest DFS rates (96.5 vs. 97.9 vs. 98.4%, p = 0.020). Conclusion: CLT is not a protective factor in PTC patients. We provide initial evidence that the preoperative TPOAb instead predicts recurrence in papillary thyroid carcinoma.

Use of 99mTc-sestamibi SPECT/CT imaging in predicting the degree of pathological hyperplasia of the parathyroid gland: semi-quantitative analysis.

BACKGROUND: Previous studies have demonstrated that 99mTc-sestamibi (99mTc-MIBI) Single-Photon Emission Computed Tomography/ Computed Tomography (SPECT/CT) imaging is an effective isotopic technique for locating the parathyroid in secondary hyperparathyroidism (SHPT). This study aimed to explore further the correlation between 99mTc-MIBI SPECT/CT imaging and SHPT to demonstrate the value of 99mTc-MIBI SPECT/CT in evaluating the degree of pathological hyperplasia of the parathyroid gland (PG). METHODS: The demographics, surgical records, and follow-up information of 91 patients were recorded and analyzed. A total of 216 paraffin-embedded PGs of 54 patients were obtained and analyzed. RESULTS: Patients with 99mTc-MIBI negative PG(s) had significantly lower preoperative serum phosphorus and higher serum calcium levels at 6 months postoperatively compared to those with 99mTc-MIBI positive PG(s) (P<0.05). We also found a higher total uptake ratio of the region of interest (URRI) and higher URRI max in the hypocalcemia group than in the non-hypocalcemia group. Both URRI total (P=0.003) and URRI max (P=0.028) were independent risk factors for hypocalcemia 6 months postoperatively. The URRI values of the PGs were significantly positively correlated with glandular weight (R2=0.343, P<0.001), glandular volume (R2=0.240, P<0.001), and degree of pathological hyperplasia (P<0.001). However, the URRI value of the PGs exhibited a notably weak correlation with proliferating cell nuclear antigen (PCNA) (R2=0.035, P=0.006). The area under the receiver operating characteristic curve showed a URRI evaluative value of 0.771 for diffuse and nodular types in 216 PGs (P<0.001). We further evaluated 167 nodular-type PGs, distinguishing between nodular hyperplasia and a single nodule; the URRI evaluative value reached 0.819, which was higher than the volume or weight (P<0.001). CONCLUSIONS: The 99mTc-MIBI SPECT/CT scintigraphy results were related to serum calcium levels at 6 months after total parathyroidectomy with autotransplantation (TPTX+AT), suggesting the occurrence of hypocalcemia (6 months after TPTX+AT). More importantly, this technique effectively evaluated the pathological hyperplasia of PGs preoperatively, and therefore, could assist surgeons in selecting the PGs with the lowest degree of hyperplasia intraoperatively.

Coexistence of papillary thyroid carcinoma in secondary hyperparathyroidism.

