Objectives: B-Raf proto-oncogene ( BRAF) and telomerase reverse transcriptase ( TERT) mutations have established roles in the management of thyroid nodules, but rat sarcoma virus oncogene homolog ( RAS) mutations’ significance is less conclusive. Integrating mutation profiles with cytological grades provides evidence to evaluate the clinical significance of molecular testing in risk stratification and diagnosis of thyroid nodules. Material and Methods: A total of 5378 nodules were subjected to fine-needle aspiration (FNA cohort) and classified according to the Bethesda system for reporting thyroid cytology. Of them, 705 were surgically removed (thyroidectomy cohort) and pathologically examined, and 53 were followed for 0.5–53.6 months (follow-up cohort) and underwent at least one follow-up FNA. Eight follow-up nodules were resected. All FNA samples were tested for mutations of the BRAF V600E , TERT , and RAS genes. Results: 640 nodules of the thyroidectomy cohort and 6 follow-up nodules were malignant. Molecular testing showed BRAF V600E had a prevalence of 41.5, 58.0, and 15.1% in FNA-, thyroidectomy-, and follow-up cohorts, respectively, but RAS mutations’ prevalence was no higher than 4% in any of them, while TERT mutations’ prevalence was <1%. Consistently, BRAF V600E had a sensitivity of 63.9% and a specificity of 100% in diagnosing malignancy in the thyroidectomy cohort, while RAS mutations had much lower sensitivity and specificity (1.88 and 7.65%, respectively). Furthermore, BRAF V600E increased the risk of malignancy of the FNA cohort to 100% regardless of cytological grades, while RAS mutations showed no increase. In the follow-up nodules, BRAF V600E was strongly associated with malignancy and elevated cytological grades, while RAS mutations showed no association. Conclusion: BRAF V600E is a robust marker for thyroid malignancy in FNA specimens, whereas RAS mutations have limited standalone diagnostic utility but may still provide adjunctive value for risk stratification when interpreted together with cytology and clinical findings.

BACKGROUND RAS , BRAF , and mismatch repair (MMR)/microsatellite instability (MSI) are crucial biomarkers recommended by clinical practice guidelines for colorectal cancer (CRC). However, their characteristics and influencing factors in Chinese patients have not been thoroughly described. AIM To analyze the clinicopathological features of KRAS , NRAS , BRAF , and PIK3CA mutations and the DNA MMR status in CRC. METHODS We enrolled 2271 Chinese CRC patients at the China-Japan Friendship Hospital. MMR proteins were tested using immunohistochemical analysis, and the KRAS/NRAS/BRAF/PIK3CA mutations were determined using quantitative polymerase chain reaction. Microsatellite status was determined using an MSI detection kit. Statistical analyses were conducted using SPSS software and logistic regression. RESULTS The KRAS , NRAS , BRAF , and PIK3CA mutations were detected in 44.6%, 3.4%, 3.7%, and 3.9% of CRC patients, respectively. KRAS mutations were more likely to occur in patients with moderate-to-high differentiation. BRAF mutations were more likely to occur in patients with right-sided CRC, poorly differentiated, or no perineural invasion. Deficient MMR (dMMR) was detected in 7.9% of all patients and 16.8% of those with mucinous adenocarcinomas. KRAS , NRAS , BRAF , and PIK3CA mutations were detected in 29.6%, 1.1%, 8.1%, and 22.3% of patients with dMMR, respectively. The dMMR was more likely to occur in patients with a family history of CRC, aged < 50 years, right-sided CRC, poorly differentiated histology, no perineural invasion, and with carcinoma in situ , stage I, or stage II tumors. CONCLUSION This study analyzed the molecular profiles of KRAS , NRAS , BRAF , PIK3CA , and MMR/MSI in CRC, identifying key influencing factors, with implications for clinical management of CRC.

BackgroundAnaplastic lymphoma kinase (ALK) rearrangement, the most common oncogenic rearrangement in lung adenocarcinoma, occurs in approximately 5% of non-small cell lung cancer (NSCLC) patients. EML4 gene is the most common partner of ALK rearrangement, and distinct EML4-ALK fusions differ in their responsiveness to ALK tyrosine kinase inhibitors. However, the concurrence of two ALK rearrangements in one patient and whose response to ALK-TKIs have rarely been reported so far.Case presentationA 47-year-old Chinese male was diagnosed with stage IV lung adenocarcinoma with multiple intracranial metastases and adrenal metastasis. After progression of two lines of chemotherapy combined with local radiotherapy regimens, his tumor tissue sample was sent to perform the DNA-based next-generation sequencing of 116 genes. Surprisingly, EML4-ALK (E13:A20) fusion and a novel SV2B-ALK (S6:A20) fusion were concurrently identified, which was confirmed using immunohistochemistry and fluorescence in-situ hybridization. Given the superior efficacy of alectinib, the patient received alectinib in the third-line setting with the progression-free survival over 14 months up to now. Moreover, through comprehensive review of previous literatures, a total of 22 patients with multiple ALK fusions and their response to ALK-TKIs were summarized.ConclusionThis is the first report of a NSCLC patient with a novel SV2B-ALK, EML4-ALK double-fusion benefiting from alectinib. Alectinib may be an effective therapeutic option for both primary and metastatic lesions including brain metastases in the late-line setting in NSCLC patients with double-ALK fusion.

