[The Polymorphism Analysis of HLA Class II Alleles Based on Next-Generation Sequencing and Prevention Strategy for Allele Dropout].

OBJECTIVE: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system. METHODS: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods. RESULTS: A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System. CONCLUSION: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

Fanconi Anemia Complementary Group A (FANCA) Facilitates the Occurrence and Progression of Liver Hepatocellular Carcinoma.

BACKGROUND: Liver hepatocellular carcinoma (LIHC) is a serious liver disease worldwide, and its pathogenesis is complicated. AIMS: This study investigated the potential role of FANCA in the advancement and prognosis of LIHC. METHODS: Public databases, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot (WB) and immunohistochemistry (IHC) were employed to measure FANCA expression between tumor and normal samples. The relationship between FANCA expression and prognosis of LIHC patients were examined. Functional enrichment of FANCA-related genes was performed. Furthermore, univariate and multivariate analyses were conducted to determine the independent prognosis value of FANCA in LIHC. Finally, influence of FANCA knockout on the proliferation, migration, and invasion of HepG2 cell was validated with cloning formation, CCK8, and Transwell assays. RESULTS: Expression analysis presented that FANCA had high expression level in LIHC tissues and cells. Receiver operating characteristic (ROC) curve analysis showed that FANCA was of great diagnosis value in LIHC. Clinicopathological analysis revealed that FANCA was significantly greater expressed in the advanced stage than in the early stage of LIHC. Univariate, multivariate, and Kaplan-Meier survival analysis confirmed that high expression of FANCA was strongly associated with poor survival of LIHC patients. In addition, high level of FANCA in LIHC showed a negative association with immunoinfiltrated B cells, T cells, and stromal scores. Moreover, Knockout of FANCA significantly inhibited HepG2 cell proliferative activity, migration, and invasion ability. CONCLUSIONS: Our data revealed that high level of FANCA was closely associated with LIHC malignant progression, suggesting its potential utility as a diagnostic, predictive indicator, and therapeutic target.

A Four Amino Acid Metabolism-Associated Genes (AMGs) Signature for Predicting Overall Survival Outcomes and Immunotherapeutic Efficacy in Hepatocellular Carcinoma.

Metabolites are important indicators of cancer and mutations in genes involved in amino acid metabolism may influence tumorigenesis. Immunotherapy is an effective cancer treatment option; however, its relationship with amino acid metabolism has not been reported. In this study, RNA-seq data for 371 liver cancer patients were acquired from TCGA and used as the training set. Data for 231 liver cancer patients were obtained from ICGC and used as the validation set to establish a gene signature for predicting liver cancer overall survival outcomes and immunotherapeutic responses. Four reliable groups based on 132 amino acid metabolism-related DEGs were obtained by consistent clustering of 371 HCC patients and a four-gene signature for prediction of liver cancer survival outcomes was developed. Our data show that in different clinical groups, the overall survival outcomes in the high-risk group were markedly low relative to the low-risk group. Univariate and multivariate analyses revealed that the characteristics of the 4-gene signature were independent prognostic factors for liver cancer. The ROC curve revealed that the risk characteristic is an efficient predictor for 1-, 2-, and 3-year HCC survival outcomes. The GSVA and KEGG pathway analyses revealed that high-risk score tumors were associated with all aspects of the degree of malignancy in liver cancer. There were more mutant genes and greater immune infiltrations in the high-risk groups. Assessment of the three immunotherapeutic cohorts established that low-risk score patients significantly benefited from immunotherapy. Then, we established a prognostic nomogram based on the TCGA cohort. In conclusion, the 4-gene signature is a reliable diagnostic marker and predictor for immunotherapeutic efficacy.

The novel HLA-B*13:179 variant detected by next generation sequencing in a Chinese individual.

HLA-B*13:179 differs from HLA-B*13:99 by one nucleotide substitution at position 829(A>G) in exon 4.

Identification of the novel HLA-B*46:01:33 allele by next generation sequencing in a Chinese individual.

B*46:01:33 differs from B*46:01:01:01 by one nucleotide change at nucleotide 105 in exon 2 from C to T.

