Neuropathologist-level integrated classification of adult-type diffuse gliomas using deep learning from whole-slide pathological images.

Current diagnosis of glioma types requires combining both histological features and molecular characteristics, which is an expensive and time-consuming procedure. Determining the tumor types directly from whole-slide images (WSIs) is of great value for glioma diagnosis. This study presents an integrated diagnosis model for automatic classification of diffuse gliomas from annotation-free standard WSIs. Our model is developed on a training cohort (n = 1362) and a validation cohort (n = 340), and tested on an internal testing cohort (n = 289) and two external cohorts (n = 305 and 328, respectively). The model can learn imaging features containing both pathological morphology and underlying biological clues to achieve the integrated diagnosis. Our model achieves high performance with area under receiver operator curve all above 0.90 in classifying major tumor types, in identifying tumor grades within type, and especially in distinguishing tumor genotypes with shared histological features. This integrated diagnosis model has the potential to be used in clinical scenarios for automated and unbiased classification of adult-type diffuse gliomas.

Undifferentiated colonic neoplasm with SMARCA4 germline gene mutation and loss of SMARCA4 protein expression: a case report and literature review.

BACKGROUND: Nonsense mutation or inactivation of SMARCA4 (BRG1) is associated with a monomorphic undifferentiated histological appearance in tumors at different sites. The association between SMARCA4 alteration and undifferentiated colonic carcinoma needs to be further elucidated. METHODS: A 61-year-old male patient presented to the hospital with intermittent epigastric pain in the right upper abdomen and abdominal distension. The enhanced computed tomography detected a mass in the hepatic flexure of the colon and multiple liver metastases. RESULTS: The right hemicolectomy contained a 4.5-cm undifferentiated malignancy with cells arranged in sheets, abundant necrosis, and areas showing rhabdoid morphology. The immunohistochemistry result showed that these tumor cells were focally positive for cytokeratin (CK), CK8, and CK18; however, diffusely positive for vimentin, P53, Fli-1, and SALL-4. Notably, tumor cells showed a heterogeneous loss of SMARCA4 expression pattern and intact SMARCB1 expression. Next-generation sequencing showed a germline SMARCA4 c.3277C>T(p.R1093*)mutation, somatic APC mutation, and no abnormal SMARCB1 gene. The tumor exhibited microsatellite stability, negative PD-L1 expression, and few infiltrating CD8 + T cells. The patient died a month later after surgery. CONCLUSIONS: We presented a rare case of undifferentiated colonic neoplasm with loss of SMARCA4 protein expression and germline SMARCA4 mutation. Moreover, the role of SMARCA4 alterations in tumor diagnosis and treatment was also summarized.

DNA Methylation Haplotype Block Markers Efficiently Discriminate Follicular Thyroid Carcinoma from Follicular Adenoma.

CONTEXT: Follicular thyroid carcinoma (FTC) is the second most common type of thyroid carcinoma and must be pathologically distinguished from benign follicular adenoma (FA). Additionally, the clinical assessment of thyroid tumors with uncertain malignant potential (TT-UMP) demands effective indicators. OBJECTIVE: We aimed to identify discriminating DNA methylation markers between FA and FTC. METHODS: DNA methylation patterns were investigated in 33 FTC and 33 FA samples using reduced representation bisulfite sequencing and methylation haplotype block-based analysis. A prediction model was constructed and validated in an independent cohort of 13 FTC and 13 FA samples. Moreover, 36 TT-UMP samples were assessed using this model. RESULTS: A total of 70 DNA methylation markers, approximately half of which were located within promoters, were identified to be significantly different between the FTC and FA samples. All the Gene Ontology terms enriched among the marker-associated genes were related to "DNA binding," implying that the inactivation of DNA binding played a role in FTC development. A random forest model with an area under the curve of 0.994 was constructed using those markers for discriminating FTC from FA in the validation cohort. When the TT-UMP samples were scored using this model, those with fewer driver mutations also exhibited lower scores. CONCLUSION: An FTC-predicting model was constructed using DNA methylation markers, which distinguished between FA and FTC tissues with a high degree of accuracy. This model can also be used to help determine the potential of malignancy in TT-UMP.

