An optimised tissue disaggregation and data processing pipeline for characterising fibroblast phenotypes using single-cell RNA sequencing

Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. We also assess the impact of in vitro culture conditions on stromal cell gene expression and proliferation, showing that altering these conditions can skew phenotypes

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy

Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

Neoadjuvant therapy followed by surgery is the standard of care for locally advanced esophageal adenocarcinoma (EAC). Unfortunately, response to neoadjuvant chemotherapy (NAC) is poor (20-37%), as is the overall survival benefit at five years (9%). The EAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. To identify associations between genomic events and response to NAC in EAC, a comparative genomic analysis was performed in 65 patients with extensive clinical and pathological annotation using whole-genome sequencing (WGS). We defined response using Mandard Tumor Regression Grade (TRG), with responders classified as TRG1-2 (n = 27) and non-responders classified as TRG4-5 (n =38). We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs. 1.70/Mb, p = 0.036) and elevated copy number variation in non-responders (282 vs. 136/patient, p < 0.001). We identified copy number variants unique to each group in our cohort, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of note, NAV3 mutations were exclusively present in the non-responder group with a frequency of 22%. Thus, lower mutation burden, higher chromosomal instability and specific copy number alterations are associated with resistance to NAC

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

Tracking subclonal mutations in IGHV-mutated CLL with progressive disease

Most CLL is diagnosed with a low tumor burden with no indication for therapy. Biomarkers, such as unmutated IGHV genes, TP53 loss/mutation and raised β2M predict short time to first treatment and overall survival; however there remain patients with good risk biomarkers who nevertheless develop progressive disease. Advances in genomics and immunogenetics have lead to the discovery of new biomarkers and their integration with cytogenetic data refines outcome prediction. However these novel markers are predominantly found in IGHV unmutated cases (U-CLL).To identify novel genetic mechanisms that may contribute to progression, we have studied 13 patients (pts) presenting with cMBL (n=3) or Stage Binet A/Rai 0 disease and good risk markers: IGHV-mutated, excluding poor risk stereotypes (n=13), no 17p or 11q deletion by FISH, (n=13), sole del13q14 (n=8), low CD38 expression (n=13) who all developed lymphocytosis (n=13), between two untreated timepoints (TP1 &amp; TP2), 10 of whom subsequently required treatment.Copy number analysis (SNP6), whole exome sequencing (WES; Agilent SureSelect &amp; Illumina sequencing) of tumour-germline pairs and targeted deep sequencing (TDS; Haloplex, Agilent) of the the WES-identified variants and the 22 most frequently mutated genes in CLL, to a mean depth of 3681 fold, were performed at TP1 and TP2. TP1 was close to diagnosis (median of 1 yr, range 0.11-7.33) with a median time to TP2 of 4.5 yrs (0.2-8.9). In addition, TDS was undertaken at later time points in one patient described in point 4), who relapsed and ultimately transformed.Our analysis shows the following potential mechanisms:1. Our germline WES data revealed 5 heterozygous missense/frameshift variants in 5 genes in 5 pts, also known to be targeted by somatic mutation in CLL (eg: FBXW7, POT1, SAMHD1. Fig1).2. We then established the somatically-acquired mutation profile of each patient. We validated 72% (224/312) of the mutations discovered by WES using TDS and identified clinically relevant mutations earlier on in disease, supporting the hypothesis that sub-clonal mutations in genes in addition to TP53 may drive a progressive clinical course. At diagnosis (TP1) by WES/TDS, 5/13 pts had mutations in CLL driver genes (ATM, NOTCH1, SF3B1, TP53) and 2/13 pts had mutations in genes of undetermined clinical significance (CHD2, NFKBIE, ZMYM3). One patient was MYD88 mutated at TP1 and remains untreated after follow up of 12 yrs. In total, the following 9 genes (ATM, CHD2, DDX3X, MYD88, NOTCH1, NFKBIE, SF3B1, TP53 &amp; ZMYM3) were mutated in 62% (8/13) pts at TP1.3. Of the remaining 5/13 pts lacking a detectable mutation in any of the established CLL genes, we observed on average 7 mutations/patient in genes involved in cancer and each patient harboured one or more mutated genes with a role in haematological malignancy (eg. ITGA6, KLHL6, LTF, TNFAIP3).4. One patient exhibited a remarkable temporal shift in copy number changes and mutations. At TP2, SNP6 analysis could not detect the del13q observed at TP1, and a clonal trisomy 12 had emerged, along with several mutations associated with progressive disease (BIRC3, IRF4, NOTCH1), that predominate in U-CLL. As a consequence we re-analysed the IGHV mutational status at TP2, and showed that rather than the IGHV3-48 with 92% germline identity identified at diagnosis, our patient exhibited an additional and dominant IGHV5-10-1*01 (100% identity) clone at TP2, 8 yrs after TP1. Additional analysis of intermediate samples detected the unmutated clone as far back as 4 yrs post diagnosis, and TDS analysis showed the NOTCH1 mutation was a minor subclone at diagnosis (0.06% VAF). Ultimately, this patient developed Richters syndrome with expansion of the NOTCH1 mutation (27% VAF). Retrospective sequential immunogenetic analysis of the other 12 cases yielded no other example of this phenomenon.In summary, IGHV-mutated cMBL/early stage CLL with a progressive outcome can be associated with, the presence of germline or subclonal gene mutations of known or putative importance in CLL, or the emergence of a IGHV-unmutated clone. Our data supports deep sequencing in the clinical setting for earlier detection of pathogenetic mutations and emerging immunogenetically distinct subclones in patients with early stage 'good risk’ disease