Evaluation of high-throughput genomic assays for the Fc gamma receptor locus

Cancer immunotherapy has been revolutionised by the use of monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics

Fcγ receptors: genetic variation, function, and disease

Fcγ receptors (FcγRs) are key immune receptors responsible for the effective control of both humoral and innate immunity and are central to maintaining the balance between generating appropriate responses to infection and preventing autoimmunity. When this balance is lost, pathology results in increased susceptibility to cancer, autoimmunity, and infection. In contrast, optimal FcγR engagement facilitates effective disease resolution and response to monoclonal antibody immunotherapy. The underlying genetics of the FcγR gene family are a central component of this careful balance. Complex in humans and generated through ancestral duplication events, here we review the evolution of the gene family in mammals, the potential importance of copy number, and functionally relevant single nucleotide polymorphisms, as well as discussing current approaches and limitations when exploring genetic variation in this region

Inhibitory FcγRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity

Key PointsFcγRIIb-dependent internalization of therapeutic mAbs is dependent on antibody specificity. FcγRIIb can be activated in both cis and trans configurations.</jats:p

Genomic dissection of the Fcγ receptor region in the context of monoclonal antibody therapy

Development of the anti-CD20 antibody, rituximab, heralded the start of monoclonal antibody (mAb) therapy as an effective means of treating cancer. Despite its undoubted impact, clinical responses remain variable and cures are rarely achieved. Evidence from pre-clinical models and human trials indicates that mAbs primarily act through engaging low-affinity Fc gamma receptor (FcγR)-expressing effector immune cells. The low-affinity FcγR genes, FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B, are located within a highly homologous 200 kb region at 1q23, which is the result of an ancestral segmental duplication event (Figure 1). The locus also contains numerous single nucleotide polymorphisms (SNPs), many of which can affect receptor affinity and/or function and are associated with differential responses following mAb immunotherapy. Moreover, the region contains extensive copy number variation (CNV) that also can affect the expression and function of these receptors, but its impact on mAb immunotherapy remains unknown. To investigate the full impact of SNPs and CNV in the FcγR locus, we have optimised a number of sensitive and specific assays which are amenable to formalin fixed paraffin embedded (FFPE) material in order to apply them to clinical trial samples in a high-throughput manner. Initially we assessed the accuracy of established TaqMan and novel allele-specific (KASP) genotyping assays for FCGR2A-131H/R (rs1801274), FCGR3A-158F/V (rs396991) and FCGR2B-232I/T (rs1050501) SNPs by analysing 2085 DNA samples derived from peripheral blood lymphocytes (PBL) from a large, multi-centre cohort. Our data showed that although clear discrimination was possible at the FCGR2A-131H/R SNP, we needed additional selective Sanger sequencing to discriminate the FF/FV and IT/TT genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs, respectively. This difficulty in genotype discrimination in the cases of FCGR3A and FCGR2B is likely due to sequence homology with other genes and CNV in the gene regions which complicate assay design and interpretation of certain genotypes. Secondly, we applied a combined KASP genotyping and Sanger sequencing approach to matched PBL DNA and FFPE-extracted DNA from follicular lymphoma (FL) patients [n=14] and showed that while FFPE material was more likely to fail genotyping, successfully genotyped cases were concordant with the matched genomic DNA samples. FFPE samples which failed to amplify PCR products of at least 100 bp using the BIOMED-2 multiplex PCR protocol were more likely to fail genotyping assays. Finally, we assessed the ability of a multiplex ligation-dependent probe amplification (MLPA) assay to concurrently determine SNP genotype and CNV in the low-affinity FCGR locus in a cohort of 155 normal donors and DNA from seven matched PBL-/FFPE-derived FL cases. We employed a paralog ratio test (PRT) assay for FCGR3A and FCGR3B CNV confirmation. In our normal donors, MLPA and PRT results were concordant. 16% of normal donors harboured a deletion [n=15] or duplication [n=10] affecting the FCGR2C locus (A summary of regions of CNV is shown in Figure 1). CNV of FCGR3B was associated with variation at FCGR2C and no CNV was observed in FCGR2A and FCGR2B. CNV affecting FCGR3A was observed in 5% of donors with deletions and duplications in 4 and 5 donors, respectively. In the FFPE-derived DNA samples, we observed elevated variability in data quality that was most noticeable in probes targeting HSPA6, FCGR2C exon 4 and HSPA7. Poor quality data correlated with samples that failed to amplify at least the 100 bp PCR product using the BIOMED-2 multiplex PCR protocol. As such, the preclusion of HSPA6, FCGR2C exon 4 and HSPA7 probes and FFPE samples that failed to amplify any BIOMED-2 PCR product from the analysis permitted the production of high-quality MLPA data. Finally, we designed, and are currently optimising, a targeted re-sequencing platform (Haloplex, Agilent) to interrogate informative regions of the FcγR region, which includes those with unique sequence identify for CNV analysis, and those that include known SNPs. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of antibody therapy. This work will ultimately provide a detailed architecture of the region and establish the importance of FcγR genetics in predicting response to antibody therapeutics

Utility of cerebrospinal fluid liquid biopsy in distinguishing CNS lymphoma from cerebrospinal infectious/demyelinating diseases

Abstract Background Distinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique. Methods In this study, we extracted cell‐free DNA from the CSF (CSF‐cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet‐digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease. Results Blood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF‐cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF‐cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18–93 days). Conclusions These results suggest that mutation analysis using CSF‐cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform

Correction: Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus.

[This corrects the article DOI: 10.1371/journal.pone.0142379.