Mutational profiling of poorly differentiated and anaplastic thyroid carcinoma by the use of targeted next-generation sequencing.

AIMS: To characterise the mutational profiles of poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) and to identify markers with potential diagnostic, prognostic and therapeutic significance. METHODS AND RESULTS: Targeted next-generation sequencing with a panel of 18 thyroid carcinoma-related genes was performed on tissue samples from 41 PDTC and 25 ATC patients. Genetic alterations and their correlations with clinicopathological factors, including survival outcomes, were also analysed. Our results showed that ATC had significantly higher mutation rates of BRAF, TP53, TERT and PIK3CA than PDTC (P = 0.005, P = 0.007, P = 0.005, and P = 0.033, respectively). Nine (69%) ATC cases with papillary thyroid carcinoma (PTC) components harboured BRAF mutations, all of which coexisted with a late mutation event (TP53, TERT, or PIK3CA). Nine cases with oncogenic fusion (six RET cases, one NTRK1 case, one ALK case, and one PPARG case) were identified in 41 PDTCs, whereas only one case with oncogenic fusion (NTRK1) was found among 25 ATCs. Moreover, all six cases of RET fusion were found in PDTC with PTC components, accounting for 33%. In PDTC/ATC patients, concurrent TERT and PIK3CA mutations were associated with poor overall survival after adjustment for TNM stage (P = 0.001). CONCLUSIONS: ATC with PTC components is typically characterised by a BRAF mutation with a late mutation event, whereas PDTC with PTC components is more closely correlated with RET fusion. TERT and concurrent PIK3CA mutations predict worse overall survival in PDTC/ATC patients.

Mutation profiles of follicular thyroid tumors by targeted sequencing.

BACKGROUND: One of the major challenges remaining in the classification of thyroid tumor is the determination of whether a nodule is benign or malignant. We aimed to characterize the mutational profiles of follicular thyroid tumor and to identify markers with potential diagnostic and prognostic implications. METHODS: Targeted sequencing with a panel of 18 thyroid cancer-related genes was performed on 48 tissue samples from follicular thyroid adenoma (FTA), 32 follicular tumors of uncertain malignant potential (FT-UMP), 17 well-differentiated tumors of uncertain malignant potential (WDT-UMP) and 53 samples from follicular thyroid carcinoma (FTC). The correlation of mutation profiles and clinicopathological features and prognosis were also analyzed. RESULTS: We identified 95 nonsilent mutations spanning 14 genes. Specifically, TERT promoter (TERTp) mutations were exclusively detected in FTC. A total of 80% EIF1AX exon 2 mutations (4/5) and 75% TSHR mutations (3/4) occurred in FTA, whereas the rest of them occurred in FT-UMP. KRAS mutations and TP53 mutations were only presented in borderline or malignant tumors. H/N-RAS mutations were detected in all four subtypes, but were most commonly found in WDT-UMP (p = 0.031). All N-RAS mutations were located at codon 61. BRAF V600E and RET fusion were absent in the entire cohort. In FTC cases, EIF1AX mutations were all located at intron 5/exon 6 and correlated with advanced disease (p = 0.032). Both EIF1AX and TERTp mutations predicted shorter disease-free survival (p = 0.007, p = 0.024, respectively). Further analysis revealed that TERTp mutations were correlated with shorter disease-free survival in patients with minimally invasive /encapsulated angioinvasive FTC (p = 0.017), but not in those with widely invasive FTC (p = 0.297). CONCLUSION: TERTp, EIF1AX, TSHR, H/N/K-RAS and TP53 mutations may have diagnostic or prognostic potential in follicular thyroid tumors. TERTp mutations may predict a poor outcome in patients with minimally invasive/encapsulated angioinvasive FTC.

Hobnail variant of papillary thyroid carcinoma: molecular profiling and comparison to classical papillary thyroid carcinoma, poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma.

