2022年 · 第05卷第04期

To the editor:

Along with the advances in cancer genomics and the development of targeted therapies, the field of molecular diagnostics has undergone rapid evolution to meet the growing needs associated with patient care. Here, we review the past, present, and possible future of molecular diagnostics, including technologies and testing principles, to provide a comprehensive landscape of molecular diagnostic technologies, testing platforms, and applications. This review is based on the US Food and Drug Administration publications, the National Comprehensive Cancer Network guidelines, and the peer-reviewed English literature published between 2003 and 2021. We conclude that molecular diagnostics has changed dramatically during the past two decades. Next-generation sequencing–based comprehensive genomic profiling has replaced single-gene/single-locus testing for simultaneous detection of mutations, copy number alterations, structural variants, and mutational signatures to facilitate cancer diagnosis, prognosis prediction, targeted therapies, and immunotherapies. Laboratory-developed tests and companion diagnostics approved by the US Food and Drug Administration both play important roles in cancer patient management.

Glioma is the most aggressive brain tumor having invasive ability and a highly heterogeneous phenotype. Many patients with glioma respond poorly to traditional surgery or temozolomide-based chemotherapy. Over the past few decades, developments in immunotherapeutic strategies have provided newer insights into the treatment of gliomas. Immunotherapy is based on the principle of normalization or recovery of T cell-mediated anti-tumor immunoreaction. Different innovative strategies have been used; these include enhancement of immunogenicity by administration of tumor antigens or dendritic cell vaccines, replenishment of cytotoxic T cells by adoptive T cell transfer, repair of exhausted T cells by immune checkpoint inhibitors, and the use of other immune activators such as oncolytic viruses. However, many immunotherapy-based clinical trials did not meet the expected therapeutic endpoints in patients with glioma. Gliomas use unique strategies to generate an immune-suppressive microenvironment; these include limiting immunogenicity and repressing T cell infiltration or activation. This may be addressed by the incorporation of immunotherapy with standard therapy or by use of certain innovative approaches such as tumor-treating fields. In this review, we summarize the updated immunotherapies in glioma and discuss current limitations and future prospects.

Emerging data have shown that non-coding RNAs (ncRNAs) can encode micro-peptides (≤100 amino acids) that play an important role in regulating physiological and pathological processes. Herein, we explored ncRNAs that may encode micro-peptides that are involved in the development of hepatocellular carcinoma (HCC).

High-throughput sequencing of ribosomal protein S6 (RPS6) was performed in four cancer cell lines using RNA-immunoprecipitation (RIP). UCSC databases obtained the full length of the gene sequences and quantitative polymerase chain reaction (qPCR) was used to evaluate expression levels of ncRNAs of interest. The coding activity of ncRNA was assessed in vitro by co-immunoprecipitation, plasmid transfection, western blot, immunofluorescence and RNA fluorescence in situ hybridization. Mass spectrometry was performed to explore the potential functions of candidate micro-peptide in HCC. This study involving human tissue specimens was conducted in accordance with Declaration of Helsinki and approved by the Institutional Review Board of Changhai Hospital, Naval Military Medical University, China (approval No. CHEC2020-081) on June 6, 2020.

We performed RIP assay using primary antibodies for RPS6 and high-throughput sequencing. A total of 223 overlapping genes were captured by RPS6-RIP. Venn diagram analysis revealed that 60 overlapping genes were detected in four cancer cell lines. QRT-PCR showed that six of the candidate genes (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, HCP5, RP11-517B11.7) were expressed in Huh7 and Hep3B cells. Further analysis of these six candidate genes and found that five (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11-517B11.7) displayed higher expression levels in HCC cell lines (Huh7, Hep3B) and tumor tissues than in liver cell lines (L-02, QSG-7701) and non-tumor tissues, respectively. Performed additional RIP assays and confirmed that four of the genes (RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11-517B11 .7) bound RPS6. We obtained the full length of the four gene sequences from the UCSC database and analyzed the open reading frames by ORF Finder; to determine the translation potential of the four candidate small open reading frames (smORFs), we subcloned a FLAG epitope tag into the C-terminal of the four selected smORFs before the stop codon, and the fusion sequences were then cloned into three different plasmid vectors (pSPT19, pcDNA3.1, and PEGFP-N1). We performed coupled transcription and translation reactions and found that the pSPT19 plasmids encoded small peptides in vitro. After then transfected the pcDNA3.1 constructs into Huh7 cells, and a single 7.2 kDa micro-peptide was encoded from the candidate smORF of RP11.119F7.5. We transfected the recombinant pEGFP-N1 plasmids with smORFs in HCC cells, and western blot analysis revealed a band above GFP in the RP11.119F7.5 recombinant plasmid lane. The coding potential of the RP11-119F7.5 vector was also confirmed by immunofluorescence assay. Fluorescence in situ hybridization assay revealed that RP11-119F7.5 was localized in the cytoplasm and nucleoplasm of HCC cells. Gene ontology enrichment analysis showed that the micro-peptide–interacting proteins were mainly involved in extracellular exosomes. We also found the identified proteins were involved in several biological functions like protein binding, poly(A) RNA binding, translational initiation, and the nuclear-transcribed mRNA catabolic process. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed the peptide-interacting proteins might participate in several critical pathways including ribosome, biosynthesis of amino acids, carbon metabolism, biosynthesis of antibiotics, glycolysis and gluconeogenesis, pathogenic Escherichia coli infection and influenza A.

