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Aim and Objective: A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining hypaphorine, a potential agent for treating osteoclast- based bone loss, was developed and validated in rat plasma. Materials and Methods: Plasma samples were pretreated by the protein precipitation. Chromatographic separation was performed using an Inertsil ODS-3 column (50 mm × 4.6 mm, 5 μm). The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile in a gradient mode at a flow rate of 0.5 mL/min. The transitions from protonated precursor ion [M + H]+ to the particular daughter ion were acquired using selected reaction monitoring (SRM). The mass transitions of hypaphorine and IS were found to be 247 → 188 and m/z 219 → 188, respectively. The method was validated in terms of selectivity, linearity, accuracy and precision, extraction recovery and matrix effect, stability, and carryover. Results: It showed good linearity over the range of 1-2000 ng/mL (R2 = 0.9978). The intra-batch accuracy was within 93.95-105.81%, and the precision was within 4.92-11.53%. The inter-batch accuracy was within 96.18-100.39% with a precision of 6.22-11.23%. The extraction recovery and matrix factors were acceptable. Conclusion: The simple and rapid method was successfully applied to the pharmacokinetic study in rats following oral administration of hypaphorine at the doses of 0.5, 1.5, and 4.5 mg/kg.

Aim and Objective: An analytical method for the determination of mobocertinib, an investigational tyrosine kinase inhibitor, was developed and optimized by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in rat plasma. Materials and Methods: Plasma samples were pretreated by the protein precipitation method with a methanol solution of osimertinib as the internal standard (IS). Chromatographic separation was performed using an Inertsil ODS-3 column (50 mm × 4.6 mm, I.D. 5 μm) column with the temperature maintained at 40 °C. The mobile phase consisted of water (containing 0.1% formic acid) and methanol in a gradient mode at a flow rate of 0.5 mL/min. Mass spectrometric detection was carried out in the selected reaction monitoring (SRM) mode with positive electrospray ionization, and the mass transitions of mobocertinib and osimertinib were m/z 587.01 → 71.88 and m/z 499.80 → 71.94, respectively. The method was validated in terms of selectivity, linearity, accuracy and precision, extraction recovery and matrix effect, stability and carryover as per the guidelines for bioanalytical method validation (FDA, 2018). The method was applied to the pharmacokinetic study of mobocertinib in rats by oral gavage at the doses of 2, 6, and 18 mg/kg. A total of 216 plasma samples from 18 rats were analyzed. Results: It showed good linearity over the range of 1-1000 ng/mL (R2 = 0.9957). The intra-batch accuracy was within 94.65-102.59% and the precision was within 5.49-10.46%. The inter-batch accuracy was within 97.08-102.25% with a precision of 7.54-10.13%. The extraction recovery and matrix factor were acceptable for the bioanalysis of mobocertinib. Additionally, mobocertinib was found to be stable under the detected conditions. Mobocertinib showed linear pharmacokinetic characteristics following oral administration to rats at 2.0-18.0 mg/kg. Conclusion: The developed and validated method was successfully employed in the pharmacokinetic study in rats following oral administration of mobocertinib at the doses of 2, 6, and 18 mg/kg.

Background: Identification of genomic markers using NGS (next-generation sequencing) technology would be valuable for guiding precision medicine treatments for pancreatic cancers. Traditional somatic mutation methods require both tumor and matched non-tumor samples. However, only tumor samples are available mostly, especially in retrospective studies. In this study, we tried to analyze the associations between clinical features and oncogenic somatic mutations in genome-wide tumor-only samples. Methods: Fifty-four tumor-only samples derived from pancreatic cancer patients were used for whole-exome sequencing. An approach involving SNP filtering of variants included in the Catalogue of Somatic Mutations in Cancer (COSMIC) database was used to identify oncogenic somatic mutations. The relationships between oncogenic mutations and clinical features were analyzed and simultaneously compared with those from the TCGA database. Results: By analyzing the mutations from tumor only samples, divergent mutation profiles were observed in different locations (head vs. body/tail) of pancreatic tumors. The divergences between pancreatic head and body/tail cancers were also confirmed by the TCGA data. Furthermore, mutations of several genes were found to be significantly associated with clinical features, such as pathological stage and the degree of tumor differentiation. Conclusion: The results confirmed the efficiency of our approach in identifying oncogenic somatic mutations from tumor only samples and revealed the associations between somatic mutations and clinical features in pancreatic cancer.