e15532 Background: Microsatellite instability (MSI) is a hypermutated phenotype primarily caused by the deficiency of DNA mismatch repair activity. Conventional MSI detection platforms are time consuming and need extra normal tissue. Due to the short DNA sequences of microsatellite loci in MSI samples, the method can be simplified to a one-step approach to differentiate MSI status by observing the melting curve. Herein a novel feasible MSI status detection method based on one step-PCR in single tumor tissue was developed, and validated in colorectal cancer samples. Methods: Specific microsatellite sites shorter than 12 bp in length (feasibility of fluorescence probes) were selected from public databases for analysis. In tumor tissues from 98 CRC patients with known MSI status, microsatellite sites with the highest receiver operating characteristic (ROC)-area under the ROC curve (AUC) were eventually selected for the final PCR panel design. The cut-off value of this assay was defined by the number of melting peaks in a specific Tm value range. An independent set of 199 tumor tissues from CRC patients with known MSI by Sanger sequencing was recruited to validate the performance of this novel PCR MSI kit. Results: The ROC-AUCs of the pre-selected eight mononucleotide repeats were 0.82, 0.93, 0.79, 0.78, 0.98, 0.95, 0.92, and 0.79. When 0 or 1 marker had a melting peak within the specified Tm value range, the sample was identified as "MSS/MSI-low (MSI-L)"; when 2 or more markers had melting peaks within the specified Tm value range, the sample was determined to be "MSI-high (MSI-H)". In the clinical sample set, compared with the results given by conventional MSI detection method, the sensitivity of the 8-gene panel for MSI-H was 99.4% (166/167), the specificity of the 8-gene panel was 100%, and the overall concordance reached to 99.5%. Conclusions: The novel PCR-MSI detection method using 8-gene panel developed in this study was feasible and accessible in clinical practice, and a large-scale clinical validation is in progress. [Table: see text]

6054 Background: Medullary thyroid carcinoma (MTC) is a rare malignancy, and its molecular pathogenesis is far from being understood. Therefore, a relatively comprehensive and accurate genomic profiling is needed for the future molecular diagnosis, treatment and early prevention of MTC patients. Methods: In this study, we investigated both the somatic and germline mutational spectrum of MTC, whole-exome sequencing (WES) was performed on tumor samples and matched noncancerous tissues from 30 MTC patients. Genetic alterations were defined and analyzed using MutSigCV and novel germline mutations were identified using MutationTaster and cross-referenced in PubMed. Results: In somatic mutational spectrum we found the top three significantly mutated genes (SMGs) in our MTC samples were RET, FAM186A and PRG4 genes (33%, 33% and 27%, respectively). For the germline mutations, FAT4 was detected as the most common mutated genes, following by RET and FAT1. Besides, we identified 30 novel germline mutations such as IGF1R, PDK1, NOTCH1, RPTOR, MPL, SETD2 and ARID2. Those mutated genes have been previously associated with neurofibromatosis, Lynch syndrome and other diseases respectively and now predicted potential functional pathogenicity in the patients with MTC. Conclusions: The study elucidated a relatively more comprehensive genomic landscape of MTC. In addition to the RET gene, which has been studied extensively, other SMGs may also be of interest including novel germline mutations. These findings may indicate the potential basis of molecular diagnosis, early prevention and targeted therapeutic options for patients with MTC in the future.

