BRAF^V600E Wild-type Papillary Thyroid Carcinomas:Clinicopathologic Characteristics And Genetic Profiles

Papillary thyroid carcinoma（PTC）is the most common thyroid malignancy,accounting for 85%-90%of all thyroid cancers.BRAF point mutation（c.1799 T>A,p.V600E）is the most common gene variation in PTC.Previous studies have showed that BRAF^V600E is associated with the highly aggressive biological behavior of PTC,and is an independent prognostic factor for PTC,which can be used as a reference index for the ¹³¹I treatment stratification system.However,in recent years,it has also been reported that BRAF^V600E mutation is associated with the histological subtype of PTC,but not with some aggressive clinicopathological features,and cannot be used as an independent prognostic factor.In addition to BRAF^V600E,there are also some other important gene mutations in PTC,such as RAS mutation,RET,NTRK,ALK rearrangement,TERT promoter mutation,etc.These mutation genes are related to the histological type and prognosis of PTC.There are few reports on BRAF^V600E wild-type PTC so far,and there is insufficient understanding of the clinicopathologic features and genetic variations for these cases.In this study,BRAF^V600E wild-type PTC was used as the research object,and the clinicopathological characteristics were retrospectively studied.RAS and TERT promoters were detected by Sanger sequencing,and RET and ALK were detected by fluorescence in situ hybridization（FISH）.The expressions of ALK,pan-TRK,PD-L1,and mismatch repair proteins were detected by immunohistochemistry.Some cases are selected for next generation sequencing（NGS）,and the interested genetic variants were detected by Sanger sequencing.In addition,we analyzed the pathological characteristics of PTC with certain gene mutations,and try to develop rationales for the diagnosis and treatment of related PTC.Part Ⅰ Clinicopathologic characteristics of BRAF^V600E wild-type papillary thyroid carcinomasObjective To analyze the clinicopathological and prognostic characteristics of BRAF^V600E wild-type PTC.Methods A total of 1564 PTC cases which were surgically resected between November 2016 and November 2021 at the Affiliated Hospital of Qingdao University were collected（in order to ensure sufficient tumor tissues,only those with a maximum diameter≥1 cm were selected）,and the status of BRAF^V600E was determined by immunohistochemical staining（negative cases were defined as wild-type,positive was mutant-type）.The clinicopathological characteristics of BRAF^V600E wild-type PTC,including gender,age,number of lesions,lymph node metastasis,concomitant lesions,and histological type,were collected,and chi-square test was used to analyze the difference of these clinicopathological characteristics between BRAF^V600E wild-type cases and mutant-type cases.Results Among the 1564 PTC cases,1261 were BRAF^V600E mutant-type（80.6%）and303 were BRAF^V600E wild-type（19.4%）.In this cohort,there were 67 males and 236 females（male to female ratio:1:3.5）that aged from 11 to 70 years old（mean:42 years）.The tumor size ranged from 1 cm to 7.5 cm（mean:1.8 cm）.A total of 198 cases had capsule invasion（or occurred under the capsule）（65.3%）,207 cases had regional lymph node metastasis（68.3%）,120 cases had Hashimoto’s thyroiditis（39.6%）,and 80 cases were multiple lesions（26.4%）.Among 1261 cases of BRAF^V600E mutant PTC,there were298 males and 963 females（male to female ratio 1:3.2）that aged 17-77 years（average age 44 years,median age 43 years）.The tumor size was 1-8 cm（average 1.6 cm）.900 cases had capsule invasion or occurred under the capsule（71.4%）,717 cases had regional lymph node metastasis（56.9%）,221 cases had Hashimoto’s thyroiditis（17.5%）,and 411cases had multiple lesions（32.6%）.Compared with BRAF^V600E mutant PTC,BRAF^V600Ewild-type PTC was more likely to be accompanied by Hashimoto’s thyroiditis（P=0.00）,and the rate of regional lymph node metastasis was higher（P=0.00）.Conclusions This study is currently one of the largest case-control studies on BRAF^V600E wild-type PTC in China.Some BRAF^V600E wild-type PTCs also have strong aggressive biological behaviors,and may be closely related to Hashimoto’s thyroiditis.BRAF mutation status can not be served as the only marker to evaluate the aggressive behaviors of PTC.Part Ⅱ Identification of genetic variants in BRAF^V600E wild-type papillary thyroid carcinomasObjective To investigate the genetic variants in BRAF^V600E wild-type PTC.Methods A total of 118 cases of PTC confirmed by immunohistochemistry and ARMS-PCR without BRAF^V600E mutation were selected for detailed analysis.Sanger sequencing was performed in 99 cases to detect the 12/13 codons of exon 2 and the 61codon of exon 3 of KRAS/NRAS/HRAS and TERT promoter（c.