Correlation between NGS panel-based mutation results and clinical information in colorectal cancer patients.

Early mutation identification guides patients with colorectal cancer (CRC) toward targeted therapies. In the present study, 414 patients with CRC were enrolled, and amplicon-based targeted next-generation sequencing (NGS) was then performed to detect genomic alterations within the 73 cancer-related genes in the OncoAim panel. The overall mutation rate was 91.5 % (379/414). Gene mutations were detected in 38/73 genes tested. The most frequently mutated genes were TP53 (60.9 %), KRAS (46.6 %), APC (30.4 %), PIK3CA (15.9 %), FBXW7 (8.2 %), SMAD4 (6.8 %), BRAF (6.5 %), and NRAS (3.9 %). Compared with the wild type, TP53 mutations were associated with low microsatellite instability/microsatellite stability (MSI-L/MSS) (P = 0.007), tumor location (P = 0.043), and histological grade (P = 0.0009); KRAS mutations were associated with female gender (P = 0.026), distant metastasis (P = 0.023), TNM stage (P = 0.013), and histological grade (P = 0.004); APC mutations were associated with patients <64 years of age at diagnosis (P = 0.04); PIK3CA mutations were associated with tumor location (P = 4.97e-06) and female gender (P = 0.018); SMAD4 mutations were associated with tumor location (P = 0.033); BRAF mutations were associated with high MSI (MSI-H; P = 6.968e-07), tumor location (P = 1.58e-06), and histological grade (P = 0.04). Mutations in 164 individuals were found to be pathogenic or likely pathogenic. A total of 26 patients harbored MSI-H tumors and they all had at least one detected gene mutation. Mutated genes were enriched in signaling pathways associated with CRC. The present findings have important implications for improving the personalized treatment of patients with CRC in China.

Establishment and evaluation of digital PCR methods for HER2 copy number variation in breast cancer.

Accurate measurement of human epidermal growth factor receptor 2 (HER2) copy number variation (CNV) is very important for guiding the tumor target therapy in breast cancer. Digital PCR (dPCR) is a sensitive and an absolute quantitative method, which can be used to detect HER2 CNV. Three HER2 exon-specific digital PCR assays along with three new reference genes assays (homo sapiens ribonuclease P RNA component H1 (RPPH1), glucose-6-phosphate isomerase (GPI), and chromosome 1 open reading frame 43 (C1ORF43), on different chromosomes) were established and validated by using standard reference material, 8 different cell lines and 110 clinical Formalin-fixed and paraffin-embedded (FFPE) samples. DPCR can achieve precise quantification of HER2 CNV by calculating the ratio of HER2/reference gene. The positive and negative coincidence rates were 98% (53/54) and 95% (53/56), respectively, compared with fluorescence in situ hybridization (FISH) diagnostic result 110 of FFPE samples. The common reference gene CEP17 used for FISH diagnostic was not suitable as single reference gene for HER2 CNV measurements by dPCR. The best practice of HER2 CNV determination by dPCR is to conduct the three duplex assays of H1 (HER2 exon 4) with the proposed three new reference genes, with a positive cut-off value of H1/RPPH1 ≥ 2.0 or H1/averaged reference gene ≥ 2.0. The proposed dPCR method in our study can accurately provide absolute copy number of HER2 and reference gene on an alternative chromosome, thus avoiding false negative caused by polysomy of chromosome 17. The improved molecular typing and diagnosis of breast cancer will better guide clinical medication.

Non-classical phenotypes of mismatch repair deficiency and microsatellite instability in primary and metastatic tumors at different sites in Lynch syndrome.

