Comprehensive evaluation of risk factors for lymph node metastasis with papillary thyroid carcinoma in Southwest China patients

Background: With the increasing incidences of papillary thyroid cancer(PTC), it is important to risk-stratify patients who may have more aggressive tumor biology. This study aimed to evaluate the risk factors for lymph node metastasis with PTC in Southwest China Patients which may provide a substantial reference for clinical diagnosis and treatment. Methods: 1045 PTCs (313 PTMC and 732 non-PTMC) between August 2016 and August 2019 were examined totally (including one Tibetan). BRAF V600E mutation was tested in all samples. The clinical data (gender, age, tumor location, sample source and pathological features) were retrospectively analyzed. Logistic regression analysis was performed to evaluate independent risk factors for LNM. Results: 181 out of 313 PTMC cases (57.8%), 145 out of 732 non-PTMC cases (19.8%) had BRAF V600E mutation, the Tibetan had a double mutation of BRAF L597Q and V600E in two separate lesions. In PTMC, significant difference in gender and sample source was found (BRAF V600E mutation vs. wild-type). In non-PTMC, significant difference in gender was found (BRAF V600E mutation vs. wild-type). The female (OR=1.952; 95% CI= 1.373-2.774; P= 0.00), age (31-59 years) and diameter of tumor ≤1cm (OR=3.273; 95% CI= 2.417-4.432; P= 0.000) were significant independent predictors of LNM in all PTCs. In PTMC, the female (OR= 3.002; 95% CI= 1.654-5.446; P= 0.00) was a significant independent predictor of LNM. The tumor in left and right lobes simultaneously was an independent protective factor of LNM in each group (PTCs: OR=0.287; PTMC: OR=0.170; non-PTMC: OR=0.441, respectively). The BRAF V600E mutation rate of US-FNAC was much higher than FFPE in PTMC ( P= 0.018). gene logistic regression analysis were performed to assess independent risk factors for presence of LNM in PTCs, results are reported as odds ratios (OR) with 95% confdence intervals (CI). A p-value less than 0.05 was considered statistically significant. FFPE: paraffin-embedded; FNAC: fine-needle aspiration cytology; HE: hematoxylin-eosin staining; IHC: immunohistochemical analysis; PCR: polymerase chain reaction; LNM: lymph node metastasis; OR: odds ratios.

As in PTC in general, lymph node metastasis has been reported to be a risk factor for increased tumor recurrence rates and also be closely in connection with reduced survival rate [19] . In addition, in 2018, Lutz. et al [20] reported that an imbalance in DNA repair gene expression is associated with aggressive clinicopathological features in PTCs. Given the controversies above, a total of 1045 PTCs patients were enrolled in this study, including 313 patients with PTMC and 732 patients in non-PTMC (diameter of tumor 1 cm). The aim of this retrospective observational study was to verify the associations of BRAF V600E mutations with clinicopathologic features and next to identify the risk factors for LNM of PTC patients. One case of solitary brain metastasis from occult papillary thyroid carcinoma in the Chinese Tibetan population was reported firstly in detail. This information may bring benefits for clinicians to make correct clinical decisions for PTC patients in future.
Methods independently in a blinded manner. The inconsistent diagnostic cases have been discussed with a third pathologist.

Preoperative skull CT/MRI scan and molecular pathology diagnosis
The CT, MRI, color ultrasound diagnosis, ultrasound-guided fine-needle aspiration (US-FNA) and HE staining were used for morphological detection. The specific expression of thyroid cancer-related proteins (cytokeratin, CK; thyroglobulin, Tg and thyroid transforming factor-1, TTF-1) were detected by the immunohistochemical (IHC) analysis.  (Table 7), the forward primer of BRAF in Sanger sequencing: 5'-GCTTGCTCTGATAGGAAAATGAG-3', the reverse primer of BRAF in Sanger sequencing: 5'-GGGCCAAAAATTTAATCAGTGG-3' and the primers were synthesized by Invitrogen Bio-Tech Co., Ltd.

Sample collection, DNA extraction and Mutation Screening
(Shanghai, China).

Statistical analysis
Statistical analysis was performed using IBM SPSS 22.0 software (IBM Corp., Version 22.0, Armonk, NY, USA). Quantitative data were expressed as mean ± SD. Qualitative data were represented as a percentage or frequency. The Chi square test or Fisher's exact test was used to evaluate the difference in clinical features between two different groups. The univariate and multivariate logistic regression analysis were performed to assess independent risk factors for presence of LNM in PTCs, results are reported as odds ratios (OR) with 95% confdence intervals (CI). A p-value less than 0.05 was considered statistically significant.  Table 1). The BRAF V600E mutation rate in PTMC group was much higher than the non-PTMC group (P=0.00). The frequency of lymph node metastasis in PTMC group was also significantly higher than the non-PTMC group (P=0.00). Other clinical parameters showed no significant differences between the two groups.