BACKGROUND: As a rare but aggressive papillary thyroid carcinoma (PTC) variant, the genetic changes of hobnail variant of PTC (HVPTC) are still unclear. RESULTS: The prevalence of HVPTC was 1.69% (18/1062) of all PTC diagnosed in our cohort. 73 samples from 55 patients (17 HVPTC, 26 CPTC, 7 PDTC and 5 ATC) were successfully analyzed using targeted NGS with an 18-gene panel. Thirty-seven mutation variant types were identified among 11 genes. BRAF V600E mutation was the most common mutation, which is present in almost all HVPTC samples (16/17, 94%), most CPTC samples (20/26, 77%), and none of the ATC and PDTC samples. TERT promoter mutation (C228T) was identified in 2 ATC and one HVPTC patient. RAS and TP53 mutation are almost exclusively present among ATC and PDTC samples although TP53 mutation was also observed in 3 HVPTC patients. Six different GNAS mutations were identified among 8 CPTC patients (31%) and none of the HVPTC patients. The only patient who died of disease progression harbored concomitant TERT C228T mutation, BRAF V600E mutation and TP53 mutation. METHODS: HVPTC cases were identified from a group of 1062 consecutive surgical specimens diagnosed as PTC between 2000 and 2010. Targeted next-generation sequencing (NGS) was applied to investigate the mutation spectrum of HVPTC, compared to classical PTC (CPTC), poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC). CONCLUSION: As an aggressive variant of PTC, HVPTC has relatively specific molecular features, which is somewhat different from both CPTC and ATC/PDTC and may underlie its relatively aggressive behavior.

Expression of amphiregulin predicts poor outcome in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: The validation of novel diagnostic, prognostic and predictive biomarkers in cancer is crucial for optimizing the choice and efficacy of personalized therapies. The aim of this study was to determine the epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII) and amphiregulin (AREG) protein expression levels and to evaluate the prognostic significance of EGFR, EGFRvIII and AREG in pancreatic ductal adenocarcinoma (PDAC). METHODS: The EGFR, EGFRvIII and AREG protein levels in PDAC (n = 92) were examined by using immunohistochemistry. The associations between EGFRvIII expression, AREG expression, AREG/EGFR co-expression and clinicopathological factors were assessed, the correlation between AREG and EGFR expression was analyzed and the survival analyses were performed. RESULTS: Among the lesions of PDAC, 12 (13 %) stained positive for EGFRvIII, 49 (53.3 %) stained positive for AREG and 22(23.9 %) stained double positive for AREG/EGFR. The relationships between each protein expression level and the clinicopathologic factors were examined, only AREG/EGFR co-expression was significantly related to tumor differentiation (P = 0.032). The correlation between AREG and EGFR expression was statistically insignificant (P = 0.709). Univariate survival analysis proved that high tumor-node-metastasis (TNM) stage, poor tumor differentiation and AREG expression were significant poor prognostic factors for disease-free survival (DFS) and overall survival (OS). By multivariate survival analysis, tumor differentiation was an independent poor prognostic factor for DFS (HR = 1.785, P < 0.05), whereas high TNM stage (HR = 2.25, P < 0.05), poor tumor differentiation (HR = 2.125, P < 0.01), positive resection margins (HR = 1.84, P < 0.05), and AREG expression (HR = 1.822, P < 0.05) were all independent poor prognostic factors for OS. CONCLUSIONS: In conclusion, our data indicate that AREG expression is an important prognostic biomarker in PDAC .

Genetic alterations and protein expression in combined small cell lung cancers and small cell lung cancers arising from lung adenocarcinomas after therapy with tyrosine kinase inhibitors.

There are 2 hypotheses regarding the mechanism underlying the adenocarcinoma (AD) to small cell lung cancer (SCLC) transition in patients receiving Tyrosine kinase inhibitor (TKI) therapy: 1) AD gives rise to SCLC owing to the pressure of the TKI therapy, and 2) the SCLC coexists with the AD de novo, but is not detected in biopsy specimens of the heterogeneous tumor. In this study, we try to address this issue by examination the genetic alteration and protein expression profile between SCLC arising from AD, and SCLC in combined small cell lung cancers (CSCLC). In the former, the SCLC had the same genetic profile as the AD, and we strongly suggest that the transition was a consequence of TKI therapy. In the latter, genetic alterations and protein expression tended to differ between the NSCLC and SCLC components of the CSCLC. The results showed that EGFR and KRAS mutation were found in 1 but not both component of CSCLC, and the NSCLC component usually expressed the EGFR and RB1 proteins, whereas the SCLC component did not. This finding indicates that the NSCLC and SCLC components arose separately and that CSCLC are unsuitable for TKI therapy despite the presence of sensitive EGFR mutations.