A rapid classification method was developed for the malignant and benign thyroid nodules with ultrasound guided-fine needle aspiration biopsy (FNAB) samples. With probe electrospray ionization mass spectrometry, the mass-scan data of FNAB samples were used as datasets for machine learning. The patients were marked as malignant (98 patients), benign (110 patients) or undetermined (42 patients) by experienced doctors in terms of ultrasound, the B-Raf (BRAF) gene, and cytopathology inspections. Pairwise coupling was performed on 163 ions to generate 3630 ion ratios as new features for classifier training. With the new features, the performance of the multilayer perception (MLP) classifier is much better than that with the 163 ions as features directly. After training, the accuracy of the MLP classifier is as high as 92.0%. The accuracy of the single-blind test is 82.4%, which proved the good generalization ability of the MLP classifier. The overall concordance is 73.0% between prediction and six-month follow-up for patients in the undetermined group. Especially, the classifier showed high accuracy for the undetermined patients with suspicious for papillary carcinoma diagnosis (90.9%). In summary, the machine learning method based on FNAB samples has potential for real clinical applications.

Frozen section examination could provide pathological diagnosis for surgery of thyroid nodules, which is time-consuming, skill- and experience-dependent. This study developed a rapid classification method for thyroid nodules and machine learning. Total 69 tissues were collected including 43 nodules and 26 nodule-adjacent tissues. Intraoperative frozen section was first performed to give accurate diagnosis, and the rest frozen specimen were pretreated for probe electrospray ionization mass measurement. By multivariate analysis of mass scan data, a series compounds were found downregulated in the extraction solution of papillary thyroid carcinoma (PTC), but some were found upregulated by mass spectrometry imaging. m/z 758.5713 ([PC[34:2] + H]+ ), m/z 772.5845 ([PC[32:0] + K]+ ), and m/z 786.6037 ([PC[36:2] + H]+ ) were firstly identified as potential biomarkers for nodular goiter (NG). Machine learning was employed by means of support vector machine (SVM) and random forest (RF) algorithms. For classification of PTC from NG, SVM and RF algorithms exhibited the same performance and the concordance was 94.2% and 94.4% between prediction and pathological diagnosis with positive and negative mass dataset, respectively. For the classification of PTC from PTC adjacent tissues, SVM was better than RF and the concordance was 93.8% and 83.3% with positive and negative mass dataset, respectively. With the identified compounds as training features, the sensitivity and specificity are 87.5% and 88.9% for the test set. The developed method could also correctly predict the malignancy of one medullary thyroid carcinoma and one adenomatous goiter (benign). The diagnosis time is about 10 min for one specimen, and it is very promising for the intraoperative diagnosis of papillary thyroid carcinoma.

Objective:To analyze the value of folate receptor-positive circulating tumor cells (FR +-CTC) in the diagnosis and efficacy evaluation of patients with small cell lung cancer (SCLC). Methods:The data of 59 patients with SCLC and 14 patients with benign pulmonary diseases treated in China-Japan Friendship Hospital from May 2017 to October 2019 were retrospectively analyzed. Folate receptor targeted detection was used to detect the level of FR +-CTC in the blood of SCLC patients. The levels of serum progastrin-releasing peptide (Pro-GRP), neuron-specific enolase (NSE), cytokeratin 19 fragment 21-1 (Cyfra21-1) , and carcinoembryonic antigen (CEA) were detected by using chemiluminescence. The median ( P25, P75) was used as all the detection indexes. Mann-Whitney U test was used for pairwise comparison, Spearman correlation test was used to analyze the correlation between two variables, and receiver operator characteristic (ROC) curve was used to evaluate the diagnostic efficacy. Results:The level of FR +-CTC in 59 patients with SCLC was 11.00 FU/3 ml (7.10 FU/3 ml, 14.50 FU/3 ml), and the positive rate of FR +-CTC in patients with SCLC was 66.10% (30/59); the level of FR +-CTC in 14 patients with benign pulmonary diseases was 6.75 FU/3 ml (5.03 FU/3 ml, 7.85 FU/3 ml), and the positive rate of FR +-CTC in 14 patients with benign pulmonary diseases was 14.29% (2/14). The level of FR +-CTC in patients with SCLC was higher than that in patients with benign pulmonary diseases, and the difference was statistically different ( U = 33.50, P < 0.01). The expression level of FR +-CTC was not related to age, gender and smoking history in SCLC patients (all P>0.05). The expression level of FR +-CTC in patients with extensive-stage was higher than that in patients with limited-stage, and the difference was statistically significant ( P < 0.05). Tumor markers Pro-GRP, NSE, Cyfra21-1 and CEA were compared with FR +-CTC, and the ROC curve was drawn; the results showed that FR +-CTC had better sensitivity (71.2%) and specificity (92.90%) in the diagnosis of SCLC. For SCLC patients who received chemotherapy, the decrease range of FR +-CTC in patients with partial remission and stable disease was greater than that in patients with the progression of disease, and the differences were statistically significant (all P < 0.05). Conclusion:FR +-CTC can assist the diagnosis and disease staging of SCLC. For patients receiving chemotherapy, continuous detection of circulating tumor cells can help to evaluate the efficacy of chemotherapy and provide a reference for the choice of clinical treatment.

