Functional impact of rs2981578 allelic modification in a panel of heterozygous and control MCF7 clones.

<p>A) Morphological appearance of a control versus a heterozygous clone (Scale bar: 50 µm) and expression of ERα in control and heterozygous clones. HSC70 was used as loading contol. Western blot is representative of three independent experiments. B) Quantitative RT-PCR of ERα expression level upon exposure to 1 µM Tamoxifen relative to control (vehicle, EtOH) for 48 h. C) Quantitative RT-PCR of PS2 and cMyb expression level, two target genes of ERα, upon exposure to 1 µM Tamoxifen relative to control (vehicle, EtOH) for 48 h. mRNA levels are shown relative to GAPDH expression, and normalized over untreated cells. Mean ± SEM of three independent experiments are presented. Two-way ANOVA showed no significant difference in expression levels between the control and heterozygous clones (ERα: p = 0.6491; PS2: p = 0.1098; cMyb: p = 0.2304). D) Expression of FOXA1 in the controls and heterozygous clones. HSC70 was used as loading control (western blot is representative of three independent experiments). FOXA1 ChIP in one control and one heterozygous clone following exposure to oestrogen (E2) (full medium, E2 starvation or E2 stimulation). Primers recognizing the rs2981578 locus and a positive control (<i>Greb1</i> promoter) were used. ChIP was performed in triplicate and the amount of precipitated DNA were normalized to the input DNA and a negative control (<i>CCDN1</i> intron). Student’s two-tailed t-Test was used to analyse significance.</p

Assessment of ASE in breast cancer.

<p>A) Cartoon representing ASE where a cis-regulatory difference exists between G (blue) and A (grey) alleles. The activity of allele G is higher because of the differential binding of a transcription factor (yellow), which results in a relative abundance of blue mRNA transcript. A marker SNP, located in the coding region of the gene and with a heterozygous genotype, is used to differentiate the origin of each mRNA transcript synthesized (C and U). B) Allele frequencies for rs2981578 and two marker SNPs measured in a panel of 72 ERα positive breast cancer samples. C) Absolute ΔCt was measured in five samples that were heterozygous both for rs2981578 and rs1047100 (marker SNP) and compared to 11 controls in which rs2981578 was homozygous. Mann Whitney test revealed no statistical differences between the two groups.</p

FGFR2 expression and signaling.

<p>A) Quantitative RT-PCR of FGFR2 isoforms (FGFR2a, FGFR2b and FGFR2c) in control and heterozygous clones. Error bars represent SEM of three independent experiments. Two-way ANOVA showed no statistical significance in expression of the receptors in control versus heterozygous clones. B) Representative western blots of ERK phosphorylation following stimulation of control and heterozygous clones with 100 ng/ml FGF7 and FGF10. HSC70 is used as loading control. C) Representative western blots of ERK phosphorylation following stimulation of the MCF7 clones with different amount of FGF7 and FGF10. HSC70 is used as loading control. The stimulations were performed in triplicate.</p

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution

<div><p>Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10⁻². By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.</p></div

Flow-sorting of the 4 different B cell sub-populations.

<p>Lymph nodes cell suspensions from sequential biopsies obtained from patients with FL/t-FL were stained and sorted in 4 different populations according to the expression of IgD, CD38, CD10 and CXCR4. ME: IgD-CD38-; PGC: IgD+CD38+, CB: IgD-CD38+CD10+CXCR4+; CC: IgD-CD38+CD10+CXCR4-. Flow-cytometric identification of the 4 sub-populations in the sample R8403, patient 3 (top), sample R0012, patient 1 (middle) and tonsils (bottom). In the 2 patients samples the numbers indicate the percentage of the gated and sorted subsets whilst in the tonsil example the numbers are representative of 8 independent experiments on as many different biopsies.</p

Characteristics of the patients and samples included in the study.

<p>(A) Three patients were included in the analysis: pt1 and pt2 displaying a pattern of direct evolution and pt3 of evolution through a CPC based on HH analysis. Biopsies were collected at different time points: at relapse/transformation (pt1 and pt2) and after a watch wait period (pt3). In pt 1 two additional biopsies (AD1548 and 1787) analyzed only on the SNP array are shown. In pt1 all samples had an identical MC whilst in pt2 the MC sequences from samples R1655 FL1 and R3878 FL2 showed 2 different dominant clones carrying 3 discriminating mutations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134833#pone.0134833.s007" target="_blank">S7 Fig</a>). Because these 3 bases also differ from the germline sequence a pattern of direct evolution could not be excluded. (B) Twenty-two samples, from 4 flow-sorted sub-populations, included in the deep sequencing study: pre-germinal center (PGC) (IgD+CD38+) white circles, the germinal center centroblasts (CB) (IgD+CD38+CD10+CXCR4+) and centrocytes, (CC) (IgD+CD38+CD10+CXCR4-) light grey circles and memory enriched (ME) (IgD-CD38-) dark grey circles. The 2 CC CD77- sorted sub-population from R0012 t-FL and R1381 FL1 biopsies, which were part of the deep sequencing study but not included in the final analysis are not shown. The FL sequences are depicted as white circles, the t-FL clones as grey circles, the germline sequence as black circles and the putative CPC sequence as a dashed grey circle. The 2 samples not included in the deep sequencing of IgH-VH gene are depicted as dotted circles. Each vertical arrow represents a line of therapy; the left hand vertical bar shows the time of diagnosis and the right hand vertical bar the time of death. FL = Follicular Lymphoma; t-FL = transformed Follicular Lymphoma; WW = watch and wait.</p

