DOI: https://doi.org/10.21203/rs.3.rs-2718877/v1
The incidental finding of thyroid inclusions in lymph nodes of neck dissections of non-thyroid cancer patients is an unusual event. It is still controversial for pathologists for whether this represents benign inclusions or metastatic papillary thyroid carcinoma (PTC). This study is to analyze clinicopathological features of such cases in attempt to explore their clinical implications.
Pathological data were searched for incidentally detected PTC of cervical lymph nodes in non-thyroid cancer cases. Clinicopathological characteristics were reevaluated and recorded. BRAF V600E protein expression and sequencing analysis was then performed in cases with sufficient tissues.
31 patients had an incidental finding of PTC in lymph nodes of patients with non-thyroid cancer. BRAF immunohistochemical staining were performed in 17 metastatic lymph nodes with sufficient tumor tissues, and 6 were positive. BRAF V600E point mutation was detected in 5 of 6 BRAF V600E positive cases. Subsequent imaging examination of the thyroid showed undetected nodules or calcifications/benign nodules in twenty patients, and suspected malignant nodules in 5 patients. 12 patients underwent total thyroidectomy or ipsilateral lobectomy, and 6 showed PTC in postoperative pathological examinations. The remaining 19 patients without surgery were kept under active surveillance, and no one had recurrence of PTC.
Incidentally discovered PTC in lymph nodes have usually interpreted as metastasis from a clinical occult thyroid primary cancer, but primary PTC was not always detected. This suggests it would be double occult lesions. With regards to concurrence with highly malignant tumor, most patients could keep regular surveillance.
Thyroid inclusions may be Incidentally discovered in cervical lymph nodes during histological examination of neck dissections performed in nonthyroidal cancer. These inclusions mostly show malignant features of PTC and thus to be considered as occult metastatic PTC. However, they may display benign-appearance thyroid follicles in a few cases. There are conflict opinions for such cases. Some authors believe these thyroid benign-looking follicles are well-differentiated metastatic PTC from primary thyroid carcinoma, but others consider to be benign thyroid heterotopia[1–3]. In addition to diagnostic dilemma, clinical management on these patients is also controversial. The focus of arguments is whether the clinicians should choose surgery or keep active surveillance. PTC patients usually have a good prognosis even the presence of lymph nodes metastasis. As the occult metastatic PTC often occurs in the context of more aggressive squamous cell carcinomas, the clinical significance and management of incidentally detected metastatic PTC have not been well defined. Especially when imaging finding is negative, the focus might be extremely small in gross examination, resulting in difficulty to find the primary thyroid tumor in some cases.
Mutations of the BRAF gene is the most common genetic event in PTC, and it has been found in more than 40% of these tumors. BRAF V600E point mutation is accounting for 98% of all BRAF point mutation and plays an important role in the pathogenesis and progression of PTC. BRAF V600E point mutation is reported to be present in only PTC and its congeners, but not in benign thyroid lesions[4]. Therefore, detection of BRAF V600E mutation is of great helpful for diagnosis of PTC, while immunohistochemical staining of BRAF V600E can be used as an effective substitution for molecular detection[5–8].
In this study, we retrospectively reviewed the pathological database from patients undergoing surgery of neck dissection of non-thyroid carcinomas, analyzed the clinicopathological features of the Incidentally detected PTC in cervical lymph nodes, detected BRAF V600E expression and mutations in cases with sufficient tumor tissues, and further tracked their imaging status and postoperative pathology of corresponding thyroid tissue, in attempt to explore their clinical implications and significance.
The study was approved by the ethics committee and institutional review board of Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS, 21/517–3188), and all patients were exempt from informed consent. We retrospectively searched the pathological database from patients undergoing surgery of neck dissection in national cancer center during the period from 2011 to 2021. 2650 neck dissections were performed in patients with head and neck non-thyroid carcinoma, and 31 patients with an incidental finding of PTC in cervical lymph nodes were identified. Clinicopathological characteristics of these cases including their corresponding cancers were reevaluated and recorded by two senior pathologists. Immunohistochemical staining for one or several thyroid related markers (PAX8/TTF1/TG/CK19) were carried out in cases with available tumors to confirm their thyroid origin. BRAF V600E staining was performed in 17 lymph nodes. To further detect BRAF mutation, 6 cases with BRAF V600E positive expression were then examined by using amplification refractory mutation system (ARMS). Subsequently, imaging examination and postoperative pathology of primary thyroid was also reviewed and evaluated.
