Change in CD3 positive T-cell expression in psoriatic arthritis synovium correlates with change in DAS28 and magnetic resonance imaging synovitis scores following initiation of biologic therapy-a single centre, open-label study

Eliza K Pontifex
Danielle M Gerlag
Martina Gogarty
Marjolein Vinkenoog
Adrian Gibbs
Ilse Burgman
Ursula Fearon
Barry Bresnihan
Paul Peter Tak
Robin G Gibney
Douglas J Veale
Oliver Fitzgerald
Change in CD3 positive T-cell expression in psoriatic arthritis synovium correlates with change in DAS28 and magnetic resonance imaging synovitis scores following initiation of biologic therapy-a single centre, open-label study
R E S E A R C H A R T I C L E Open Access
Introduction: With the development of increasing numbers of potential therapeutic agents in inflammatory disease comes the need for effective biomarkers to help screen for drug efficacy and optimal dosing regimens early in the clinical trial process. This need has been recognized by the Outcome Measures in Rheumatology Clinical Trials (OMERACT) group, which has established guidelines for biomarker validation. To seek a candidate synovial biomarker of treatment response in psoriatic arthritis (PsA), we determined whether changes in immunohistochemical markers of synovial inflammation correlate with changes in disease activity scores assessing 28 joints (ΔDAS28) or magnetic resonance imaging synovitis scores (ΔMRI) in patients with PsA treated with a biologic agent. Methods: Twenty-five consecutive patients with PsA underwent arthroscopic synovial biopsies and MRI scans of an inflamed knee joint at baseline and 12 weeks after starting treatment with either anakinra (first 10 patients) or etanercept (subsequent 15 patients) in two sequential studies of identical design. DAS28 scores were measured at both time points. Immunohistochemical staining for CD3, CD68 and Factor VIII (FVIII) was performed on synovial samples and scored by digital image analysis (DIA). MRI scans performed at baseline and at 12 weeks were scored for synovitis semi-quantitatively. The ΔDAS28 of the European League Against Rheumatism good response definition (>1.2) was chosen to divide patients into responder and non-responder groups. Differences between groups (Mann Whitney U test) and correlations between ΔDAS28 with change in immunohistochemical and MRI synovitis scores (Spearman's rho test) were calculated. Results: Paired synovial samples and MRI scans were available for 21 patients (8 anakinra, 13 etanercept) and 23 patients (8 anakinra, 15 etanercept) respectively. Change in CD3 (ΔCD3) and CD68 expression in the synovial sublining layer (ΔCD68sl) was significantly greater in the disease responders compared to non-responders following treatment (P = 0.005 and 0.013 respectively). ΔCD3, but not ΔCD68 or ΔFVIII, correlated with both ΔDAS28 (r = 0.49, P = 0.025) and ΔMRI (r = 0.58, P = 0.009). Conclusions: The correlation of ΔCD3 with ΔDAS28 and ΔMRI following biologic treatment in this cohort contributes to the validation of ΔCD3 as a synovial biomarker of disease response in PsA, and supports the further evaluation of ΔCD3 for predictive properties of future clinical outcomes.

Introduction

Psoriatic arthritis is a chronic and debilitating inflammatory arthropathy. It accounts for 15% of referrals to early arthritis clinics, and has considerable morbidity [1]. The Outcome Measures in Rheumatology Clinical Trials (OMERACT) PsA working group has identified a hierarchy of domains to be included in PsA clinical trials [2], which includes tissue analysis and magnetic resonance imaging (MRI) in the outer domain, on the research agenda. Utilizing these two domains, we have sought a potential synovial biomarker of treatment response in PsA. A biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of a normal biologic process, a pathophysiologic process, or a pharmacological response to therapeutic intervention [3].