BACKGROUND: The coexistence of primary hyperparathyroidism and papillary thyroid carcinoma (PTC) is common and may be associative with more aggressive PTC, with higher rates of extrathyroidal extension and multicentricity. However, it is unclear whether secondary hyperparathyroidism (SHPT) is associated with more invasive PTC in terms of morbidity, tumor pathological characteristics, and prognosis. The aim of this study was to evaluate the rate and tumor characteristics of PTC in patients with SHPT. METHODS: A total of 531 patients diagnosed with SHPT who underwent surgery from August 2013 to December 2018 at the First Affiliated Hospital of Zhejiang University were evaluated retrospectively. Patient demographics, surgical records, and follow-up information were recorded and analyzed. Control subjects were matched to the enrolled patients in a 1:4 ratio in terms of age, sex and pathological subtype. RESULTS: Among the 531 patients with SHPT who underwent surgery, 34 had coexisting PTC and PTC + SHPT (6.4%). The mean tumor diameter in the PTC + SHPT group was smaller than that in the PTC group (5.57 mm vs 9.00 mm, p < 0.001). The proportion of papillary thyroid micro-carcinoma in the PTC + SHPT group was significantly higher than that in the PTC group (29 [85.29%] vs. 86[63.24%], p = 0.014). There were no statistically significant differences between groups in terms of tumor multicentricity (15 [44.12%] vs 39 [28.68%], p = 0.066), tumor bilaterality (9 [26.47%] vs. 29 [21.32%], p = 0.499), tumor extrathyroidal extension (2 [5.88%] vs. 19 [13.97%], p = 0.255), or lymph node (LN) metastasis rate (12 [35.29%] vs. 49 [36.03%], p = 1.000). However, the PTC + SHPT and PTC groups were significantly different in terms of contralateral thyroidectomy (10 [29.41%] vs. 70 [51.47%], p = 0.023) and lymph node dissection (22 [64.71%] vs. 125 [91.91%], p < 0.001).There was no significant difference between the PTC + SHPT and PTC groups in terms of prognostic staging (33 [97.06%] vs. 122 [89.71%], p = 0.309) or recurrence (mean follow-up time: 36 months vs. 39 months, p = 0.33). CONCLUSIONS: The prevalence of PTC is high in patients with SHPT; compared with PTC in the general population, most papillary thyroid carcinomas with SHPT are occult thyroid carcinomas and present no significant difference in terms of tumor pathological features and prognostic staging. It is necessary for surgeons to perform more adequate preoperative examination and be more careful during surgery to avoid missing the coexistence of PTC in patients with SHPT.

A Comparison of the Seventh and Eighth Editions of the AJCC Staging Systems to Predict Recurrence in Papillary Thyroid Microcarcinoma.

BACKGROUND: The incidence of papillary thyroid microcarcinoma has been constantly rising in recent decades. The tumor, node, metastasis staging system is designed to predict prognosis in patients with papillary thyroid carcinoma. Recent studies have shown that the American Joint Committee on Cancer (AJCC) 8th edition is superior to the 7th edition for predicting tumor recurrence in PTC patients. To date, whether the 8th edition is also better able to predict recurrence in papillary thyroid microcarcinoma (PTMC) remains unclear. METHOD: We enrolled 1007 cases from our thyroid cancer database in the First Affiliated Hospital, Zhejiang University School of Medicine, from 1997 to 2011. Univariable and multivariate Cox hazard regression analyses were used to identify the association between variables and recurrence. Disease-free survival was calculated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: A total of 1007 PTMC patients were enrolled, with a median follow-up of 67 months. Of 93 (9.2%) patients downstaged by the changes in versions, 49 (52.7%) were downstaged because the age-at-diagnosis cut-off used for staging increased from 45 to 55 years, while 35 (37.6%) were downstaged due to the weakening of the effects of lymph node metastasis. The recurrence rate of PTMC was 4.17%. Univariate Cox hazards regression analyses showed that TNM stage according to the AJCC 8th edition was significantly associated with recurrence, while the recurrence survival curves showed that TNM stage (stage I vs. stage II-IV) according to the AJCC 8th edition, but not the 7th edition, was significantly associated with disease-free survival (p < 0.05). CONCLUSIONS: The AJCC 8th edition has better ability to predict recurrence in PTMC patients than the 7th edition.

VCAM-1 Upregulation Contributes to Insensitivity of Vemurafenib in BRAF-Mutant Thyroid Cancer.