Immunohistochemical staining (IHC)
BRAF V600E (Ventana VE1 Mouse Monoclonal Primary Antibody) IHC staining was performed on 4 µm sections of formalin-fixed, paraffin-embedded tissues by using Ventana Benchmark IHC automated slide strainer in combination with the OptiView DAB IHC detection kit as previously described [9]. The cytoplasmic staining of tumor cells was considered as positive BRAF V600E staining.
Amplification refractory mutation system (ARMS)
The mutational status of BRAF V600E was detected using the Human BRAF Gene Mutation Detection Kits (ACCB, Beijing, China). The kits applied quantitative real time PCR (qRT-PCR) platform combining ARMS primers and Taq Man probes, and testing was conducted as previously described[10]. Briefly, 5 ng of genomic DNA and PCR master mix were added and proceeded for Real-time PCR analysis which was performed for 5 min at 95°C, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Mutation subtypes were determined according to threshold count (Ct, mutations were identified when the Ct value < 36), following the manufacturer's instruction.
31 patients with an incidental finding of PTC in lymph nodes were identified in 2650 patients, indicating a prevalence of 1.2% in this cohort. 27 were male and 4 were female. The median age was 62 (range 39 to 70 years). 29 cases of primary tumor were squamous cell carcinoma (9 larynx, 6 hypopharynx, 6 esophagus, 4 oral cavity, 2 oropharynx and 2 salivary gland), and 1 was low grade polymorphous adenocarcinoma of oropharynx, and 1 was myoepithelial carcinoma of oral cavity. Regarding the clinical stage of primary tumor, 4 were stage Ⅰ, 5 stage Ⅱ, 11 stage III and 11 stage IV (Tables 1 and 2).
| Location | N(%) |
|---|---|
| Larynx | 9(29%) |
| Hypopharynx | 6(19.4%) |
| Esophagus | 6(19.4%) |
| Oral cavity | 5(16.1%) |
| Oropharynx | 3(9.7%) |
| Salivary gland | 2(6.4%) |
| Characteristics | N (%) | |
|---|---|---|
| Age (years) | 62 ± 8.47 | |
| Sex | ||
| Male | 27(87.1%) | |
| Female | 4(12.9%) | |
| Histology of initial carcinomas | ||
| Squamous cell carcinoma | 29(93.5%) | |
| Low grade adenocarcinoma | 1(3.25%) | |
| Myoepithelial carcinoma | 1(3.25%) | |
| Clinical stage | ||
| Stage Ⅰ | 4(12.9%) | |
| Stage Ⅱ | 5(16.1%) | |
| Stage Ⅲ | 11(35.5%) | |
| Stage Ⅳ | 11(35.5%) |
A total of 57 lymph nodes were involved by PTC, of which 20 were level VI, 12 were level III, 10 were level Ⅱ, 8 were level IV, 2 were level Ⅶ, and 5 were unknown location (Table 3). 11 patients presented ≥ 2 nodes involvement, and 20 patients presented only single node involvement. Papillary structure and nuclear feature were observed in 15 patients (Fig. 1). Psammoma bodies were found in 5 patients. One or several thyroid related markers (PAX8/TTF1/TG/CK19) were stained in most cases and confirmed thyroid origin. BRAF V600E immunohistochemical staining were performed in 17 metastatic lymph nodes with sufficient tumor tissues, and 6 were positive (Table 4). BRAF V600E point mutation was detected in 5 of 6 BRAF V600E positive cases and was not detected in one BRAF V600E positive case (Fig. 2). Regarding the inconsistent result with IHC staining, we reexamined the HE slides and found less than 10% tumor cell content in the nodes. Therefore, the negative molecular finding might be caused by the too small population of tumor cells.