It has already been established in rheumatoid arthritis (RA) that the mean change in DAS28 correlates with the mean change in synovial sublining CD68 expression across several RA patient cohorts receiving different therapeutic agents [4][5][6][7]. Few studies have correlated clinical composite scores with changes in PsA synovial cell populations however. One of the reasons for this is that no composite score has yet been validated in PsA, although such work is currently in progress [8]. DAS28, a score validated in RA [9], has proven to be a highly effective tool in previous studies of PsA and biologic agents [10][11][12] and is suitable for studies involving synovial tissue analysis as it focuses on articular involvement. In the synovial tissue of our patient cohort, we measured the expression of CD68, a macrophage marker, given the clinical correlations found in RA; FVIII, an endothelial cell marker, due to the hypervascularity and vessel tortuosity evident in inflammed PsA synovium compared to that of RA [13][14][15][16]; and CD3, a T-cell marker. Importantly, a previously published study which utilized DAS28 found a significant correlation between ΔDAS28 and ΔCD3 in the synovium of patients with PsA after adalimumab treatment [12]. Should this finding prove reproducible, particularly if different therapeutic agents are used, ΔCD3 may meet the discrimination criterion of the OMERACT biomarker validation filter [17] and the exploration of ΔCD3 as a predictive biomarker of future treatment response in PsA would be supported. ΔCD3 could be used to determine the potential efficacy of new therapeutic agents in PsA at an early stage, as is already happening in RA clinical trials of novel therapeutic compounds, where synovial sublining ΔCD68 measurements are being observed to reflect clinical response [18,19].

While MRI has been used to highlight the importance of bone marrow oedema and entheseal sites of inflammation in PsA [20,21], to date there have been no studies comparing histological change with quantified synovitis by dynamic or static MRI. In this study we examine the relationship between clinical scores and both immunohistochemical (IHC) and MRI measures of synovitis following biologic treatment in PsA to help identify a potential biomarker of treatment response.

Materials and methods

Study protocol

Twenty-five patients who met the CASPAR classification criteria for PsA [22] were enrolled in two sequential studies of identical design. The first 10 consecutive patients received anakinra, an IL-1 receptor antagonist, 100 mg by subcutaneous injection (SC) daily, followed by 15 consecutive patients who received etanercept, a TNF receptor antagonist, 25 mg twice weekly SC. Both were 12-week, single centre, open-label studies undertaken at St. Vincents University Hospital, Dublin. Ethical approval was obtained by the hospital's ethics committee and written informed consent was provided by all patients. At the time of enrolment, each patient had to have a diagnosis of PsA for at least three months, and at least three tender and three swollen joints (one of which was a knee), of a 68-joint assessment,.

Clinical parameters were measured at weeks 0 and 12, including 28 and 68 tender (TJC) and 28 and 66 swollen joint counts (SJC), patient pain and disease 0 to 100 mm visual analogue scale (VAS) and the Health Assessment Questionnaire (HAQ). Serum erythrocyte sedimentation rate (ESR) (Test-1, Ali Sax) and C-reactive protein (CRP) levels (nephelometry) were also measured. A DAS28 score was calculated at each time point. To look for changes in cell marker expression and MRI synovitis scores reflecting change in clinical activity, the change in DAS28 of the EULAR definition of good response (>1.2) [23] was chosen to divide the cohort into two groups, labelled here as responders (ΔDAS28 >1.2) and non-responders (ΔDAS28 ≤1.2).

To compare the single joint MRI synovitis scores with a single joint clinical measure, a more detailed clinical assessment was performed of each patient's involved knee. It was scored in the manner of the first published study of PsA and a biologic agent in which pain and swelling were evaluated separately on a scale of 0 to 3, where 0 represents the absence of pain or swelling [24]. The sum of these is the final score for a given joint, which will range from 0 to 6. A patient was defined as a knee responder in this study if there was a reduction in their involved knee score following treatment at Week 12.