PTMC
The relationship of BRAF mutation status and clinical characteristics of 313 PTMC patients were analyzed in this study. The BRAF V600E mutation showed significant association with male gender (P=0.026) and sample source from FFPE tissues (P=0.018) compared with the BRAF V600E wild-type in PTMC patients. However, there was no difference in lymph node metastases, age and location of thyroid tumor between the BRAF V600E mutation and the BRAF V600E wild-type ( Table 2).
The BRAF V600E mutation showed significant association with male gender (P=0.003) compared with the BRAF V600E wild-type in non-PTMC patients. However, there was no difference in other clinical features between the BRAF V600E mutation and the BRAF V600E wild-type ( Table 2).
The clinicopathological characteristics and sample source in all BRAF V600E mutation patients were compared additionally (Table 3). A lower rate of lymph node metastasis (P=0.00, χ 2 =42.369) was presented in PTMC than in non-PTMC. The number of middle subgroup (31-59, P=0.004, χ 2 =11.306) had statistical significance between PTMC and non-PTMC. However, there was no difference in other clinical features between PTMC and non-PTMC.
In non-PTMC (Table 6), the tumor in left and right lobes simultaneously (OR =0.441; 95% CI= 0.220-0.882; P= 0.00) was characterized as a protective factor for LNM. The gender, age and BRAF V600E mutation did not show statistical differences with LNM (P> 0.05).

The BRAF L597Q mutation of PTC in one Tibetan
A 57-year-old Tibetan male patient (with a protruding mass on the left forehead) came to hospital with numbness in the right limb for three weeks. The preoperative skull CT and MRI scan showed a 9.3 cm × 8.1 cm mass in the left frontal ( Fig. 1A and B). The skin on the surface of the mass was normal and hard in texture. The preoperative conventional color ultrasound diagnosis showed that the size of the bilateral thyroid was normal, and the echo in the right lobe was uneven. A mass (1.2 cm × 0.7 cm) was visible, with oval shape and clearly defined border. The "left frontal lobe occupancy and skull tumor resection" surgical plan was suggested for implementation by the multi-disciplinary team (MDT). Intraoperative display showed that the scalp tissue and the left frontal skull had obvious adhesion, and the protruding bone tissue surface was uneven and loose. The hyperplasia of the inner and outer plates of the skull was obvious (Fig. 1C). The tumor tissue was white and solid, and there was no adhesion with the surrounding brain tissue (Fig. 1D). The preoperative conventional color ultrasound diagnosis showed that the size of the bilateral thyroid was normal, and the echo in the right lobe was uneven. A mass (0.7 cm × 0.5 cm) was visible, with oval shape and clearly defined border ( Fig. 2A). The US-FNA was used to biopsy the thyroid nodules, the tumor cells were relatively uniform in size, with round nuclei, small nucleoli visible in part (Fig. 2B).
Some nucleus of cancer cells showed Ground Glass Opacity (GGO) by HE staining. The nuclear grooves and pseudoinclusions in nucleus were observed clearly, and psammoma bodies was seen in interstitial tissue (Fig. 3A). The cytokeratin (CK), thyroglobulin (Tg) and thyroid transforming factor-1 (TTF-1) were immunoreactive (Fig. 3B, C and D) by IHC. The ARMS-PCR, NGS and Sanger sequencing analysis showed that the patient had a double mutation of BRAF L597Q and V600E in two separate lesions (Fig. 4). The BRAF V600E (chr7:140453136 c.1799T>A) mutation was located in situ ( Fig. 4A and C), but the BRAF L597Q (chr7:140453145 c.1790T>A) mutation was located in the intracranial metastases ( Fig. 4B and D). The abundances of BRAF L597Q and V600E were 36.9% and 8.1% respectively, as determined by NGS, and which were successfully verified by Sanger sequencing (the gold standard of gene sequencing).