BRAF V600E and TERT Promoter Mutations in Papillary Thyroid Carcinoma in Chinese Patients.

BACKGROUND: The BRAF V600E and telomerase reverse transcriptase (TERT) promoter mutations have been reported in papillary thyroid carcinoma (PTC). The aim of this retrospective cross-sectional study was to add further information regarding the prevalence of the BRAF V600E and TERT promoter mutations in Chinese PTC and their clinicopathological associations. METHODS: We detected the BRAF V600E mutation and TERT promoter mutations in 455 Chinese PTC patients and analyzed the association of these mutations with several clinicopathological features. RESULTS: The BRAF V600E mutation was detected in 343 (75.4%) of 455 cases and was significantly associated with older age (p<0.001) and conventional subtype (p = 0.003). TERT promoter mutations were detected in 19 (4.4%) of 434 PTCs and were associated with older age (p<0.001), larger tumor size (p = 0.024), and advanced TNM stage(p<0.001). Of the 19 patients that were positive for TERT promoter mutations, 18 (94.7%) also harbored the BRAF V600E mutation. CONCLUSION: We determined the prevalence and clinicopathological associations of BRAF V600E and TERT promoter mutations in Chinese PTC patients. TERT promoter mutations but not the BRAF V600E mutation were associated with more advanced TNM stage upon diagnosis.

Screening for major driver oncogene alterations in adenosquamous lung carcinoma using PCR coupled with next-generation and Sanger sequencing methods.

We investigated the frequency of major driver oncogenes in lung adenosquamous cell carcinoma (ASC) cases. Frequency of EGFR, K-Ras, B-Raf, PIK3CA, DDR2, ALK, and PDGFRA gene mutations was examined in 56 patients using next-generation sequencing, polymerase chain reaction, and Sanger sequencing. Macrodissection or microdissection was performed in 37 cases to separate the adenomatous and squamous components of ASC. The overall mutation rate was 64.29%, including 55.36%, 7.14%, and 1.79% for EGFR, K-Ras, and B-Raf mutations, respectively. PIK3CA mutation was detected in three cases; all involved coexisting EGFR mutations. Of the 37 cases, 34 were convergent in two components, while three showed EGFR mutations in the glandular components and three showed PIK3CA mutations in the squamous components. With respect to EGFR mutations, the number of young female patients, nonsmokers, and those with positive pleural invasion was higher in the mutation-positive group than that in the mutation-negative. K-Ras mutation was significantly associated with smoking. Overall survival in the different EGFR mutation groups differed significantly. The frequency and clinicopathological characteristics of EGFR- and K-Ras-mutated adenosquamous lung carcinoma were similar to that noted in Asian adenocarcinomas patients. The high convergence mutation rate in both adenomatous and squamous components suggests monoclonality in ASC.

Validation of targeted next-generation sequencing for RAS mutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR.

OBJECTIVE: To validate the targeted next-generation sequencing (NGS) platform-Ion Torrent PGM for KRAS exon 2 and expanded RAS mutations detection in formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) specimens, with comparison of Sanger sequencing and ARMS-Scorpion real-time PCR. SETTING: Beijing, China. PARTICIPANTS: 51 archived FFPE CRC samples (36 men, 15 women) were retrospectively randomly selected and then checked by an experienced pathologist for sequencing based on histological confirmation of CRC and availability of sufficient tissue. METHODS: RAS mutations were detected in the 51 FFPE CRC samples by PGM analysis, Sanger sequencing and the Therascreen KRAS assay, respectively. Agreement among the 3 methods was assessed. Assay sensitivity was further determined by sequencing serially diluted DNA from FFPE cell lines with known mutation statuses. RESULTS: 13 of 51 (25.5%) cases had a mutation in KRAS exon 2, as determined by PGM analysis. PGM analysis showed 100% (51/51) concordance with Sanger sequencing (κ=1.000, 95% CI 1 to 1) and 98.04% (50/51) agreement with the Therascreen assay (κ=0.947, 95% CI 0.844 to 1) for detecting KRAS exon 2 mutations, respectively. The only discrepant case harboured a KRAS exon 2 mutation (c.37G>T) that was not covered by the Therascreen kit. The dilution series experiment results showed that PGM was able to detect KRAS mutations at a frequency of as low as 1%. Importantly, RAS mutations other than KRAS exon 2 mutations were also detected in 10 samples by PGM. Furthermore, mutations in other CRC-related genes could be simultaneously detected in a single test by PGM. CONCLUSIONS: The targeted NGS platform is specific and sensitive for KRAS exon 2 mutation detection and is appropriate for use in routine clinical testing. Moreover, it is sample saving and cost-efficient and time-efficient, and has great potential for clinical application to expand testing to include mutations in RAS and other CRC-related genes.