在中国,乳腺癌的发病率居女性恶性肿瘤首位,且其发病率呈逐年增长趋势,严重威胁着广大女性的生命健康[1].目前,手术切除是临床治疗乳腺癌的最常见方式,术中快速冷冻病理诊断在临床上运用频繁,可对患者乳腺肿块性质进行快速诊断[2].但冷冻切片在质量控制上仍有不足,尤其是富含脂肪组织多的冷冻切片有时制片效果并不理想,存在漏诊的情况.通过实验观察将脂肪含量不同的乳腺组织在低温条件下,分设不同冷冻时间以及冷冻温度、切片厚度,对比冷冻切片制片效果.现对我院快速冷冻乳腺组织切片实验对比进行分析,报道如下.

目的:分析非小细胞肺癌罕见驱动基因突变特点及其与临床病理特征的相关性.方法:收集中日友好医院病理科2018年11月～2019年12月间送检的630例非小细胞肺癌患者手术切除标本.使用荧光定量PCR法对标本的驱动基因进行联合检测,包括EGFR、KRAS、NRAS、BRAF、PIK3CA、HER2基因突变,以及ALK、ROS1、RET基因融合.结果:标本整体基因突变阳性率为63.49％,融合阳性率为6.35％,全部野生型的病例占30.16％.罕见驱动基因改变占全部驱动基因改变的27.27％ (120/440).EGFR突变病例中,罕见突变位点占9.63％ (31/322).EGFR20外显子插入突变及HER2突变均与患者年龄负相关(r=-0.108、r=-0.191,P＜0.05).RAS突变与肿瘤大小正相关(r=0.159,P＜0.05),并与性别及组织学分型具有相关性(P＜0.05).ALK、ROS1、RET融合均与组织学包含微乳头成分正相关(r=0.097、r=0.105、r=0.136,P＜0.05).ALK融合与组织学分型以及淋巴结转移的相关性具有统计学意义(P＜0.05).结论:非小细胞肺癌罕见驱动基因突变虽然发生率较低,但包含一种以上罕见突变的患者比例较高.这些驱动基因突变与临床病理特征的关系多种多样,大多与组织学分型相关.

目的 明确KRAS、NRAS、HRAS、BRAF、TERT基因在甲状腺细针穿刺标本中的表达特点,并评价这些基因对细胞学辅助诊断方面的应用价值.方法 回顾性研究中日友好医院病理科2019年3月至8月期间送检的甲状腺细针穿刺标本360例,每例标本均包含细胞涂片以及穿刺组织1管.涂片经95％乙醇固定后,行巴氏染色,细胞学结果依据Bethesda分级系统读片.另一瓶穿刺组织保存在生理盐水中,经核酸提取,并用实时定量PCR法进行基因检测.结果 标本无法诊断或不满意43例(11.94％),良性病变67例(18. 61％),意义不明确的细胞非典型病变或意义不明确的滤泡性病变37例(10. 28％),滤泡性肿瘤或可疑滤泡性肿瘤17例(4.72％),可疑乳头状癌34例(9.44％),乳头状癌162例(45.00％).BRAF V600E突变166例(46.11％),KRAS 2号外显子突变1例(0.28％),NRAS3号外显子突变12例( 3. 33％), HRAS 3号外显子突变2例(0. 56％) ,TERT启动子突变3例(0.83％).其中NRAS突变伴有BRAF突变1例,TERT启动子突变3例均伴有BRAF突变.BRAF基因突变与患者细胞学诊断结果的相关性具有统计学意义(P＜0.05);TERT基因与性别及淋巴结转移与否的相关性具有统计学意义(P＜0.05);NRAS突变与细胞学诊断结果的相关性具有统计学意义(P＜0.05).结论 几种基因突变中,以BRAF基因突变最为常见,且对于辅助诊断的应用价值较高;在RAS基因中又以NRAS突变居多;而TERT启动子突变一般与BRAF基因突变伴随.

结核病是由结核分枝杆菌感染引起的慢性传染病,结核菌可能侵入人体全身各种器官,主要侵犯肺脏,称为肺结核病,对人体危害很大.长期以来,结核病由于其强烈传染性,一直困扰大众.组织标本应用抗酸染色法检测结核分枝杆菌,其敏感性低,使得检出的阳性率低,难以满足患者和临床的精准诊疗需求[1].近年来,随着分子生物学技术的高速发展,因其具有较高的敏感性和特异性而广泛应用于各种病原体的核酸检测[2].