Patients were excluded if they had received a biologic agent within 12 weeks, cyclosporin or leflunomide within 8 weeks, or methotrexate or sulfasalazine within 4 weeks of enrolment into the study. Patients taking ≥10 mg of prednisolone or those who had a prednisolone dose change within four weeks of study Day 1 were also excluded, as were those who were pregnant, breastfeeding, had significant liver, renal or haematological abnormalities, or a history of cancer within five years of the study's commencement. Prior to receiving etanercept, patients were screened for latent tuberculosis with a chest X-ray and Mantoux test.

Arthroscopy

Arthroscopy and synovial biopsy of the involved knee joint was performed at two time points (weeks 0 and 12), with a Storz arthroscope and 1.5 mm grasping forceps. Biopsy samples were obtained from all knee joint compartments, embedded in TissueTek OCT compound (Sakura, Alphen aan den Rijn, Netherlands) and stored in liquid nitrogen. In the majority of cases, six individual biopsies were included together in one OCT mould for cutting and analysis. Seven μm thick sections were cut using a cryostat, placed on glass slides coated with 2% 3-amino-propyl-triethoxy-silane (Sigma-Aldrich Ireland Ltd, Dublin, Ireland) and dried overnight at room temperature. Sections were stored at -80°C until required for staining.

Immunohistochemistry

A routine three-stage immunoperoxidase labeling technique was used. Tissue sections were allowed to reach room temperature, were fixed in acetone for 10 minutes and then air-dried. The remaining steps were performed in an autostainer using reagents from an Envision+ system-HRP (AEC) kit (Dako, Glostrup, Denmark). Following quenching of endogenous peroxidase activity, the synovial sections were incubated with mouse monoclonal antibodies against cell specific markers CD3, CD68, and FVIII (Dako, Glostrup, Denmark) for 30 minutes. Incubation with a labelled polymer-HRP anti-mouse antibody followed, and colour was then developed with amino-ethylcarbazole (AEC). Slides were counterstained with Mayer's haematoxylin (BDH Laboratories, Poole, UK) and mounted.

Digital image analysis

All slides were randomly assigned code numbers for analysis and only tissue samples with a clearly identifiable intimal lining layer were included. All analysis was undertaken by EKP. Eighteen high power images were taken per slide for each of the three cell specific markers stained. In the case of CD68, the intimal lining layer was highlighted manually per image, such that staining could be quantified in two areas -the intimal lining (ll) and synovial sublining (sl) layers. Analysis was performed using the Qwin analysis system (Leica, Cambridge, UK) as has been previously described [25,26]. Results are expressed as the number of positively stained cells/mm 2 of tissue for CD3 and CD68, and by integrated optical density (IOD)/mm 2 for FVIII. The average value over all six biopsies per patient per time point was used for analysis.

MRI

An MRI scan of the same involved knee was performed for each patient the day prior to arthroscopy at weeks 0 and 12, using a 1.5T Signa Excite HD MRI scanner (General Electric Healthcare, Chalfont St Giles, Buckinghamshire, UK) and a dedicated eight channel array HD knee surface coil with patients lying supine. The examinations performed included intravenous contrast enhanced (Gadoteric acid, Dotarem, 0.5 mmol/mL, Guerbet Laboratories, Birmingham, UK); 10 mls in all examinations by slow hand injection) T1-weighted fat suppressed pulse sequences in coronal, sagittal and axial planes. Scanning parameters were as follows: Once complete, the scans were arranged into pairs of pre-and post-treatment images for each patient. These were scored semi-quantitatively by one consultant radiologist with a special interest in musculoskeletal radiology who was blind to patient identity and scan chronology. Each knee was divided into four anatomical regions (medial and lateral parapateller recesses, intercondylar notch and suprapatellar pouch) and a synovitis score ranging from 0 to 3 was assigned to each region (0 = normal synovium, 1 = diffuse, even thickening, 2 = nodular thickening, 3 = gross, nodular thickening) based on the overall impression of the severity of synovial abnormality in the three orthogonal scanning planes. This method has been described and validated for synovitis in knee osteoarthritis by Rhodes et al. [27]. The regional scores were added for a final synovitis score per knee ranging from 0 to 12.

