Rebecca Hands

Colorectal cancers with microsatellite instability display mRNA expression signatures characteristic of increased immunogenicity

BackgroundColorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared to microsatellite stable (MSS) cancers. The observation of pronounced lymphocytic infiltrates suggests that MSI-H cancers are inherently more immunogenic. We aimed to compare the gene expression profiles of MSI-H and MSS cancers to provide evidence for an activated immune response in the former.ResultsWe analysed tissue from 133 colorectal cancer patients with full consent and Local Ethics Committee approval. Genomic DNA was analysed for microsatellite instability in BAT-26. High-quality RNA was used for microarray analysis on the Affymetrix® HG-U133A chip. Data was analysed on GeneSpring software version 6.0. Confirmatory real-time RT-PCR was performed on 28 MSI-H and 26 MSS cancers. A comparison of 29 MSI-H and 104 MSS cancers identified 2070 genes that were differentially expressed between the two groups [P < 0.005]. Significantly, many key immunomodulatory genes were up-regulated in MSI-H cancers. These included antigen chaperone molecules (HSP-70, HSP-110, Calreticulin, gp96), pro-inflammatory cytokines (Interleukin (IL)-18, IL-15, IL-8, IL-24, IL-7) and cytotoxic mediators (Granulysin, Granzyme A). Quantitative RT-PCR confirmed up-regulation of HSP-70 [P = 0.016], HSP-110 [P = 0.002], IL-18 [P = 0.004], IL-8 [0.002] and Granulysin [P < 0.0001].ConclusionsThe upregulation of a large number of genes implicated in immune response supports the theory that MSI-H cancers are immunogenic. The novel observation of Heat Shock Protein up-regulation in MSI-H cancer is highly significant in light of the recognised roles of these proteins in innate and antigen-specific immunogenicity. Increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators also indicate an activated anti-tumour immune response.

Quantification of cytokeratin 20, carcinoembryonic antigen and guanylyl cyclase C mRNA levels in lymph nodes may not predict treatment failure in colorectal cancer patients

Conventional histopathologic staging of primary colorectal cancers does not allow accurate prognostic stratification within a given tumour stage. Therefore, PCR‐based assays are increasingly used to try to predict more accurately the likelihood of disease progression for the individual patient. Real‐time reverse transcription PCR (RT‐PCR) assays were used to detect and quantitate cytokeratin 20 (ck20), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC) mRNA in 149 lymph nodes (LN) from 17 patients with benign disease and 302 LN from 42 patients with colorectal cancer who had curative (R0) resections. None of the markers were specific, with ck20, CEA and GCC mRNA detected in 47%, 89% and 13% of 149 LN, respectively, from patients with benign disease. The sensitivity of all 3 markers was very high, with mRNA detected in 93%, 100% and 97% of 30 histologically involved LN, respectively. There was significant overlap in the mRNA levels of all 3 markers between histologically involved and uninvolved LN. There was no association between mRNA levels and distant recurrence (median follow‐up: 3.94 years, range 3.35–5.12). We conclude that the use of molecular techniques to detect occult disease in LN may suffer from the same limitations as conventional methods. Instead, accurate prognostic stratification requires careful assessment of the likely metastatic potential of the primary cancer.

Angiogenic gene expression and vascular density are reflected in ultrasonographic features of synovitis in early rheumatoid arthritis: an observational study