| Clinical levels | N(%) |
|---|---|
| Level Ⅱ | 10(17.5%) |
| Level Ⅲ | 12(21.1%) |
| Level Ⅳ | 8(14%) |
| LevelⅤ | 0(0%) |
| Level Ⅵ | 20(35.1%) |
| Level Ⅶ | 2(3.5%) |
| Unknown | 5(8.8%) |
| Clinical features | N(%) |
|---|---|
| Levels of lymph nodes | |
| Central | 22(38.6%) |
| Lateral | 30(52.6%) |
| Unknown | 5(8.8%) |
| Number of foci | |
| Unifocal | 20(64.5%) |
| Multifocal | 11(35.5%) |
| Structure of morphology | |
| Papillary | 10(45.5%) |
| Follicular | 12(54.5%) |
| Nuclear feature of PTC | |
| Yes | 10(45.5%) |
| No | 12(54.5%) |
| Psammoma bodies | |
| Yes | 4(18.2%) |
| No | 18(81.8%) |
| BRAF V600E staining | |
| Positive | 6(35.3%) |
| Negative | 11 (64.7%) |
Subsequent ultrasound of the thyroid showed undetected nodules or calcifications/benign nodules in twenty patients, and suspected malignant nodules in 5 patients. 12 patients underwent total thyroidectomy or ipsilateral lobectomy. After a thorough sampling, pathological finding showed PTC in 6 patients. Among them, the largest diameter of PTC tumor was 10mm in one patient, 5mm in one patient, and less than 1mm PTC in 4 cases (Table 5). The remaining 19 patients without surgery were kept under active surveillance, and no one had any kind of recurrence of PTC.
| Thyroid status | N(%) |
|---|---|
| Imaging results | |
| Positive | 5(16.1%) |
| Negative | 20(64.5%) |
| Unknown | 6(19.4%) |
| Thyroidectomy | |
| Yes | 12(38.7%) |
| No | 19(61.3%) |
| Primary thyroid PTC | |
| Positive | 6(50%) |
| Negative | 6(50%) |
The incidental finding of thyroid tissue in cervical lymph nodes during neck dissections performed in nonthyroidal cancer is rare. The prevalence reported in several previous studies ranged from 0.3%-1.6%[11–13]. We observed a prevalence of 1.2% (31/2650) in a large cohort of head and neck non-thyroid carcinoma. All the inclusions were found incidentally in the pathological examination of the neck dissection specimen, and no clinical history and suspicion of PTC was reported in clinical records. Among all the 57 involved lymph lodes, central neck level (level Ⅵ) was most frequently involved, accounting for 35.1%. It is not surprising, as Level Ⅵ is also the most common site and the first echelon for metastasis of papillary PTC. Of the 31 patients in our cohort, 27 were male and the median age were 62, indicating an older male predominance, which is very different from primary PTC that presents young female predominance. As for the initial non-thyroid tumors, 93.5% (29/31) were squamous cell carcinomas. These findings could be interpreted by the high incidence of squamous cell carcinomas in head and neck of male.
Although the incidental discovery of thyroid tissue in cervical lymph nodes is generally regarded as a metastatic lesion derived from thyroid gland, sometimes it remains controversial on the diagnosis between metastatic PTC and benign thyroid inclusions. Especially for the morphologically benign appearing thyroid follicles in lymph nodes, it is not easy to determine metastases or benign heterotopia. As salivary glands, naevus cells and Müllerian epithelia aberrantly occurred in lymph nodes have been well-established, the possibility of benign thyroid heterotopia has also been proposed[14–16]. In addition, diagnostic criteria have been suggested for benign heterotopia in lymph nodes: 1) adjacent to or in the lymph node capsule, 2) no more than two nodes involvement, 3) morphology of thyroid follicles and absence of nuclear features of thyroid papillary carcinoma, 4) absence of psammoma bodies and desmoplastic stroma, 5) inconsistent immunohistochemical and molecular profiling for thyroid cancer[14, 15]. The diagnosis of benign heterotopia might be considered only when all the conditions mentioned above are fulfilled. However, some pathologists contest the opinion of benign heterotopia in lymph nodes as no confirmed embryological or anatomic evidence for such inclusions. Moreover, it is not uncommon that metastatic well-differentiated follicular variant of PTC present normal like appearance in cervical lymph nodes. In the context of molecular evidence, the BRAF V600E point mutation has been reported in morphologically benign appearing thyroid inclusions of cervical lymph nodes[17]. This finding would question the notion of benign nodal thyroid heterotopia. Taken together, occult metastatic carcinoma in cervical lymph nodes must always be taken into consideration even if histopathologic studies do not reveal features of PTC. In our present study, 22 cases were available for morphological evaluation, 10 showed normal looking thyroid follicular morphology, and 12 presented at least one of the following feathers: papillary structure, nuclear features of PTC and psammoma bodies. Among the 10 cases with normal looking thyroid follicular morphology, 2 underwent thyroidectomy and PTC were found after a thorough sampling. It has been generally believed that a primary PTC is undoubtedly necessary for the development of an occult metastatic thyroid carcinoma. Identification of primary PTC in the 2 postoperative thyroid specimens in our study indicates the possibility of an occult metastasis rather than an ectopia of benign appearing thyroid follicles in cervical lymph nodes. It must be mentioned that the two primary PTC was considerably small (less than 1 millimeter in size), as most PTCs are not highly aggressive, it seems to be unusual for small tumors to metastasize. However, our observation emphasizes that no matter how small the tumor is, even less than 1 millimeter, it might have a significant potential to metastasis.