Vemurafenib, an inhibitor of mutant BRAF activity, is a promising anticancer agent for patients with BRAF-mutant metastatic melanoma. However, it is less effective in BRAF-mutant thyroid cancer, and the reason for this discrepancy is not yet fully elucidated. By RNA sequencing analysis, we identified vascular cell adhesion molecular-1 (VCAM-1) to be highly upregulated in both time- and dose-dependent manners during BRAF inhibition (BRAFi) in a BRAF-mutant papillary thyroid cancer cell line (BCPAP). Cell cytotoxicity and apoptosis assays showed that knockdown of the induced VCAM-1 in BCPAP cells augmented the antitumor effects of vemurafenib, with decreased IC50 values of 1.4 to 0.8 µM. Meanwhile, overexpression of VCAM-1 in a BRAF-mutant anaplastic thyroid cancer cell line (FRO) reduced the sensitivity to vemurafenib, with increased IC50 values of 1.9 to 5.8 µM. Further investigation showed that PI3K-Akt-mTOR pathway was activated during BRAFi. Co-treatment with Akt signaling inhibitor MK2206 decreased the induced expression of VCAM-1 during BRAFi. This combination further improved the efficacy of vemurafenib. Moreover, VCAM-1 promoted migration and invasion in thyroid cancer cells in vitro, which was also indicated in thyroid cancer patients. The present study is the first to demonstrate that VCAM-1 is upregulated in thyroid cancer cells treated with vemurafenib and contributes to vemurafenib resistance in BRAF-mutant thyroid cancer cells. Targeting the PI3K-Akt-mTOR pathway-mediated VCAM-1 response may be an alternative strategy to sensitize BRAF-mutant thyroid cancers to vemurafenib.

Nomogram-Based New Recurrence Predicting System in Early-Stage Papillary Thyroid Cancer.

BACKGROUND AND OBJECTIVES: The clinicopathological risk factors to predict recurrence of papillary thyroid cancer (PTC) patients remain controversial. METHODS: PTC patients treated with thyroidectomy between January 1997 and December 2011 at the First Affiliated Hospital of Zhejiang University (Zhejiang cohort) were included. Multivariate Cox regression analysis was conducted to identify independent recurrence predictors. Then, the nomogram model for predicting probability of recurrence was built. RESULTS: According to Zhejiang cohort (N = 1,697), we found that the 10-year event-free survival (EFS) rates of PTC patients with early-stage (TNM stages I, II, and III) were not well discriminated (91.6%, 89.0%, and 90.7%; P=0.768). The multivariate Cox model identified age, bilaterality, tumor size, and nodal status as independent risk factors for tumor recurrence in PTC patients with TNM stages I-III. We then developed a nomogram with the C-index 0.70 (95% CI, 0.64 to 0.76), which was significantly higher (P < 0.0001) than the AJCC staging system (0.52). In the validation group, the C-index remained at a similar level. CONCLUSIONS: In this study, we build up a new recurrence predicting system and establish a nomogram for early-stage PTC patients. This prognostic model may better predict individualized outcomes and conduct personalized treatments.

Clonal analysis of early-stage bilateral papillary thyroid cancer identifies field cancerization.

INTRODUCTION: Bilaterality is a newly identified indicator for aggressive tumor behavior and poor outcome in papillary thyroid cancer. However, the clonal origin of these bilateral tumors remains unclear. METHODS: Here we analyzed 28 pairs of early-stage papillary thyroid cancers (stage I-II without extra-thyroidal extension, lymph node metastasis or distant metastasis) that underwent surgery at First Affiliated Hospital of Zhejiang University School of Medicine (Hangzhou, China). Genomic DNA was extracted from paraffin-embedded tissues after microdissection and analyzed for BRAF mutation and X-chromosome inactivation. RESULTS: A total of 16 patients (16/28, 57.1%) harbored different BRAF status in bilateral tumors. Fourteen patients were available for X-chromosome inactivation assay and 10 of them achieved informative results. Bilateral tumors from four cases had distinct patterns of X-chromosome inactivation. Combining the results of X-chromosome inactivation and BRAF analysis, we demonstrated that at least 64.3% (18/28) cases harbored discordant X-chromosome inactivation or BRAF status, indicating their independent clonal origin in bilateral tumors. CONCLUSIONS: The present study confirms "field cancerization" in early-stage bilateral thyroid cancers, suggesting that these subtype papillary thyroid cancers should be treated as independent and localized tumors.

Mitochondrial DNA depletion, mitochondrial mutations and high TFAM expression in hepatocellular carcinoma.