-146 C>T and c.-124 C>T）.FISH was performed to detect RET and ALK gene rearrangements.Immunohistochemistry was carried out to detect ALK（D5F3）,pan-TRK,PD-L1（22C3）,MLH1,PMS2,MSH2,MSH6.For those PTC cases with special histological subtypes,pan-TRK and ALK immuno-positivity,or high quality of extracted genomic DNA,further NGS detection was performed（Novogene ALL-IN-ONE cancer gene panel was used in15 cases and Beijing Geneis thyroid cancer 18-gene panel was used in 9 cases）,The detected point mutations were then verified in other PTC cases by Sanger sequencing.Results Among the 118 PTC cases,74 cases belonged to classic subtype,37 were infiltrating follicular subtype,4 were diffuse sclerosing subtype,and 3 were other special types.Some morphological features suggested BRAF wild-type PTC.104 PTCs had follow-up records of 4 to 67 months,and recurrence and metastasis occurred in 10 cases（9.6%）.Sanger sequencing showed that 7 cases exhibited TERT promoter mutation（c.-124 C>T,7.1%,7/99）,and all cases had no RAS mutation.The FISH study showed that RET gene rearrangement occurred in 10 cases（12.5%,10/80,and the other 19 cases could not be interpreted）,and ALK rearrangement was not detected in all cases.Immunohistochemistry staining showed that only one case was positive for ALK（1.0%,1/99）and 6 cases were positive for pan-TRK（6.1%,6/99）.Immunohistochemistry showed 1 case was positive for ALK（1.0%,1/99）,6 cases positive for pan-TRK（6.1%,6/99）,and 6 cases positive for PD-L1（6.1%,6/99,of which only 1 case was diffusely positive）,while microsatellite instability was not found in all cases.NGS results showed that 1 case had NIN nonsense mutation,1 case had CHIC2,KAT6B,AXIN1 missense mutations,and 1 case had LPHN3 missense mutation and DOCK8 frameshift mutation.Sanger sequencing proved that these mutations did not appear in other cases.In addition,3 cases had RET rearrangement（demonstrated by FISH,all were CCDC6::RET fusion）,1case had NTRK1 rearrangement（TPM3::NTRK1 fusion）,and 1 case had ALK point mutation（c.2919G>A and c.3541C>T）.Of the 6 cases with positive pan-TRK immunohistochemistry,5 cases underwent NGS testing which showed that 4 cases had no NTRK1/2/3-related abnormalities.Conclusions Infiltrative follicular subtype,classic subtype lacking stroma or with a large number of follicular structures,or PTCs accompanied with obvious Hashimoto’s thyroiditis may be BRAF^V600Ewild-type.RET rearrangement and TERT promoter mutation were relatively common in BRAF^V600Ewild-type,while RAS and other gene mutations were relatively rare.BRAF^V600Eimmunohistochemical staining and ALK immunohistochemical staining were consistent with genetic sequencing results,while pan-TRK immunohistochemical staining and sequencing results had a low coincidence rate.Part Ⅲ Clinicopathological features of papillary thyroid carcinoma with receptor tyrosine kinase translocationObjective To investigate the clinicopathological features of PTC with receptor tyrosine kinase（RTK）translocation.Methods A total of 9 cases of BRAF^V600E wild-type PTC with RTK gene translocation confirmed by NGS detection in the second part and NGS detection in our institution,including 4 cases of RET rearrangement,3 cases of ALK rearrangement,and 2cases of NTRK1 rearrangement,were carefully reviewed,and the clinicopathological features were summarized.Results In this cohort,2 cases were diffuse sclerosing subtype,5 cases were classic subtype（1 case with high-grade differentiated thyroid cancer,and 1 case with undifferentiated carcinoma）,2 cases were infiltrative follicular subtype（1 case with high-grade grade differentiated thyroid carcinoma）.RTK-translocated PTCs often exhibited highly aggressive histological features:diffuse sclerosis,vascular invasion,intrathyroid dissemination,tumor necrosis,complex cribriform structure,or cervical lymph node metastasis.Lung metastasis was presented in 2 cases at the time of thyroidectomy.During the follow-up period（3 to 53 months）,3 cases developed tumor progression.Conclusions PTCs with RTK gene translocation usually had highly aggressive biological behaviors.