Background The status of targeted genes and the association between targeted genes and clinicopathological features in Chinese lung cancer patients remains to be elucidated. Methods The status of 10 targeted genes was evaluated by next‐generation sequencing (NGS) in 884 non‐small cell lung cancer (NSCLC) patients. The relationship between gene alterations and clinicopathological characters was analyzed. Results Overall, 684 (77.4%) patients harbored gene alterations, and EGFR (510, 57.7%) was found to be the most common type of mutation followed by KRAS (91, 10.3%), HER2 (38, 4.3%), PIK3CA (32, 3.6%), ALK (21, 2.4%), BRAF (10, 1.1%), ROS1 (5, 0.6%), RET (5, 0.6%), MET (4, 0.5%) and NRAS (1, 0.1%). Gene alterations were more frequent in females, non‐smokers and adenocarcinoma (P, In this research, we investigated the frequency of common driving genes in 884 patients with non‐small cell lung cancer and analyzed their association with clinicopathologic parameters in in eastern China. Most patients had at least one genetic alteration, and individual patients harbored synchronous mutation. Each gene alteration has unique clinicopathologic characteristics.

BackgroundLung cancer is a major health concern worldwide because of its increasing incidence and mortality. This study aimed to clarify the association between mesenchymal-epithelial transition (MET) genomic alterations and clinical characteristics of lung cancer.MethodWe collected data from 5,008 patients with lung cancer diagnosed and treated between January 2017 and July 2021 at the Affiliated Hospital of Qingdao University. Genomic alterations in the MET gene, including the exon 14 skipping mutation and amplification, were detected using amplification refractory mutation system-polymerase chain reaction (2,057 cases) and next-generation sequencing (2,951 cases). Clinical characteristics such as age, sex, tumor location, tumor stage, smoking, pleural invasion, and histology were statistically analyzed for MET exon 14 skipping mutation and amplification. The DNA splicing sites causing the MET exon 14 skipping mutation at the mRNA level were also investigated.ResultsThe incidence of the MET exon 14 skipping mutation was 0.90% (41/4,564) in adenocarcinoma, 1.02% (3/294) in squamous cell carcinoma, and 8.33% (1/12) in sarcomatoid carcinoma specimens. It was more frequently observed in patients over 60 years of age than the MET exon 14 skipping mutation wildtype. The MET exon 14 skipping mutation co-occurred with epidermal growth factor receptor (EGFR) L858R, EGFR 19-Del, and BRAF V600E mutations. At the DNA level, single nucleotide mutation and small fragment deletion (1–38 base pairs) upstream and downstream of MET exon 14 led to MET exon 14 skipping mutation at the mRNA level. MET amplification occurred in 0.78% (21/2,676) adenocarcinoma and 1.07% (2/187) squamous cell carcinoma specimens and was significantly associated with advanced tumor stages (III + IV) compared to the MET amplification wildtype. MET amplification primarily co-occurred with the EGFR mutation.ConclusionsOur study found that MET genomic alterations were statistically related to age and tumor stage and co-existed with mutations of other oncogenic driver genes, such as EGFR and BRAF. Moreover, various splicing site changes at the DNA level led to the exon 14 skipping mutation at the mRNA level. Further studies are required to clarify the association between MET genomic alterations and prognosis.