A novel HLA-C null allele, HLA-C*08:236N, identified by next-generation sequencing in a Chinese individual.

HLA-C*08:236N differs from C*08:01:01 by a single nucleotide exchange in exon 5 at position 1991.

Full-length sequence of the novel HLA-C*03:04:74 allele by next generation sequencing in a Chinese individual.

C*03:04:74 differs from C*03:04:01:02 by one nucleotide change at nucleotide 1047 in exon 6 from G to A.

[Exclusion of HLA-C Genotype with Zero Mismatched PCR-SBT Results by Next Generation Sequencing].

OBJECTIVE: Three cases of rare alleles of HLA-C with zero mismatched PCR-SBT results were analyzed by full-length sequencing to determine the true genotypes. METHODS: Three rare HLA-C alleles with zero mismatched PCR-SBT results were screened from clinical transplant matching samples, and the full-length sequence was detected by next-generation sequencing technology. RESULTS: The results of PCR-SBT typing of 3 samples were: HLA-C*03:04, 12:167; HLA-C*07:291, 15:02; HLA-C*01:43, 08:16. Other alleles were not in the CWD table of common and confirmed HLA alleles in China (version 2.3) except common allele HLA-C*03:04, HLA-C*15:02. NGS full-length sequencing revealed that the HLA-C genotypes of the three samples were a combination of common alleles and novel alleles, and the three novel alleles had a base mutation in exons 6, 2, and 4, respectively. The novel allele sequences have been submitted to the Genbank database (MK629722, MK335474, MK641803), which were officially named HLA-C*03:04:74, HLA-C*15:192, HLA-C*08:01:25 by the WHO HLA Nomenclature Committee. The HLA high-resolution typing results of 3 samples were: HLA-C*03:04:74, HLA-C*12:03; HLA-C*07:02, HLA-C*15:192; HLA-C*01:02, HLA-C*08:01:25. CONCLUSION: HLA typing results containing rare alleles should be treated cautiously, and the full-length sequence should be verified by NGS or cloning. The laboratory finally confirmed that the 3 cases of PCR-SBT zero mismatch HLA-C genotypes are the combination of common alleles and novel alleles by NGS sequencing, which provides an accurate basis for clinical transplantation matching and enriches the human HLA genetic database.

Discovery of the HLA-C*08:99 allele in a Chinese individual.

HLA-C*08:99 differs by one non-synonymous nucleotide from C*08:01:01 in exon 5, codon 288 GTT>ATT.

Discovery of the HLA-C*03:561 allele, a variant of HLA-C*03, in a Chinese individual.

HLA-C*03:561 differs from HLA-C*03:02:02:01 by one nucleotide change in exon 4 at position 862 (G>A).

Identification of the novel HLA-DRB3*02:02:19 allele.

The HLA-DRB3*02:02:19 allele differs from DRB3*02:02:01:02 by a single nucleotide change in exon 2.

Discovery of the novel HLA-C*01:179 allele in a southern Chinese patient.

One nucleotide substitution in codon 189 of HLA-C*01:02:01:01 results in a novel allele, HLA-C*01:179.

A substitution in exon 2 resulted in the novel HLA-A*30:140 variant identified in a Chinese individual.

HLA-A*30:140 differs from HLA-A*30:01:01 by one nucleotide change in exon 2 at position 341 (C > A).

Identification of HLA-DPB1*1104:01 by next-generation sequencing in a Chinese individual.

HLA-DPB1*1104:01 differs from HLA-DPB1*540:01 by a single nucleotide change in exon 2.

Identification of the novel HLA-B*46:78 allele by next generation sequencing in a Chinese individual.

HLA-B*46:78 differs from HLA-B*46:01:01 by one nucleotide substitution at position 205 in exon 2.

Characterization of the novel HLA-DQB1*06:01:22 allele by next-generation sequencing.

HLA-DQB1*06:01:22 differs from HLA-DQB1*06:01:01:01 by one nucleotide substitution in codon 189 in exon 4.

Characterization of the novel HLA-B*15:435 allele by next-generation sequencing in a Chinese family.