IntroductionNeovascularization contributes to the development of sustained synovial inflammation in the early stages of Rheumatoid Arthritis. Ultrasound (US) provides an indirect method of assessing synovial blood flow and has been shown to correlate with clinical disease activity in patients with Rheumatoid Arthritis. This study examines the relationship of US determined synovitis with synovial vascularity, angiogenic / lymphangiogenic factors and cellular mediators of inflammation in a cohort of patients with early Rheumatoid Arthritis (RA) patients prior to therapeutic intervention with disease modifying therapy or corticosteroids.MethodsAn ultrasound guided synovial biopsy of the supra-patella pouch was performed in 12 patients with early RA prior to treatment. Clinical, US and biochemical assessments were undertaken prior to the procedure. Ultrasound images and histological samples were obtained from the supra-patella pouch. Histological samples were stained for Factor VIII and a-SMA (a-smooth muscle actin). Using digital imaging analysis a vascular area score was recorded. QT-PCR (quantitative-PCR) of samples provided quantification of angiogenic and lymphangiogenic gene expression and immunohistochemistry stained tissue was scored for macrophage, T cell and B cell infiltration using an existing semi-quantitative score.ResultsPower Doppler showed a good correlation with histological vascular area (Spearman r - 0.73) and angiogenic factors such as vascular endothelial growth factor- A (VEGF-A), Angiopoietin 2 and Tie-2. In addition, lymphangiogenic factors such as VEGF-C and VEGF-R3 correlated well with US assessment of synovitis. A significant correlation was also found between power Doppler and synovial thickness, pro-inflammatory cytokines and sub-lining macrophage infiltrate. Within the supra-patella pouch there was no significant difference in US findings, gene expression or inflammatory cell infiltrate between any regions of synovium biopsied.ConclusionUltrasound assessment of synovial tissue faithfully reflects synovial vascularity. Both grey scale and power Doppler synovitis in early RA patients correlate with a pro-angiogenic and lymphangiogenic gene expression profile. In early RA both grey scale and power Doppler synovitis are associated with a pro-inflammatory cellular and cytokine profile providing considerable validity in its use as an objective assessment of synovial inflammation in clinical practice.

Differential Expression Patterns of the Insulin-Like Growth Factor 2 Gene in Human Colorectal Cancer

Tumour development and metastasis are associated with altered gene expression profiles. The aim of this study was to identify the transcriptional differences in normal, tumour and metastatic tissue. We used oligonucleotide arrays to identify differential expression patterns of insulin-like growth factor 2 (IGF 2) between 139 primary colorectal tumour specimens and 42 tumour-adjacent mucosa specimens from colorectal cancer (CRC) patients. The expression levels of the IGF 2 gene were significantly increased in primary tumours compared with adjacent mucosae. This was concordant with our real-time RT-PCR quantification of 48 matched tumour mucosa samples. IGF 2 expression levels were also measured by RT-PCR quantitative analysis in 18 liver metastases and 10 normal tissues from patients without cancer. The mRNA levels were significantly under-expressed in liver metastases compared with either colorectal tumours or adjacent normal mucosae. The non- malignant normal tissue expressed significantly lower IGF 2 levels than adjacent normal tissue, and this was not due to a field effect originating from the tumour. In addition, our microarray data demonstrated that IGF 2 expression was down-regulated in sporadic microsatellite instability (MSI-H) CRC and parallels under-expression of hMLH1 and IGF 2 receptor genes in these patients. We conclude that IGF 2 plays an important role in CRC development. Also, individuals with loss of genomic imprinting (LOI) causing over-expression of IGF 2 may be at greater risk of developing CRC. However, this LOI may be reversed in MSI-H patients.

Use of ultrasound-guided small joint biopsy to evaluate the histopathologic response to rheumatoid arthritis therapy: recommendations for application to clinical trials.

To examine in a cohort of rheumatoid arthritis (RA) patients undergoing serial ultrasound (US)–guided biopsies of small joints in the context of clinical trials whether sufficient synovial tissue could be obtained at both baseline and second biopsy to: 1) accurately evaluate the synovial immune phenotype, 2) permit adequate RNA extraction to determine molecular signatures, and 3) sensitively detect change in the number of synovial sublining macrophages (CD68+) following effective therapy.

THU0087 Synovial Ectopic Lymphoid-like Structures are Associated with Diagnosis of Rheumatoid Arthritis, Disease Activity and Antibody Status in Early Arthritis Patients