Clinical and molecular features of the 3 patients investigated by high-throughput sequencing.

<p>Three patients, 2 showing a pattern compatible with a direct evolution and 1 consistent with the existence of a CPC, all having a IgH-VH3 family clonal rearrangement were included in the study.</p><p>Samples AD1548 and 1787 from pt1, shown in italic, were not included in the high-throughput sequencing analysis of IgH-VH but only in the SNP CNV study.</p><p>^±The sample ID corresponds to the cell suspension vial ID or DNA identification number.</p><p>P = progression, R = relapse, SD = stable disease; subsequent number relate to episode e.g. R2 = second relapse</p><p>N.A. = not available SHM = somatic hypermutation</p><p>FL = Follicular Lymphoma; t-FL = transformed Follicular Lymphoma</p><p>MBR stands for Major Breakpoint Region and mcr for minor cluster region</p><p>* Pt2 has 2 different mutations at the same base (a pattern compatible with direct evolution)</p><p>^# Samples R8403FL1 and R9129FL2 had 2 different DH/JH rearrangements but when aligned they showed the a similar CDR3 region, with additional 21 insertions in the sample R8403FL1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134833#pone.0134833.s006" target="_blank">S6 Fig</a>)</p><p>Clinical and molecular features of the 3 patients investigated by high-throughput sequencing.</p

Models of clonal evolution developed by the CNV analysis.

<p>FL and t-FL samples are represented in white and dark grey ovals respectively. The CNVs occurring at each stage are shown below the ovals. (A) In pt1 two additional samples (AD1548 FL and 1787 t-FL) that were not investigated during the 454 deep sequencing experiments were analyzed and are shown as dotted ovals. We observed a model of clonal selection, where more primitive ancestor cells not detected in the first biopsy (depicted in light grey) later become the R0012 t-FL and R2005 FL dominant clones. The time course between clinical episodes (bottom) indicates that these precursor cells can persist for many months before disease presents clinically. The dotted arrows represent the evolution from an ancestor subclone into a more mutated tumor cell, whilst the bold arrows show the emergence of an ancestor cell, by clonal selection, identical to the R0012 t-FL and R2005 FL respectively. (B) In pt3 we observe a model of divergent evolution. The more primitive progenitor cell (light grey oval) has already acquired 2 CNV and subsequently acquires the loss of 6q and the loss of 15q arms captured within the R8403 FL1 and R9129 FL2 tumors respectively.</p

Merged Lineage Trees showing the dynamics of evolution of the sub-clones.

<p>(A) Merged lineage trees generated from the 2 biopsies R1655 FL1 and R3878 FL2 from pt2. G.L. means germline; the dark grey circles represent the CB clones, the medium grey circles the CC, the light grey circles the clones shared (S) among 2 or more populations and the black circles those from the not sorted (NS) whole biopsy. (B) Merged lineage trees obtained from the combination of the CB and CC libraries from the samples R0012 t-FL and R2005 FL2 from pt1. The dark grey circles represent the CB subclones, the medium grey circles the CC and the light grey circles the clones shared (S) by CB and CC. The white circles represent subclones not detected with the 454 sequencing. Squares highlight the MC.</p

High throughput sequencing results and analysis of the tumor related clone.

<p>(A) Histogram showing the tumor related reads generated in the 29 different sub-sets that passed the filtering process. In black are shown the not tumor related reads, generated using VH3 family primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134833#pone.0134833.s008" target="_blank">S1 Table</a>), in white the reads identical to the MC and in grey reads that are clonally related to the MC. * indicates the library prepared performing 6 independent PCRs, using the same primer whilst ** prepared by performing 2 independent PCR and using 2 different MID primers (B) Histogram showing the number of MC related subclones generated per library. * symbol indicates the library prepared by performing 6 independent PCRs with the same primer whilst ** symbol refers to the library prepared by performing 2 independent PCR and using 2 different MID primers. (C) Histogram showing the frequency of the clones per library. The black squares represent the number of clones detected at a frequency between 0.1 and 0.01, the dark grey squares the clones detected at a frequency between 0.01 and 0.001 and the light grey squares the number of clones detected at a frequency between 0.001 and 0.0001.</p