Discussion
With the increasing incidences of non-PTMC and PTMC, it is important to risk-stratify patients who may have more aggressive tumor biology in what is traditionally thought to be a more indolent disease, which will have management implications including whether or not to observe, the extent of surgical resection, the use of RIA therapy and the frequency of follow-up [21] . Li [22] have reported that the PTMC had an indolent course and excellent prognosis, while our results demonstrated that the incidence of LNM was more frequent in PTMC than in non-PTMC. The correlation analysis revealed that the incidence of BRAF V600E mutation in male PTMC was much higher than in female. Lee [23] have recommended that the male could be as an independent prognostic factor for recurrence in non-PTMC, but it was not a prognostic factor in PTMC. In our study, the incidence of BRAF V600E mutation in FNAC was much higher than in FFPE which was consistent with some previous studies [24,25] , however, the LNM was related to BRAF V600E wild-type in PTMC which was inconsistent with other study [26] . Thus, our findings demonstrated that the BRAF V600E mutation was more likely to manifest among male patients and more easily to be detected in FNAC of PTMC.
To further analyze the difference between PTMC and non-PTMC in the biological behavior, the clinicopathological characteristics and sample source in BRAF V600E mutation patients were also compared. Although several studies have reported that BRAF V600E mutation in PTCs was associated with aggressive pathological features, negative influence on 131 I avidity, reduced thyroperoxidase, the increased risk of lymph node metastasis and recurrence after treatment [27][28][29] , the clinical implications and clear mechanisms in PTMC and non-PTMC was contradictory. Zheng [30] have reported that tumor diameter (>0.5 cm) was an independent risk factor correlated with LNM in PTCs.
Interestingly, our findings definitely demonstrated that the LNM rate was much higher and was correlated with BRAF V600E wild-type in PTMC, which possibly indicated that BRAF V600E mutation in PTMC was less aggressive which was differ from a previous meta-analysis [22] . A study [31] have reported that most PTMC with BRAF V600E mutation did not display BRAF V600E protein expression.
Correct preoperative diagnosis is of importance. It is generally agreed that better knowledge about predictive risk factors for LNM may guide clinical decisions, while the greater risk of LNM remains debatable. After adjusting for other significant preoperative clinical factors, univariate and multivariate analysis was performed to identify the risk factors for LNM. Gender is a prominent patient background parameter for PTC. In recent years, the association between gender and recurrence or survival of PTC has been debated. A previous study reported that the male was an independent clinical prognostic factor of poor outcome in PTC [32] , but not in PTMC [23] . Recently, Roh [33] have reported that there was no association between gender and LNM. In our study, although the female had a lower BRAF V600E mutation than the male, multivariate analysis demonstrated that the female was a risk factor of LNM in PTMC and non-PTMC, which was different from above studies. A previous study [34] also have explored that the female was an independent predictive risk factor of CLNM in PTC. Controversies in different studies could be related to different sample source, sample size and detection techniques. It has been reported that the female had an earlier age of onset, but the male had a higher mortality [35,36] . It is recommended that the molecular mechanisms between LNM and gender in PTC patients should be explored in future.
In our study, the middle age (31-59) group was about 1.56 times the young age (≤30) group for LNM in PTCs, which was inconsistent with other study [37] . Our results have showed that the location of tumor in bilateral thyroid simultaneously was a protective factor for LNM in PTMC and non-PTMC, which was also inconsistent with a retrospective cohort study [38] .
The question is to clarify whether or not, after controlling the clinical importance of BRAF V600E mutation, to take into account that some other BRAF mutations deserve thoughtful analysis. The BRAF L597Q mutation incidence was less than 1% [39] such as in childhood acute lymphoblastic leukemia [18] , which might be associated with aggressive clinicopathologic features, however, the potential role of the peculiar BRAF L597Q mutation of PTC was unclear. To the best of our knowledge, we have reported the first case of solitary brain metastasis from occult papillary thyroid carcinoma in the Chinese Tibetan population in detail, as all known, the common metastatic sites of thyroid cancer are lung and bone, skull metastases are extremely rare [40] . Our results have demonstrated that the rare BRAF L597Q point mutation might play a specific role in inducing the solitary intracranial metastasis of occult papillary thyroid carcinoma in the Chinese Tibetan population. In addition, in this case, the biopsy or traditional gene sequencing technologies (such as the ARMS-PCR, solely for detecting the BRAF V600E mutation) had certain limitations, the combination of NGS and Sanger sequencing should help in detecting the rare gene mutation (e.g BRAF L597Q mutation), which was greatly recommended to improve the diagnostic accuracy and molecular mechanism analysis in rare PTC cases. It is concluded that the multilevel gene analysis may be a great substitute for the traditional gene testing [41,42] .
Nonetheless, our retrospective study had several potential limitations. Firstly, the number of the rare cases (BRAF L597Q mutation with skull metastases) was few. Secondly, the detailed molecular

Conclusions
In conclusion, we have demonstrated that the female, middle age (31-59 years) and PTMC were independently correlated with LNM in PTCs, while the tumor in left and right lobes simultaneously was a protective role in LNM. Meanwhile, our study demonstrated a negative result between BRAF V600E and LNM. Also, FNAC from tumor samples had a higher rate of BRAF V600E mutation than FFPE in PTMC, which confirmed that FNAC might be a reliable intervention to detect BRAF V600E mutation.
Moreover, to our knowledge, a possible association between the rare BRAF L597Q mutation and intracranial metastasis of occult PTC in the Chinese Tibetan population was reported firstly which may be used as a therapeutic target in future. Therefore, we suggest that clinicians should make a comprehensively consideration of clinical features: sample source, BRAF mutation, tumor size, gender, location of thyroid tumor and multilevel gene sequencing technologies and therapeutic schedule in order to achieve a relatively good prognosis.

Disclosure of Potential Conflflicts of Interest
The authors declare that they have no competing interests.    Comparison of mutation rates between PTMC and non-PTMC (Comparison of mutation rates between PTMC and non-PTMC over 3 years; Data were showed as N (%); The difference in mutation rate between PTMC groups and non-PTMC groups were highly significant.)