Background The presence of ectopic lymphoid-like structures (ELS) within the synovial tissue of a cohort of patients with inflammatory arthritis is well recognised. There is also data supporting the concept that these structures are immunologically competent and can support chronic inflammation within the joint. There is limited data however examining whether the presence of ELS associates with specific clinical phenotypes in early arthritis. Objectives The aim of this study was therefore to investigate whether synovial ELS associated with specific clinical phenotypes in an early arthritis cohort. Methods A cohort of DMARD-naïve early arthritis (<12 months duration, at least 1 swollen joint) patients recruited at Barts and The London Hospital, as part of the MRC-funded Pathobiology of Early Arthritis Cohort (PEAC) http://www.peac-mrc.mds.qmul.ac.uk/ were included in the analysis. The study was approved by the ethics committee (REC). Baseline disease characteristics assessed included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, rheumatoid factor (RF), anti-cyclic-citrullinated peptide antibodies (anti-CCP) and 28 joint-disease activity score (DAS28). All patients underwent ultrasound-guided synovial biopsy of an affected joint at baseline. Sections of paraffin embedded RA synovial tissue were stained with standard Haematoxylin and Eosin (H&E) and graded as either a diffuse or aggregate synovitis. In addition sequentially cut paraffin sections underwent immunohistochemical staining for B-cells(CD20), T cells (CD3) and macrophages(CD68) and were semi-quantitatively scored (0-4) for each marker. Results 84 sequentially recruited patients with both synovial tissue and a complete clinical data set were included within the analysis (61% female, mean age 50). 64% (n=57) of patients were classified as diffuse and 36% (n=27) as aggregate. The presence of synovial lymphocytic aggregates was significantly associated with higher synovial infiltration of T cells, B cells, plasma cells and both lining and sublining macrophages (p<0.001). Moreover, the presence of synovial aggregates was significantly associated with baseline RA diagnosis (1987 ACR criteria), positive ESR/CRP and seropositivity for RF and anti-CCP antibodies. Interestingly, there was also a significant correlation between a higher synovial infiltration of B cells and plasma cells and positivity for anti-CCP antibodies (p=0.002 and p<0.001) and RF (p<0.001 and p<0.001) in the serum. Conclusions The significant association of synovial ELS with the diagnosis of RA and sero-positivity for RF and anti-CCP antibodies in early arthritis stronlgly supports the concept that these structures are functional and involved in disease pathogenesis. In addition these observations also suggest that baseline synovial pathotype may have a role as a prognostic biomarker in early arthritis. Disclosure of Interest None Declared

A Multicenter Retrospective Analysis Evaluating Performance of Synovial Biopsy Techniques in Patients With Inflammatory Arthritis: Arthroscopic Versus Ultrasound-Guided Versus Blind Needle Biopsy

To evaluate whether the choice of synovial biopsy technique (arthroscopy, blind needle [BN] biopsy, ultrasound [US]–guided portal and forceps [P&F], or US‐guided needle biopsy [NB]) translates to significant variation in synovial tissue quality and quantity, with the aim of informing recommendations for the choice of synovial sampling technique within clinical trials.

OP0040 Synovial cell infiltration in acpa-ve patients displays similar signatures to other seronegative inflammatory arthritis. results from the pathobiology of early arthritis cohort (PEAC)