Background: Lynch syndrome is a genetic disease characterized by abnormal DNA replication caused by germline variation in the mismatch repair (MMR) gene. There are rare non-classical phenotypes with loss of MMR protein expression and inconsistent microsatellite stability (MSS) in Lynch syndrome-related colorectal cancers. However, the difference between microsatellite instability (MSI) of extraintestinal tumors in a patient with Lynch syndrome has been closely studied. Herein, we reported the non-classical phenotypes of mismatch repair deficiency (dMMR) and MSI in four cases of Lynch syndrome in patients with colorectal cancer and other primary and metastatic tumors. Methods: A retrospective analysis was conducted on four patients diagnosed with Lynch syndrome between 2018 and 2022 in the Department of Pathology of the Rocket Forces Specialized Medical Center. A one-step immunohistochemical (IHC) assay was employed to detect loss in the expression of Lynch syndrome-associated MMR proteins (MLH1, PMS2, MSH2, and MSH6). MSI detection was performed in both primary and metastatic tumors at different sites in the four patients using NCI 2B3D (BAT25, BAT26, D2S123, D17S250, and D5S346) and single nucleotide site (BAT25, BAT26, NR21, NR24, NR27, and MONO27) methods. In addition, related MMR gene germline variation, somatic mutations, and MLH1 gene promoter methylation were analyzed using next-generation sequencing and TaqMan probe-based methylation-specific polymerase chain reaction (MethyLight). Results: Two of the four patients were heterozygous for MSH6 germline pathogenic variation, and the other two were heterozygous for MSH2 germline pathogenic variation. In all cases, IHC detection of protein expression of the MMR gene with germline variation was negative in all primary and metastatic tumors; non-classical phenotypes of dMMR and MSI were present between primary and metastatic tumors at different sites. dMMR in Lynch colorectal cancer demonstrated high MSI, whereas MSI in primary and metastatic tumors outside the intestine mostly exhibited MSS or low MSI. Conclusions: The non-classical dMMR and MSI phenotype are mostly observed in Lynch syndrome, even in the context of MMR protein expression loss. Extraintestinal tumors infrequently present with a high degree of MSI and often exhibit a stable or low degree of MSI.

Relationship between CpG island methylation phenotype, microsatellite instability phenotype and mutation of KRAS, NRAS, and BRAF genes in colorectal cancer.

OBJECTIVE: CpG island methylation phenotype (CIMP) and microsatellite instability (MSI) are two different molecular mechanisms in colorectal cancer (CRC). Proto-oncogene KRAS, mutations in NRAS and BRAF play an important role in the formation of colorectal cancer. The correlation between the molecular typing of CIMP and MSI and the genes of KRAS, NRAS and BRAF was explored in this study. METHODS: A total of 110 paraffin-embedded specimens of colorectal cancer were collected from the Chinese People's Liberation Army Rocket Army Special Medical Center during the period from May 2017 to September 2018. CIMP were detected by DNA methylation quantitative PCR (Methylight). Mutations in KRAS, NRAS, and BRAF genes were detected by realtime fluorescence quantitative PCR (qPCR); MSI typing was detected by sequencing. RESULTS: Of the 110 colorectal cancer samples, 11 cases (10%) were CIMP-H, 92 cases (83.64%) were CIMP-L, and 7 cases (6.36%) were CIMP-0. 10 cases (9.09%) were MSI-H, and 100 cases (90.91%) were MSS and MSI-L. The mutation rates of KRAS, NRAS and BRAF genes were 50% (55 cases), 6.36% (7 cases) and 5.45% (6 cases), respectively. There was no significant correlation between CIMP group and MSI group (P > 0.05). Also, no significant differences were found in the mutations between the three subtypes of the CIMP group and the KRAS, NRAS genes (P > 0.05), while there was a statistically significant difference among the three subtypes of the BR and the BRAF gene mutations (P < 0.0001). There were no obvious differences between the three states of microsatellites and the mutations of KRAS and NRAS genes (P > 0.05), and the differences between them and BRAF gene mutations were statistically significant (P < 0.01). CONCLUSIONS: The BRAF gene mutation is closely related to the two types of CIMP and MSI, which may be an important part of the above two molecular mechanisms, and provide a reference for the treatment of the patients with CIMP-H and MSI-H.

Selective expression of transthyretin in subtypes of lung cancer.