We investigated the role of mitochondrial genetic alterations in hepatocellular carcinoma by directly comparing the mitochondrial genomes of 86 matched pairs of HCC and non-tumor liver samples. Substitutions in 637 mtDNA sites were detected, comprising 89.80% transitions and 6.60% transversions. Forty-six somatic variants, including 15 novel mutations, were identified in 40.70% of tumor tissues. Of those, 21 were located in the non-coding region and 25 in the protein-coding region. Twenty-two somatic nonsynonymous changes were identified as putative pathogenic variants, including 4 truncating mutations produced by three frameshifts (MT-ATP6 8628 insC; MT-ND5 13475 T-del, and MT-CYB 14984 insA) and 1 nonsense mutation in MT-CO3 9253 G>A. Among the somatic variants, only m.13676 A>G (MT-ND5), found in only 1 tumor, was heteroplasmic. Both inherited and somatic variants were predominately located in the D-loop region and the MT-ND5 gene. Tumor/non-tumor paired analysis showed that 69% of HCC samples contained significantly reduced mtDNA, compared with 49.0% of non-tumor counterparts. In 81.40% of HCC samples, mitochondrial transcription factor A (TFAM) was enriched in tumor cells but not in adjacent non-tumor cells. Neither mtDNA depletion nor TFAM overexpression correlated with the degree of cell differentiation, though TFAM expression correlated with tumor size.

Maternally inherited diabetes is associated with a homoplasmic T10003C mutation in the mitochondrial tRNA(Gly) gene.

In this report, we investigate molecular pathogenic mechanism of a diabetes-associated homoplasmic mitochondrial tRNA mutation in a Han Chinese family with maternally transmitted diabetes mellitus. Of 10 adult matrilineal relatives, 5 individuals suffered from diabetes (4 subjects with only diabetes, one subject with both diabetes and hearing impairment), while other five matrilineal relatives (one with hearing loss) had glucose intolerance. The average age at onset of diabetes in matrilineal relatives was 50 years. Molecular analysis of their mitochondrial genomes identified the novel homoplasmic T10003C mutation in the tRNA(Gly) gene belonging to haplogroup M11b. The T10003C mutation is expected to form a base-pairing (13C-22G) at the highly conserved D-stem of tRNA(Gly), thereby affecting secondary structure and function of this tRNA. A tRNA Northern analysis revealed that the T10003C mutation caused ~70% reduction in the steady-state level of tRNA(Gly). An in vivo translation analysis showed ~33% reduction in the rate of mitochondrial translation in mutant cells. Oxygen consumption analysis showed the defects in overall respiratory capacity or the ATP-linked, proton leak, and maximal respiration in mutant cells. As a result, the cellular energy deficiency contributes to the development of diabetes in subjects carrying the T10003C mutation. These data provide the first direct evidence that the tRNA(Gly) mutation might be associated with diabetes. Thus, our findings may provide new insights into the understanding of pathophysiology of maternally inherited diabetes.

Aminoglycoside stress together with the 12S rRNA 1494C>T mutation leads to mitophagy.

Aminoglycosides as modifying factors modulated the phenotypic manifestation of mitochondrial rRNA mutations and the incomplete penetrance of hearing loss. In this report, using cybrids harboring the m.1494C>T mutation, we showed that gentamycin aggravated mitochondrial dysfunction in a combination of the m.1494C>T mutation. The m.1494C>T mutation was responsible for the dramatic reduction in three mtDNA-encoded proteins of H-strand, with the average of 39% reduction, except of the MT-ND6 protein, accompanied with 21% reduction of ATP production and increase in mitochondrial reactive oxygen species, compared with those of control cybrids. After exposure to gentamycin, 35% reduction of mitochondrial ATP production was observed in mutant cybrids with a marked decrease of the mitochondrial membrane potential. More excessive cellular reactive oxygen species was detected with stimulus of gentamycin than those in mutant cells. Under gentamycin and m.1494C>T stress together, more dysfunctional mitochondria were forced to fuse and exhibited mitophagy via up-regulated LC3-B, as a compensatory protective response to try to optimize mitochondrial function, rather than undergo apoptosis. These findings may provide valuable information to further understand of mechanistic link between mitochondrial rRNA mutation, toxicity of AGs and hearing loss.