Background There is increasing evidence to suggest that ACPA +ve and ACPA-ve RA are distinct diseases. Current data demonstrates overlap in classification criteria between ACPA-ve RA and other sero negative inflammatory arthritidies such as PsA. Associated with this is a variable prognosis and response to treatment for patients with ACPA-ve RA. Biomarkers capable of refining diagnosis and improving on current classification criteria early in the disease course for patients with ACPA-ve RA are thus urgently needed. Data examining the synovial pathophysiological relationship between PsA and ACPA ±RA is currently limited although has the potential to identify disease specific synovial cellular and molecular signatures. Objectives Therefore, the aim of this study is to examine in a cohort of therapy naïve, early inflammatory arthritis patients, whether ACPA-ve RA can be defined at disease initiation according to synovial pathobiological signatures. Methods A total of 186 consecutive DMARD naïve inflammatory arthritis patients (disease duration <1 year) recruited as part of the multicentre PEAC study at Barts Health NHS Trust were evaluated. All patients underwent a baseline synovial biopsy of a clinically active joint along with collection of inflammatory markers (CRP). Following H and E staining, sections underwent immumohistochemical staining and semi-quantitative scoring (0–4) to determine the degree of CD20 +Bcells, CD3 +T cells, CD68 +lining (l) and sublining (sl) macrophage and CD138 +plasma cell infiltration. Sections were categorised into three pathotypes: (i) Fibroid(F):(CD68 SL <2 and or CD3, CD20, CD138 <1), (ii) Myeloid(M):(CD68SL >2, CD20 <1 and or CD3 >1) and (iii) Lymphoid(L):(grade 2–3 CD20 +aggregates, CD20 >2). Results 90/186 patients were classified as ACPA+ve RA, 55/186 as ACPA-ve RA and 41/186 as PsA. 80% of synovial samples were collected from small joints (wrist, MCP, PIP). All 186 samples were suitable for analysis. Results confirmed that C-reactive protein (CRP) as inflammatory marker does not differentiate between subgroups (p 0.41). Significantly higher degree of immune cell infiltration was seen between ACPA+ve vs ACPA-ve and ACPA+ve vs PsA but not between ACPA-ve and PsA (figure 1). When grouping patient between clinical subgroups (ACPA+ve vs ACPA-ve vs PsA) and pathotypes (fibroid, myeloid and lymphoid) (table1) we demonstrated a significantly higher prevalence of a lymphoid pathotype in ACPA+ve RA vs ACPA-ve or PsA. RA acpa +N909ungraded RA acpa-N5512ungraded PsAN410ungraded P value fisher test P value acpa+vs acpa- P value acpa+vs PsA P value acpa- vs PsA F 15 (16%) 17 (31%) 15 (36%) 0.01* 0.03* 0.005* 0.41 M 25 (28%) 14 (25%) 11 (27%) L 41 (45%) 12 (22%) 10 (24%) Conclusions Our results suggest that the synovial cell infiltrate (B cells, T cells, macrophages and plasma cells) in ACPA-ve RA is significantly different from ACPA +ve patients. They also suggest shared pathophysiological mechanisms between PsA and ACPA-ve RA and support a role for future refinement of diagnosis of ACPA-ve RA according to synovial pathobiology. Disclosure of Interest None declared

THU0667 A qualitative and quantitative comparison of synovial biopsy techniques during clinical trials of inflammatory arthritis

Background Synovial tissue is an attractive area of research for biomarkers of disease outcome in RA. Currently acquisition of synovial tissue using an arthroscopic approach in clinical trials is recommended though two US-guided techniques have been described, a portal and forceps (P&F) approach and an adaptation using a quick core needle (NB). However before US-guided biopsy techniques are widely adopted into clinical trials validation of performance against arthroscopy is required. Objectives To evaluate whether there were significant differences in synovial sampling quality and quantity between arthroscopic, US-P&F and US-NB procedures within the context of clinical trials. Methods This was a multicentre retrospective analysis of inflammatory arthritis patients recruited to clinical trials utilizing US-guided NB (Barts Health NHS Trust), US-guided P&F (ICRSS Policlinico San Matteo and University Hospital Birmingham) and arthroscopic biopsy (Repatriation General Hospital). Paraffin embedded synovial sections from each procedure underwent H&E staining and sections examined for intact cell lining layer (graded sections). Biopsy procedures were segregated into large (knee) and small joint procedures (wrist/MCP) for analysis. Proportion of samples yielding graded tissue per procedure was recorded. Using CellSens Dimensions software the mean area of synovial tissue obtained per procedure was determined. In addition the degree of synovitis was assessed using semi-quantitiative scoring (0–9). Results 78 patient procedures were evaluated, 22 on small joints (11 US-NB, 11 US P&F) and 56 on large joints (11 US-NB, 35 US-P&F, 10 arthroscopic). 47 patients had RA, 11 undifferentiated arthritis and 10 psoriatic arthritis. Arthroscopic sampling resulted in a significantly higher area of tissue retrieved per procedure than US P&F or US-NB. There were no significant differences in proportion of graded samples per procedure suggesting quality of synovial tissue was preserved between techniques. Finally no significant differences in degree of histological synovitis were demonstrated between sampling techniques.Table 1 Mean (SD) Small joint biopsies Large joint biopsies US –NB (n=11) US P&F (n=11) P value US-NB (n=11) US P&F (n=35) Arthroscopic (n=10) P value Joint biopsied 7 wrist 1 wrist 10 knee 32 knee 10 knee 5 MCP 10 MCP 3 ankle % graded samples per procedure 82.3 (22.4) 91 (10) 0.34 89.8 (17.5) 83.23 (19) 97 (11) 0.08 Mean area of tissue (mm2) per procedure 3.19 (1.75) 7.95 (4.91) <0.005* 4.66 (3.19) 8.8 (8.17) 17.64 (7.56) <0.0001* Synovitis score 4.9 (3.2) NA NA 3.8 (2.6) 4.53 (2.26) 4.5 (1.71) 0.35 Conclusions The results suggest that US-guided biopsy provides a reliable method for sampling synovial tissue of comparable quality to that obtained from arthroscopy. However when sampling large joints arthroscopic techniques, and when sampling small joints US-P&F yield a significantly higher quantity, though not quality, of tissue per procedure than US-NB. These results may influence choice of biopsy technique when designing clinical trial protocols in inflammatory arthritis. Disclosure of Interest None declared