Transthyretin (TTR) is expressed primarily in liver, choroid plexus of brain and pancreatic islet A and B cells. It is also synthesized in some endocrine tumors. In the present study, the protein expression of TTR in lung cancer tissues and cell lines was investigated by western blot. The mRNA expression of TTR in 24 pairs of frozen lung cancer tissues was examined by RT-PCR. The specific expression and cellular distribution of TTR were also evaluated in 104 paraffin-embedded lung cancer samples and 3 normal lung tissues by immunohistochemistry. Similarly, the subcellular localization and expression of TTR were further analyzed in lung cancer cell lines. With the exception of mucinous adenocarcinoma, the expression of TTR protein was observed in all tested subtypes of lung carcinoma. Adenocarcinoma displayed the highest positive expression rate of TTR, accounting for 84.4 %, and the positive expression rate of TTR was up to 85.7 % at stages III and IV. The secretory bubbles with strong TTR staining were observed in luminal cells of lung cancer. Furthermore, the localization of TTR in the cytoplasm of lung cancer cells and the secretion of TTR into extracellular milieu were also confirmed. Taken together, TTR is selectively synthesized in lung cancer cells and can be secreted extracellularly.

The correlation between EGFR mutation status and the risk of brain metastasis in patients with lung adenocarcinoma.

To explore the correlation between epidermal growth factor receptor (EGFR) mutation status and the risk of brain metastasis (BM) in patients with lung adenocarcinoma, the clinical data of 100 patients with pathologically confirmed lung adenocarcinoma and known EGFR mutation status at exon 18, 19, 20, or 21 were analyzed retrospectively. The incidence of BM was similar between patients with wild-type EGFR and those with EGFR mutations (p = 0.48). However, among patients with EGFR mutations, the incidence of BM was significantly higher in patients with mutation at exon 19 than in patients with mutation at other sites (p = 0.007). Besides, among patients with heterochronous BM, 66.7 % had EGFR mutations. Regarding brain-metastasis-free survival (BMFS), patients with EGFR sensitive mutations (mutation at exon 19/21/and dual mutation) had significantly shorter BMFS compared with patients with wild-type EGFR (p = 0.018). For patients treated only with chemotherapy, BM was an unfavorable prognostic factor. Patients with BM had worse overall survival compared with those without BM (p = 0.035). However, in patients with BM and EGFR sensitive mutations, those treated with tyrosine kinase inhibitors (TKIs) had significantly longer overall survival compared with those treated with chemotherapy only (p = 0.0081). In conclusion, among patients with EGFR mutations, those mutated at exon 19 had the highest incidence of BM. Furthermore, patients with EGFR mutations are more likely to develop heterochronous BM. The BMFS was significantly shorter in patients with EGFR sensitive mutations. TKIs improved the survival of patients with lung adenocarcinoma and BM who harbored EGFR sensitive mutations.

The Characteristics and Function of S100A7 Induction in Squamous Cell Carcinoma: Heterogeneity, Promotion of Cell Proliferation and Suppression of Differentiation.

S100A7 is highly expressed in squamous cell carcinomas (SCC) and is related to the terminal differentiation of keratinocytes. However, its characteristic and function in SCC is not very known. In this present study, we used immunohistochemistry to examine the expression of S100A7 in 452 SCC specimens, including the lung, esophagus, oral cavity, skin, cervix, bladder, and three SCC cell lines. We found that S100A7-positive staining showed significant heterogeneity in six types of SCC specimen and three SCC cell lines. Further examination found that S100A7-positive cells and its expression at mRNA and protein levels could be induced in HCC94, FaDu, and A-431 cells both in vitro and in vivo using immunohistochemistry, real-time PCR, and Western blotting. Notably, the upregulation of squamous differentiation markers, including keratin-4, keratin-13, TG-1, and involucrin, also accompanied S100A7 induction, and a similar staining pattern of S100A7 and keratin-13 was found in HCC94 cells both in vitro and in vivo. Further study revealed that the overexpression of S100A7 significantly increased proliferation and inhibited squamous differentiation in A-431 cells both in vitro and in vivo. Conversely, silencing S100A7 inhibited cell growth and survival and increased the expression of keratin-4, keratin-13, TG-1, and involucrin in HCC94 cells. Therefore, these results demonstrate that S100A7 displays heterogeneous and inducible characteristic in SCC and also provide novel evidence that S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation of SCC.

miR-206 inhibits metastasis-relevant traits by degrading MRTF-A in anaplastic thyroid cancer.