Objective To investigate the frequency and prognostic role of the human epidermal growth factor receptor 2 gene (HER2) and BRAF V600E gene mutation in Chinese patients with colorectal cancer (CRC). Methods Clinicopathological and survival information from 480 patients with stage I–III CRC were reviewed and recorded. HER2 amplification was analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), BRAF V600E mutation was tested by IHC and Sanger sequencing. The relationship between HER2 and BRAF V600E mutation status and clinicopathological characteristics and outcomes were determined. Results The amplification of HER2 and BRAF V600E mutation were identified in 27 of 480 (5.63%) and 19 of 480 (3.96%) CRC patients, respectively. HER2 amplification significantly correlated with greater bowel wall invasion (P = 0.041) and more advanced TNM stage (I vs. II vs. III; 0 vs 5.78% vs. 7.41%, P = 0.013). Patients suffering from tumors with poor differentiation had a higher incidence rate of BRAF V600E mutation than those with moderate/well differentiation (7.77% vs 2.92%, P = 0.04). HER2 amplification was an independent prognostic factor for worse disease-free survival (DFS) (HR = 2.53, 95% CI: 1.21–5.30, P = 0.014). Conclusion The prevalence of HER2 amplification and BRAF V600E mutation in stage I–III CRC patients in Chinese was 6% and 4%, respectively, and HER2 amplification appeared to be associated with a worse DFS. More comprehensive molecular classification and survival analysis are needed to validate our findings.

Intravenous leiomyomatosis (IVL) is a rare neoplasm that is characterized by smooth muscle cell proliferation within venous vessels. The aim of this study is to investigate the clinicopathological features, immunophenotypes, and MED12 gene mutations in IVL. Nine cases of IVL from the Affiliated Hospital of Qingdao University were collected, and the clinicopathological features were reviewed. The immunohistochemical expressions of p16, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alpha thalassemia/mental retardation syndrome X-linked (ATRX), retinoblastoma 1 (RB1), fumarate hydratase (FH), and p53, were evaluated. The mutation status of MED12 gene exon 2 was detected by Sanger sequencing. All the 9 patients were women ranging from 32 to 58 years, and uterine leiomyomas were identified in 5 patients. Immunohistochemical staining showed that all IVL and leiomyoma samples were positive for estrogen receptor and progesterone receptor, but negative for CD34. IVL displayed similar immunostaining patterns with their uterine counterparts with focal p16 immunostaining. FH, PTEN, ATRX, and RB1 were variably positive, and p53 and Ki-67 positive rates were less than 5% in all cases. Two novel genetic variations at MED12 exon 2, a synonymous mutation c.141C>T (p.Asn47=), and an in-frame deletion mutation c.133_147del15 (p.Phe45_Pro49del) were identified in two IVL cases. One missense mutation c.131G>A (p.Gly44Asp) was identified in one uterine leiomyoma. The remaining 11 tumor samples (7 IVL cases and 4 uterine leiomyomas) showed no mutations at MED12 exon 2. Our results showed two novel MED12 mutations in IVL. The MED12 mutations are different between IVL and uterine leiomyoma. These findings indicate that IVL is a unique entity and different from uterine leiomyoma.

Aims Pulmonary peripheral glandular papilloma (GP) and mixed squamous cell and glandular papilloma (MP) have very similar histological features to pulmonary ciliated muconodular papillary tumour (CMPT)/bronchiolar adenoma (BA). The underlying genetic relationships between GP/MP and CMPT/BA have rarely been characterised. We aimed to reveal the relationship between them. Methods and results We performed a clinicopathological review and next-generation sequencing (NGS) study of two GPs and five MPs. Histologically, GPs/MPs showed similar cellular and architectural features to CMPTs/BAs, such as bilayered epithelium, bronchiole-associated lesions and skipping (discontinuous) growth pattern. One MP showed partial and inconspicuous endobronchiolar growth and more glandular structures, which was very similar to the appearance of CMPT/BA. BRAF V600E mutation was detected in four papillomas (57.1%, one GP and three MPs). Conclusions Overlapping morphological features and comparable mutation profiles support that peripheral GPs/MPs and CMPTs/BAs are on the same disease spectrum. We propose expanding the concept of CMPT/BA and including GP and MP in the CMPT/BA family.