THU0100 In early inflammatory arthritis a lymphoid pathotype signficantly associates with requirement for biologic therapy at 12 months follow up: results from the pathobiology of early arthritis cohort (PEAC)

Background Early aggressive treatment in RA equates to better long term outcomes, however targeting aggressive therapies including biologics to patients with the worse prognosis is critical to deliver acceptable risk/benefit ratios and health economic improvements. Such an approach requires prognostic biomarkers, whether the well recognised heterogeneity in synovial pathobiology in early RA translates to specific disease outcomes is currently unknown. Objectives The aim of this study was to investigate whether in a treatment naïve early inflammatory arthritis cohort, baseline synovial pathotype significantly associates with disease outcome at 12 months. Methods 166 consecutive DMARD naïve patients recruited as part of PEAC at Barts Health NHS Trust with synovial tissue suitable for analysis were included. At baseline patients were classified as RA (2010 ACR/EULAR criteria) or undifferentiated (UA). All patients underwent a baseline synovial biopsy of a clinically active joint along with collection of demographic data. Patients were subsequently treated with DMARD +/- steroid therapy with aim for low disease activity (DAS <3.3). At 6 month follow up patients were escalated to biologic therapy if fulfilling UK NICE guidelines. At 12 months patients were classified as: (i) no treatment, (ii) DMARDs, and (iii) Biologic +/- DMARDs. Sequentially cut sections of baseline synovial biopsies underwent immunohistochemical staining and semi-quantitative scoring (0–4) to determine the degree of CD20+Bcells, CD3+T cells, CD68+ lining (l) and sublining (sl) macrophage and CD138+ plasma cell infiltration. Sections were categorised into 3 pathotypes, (i) Fibroid: (CD68 SL<2 and or CD3, CD20, CD138<1), (ii) Myeloid: (CD68SL>2, CD20<1 and or CD3>1) and (iii) Lymphoid: (grade 2–3 CD20+ aggregates, CD20>2). Results 79% were classified as RA and 21% as UA. Mean disease duration was 9.27 months. 92% (153/166) patients had follow-up at 12months. 29% (44/153) of patients were classified as fibroid, 34% (52/166) as myeloid and 37% (57/166) as lymphoid. At baseline patients with a lymphoid pathotype had a significantly higher CRP and DAS28 and were significantly more likely to be sero positive for for RF and ACPA (p<0.05), suggesting that a lymphoid pathotype is associated with higher levels of disease activity. At 12 months follow up a significantly higher proportion of patients classified as lymphoid vs myeloid or fibroid (58% vs 21% vs 21%) required biologic therapy. N=153 Fibroid (44) Myeloid (52) Lymphoid (57) p-value No treatment (14) n (%) 6 (42%) 6 (42%) 2 (14%) <0.05* DMARD (101) n (%) 30 (29%) 38 (37%) 33 (32%) Biologic +/− DMARD (38) n (%) 8 (21%) 8 (21%) 22 (58%) Conclusions Results demonstrate that in an early inflammatory arthritis cohort a lymphoid pathotype significantly associates with higher disease activity at baseline, sero positivity for RF and ACPA and a requirement for more aggressive therapy at 12 month. This supports a direct role for synovial lymphoid structures in disease pathogenesis and suggests a role as a prognostic biomarker facilitating early stratification of aggressive therapeutic intervention. Disclosure of Interest None declared