Subsequently, we performed BRAF V600E immunohistochemical staining in 17 cases with sufficient tumor cells, and 6 showed positive staining. Further BRAF V600E point mutation was detected in 5 of the 6 BRAF V600E positive cases. Regarding no BRAF V600E point mutation in 1 IHC positive case, we reexamined the HE slides and found less than 10% tumor cell content in the nodes. Probably, the negative molecular finding might be caused by the too small population of tumor cells. In our findings, the prevalence of BRAF V600E point mutation is slightly lower than that of primary thyroid origin PTC[4]. It is worth mentioning that one case showing bland-looking thyroid tissue was also positive for BRAF staining. This finding further supports the opinion of malignant PTC metastasis other than benign heterotopia.
When occult metastatic PTCs were identified in the cervical lymph nodes in the context of more aggressive squamous cell carcinomas, it is difficult to determine a single standard treatment as it should be based on overall survival of individual patients. Given the nature history of PTC, it is often less aggressive than the initial non-thyroid origin carcinomas of head and neck. Therefore, the prognosis of such patients usually depends on the more aggressive carcinomas but not PTC. Even so, evaluation of a thyroid tumor is also essential. When occult metastatic PTC was identified, imaging examination of thyroid should be carried out in routine clinical practice. Clinical management regarding surgery or surveillance hinges on both clinical and radiological features. In the cohort of 31 cases, imaging examination were performed in 25 patients, among them, 5 showed suspicious malignant thyroid nodules and 20 were normal or benign nodules. 12 underwent total thyroidectomy or lobectomy, and pathological results revealed PTC in 6 patients after a thorough sampling. However, among the 6 patients displaying PTC, 4 were less than 1mm in size. Moreover, PTCs were not identified in 50% (6/12) patients with surgery, suggesting that primary PTC of thyroid was not always identified in partial or total thyroid dissection specimen. Despite the negative thyroid finding of imaging or histopathology, the metastatic nature of the lymph node localization cannot be entirely excluded. The incidental discovery of PTCs in cervical lymph nodes, of course, are not always a metastasis but may occasionally be a primary lesion arising from heterotopic thyroid. On the other hand, although primary PTCs were found in 3 cases with benign imaging results, the lesions were considerably small. Therefore, if imaging shows normal or benign appearance, active surveillance could be recommended. However, when imaging reveals suspicious malignant thyroid nodules, the decision for thyroidectomy or conservative approach would be affected by multiple factors, including age, tumor size, pathological histological subtypes and clinical stage of initial non-thyroid carcinomas, as well as patient’s wishes. our results reveals that the majority of patients does not necessarily perform aggressive surgical dissection considering the minimal influence on patient outcome. These observations are also in consistent with several previous studies[18, 19].
Incidentally discovered PTC in lymph nodes have usually interpreted as metastasis from a clinical occult thyroid primary papillary carcinoma, although primary PTC of thyroid was not always detected in partial or total thyroid dissection specimen. Our findings suggest it would be double occult lesions. With regards to concurrence with highly malignant non-thyroid tumor, the incidentally detected metastatic PTC alone does not seem to influence disease progression. Clinical management regarding aggressive surgery or surveillance depends on clinical and radiological tumor features, and most patients could keep regular ultrasonic surveillance if further thyroid ultrasound does not show any malignant imaging sign.
Disclosure
The authors declare that there are no conflicts of interest.
Funding support
This work was supported by Beijing Hope Run Special Fund of Cancer Foundation of China (Grant no. LC2021A16).
Author contributions
CH carried out data acquisition, data analysis and manuscript preparation. HZ collected the paraffin blocks and tissue slides. LC cut the sections and conducted the IHC staining. TQ performed molecular detection and data analysis. HL obtained financial support and conducted this study. All authors approved the final version of the manuscript.