Reliability of panel-based mutational signatures for immune-checkpoint-inhibition efficacy prediction in non-small cell lung cancer.

OBJECTIVES: Mutational signatures (MS) are gaining traction for deriving therapeutic insights for immune checkpoint inhibition (ICI). We asked if MS attributions from comprehensive targeted sequencing assays are reliable enough for predicting ICI efficacy in non-small cell lung cancer (NSCLC). METHODS: Somatic mutations of m = 126 patients were assayed using panel-based sequencing of 523 cancer-related genes. In silico simulations of MS attributions for various panels were performed on a separate dataset of m = 101 whole genome sequenced patients. Non-synonymous mutations were deconvoluted using COSMIC v3.3 signatures and used to test a previously published machine learning classifier. RESULTS: The ICI efficacy predictor performed poorly with an accuracy of 0.51-0.09+0.09, average precision of 0.52-0.11+0.11, and an area under the receiver operating characteristic curve of 0.50-0.09+0.10. Theoretical arguments, experimental data, and in silico simulations pointed to false negative rates (FNR) related to panel size. A secondary effect was observed, where deconvolution of small ensembles of point mutations lead to reconstruction errors and misattributions. CONCLUSION: MS attributions from current targeted panel sequencing are not reliable enough to predict ICI efficacy. We suggest that, for downstream classification tasks in NSCLC, signature attributions be based on whole exome or genome sequencing instead.

Targeted next-generation sequencing using a multigene panel in myeloid neoplasms: Implementation in clinical diagnostics.

INTRODUCTION: Detection of mutations in patients with myeloid neoplasms (MNs) has shown great potential for diagnostic and prognostic purposes. Next-generation sequencing (NGS) is currently implemented for the diagnostic profiling of the four major MN subgroups. METHODS: First, we validated the targeted NGS approach using the TruSight Myeloid panel. Next, we screened 287 patients with a clinical suspicion of MN and 61 follow-up patients with documented MN. RESULTS: Validation of the NGS workflow resulted in maximal precision, accuracy, sensitivity, and specificity for gene variants with an allele frequency of at least 5% and a minimal read depth of 300. In our diagnostic screen, we identified at least one somatic mutation in 89% of patients with proven MN. Of the 155 newly diagnosed MN cases, 126 (81%) showed at least one mutation, confirming clonality. Moreover, the co-occurrence of mutated genes in the different MN subentities facilitates their classification and justifies the diagnostic use of a pan-myeloid panel. Furthermore, several of these mutations provide additional prognostic information independently of traditional prognostic scoring systems. CONCLUSION: Pan-myeloid targeted NGS fits elegantly in the routine diagnostic approach of MNs allowing for an improved diagnosis, subclassification, and prognosis.

Granulocyte chemotactic protein-2 and related CXC chemokines: from gene regulation to receptor usage.

Chemokines contribute to the inflammatory response by selective attraction of various leukocytic cell types. Human GCP-2 was originally identified by amino acid sequence analysis as a CXC chemokine co-produced with IL-8 by osteosarcoma cells. Furthermore, the complete coding domain of human GCP-2 was disclosed by means of RT-PCR. Similarly, mouse GCP-2 was isolated from fibroblastoid and epithelial cells and completely identified by sequence analysis. Human and mouse GCP-2 share 61% identical amino acids. Both chemokines occur as multiple NH2-terminally truncated forms. The shorter forms of mouse, but not those of human, GCP-2 showed a higher neutrophil chemotactic potency and gelatinase B releasing capacity. Mouse GCP-2 was a more potent neutrophil activator than human GCP-2, natural mouse KC, and MIP-2. Human GCP-2 was not chemotactic for monocytes, lymphocytes, or eosinophils. Quantitative studies of mRNA expression in diploid fibroblasts revealed GCP-2 induction by IL-1beta. Human GCP-2 induced [Ca2+]i increase in neutrophils, which was reciprocally desensitized by IL-8, GROalpha, and ENA-78. Human GCP-2 induced [Ca2+]i increases and chemotactic responses in both CXCR1- and CXCR2-transfected cells. Finally, GCP-2 provoked neutrophil accumulation and plasma extravasation in rabbit skin. In humans, GCP-2 complements the activity of IL-8 as neutrophil chemoattractant and activator but it constitutes a major neutrophil chemokine in the mouse. GCP-2 induces neutrophil chemotaxis and activation but it might also contribute to detrimental tissue damage in sepsis, acute respiratory distress syndrome, acute hypersensitivity reactions, and autoimmune diseases. It might also influence the invasive capacity of GCP-2-secreting tumor cells.

A competitive RT-PCR method for the quantitative analysis of cytokine mRNAs in mouse tissues.

The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.

Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.

Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The availability of recombinant GCP-2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP-2 during the inflammatory response.

Human monocyte chemotactic protein-2: cDNA cloning and regulated expression of mRNA in mesenchymal cells.

Stimulated MG-63 osteosarcoma cells have been used as a source to purify and identify the monocyte chemokines MCP-1, MCP-2 and MCP-3. In comparison with MCP-1, the production yields of MCP-2 and MCP-3 in these cells are rather low and variable. Although the protein sequence of human MCP-2 is identified, its DNA sequence remains elusive. A degenerate primer set was used to isolate an MCP-2 gene fragment from the chemokine YAC contig on human chromosome 17. Based on the gene sequence of MCP-2, a unique primer set was synthesized and used to screen cDNA libraries for the presence of MCP-2 transcripts by PCR. The complete MCP-2 cDNA was cloned from a human bone marrow cDNA library and sequenced. The cDNA-derived protein sequence was identical to that of purified natural MCP-2, except for Gln46 which replaced Lys46. There seem thus to exist two MCP-2 allelic variants because at position 46 the codons of two residues (Lys46 and Gln46) were detected in individual genomes. As shown by Northern hybridization, the MCP-2 steady-state mRNA levels in normal diploid fibroblasts were increased by IL-1 beta, IFN-gamma and the double-stranded RNA poly rI:rC. RT-PCR analysis showed induction of MCP-2 mRNA in MG-63 cells by IFN-gamma and IL-1 beta. The regulated production of MCP-2 by tumor cells and normal mesenchymal cells is indicative of a role in neoplasia and inflammatory host responses.

Potential therapeutic use of antibodies directed towards HuIFN-gamma.

IFN-gamma is an important regulator of immune responses and inflammation. Studies in animal models of inflammation, autoimmunity, cancer, transplant rejection and delayed-type hypersensitivity have indicated that administration of antibodies against IFN-gamma can prevent the occurrence of diseases or alleviate disease manifestations. Therefore, it is speculated that such antibodies may have therapeutical efficacy in human diseases. Since animal-derived antibodies are immunogenic in patients several strategies are being developed in order to reduce or abolish this human anti-mouse antibody (HAMA) response. In our laboratory, we have constructed a single-chain variable fragment (scFv) derived from a mouse antibody with neutralizing potential for human IFN-gamma. A scFv consists of only variable domains tethered together by a flexible linker. The scFv was demonstrated to neutralize the antiviral activity of HuIFN-gamma in vitro and therefore might be considered as a candidate for human therapy.

The human MCP-3 gene (SCYA7): cloning, sequence analysis, and assignment to the C-C chemokine gene cluster on chromosome 17q11.2-q12.

Monocyte chemotactic proteins (MCPs) are chemokines involved in macrophage recruitment during inflammation and cancer. A full-size MCP-3 cDNA was used to isolate the functional human MCP-3 gene. Based on restriction analysis, subclones were selected and the MCP-3 gene sequence was completed. In addition to a dense region with direct and inverted repeats and palindromic sequences, a double microsatellite (CA)n-(GA)n' was found at the 5'-end of the MCP-3 gene, and an RFLP was detected. The gene was regionally mapped by fluorescence in situ hybridization to human chromosome 17, subbands q11.2-q12. This site contains the MCP-subset of C-C chemokines and can be distinguished from the syntenic MIP-1 alpha locus. SCYA7 was assigned as the locus symbol of the MCP-3 gene. Double-labeling experiments confirmed the regional assignment of the MCP-3 gene close to the ERBB2 locus on human chromosome 17.

Human monocyte chemotactic protein-3 (MCP-3): molecular cloning of the cDNA and comparison with other chemokines.

When human MG-63 osteosarcoma cells are triggered with IL-1 beta, they produce, among various other cytokines, also monocyte chemotactic proteins (MCPs). Homogeneous MCP-1, MCP-2 and MCP-3 were found to induce production of gelatinase B and chemotaxis of monocytes. Based on the almost complete amino acid sequence of natural MCP-3, two sets of degenerated oligonucleotides were used for the amplification of MCP-3 cDNA. Total RNA from stimulated MG-63 cells was reverse transcribed and PCRs done on the mRNA:cDNA duplex. Using the PCR-product, several cDNAs were isolated from a cDNA library of IL-1-stimulated MG-63 cells. From the cDNA sequence the complete primary structure of the protein was deduced: MCP-3 shows 71% and 58% amino acid homology with MCP-1 and MCP-2, respectively. Our study establishes MCP-3 as an inflammatory cytokine that regulates macrophage functions. Because MCP-3 is often produced by tumor cell lines and regulates protease secretion by macrophages, its production might also contribute to invasion and metastasis of cancer cells.