Thyroid cancer develops from follicular or parafollicular thyroid cells. A higher proportion of anaplastic thyroid cancer has an adverse prognosis. New drugs are being used in clinical treatment. However, for advanced thyroid malignant neoplasm such as anaplastic thyroid carcinoma, the major impediment to successful control of the disease is the absence of effective therapies. Elucidating molecular mechanism of the disease will help us to further understand the pathogenesis and progression of the disease and offer new targets for effective therapies. In this study, we found that MRTF-A expression was upregulated in metastatic anaplastic thyroid cancer tissues, compared with primary cancer tissues and it promoted metastasis-relevant traits in vitro. miR-206 was negatively associated with metastasis in anaplastic cancer and it degraded MRTF-A by targeting its 3'-UTR in ARO anaplastic thyroid cancer cells. In addition, miR-206 overexpression inhibited invasion and migration and silencing miR-206-promoted migration and invasion in the cells. Important, restoration of MRTF-A could abrogate miR-206-mediated migration and invasion regulation. Thus, we concluded that miR-206 inhibited invasion and metastasis by degrading MRTF-A in anaplastic thyroid cancer.

RORα inhibits adipocyte-conditioned medium-induced colorectal cancer cell proliferation and migration and chick embryo chorioallantoic membrane angiopoiesis.

Lipid metabolic disturbances are related to many diseases, such as obesity, diabetes, and certain cancers. Notably, lipid metabolic disturbances have been reported to be a risk factor for colorectal cancer. Nuclear receptors act as ligand-dependent transcription regulators and play key roles in the regulation of body lipid metabolism and the development of many cancers. Retinoic acid receptor-related orphan receptor α (RORα) is a nuclear receptor and can regulate several lipid metabolism genes in certain cancers. Herein, we demonstrate that the conditioned medium from adipocytes has a proproliferative and promigratory effect on colorectal cancer cells and enhances angiogenesis in chicken embryonic chorioallantoic membranes. In addition, the conditioned medium leads to a decrease in the expression of RORα and its target genes. Meanwhile, RORα and its target gene expressions are lower in human colorectal tumor tissue compared with control colorectal tissue. Activation of RORα inhibits the effect of conditioned medium on the proliferation and migration of colorectal cancer cells as well as the angiogenesis in chicken embryonic allantoic membranes. In colorectal cancer cells, the putative ligand of RORα, cholesterol sulfate (CS), prevents cell cycle progression at the G1/S boundary and concurrently modulates the expression of cell cycle-regulatory genes in colorectal cancer cell. CS inhibits angiogenesis in chicken embryonic chorioallantoic membranes and concurrently decreases the mRNA expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α as well as the secretion of VEGF. In addition, lipogenic gene expression is higher in human colorectal tumor tissue compared with control colorectal tissue. CS inhibits the expression of lipogenic genes in colorectal cancer cells. These results suggest that RORα could represent a direct link between local lipid metabolism of colorectal tissue and colorectal cancer. Therefore, the reduction of the expression of RORα could represent a potential warning sign of colorectal cancer.

[Expression of cyclin D1 in cervical intraepithelial neoplasia and squamous cell carcinoma and its relationship with HPV16 E7 gene].

OBJECTIVE: To investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression. METHODS: Both SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma. E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively. SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7. Control cell line B3 was obtained by blank plasmid transfection into SiHa cells. RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa, B3, and cm-16 cells, respectively. RESULTS: Cyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3. Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P<0.05). Both mRNA and protein of cyclin D1 in E7(+) SiHa cells were lower than those in cm-16 and Hcc94 cells. CONCLUSION: Squamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1, suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.

Cyr61 is up-regulated in prostate cancer and associated with the p53 gene status.

Cysteine-rich 61 (Cyr61) is a member of the CCN protein family that has been implicated in diverse biological processes such as cell adhesion, proliferation, angiogenesis, and tumorigenesis. Altered expression of Cyr61 is found to be associated with human cancers. Here we show that Cyr61 was up-regulated in prostate cancer cell lines and tumor tissues. A significant correlation of Cyr61 expression was found between benign prostatic hyperplasia and prostate cancer (P = 0.002). However, there was no significant correlation between levels of PSA and Cyr61 expression (P = 0.2). Cyr61 may represent an independent prostate cancer biomarker and potentially a useful therapeutic target for prostate cancer treatment. In addition, our analysis based on published data and data present in this report indicted that levels of Cyr61 expression associated with the status of the tumor suppressor gene p53 in 32 cancer cell lines analyzed, high levels of Cyr61 expression were found in cell lines with mutant or null p53 gene, whereas lower expression levels of Cyr61 in the cell lines with wild-type p53. We further show that over-expression of dominant negative p53 or down-expression of endogenous wild-type p53 resulted in up-regulation of Cyr61 expression, suggesting a functional link between Cyr61 and p53 in cancers.