Our study revealed a novel micro-peptide translated by ncRNA RP11-119F7.5, highlighting the coding ability and potential role of ncRNAs in HCC.

The genomic landscapes of metastatic colorectal cancer (mCRC) have been extensively studied; however, the genetic mechanisms underlying the locoregional recurrence (LR) of CRC remain unclear. The objective of our study was to investigate genomic evolution during LR in CRC using high-throughput sequencing.

Twenty-three CRC patients with matched primary and LR tissues were recruited from Nanfang Hospital and Zhejiang Cancer Hospital between January 2011 and December 2018. The last date of follow-up was March 2020. Tissue samples were analyzed by whole-exome sequencing and the genomic profiles were depicted by single nucleotide variation, mutational signature, copy number variation, clonal architecture, and other features. The evolutionary process was speculated with comparison of the genetic variations between primary and LR lesions. The disseminating clusters from primary to LR lesions were identified by variant allele frequency dynamics. Furthermore, the early-recurrent biomarker was explored by comparing the indel signature between early- and late-recurrent patients. The study was approved by the Institutional Review Board of Nanfang Hospital of Southern Medical University (approval No. 2020010) on September 11, 2020.

The results highlighted distinct origins of LR between patients with high microsatellite instability and microsatellite stability. LR lesions evolved independently in patients with high microsatellite instability, while LR lesions were highly clonally related to the primary lesions in patients with microsatellite stability. Late-acquired variations in LR lesions encompassed a wide range of driver genes involved in histone methylation, DNA replication, T cell activation, PDCD1 gain, and LMNA loss. Furthermore, clonal analysis of the disseminating cells identified a dominant polyclonal seeding pattern during LR. The indel signature ID4 was associated with significantly shorter disease-free survival in patients with relapsed CRC according to a public dataset.

These findings pose a challenge for the development of new approaches targeting the interactions of multiple clones in the establishment of LR and in terms of optimizing the clinical management of susceptible patients.

To explore the antitumor and potential off-target effects of systemically delivered cholesterol-conjugated let-7a mimics (Chol-let-7a) and control mimics (Chol-miRCtrl) on hepatocellular carcinoma in vivo.

The antitumor effects of two intravenous dosing regimens of Chol-let-7a on heptocellular carcinoma growth were compared using an orthotopic xenograft mouse model. Off-targets were analyzed with histopathological and ultrapathological features of heparenal tissue and cells in the Chol-let-7a-, Chol-miRCtrl-, and saline-treated (blank) xenograft mice and normal control mice. Then, let-7a abundance in orthotopic tumors, corresponding paracancerous hepatic tissue, and normal liver tissue from healthy nude mice was examined by reverse transcription-polymerase chain reaction. The distribution of Chol-let-7a and Chol-miRCtrl in vivo was examined by whole-animal imaging and frozen-sections observation. The experiments were approved by the Institutional Research Board of Peking Union Medical College Hospital.

Continuous treatment with Chol-let-7a resulted in tumors that were 35.86% and 40.02% the size of those in the Chol-miRCtrl and blank xenograft group (P < 0.01 and P < 0.01, respectively), while intermittent dosing with Chol-let-7a resulted in tumors that were 65.42% and 56.66% the size of those in the Chol-miRCtrl and the blank control group, respectively (P < 0.05 and P < 0.05). In addition, some histopathological and ultrapathological features were only observed after treatment with the two cholesterol-conjugated molecules, however mild with intermittent dosing Chol-let-7a treatment, such as diffuse sinusoidal dilation and edema, primarily around the centrolobular vein in heptic tissues; mild hypercellularity with dilated capillary lumens in the renal tissue; and some organelle abnormalities found in heptic and renal cells. Furthermore, whole-animal imaging showed that Chol-let-7a and Chol-miRCtrl were predominantly distributed in the liver, kidney, and bladder regions after injection, and that the concentration of Chol-let-7a and Chol-miRCtrl in the kidney and the bladder decreased much slowly in the xenograft animals, especially in the Chol-miRCtrl group. Finally, RT-PCR analysis showed that let-7a levels were significantly increased in Chol-let-7a-treated xenografts compared with Chol-miRCtrl group (P=0.003) and blank xenograft group (P=0.001); however, the level was only equivalent to 50.6% and 40.7% of that in paracancerous hepatic tissue and hepatic tissue in normal mice, respectively.

Chol-let-7a, administered either continuously or intermittently, showed effective antitumor efficacy. Chol-let-7a had some off-target effects, such as mild acute hepatitis-like inflammation and non-specific drug-induced kidney injury. The intermittent dosing regimen resulted in less damage than the continuous regimen, while maintaining relatively satisfactory antitumor efficacy, which could be useful for the investigation and possible clinical use of miRNA treatment regimens in the future.