Immunohistochemistry is highly sensitive and specific for detecting the BRAF V600E mutation in papillary thyroid carcinoma.

The V600E mutation in the B-type Raf kinase (BRAF) gene is a common genetic change in cases of papillary thyroid carcinoma (PTC) that appears to play a key role in the development and progression of this disease. We sought to assess the sensitivity and specificity of immunohistochemical detection of this mutation with a V600E mutated BRAF antibody in a Chinese PTC cohort. In this study, we used fully automated immunohistochemistry (IHC) assay with a BRAF V600E (VE1) mouse monoclonal primary antibody to screen for the BRAF V600E mutation in 556 cases of PTC. Moreover, to verify the IHC staining results, real-time PCR was applied to detect this mutation in the same patient cohort. Among the 556 cases in the examined primary PTC cohort, 414 (74.5%) cases and 419 (75.4%) cases were positive for the BRAF V600E mutation by IHC staining and by real-time PCR, respectively. The real-time PCR results indicated that the sensitivity and specificity of IHC staining for the BRAF V600E mutation were 98.8% and 100%, respectively. The BRAF V600E mutation was common among Chinese patients with primary PTC, and was strongly correlated with older patient age and the conventional subtype of PTC but was not associated with parameters of clinicopathological aggressiveness. The fully automated IHC is a reliable technique that can serve as an alternative to molecular biological approaches for the routine detection of the BRAF V600E mutation in PTC patients.

Comparison of EGFR mutation status between plasma and tumor tissue in non-small cell lung cancer using the Scorpion ARMS method and the possible prognostic significance of plasma EGFR mutation status.

BACKGROUND: The aims were to compare the consistency of epidermal growth factor receptor (EGFR) mutations in the plasma and tumor tissue of NSCLC patients, and to explore the prognostic significance of plasma EGFR mutation status in tyrosine kinase inhibitors (TKIs)-treated patients with tumor EGFR mutation. METHODS: We evaluated EGFR gene (exons 18, 19, 20 and 21) mutation status in paired plasma and tumor tissue from 94 NSCLC patients before EGFR-TKIs treatments using the Scorpion amplification refractory mutation system (Scorpion-ARMS) method. RESULTS: Our results demonstrated that the rates for EGFR mutations in 94 NSCLC patients were 20% (19/94, plasma samples) and 40% (38/94, tumor tissue samples), respectively. The consistency of EGFR mutations between plasma and tissue reached 80% (75/94, P<0.001). The sensitivity of tests using plasma samples was 50% (19/38) and the specificity was 100% (49/49) compared with tissue samples. 29 of the 38 patients were treated with TKIs. Among the 29 patients, 14 patients had EGFR mutations in both plasma and tumor tissue, and these patients had a significantly shorter overall survival (OS) than those with EGFR mutations in tumor tissue only by univariate analysis (P=0.019). CONCLUSIONS: Our data demonstrated the feasibility and potential utility of plasma cell-free DNA (cfDNA) as a source of specimens for EGFR mutation detection using the Scorpion ARMS method. Moreover, plasma EGFR mutation status before TKIs therapy might be of prognostic significance for TKIs-treated NSCLC patients with tumor tissue EGFR mutation.