Optimization of FFPE preparation and identification of gene attributes associated with RNA degradation.

Formalin-fixed paraffin-embedded (FFPE) tissues are widely available specimens for clinical studies. However, RNA degradation in FFPE tissues often restricts their utility. In this study, we determined optimal FFPE preparation conditions, including tissue ischemia at 4°C (<48 h) or 25°C for a short time (0.5 h), 48-h fixation at 25°C and sampling from FFPE scrolls instead of sections. Notably, we observed an increase in intronic reads and a significant change in gene rank based on expression level in the FFPE as opposed to fresh-frozen (FF) samples. Additionally, we found that more reads were mapped to genes associated with chemical stimulus in FFPE samples. Furthermore, we demonstrated that more degraded genes in FFPE samples were enriched in genes with short transcripts and high free energy. Besides, we found 40 housekeeping genes exhibited stable expression in FF and FFPE samples across various tissues. Moreover, our study showed that FFPE samples yielded comparable results to FF samples in dimensionality reduction and pathway analyses between case and control samples. Our study established the optimal conditions for FFPE preparation and identified gene attributes associated with degradation, which would provide useful clues for the utility of FFPE tissues in clinical practice and research.

Neoadjuvant immunotherapy plus chemotherapy and adjuvant targeted therapy in ALK-positive non-small-cell lung cancer.

Neoadjuvant therapy and adjuvant therapy for locally advanced non-small-cell lung cancer (NSCLC) with ALK fusion mutation have not been thoroughly studied. Here, a stage IIIB NSCLC patient with EML4-ALK fusion mutation receiving immunotherapy plus chemotherapy as neoadjuvant treatment followed by lobectomy plus lymph node dissection is reported. The patient achieved a pathological complete response according to pathological evaluation. The patient received adjuvant crizotinib for 3 months and achieved a disease-free survival time of 36 months.

A 50-year-old man was diagnosed with lung cancer. Surgery was not initially an option due to the seriousness of the disease. He received two cycles of treatment and the tumor became obviously smaller. Then, he received an operation. No tumor cells were discovered after detection. The patient had one gene that was not normal. Consequently, he was administered medicine for 3 months. The patient has had good health for at least 36 months.

Relationship between mismatch repair protein, RAS, BRAF, PIK3CA gene expression and clinicopathological characteristics in elderly colorectal cancer patients.

BACKGROUND: Colorectal cancer (CRC) is common in elderly patients. Mismatch repair (MMR) protein deletion is one of the causes of CRC. The RAS (KRAS/NRAS), BRAF, and PIK3CA genes are important gene targets in CRC treatment and are closely related to the prognosis and survival of patients. However, little is known regarding the relationship between the expression of MMR, RAS, BRAF, PIK3CA and the clinicopathological features in CRC patients. AIM: To analyze the relationship between the expression of MMR, RAS, BRAF, PIK3CA and the clinicopathological features in CRC. METHODS: A total of 327 elderly patients with CRC were enrolled, and immuno-histochemistry was used to detect the MMR protein. Real-time quantitative polymerase chain reaction was used to detect the RAS (KRAS/NRAS), BRAF, and PIK3CA genes. The clinicopathological data of the patients were recorded and analyzed by SPSS 19.0 statistical software. RESULTS: In 327 elderly patients with CRC, the rate of MMR protein loss was 9.79% (32/327), and the deletion rate of four MMR proteins (MSH2, MSH6, MLH1, PMS2) was 1.83% (6/327), 3.06% (10/327), 7.65% (25/327), and 7.65% (25/327), respectively. There were no significant differences between MMR protein deletion and sex, pathological type, tumor morphology, differentiation degree or lymph node metastasis (P > 0.05), but there was a significant difference between MMR protein deletion and tumor diameter and tumor location (P = 0.048/P = 0.000). The mutation rates of the KRAS, NRAS, BRAF and PIK3CA genes in elderly CRC patients were 44.95% (147/327), 2.45% (8/327), 3.36% (11/327) and 2.75% (9/327), respectively; the KRAS gene mutation was closely related to tumor morphology (P = 0.002) but not to other clinicopathological features (P > 0.05), and there were no significant differences between NRAS gene mutation and clinicopathological features (P > 0.05). The BRAF gene mutation showed a significant difference in pathological type, tumor location, differentiation degree and lymph node metastasis (P < 0.05), but was not correlated with sex, tumor size and tumor morphology (P > 0.05). The PIK3CA gene mutation showed no significant differences in the above clinicopathological characteristics (P > 0.05). Significant differences were observed between MMR protein deletion and KRAS, BRAF, and PIK3CA gene mutations in elderly CRC patients (P = 0.044, P = 0.000, P = 0.003, respectively), but there was no significant difference between MMR protein deletion and NRAS mutation (P > 0.05). CONCLUSION: In elderly CRC patients, the tumor is mainly located in the right colon, and the deletion rate of MMR protein is higher when the tumor diameter is greater than or equal to 5 cm; the deletion rate of MLH1 and PMS2 is more common; the mutation rate of KRAS gene is higher than that of the NRAS, BRAF and PIK3CA genes, the BRAF gene mutation has different degrees of correlation with clinicopathological characteristics; when the MMR protein is deleted, the BRAF and PIK3CA gene mutations are often present, and the KRAS gene mutation rate is low.

Integrative genomic study of Chinese clear cell renal cell carcinoma reveals features associated with thrombus.

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with features that vary by ethnicity. A systematic characterization of the genomic landscape of Chinese ccRCC is lacking, and features of ccRCC associated with tumor thrombus (ccRCC-TT) remain poorly understood. Here, we applied whole-exome sequencing on 110 normal-tumor pairs and 42 normal-tumor-thrombus triples, and transcriptome sequencing on 61 tumor-normal pairs and 30 primary-thrombus pairs from 152 Chinese patients with ccRCC. Our analysis reveals that a mutational signature associated with aristolochic acid (AA) exposure is widespread in Chinese ccRCC. Tumors from patients with ccRCC-TT show a higher mutational burden and genomic instability; in addition, mutations in BAP1 and SETD2 are highly enriched in patients with ccRCC-TT. Moreover, patients with/without TT show distinct molecular characteristics. We reported the integrative genomic sequencing of Chinese ccRCC and identified the features associated with tumor thrombus, which may facilitate ccRCC diagnosis, prognosis and treatment.