Colorectal cancer (CRC) has become a major health concern in China due to its increasing incidence and mortality. This study aimed to clarify the relationship between tumor locations and the clinicopathological molecular marker features in eastern China CRC patients. We continuously collected data on 2,356 CRC patients who underwent surgical resection from January 2017 to April 2019. Right-sided colorectal cancer (RCC), was located from the cecum to the transverse colon and left-side colorectal cancer (LCRC) was located from the splenic flexure to the rectum. The clinicopathological indices (including age, sex, pTNM stage, mucinous production, and distant metastasis) and frequency of molecular markers such as KRAS, NRAS, BRAF, and microsatellite instability (MSI) were statistically analyzed between the RCC and LCRC groups. The associations between clinicopathological characters and molecular markers were also investigated. LCRC and RCC proportions in eastern China CRC patients were 81.75% and 18.25%, respectively. RCC (vs. LCRC) was more frequently observed with higher frequencies of MSI-high (MSI-H) and BRAF mutations in female and younger patients, and was closely associated with metastasis, poor differentiation, and mucinous tumors. Tumor location also showed significant differences in bowel wall infiltration degree and pTNM stage. Mutation rates of KRAS, NRAS, MSI, and BRAF were 40.15%, 3.85%, 6.31%, and 2.30%, respectively. Patients with a KRAS mutation tended to be female, had mucinous, perineural invasive, and polypoid tumor. Those with NRAS mutation tended to develop well-differentiated ulcerative tumors. The BRAF mutation was more relevant with lymph node involvement, deeper infiltration of the bowel wall, mucinous, poorly-differentiated tumor with thrombus, and perineural invasion. Furthermore, MSI-H was more commonly found in younger patients with deeper bowel wall infiltration and a poorly-differentiated polypoid tumor, whereas MSS patients tended to develop lymph node involvement, and a mucinous and perineural invasive tumor. In our study, we found that LCRC and RCC showed different features on the clinicopathological and molecular markers in eastern China CRC patients. Since our data differ from those of Western countries and other regions in China, further studies are required to clarify the regional differences of the clinicopathological and molecular markers in CRC patients.

Background Perturbations in key driver genes or recurrently somatic mutated genes, as well as altered signaling pathways underlying pancreatic cancer have been largely discovered with the help of massive parallel sequencing. However, to date, the relationships between somatic alterations occurrence and clinical features are still less understood in pancreatic cancer. Methods Using the genomic DNA from each sample, libraries were constructed by shearing genomic DNA and ligating Illumina paired-end adaptors first, then the constructed libraries were hybridized to Agilent Human All Exon Target Enrichment kit V1. The purified capture products were then amplified to make whole exome libraries. The qualified libraries were subjected to 150 base paired-end sequencing on the Illumina NovaSeq instrument. The Genome Analysis Toolkit (GATK) was used to call variants in the sequencing data. All the statistical analyses were performed using IBM SPSS Statistics version 23.0 software. Results In this study, a total of 54 pancreatic ductal adenocarcinoma patients were enrolled, and pancreatic tumour samples without matched normal tissues were subjected to whole-exome sequencing. Based on the high-confidence putative somatic genes identified from our tumour-only sequencing, the results revealed alterations in cancer progression- and metastatic-related signaling pathways (i.e., E-cadherin and CDC42 signaling pathways) were predominantly enriched in late-stage (stage III/IV) tumours. Moreover, mutant EHMT1, as well as KRT6C, were significantly associated with tumour stage, while mutant H3F3A, DPY19L2, ABCB5, and ASTN1 were all significantly associated with the degree of tumour differentiation. Conclusions Together, our data suggest the prevalence of association between somatic alteration at the genomic level and clinical features in pancreatic cancer. Legal entity responsible for the study The authors. Funding The National Natural Science Foundation of China. Disclosure All authors have declared no conflicts of interest.

Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (ALK+ DLBCL) is characterized by the presence of immunoblastic or plasmablastic cells with a strong ALK protein expression that is frequently associated with t(2;17)(p23;q23). The present study reports a case of ALK+ DLBCL in a 26-year-old male with a duodenal mass. Histologically, the neoplastic cells demonstrated prominent plasmablastic differentiation with abundant amphophilic cytoplasma and central nucleoli. Paraffin immunohistochemistry revealed: an exclusively cytoplasmic granular expression of ALK; CD138, immunoglobulin A (IgA) and CD79α positivity; and focal expression of multiple myeloma oncogene 1 (Mum-1), CD30 and epithelial membrane antigen (EMA). However, the immunohistochemical staining was negative for CD3, CD38 and CD20. Fluorescence in situ hybridization (FISH) analysis using an ALK break-apart probe revealed the presence of ALK gene rearrangements in the patient. To the best of our knowledge, the current case represents the first example of primary extranodal ALK+ DLBCL presenting as a duodenal mass.

Papillary renal cell carcinoma (PRCC) includes two different morphological subtypes. The differences of genetics and ultrastructure of the two subtypes have been rarely reported. Also, new biomarkers related to the diagnosis and prognosis of PRCC have still not been well elucidated. Immunohistochemistry, fluorescence in situ hybridization (FISH) and transmission electron microscopy were used systematically to determine the characteristics of 56 cases of PRCC and to reveal new diagnostic and prognostic information. Type 1 PRCC presented higher expression rates of EMA and CK7, whereas type 2 presented a higher expression rate of CD10. New immunohistochemical markers, including: p504s, PAX-2, PAX-8 and CA-IX showed extensive immunostaining in PRCC. We first revealed a distinct immunostaining pattern of CA-IX, which was located in multiple foci in PRCC. All tumors had at least one chromosomal aberration including loss of Y, gains of 7 or 17. Gain of chromosome 17 was common in type 1; losses of chromosome 18, 11 and 8 appeared in type 2. Ultrastructurally, glycogen granules and secondary lysosomes were seen in type 1, mitochondria and smooth endoplasmic reticulum were scattered in type 2. Tumor subtype, nuclear grade, TNM stage, clear cell renal cell carcinoma (CCRCC) component and sarcomatoid elements, metastasis, CAIX expression, losses of chromosome 18 and 8 were related to poor outcome of PRCC. We conclude that the two subtypes of PRCC originate from different renal cells, and arise from partially common genetic pathways. EMA, CK7, CD10, p504s, PAX-2, PAX-8 and CA-IX are helpful markers in the differential diagnosis of PRCC. CA-IX expression, losses of chromosome 18 and 8 are new prognostic factors of PRCC.

To investigate the concordance of dual-color silver enhanced in-situ hybridization (DSISH) and immunohistochemistry (IHC) in the detection of HER2 gene amplification and expression and to evaluate the values of DSISH in detecting HER2 gene status in gastric carcinoma.By using automated DSISH and IHC, HER2 gene status was detected in 230 cases of gastric cancer.Among the 230 cases of gastric carcinoma tested by DSISH, 43 cases were positive and 187 cases were negative; HER2 gene amplification rate was 18.7% (43/230). The expression of HER2 protein was negative, weakly, moderately and strongly positive in 115, 69, 15 and 31 cases, respectively, by IHC. HER2 protein positive rate was 13.5% (31/230). Of the 43 HER2 gene amplification cases by DSISH, 2, 10, 2 and 29 cases were negative, weakly, moderately and strongly positive by IHC; Of the 187 HER2 negative cases by DSISH, 113, 59, 13 and 2 cases were negative, weakly, moderately and strongly positive by IHC, respectively. The overall concordance of HER2 status in the investigation between IHC and DSIDH was 93.5% (201/215), with a high consistency (Kappa coefficient 0.767, P0.01).DSISH can be applied to detect the HER2 gene status in gastric cancer and it also has a high consistency with the result of IHC. In addition, due to frequent heterogeneous expression of HER2, cases with moderate HER2 protein expression may need further assessment by DSISH.