[Selective Expression of Calcium-Binding Protein S100A7 in Lung Cancer.].

BACKGROUND: S100A7 is a member of S100 family and its overexpression has been previously associated with carcinogenesis of certain cancers. The aim of this study is to explore the expression of calcium-binding protein S100A7 in lung cancer. METHODS: In the present study, RT-PCR, Western-blot and immunohistochemistry were used to analyze the different expression of S100A7 in lung cancer tissues and paired noncancerous lung tissues. RESULTS: The specific expression of S100A7 was found in squamous cell carcinomas and large cell lung carcinomas, whereas it was not detected in adenocarcinomas, small cell carcinomas, other cell types of lung cancer and normal lung tissues as well as paired noncancerous lung tissues. CONCLUSIONS: S100A7 was selectively expressed in squamous cell carcinomas and large cell lung carcinomas and may serve as a potential marker for lung cancer diagnosis.

[A new serum biomarker for lung cancer - transthyretin.].

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide and very few specific biomarkers could be used in clinical diagnosis at present. The aim of this study is to find novel potential serum biomarkers for lung cancer using Surface Enhanced Laser Desorption/Ionization (SELDI) technique. METHODS: Serum sample of 227 cases including 146 lung cancer, 13 pneumonia, 28 tuberculous pleurisy and 40 normal individuals were analyzed by CM10 chips. The candidate biomarkers were identified by ESI/MS-MS and database searching, and further confirmed by immunoprecipitation. The same sets of serum sample from all groups were re-measured by ELISA assay. RESULTS: Three protein peaks with the molecular weight 13.78 kDa, 13.90 kDa and 14.07 kDa were found significantly decreased in lung cancer serum compared to the other groups and were all automatically selected as specific biomarkers by Biomarker Wizard software. The candidate biomarkers obtained from 1-D SDS gel bands by matching the molecular weight with peaks on CM10 chips were identified by Mass spectrometry as the native transthyretin (nativeTTR), cysTTR and glutTTR, and the identity was further validated by immunoprecipitation using commercial TTR antibodies. Downregulated of TTR was found in both ELISA and SELDI analysis. CONCLUSIONS: TTRs acted as the potentially useful biomarkers for lung cancer by SELDI technique.

Identification and validation of S100A7 associated with lung squamous cell carcinoma metastasis to brain.

To identify potential markers associated with non-small cell lung cancer (NSCLC) metastasis to brain, comparative proteome analysis on two lung squamous cell carcinoma (SCC) cell lines, NCI-H226 and H226Br (the brain metastatic cell line of NCI-H226), was performed using two-dimensional electrophoresis (2-DE) followed by a tandem mass spectrometer with a matrix-assisted laser desorption/ionization (MALDI) source. Twenty differential proteins were identified, of which 6 proteins were up-regulated in H226Br cell compared with NCI-H226 cells, whereas 14 proteins were down-regulated. S100A7 and 14-3-3sigma, two of candidate proteins significantly upregulated and downregulated in H226Br cell, were selected to verify the liability of the differential proteins by Western blot. The results were in accordance with 2-D data. To determine whether S100A7 overexpression is actually associated with SCC metastasis to brain, S100A7 protein was testified in 10 brain metastasis tissues from NSCLC, 38 primary NSCLC tissues including half matched local positive lymph nodes, 5 primary brain tumors and 2 non-cancer brain tissues by immunohistochemistry. Of particular interest to us was that the positive staining of S100A7 could be found in 3/5 (60%) brain metastases tissue from SCC and 8/21 (38%) the primary lung SCC tissues, while no positive staining was observed in the brain metastases tissue from Ad (n=5), the primary adenocarcinoma (Ad) tissues (n=17), the primary brain tumors (n=5), all local positive lymph nodes from the primary NSCLC (n=19) and non-cancer brain tissues (n=2). These findings suggest that S100A7 expression is closely associated with SCC metastasis to brain and may be a potential biomarker for monitoring the development of SCC.