HLA-B*15:435 has 5 nt changes from HLA-B*15:09:01 in exon 3 and 4.

Coexpression of CD5 and CD43 predicts worse prognosis in diffuse large B-cell lymphoma.

Both CD5 and CD43 are expressed on the surface of B lymphocytes of definite phase and associated with the adverse outcome in diffuse large B-cell lymphoma (DLBCL). However, the relationship between CD5 and CD43 expression and the prognostic value of CD5/CD43 coexpression in DLBCL are unknown. We herein determined the correlation between CD5 and CD43 expression, as separate factors or in combination, with the clinicopathological features and survival of 200 patients with DLBCL receiving standard chemotherapy with or without rituximab. Among these DLBCL patients, CD5 expression, CD43 expression, and CD5/CD43 coexpression were detected in 18 (9%), 57 (27%), and 10 (5%) patients, respectively, and all were positively correlated with advanced age and nongerminal cell type. CD5-positive and CD43-positive DLBCL patients had poorer event-free survival (EFS, P < 0.001) and overall survival (OS, P < 0.001) than CD5-negative and CD43-negative patients, respectively. CD5/CD43 coexpression was correlated with a significantly worse prognosis than CD5 or CD43 expression alone. Univariate analysis showed that CD5 expression, CD43 expression, and CD5/CD43 coexpression were all adverse prognostic factors for DLBCL patient survival, and CD5/CD43 coexpression was associated with a greater relative risk for recurrence and death than either CD5 or CD43 expression alone. Multivariate analysis demonstrated that CD5/CD43 coexpression was an independent prognostic factor for EFS (P < 0.001) and OS (P < 0.001) in DLBCL. In conclusion, our data indicate that DLBCL patients with CD5/CD43 coexpression represent a specific subgroup with a significantly worse prognosis than those expressing either marker alone.

Giant seminoma in an undescended testicle metastasizing to the neck and liver.

Germ cell tumors account for 98% of all testicular malignancies. Delays in seeking treatment are unfortunately common and may lead to metastatic spread. The present study reported a case of a 24-year-old man with a giant 12×10 cm left inguinal mass and a left neck mass that had grown rapidly during recent months. Computed tomography confirmed that the mass measured 12.1×9.4 cm and was a left undescended testicle malignancy, and also revealed widespread metastasis to the liver and a large retroperitoneal mass (12.6×8.2 cm). Immunohistochemical staining confirmed seminoma. The patient was treated with chemotherapy with the VIP protocol (cisplatin, etoposide and ifosfamide). Following courses of chemotherapy, the patient received complete clinical remission and was disease-free at the 6 month follow-up.

Side population in hepatocellular carcinoma HCCLM3 cells is enriched with stem-like cancer cells.

Substantial evidence implicates that low-abundance cancer stem cells (CSCs) are responsible for tumor metastasis and recurrence in hepatocellular carcinoma (HCC). Side population (SP) cells possess typical CSCs-like features, and are frequently considered as a special subpopulation in which CSCs are enriched and in studies may be considered as a substitute for CSCs. The aim of the present study was to examine the abundance of SP cells in human HCC cell lines with different metastatic potentials and compare their CSC-like, tumorigenic and invasive properties with those of the main population (MP) cells. An experimental system is described for identifying SP cells and analyzing their CSC-like properties. The relative abundance of SP cells correlated directly with the metastatic potential of the HCC cell line: HCCLM3, 16.3±2.2%; MHCC97-H, 8.4±0.7%; MHCC97-L, 4.7±0.5%; and Huh7, 1.0±0.3% (P<0.05). SP cells isolated from HCCLM3 cultures showed significantly higher proliferation rates and clonogenicity than the corresponding MP cells, in addition to higher migration and invasive abilities in vitro and greater tumorigenicity in mice. Expression levels of all CSC-associated genes tested, except EpCAM and Oct4, were significantly higher in SP cells. The findings revealed that the proportion of SP cells correlates with metastatic potential, and SP cells demonstrated the characteristics expected of CSCs, implicating them in HCC metastasis. Further studies on the identification and characterization of SP cells using clinical HCC specimens will contribute to the understanding of how SP cells are involved in these disease processes.