Statistical analysis

Data was analysed with SPSS 12.0.1 for Windows (SPSS Inc, IBM, Chicago, Illinois, USA). Change in clinical parameters, IHC markers and MRI synovitis scores following treatment were evaluated using the Wilcoxon signed rank test and differences between responder and non-responder groups were determined with the Mann Whitney U test. Correlations between ΔDAS28 with change in IHC and MRI synovitis scores were calculated using Spearman's rho test.

Results

Twenty-five patients completed at least one of the IHC or MRI components of these studies (19 completed all components), and were, therefore, included for clinical analysis (10 anakinra, 15 etanercept). Patients were 50% and 66.6% female, had a mean age (range) of 43.2 (27 to 60) and 48.7 (26 to 64) years and a mean disease duration of 9.1 (1 to 42) and 7.5 (1 to 29) years in the anakinra and etanercept treated cohorts, respectively. Four patients had oligoarthritis (≤4 involved joints) at enrolment (all etanercept treated); the remaining 21 patients had polyarticular disease (mean SJC66 17, SD 9.2, range 6 to 43).

Five of the anakinra patients (50%) and 11 of the etanercept patients (73.3%) were taking a non-steroidal anti-inflammatory drug (NSAID), 2 and 4 of whom were also on a stable dose of prednisolone ≤10 mg.

Clinical responses

Changes in clinical parameters following treatment are shown in Table 1. In both studies, the DAS28 was reduced significantly after treatment; the changes were more pronounced in the etanercept group compared to the anakinra group. Nineteen of the 25 patients achieved an improvement in DAS28 of >1.2 and are labelled responders (5 anakinra and 14 etanercept), and 6 patients did not and are labelled non-responders (5 anakinra, 1 etanercept).

Twenty-three patients had involved knee scores available. There was a significant difference between the knee scores of the knee responders (n = 16, 8 anakinra, 8 etanercept) at Week 0 and Week 12 (3 (1 to 6) and 1 (0 to 4) respectively, median (range), P = 0.00), and not between the non-responders (n = 7, 2 anakinra, 5 etanercept), (2 (2 to 3) and 3 (2 to 4) respectively, P = 0.1).

Immunohistochemistry

Synovial biopsies at baseline and 12 weeks were available for 21 patients (8 anakinra, 13 etanercept).

Combining the total patient cohort, there was a significant reduction in CD3 expression following treatment in the responder group (28 (1 to 1,344) at Week 0 to 17.5 (0.5 to 734) at Week 12, P = 0.026, median (range)), but not in the non-responder group (68 (13 to 265) at Week 0 and 217 (14 to 389) at Week 12, P = 0.080) ( Figure 1A). A reduction in expression was not observed for any of the other cell markers following treatment in either the responder or non-responder groups. Of interest, however, there was a significant increase in CD68sl expression in the non-responder group at Week 12 (1,835 (1 667 to 2,218)) compared to Week 0 (1,409 (494 to 1,795)), (P = 0.043).

The degree of change in cell marker expression following treatment was significantly greater for ΔCD3 in the group of responders (19 (-100 to 1,031)) than the non-responders (-109.3 (-376 to 3)), P = 0.005, ( Figure  1C). This was also the case for ΔCD68sl (-53 (-1,336 to 2,178)) among the responders compared to the nonresponders (-382 (-1,247 to -127)), P = 0.013.

Looking at the individual treatment groups separately, there was a significant reduction in CD3 expression in the etanercept treated responders at Week 12 (n = 16), but not CD68sl, CD68ll or FVIII ( Figure 1B and Table 2). In the anakinra treated patients, there was no change within the responder group in CD3 expression, but there was a non-significant increase in the non-responder group following treatment.

MRI

Paired baseline and 12-week scans were available for 23 patients (8 anakinra, 15 etanercept).

There was no change in MRI detected synovitis following treatment in the combined cohort of responders (5 (4 to 12) at Week 0 to 5 (2 to 11) at Week 12, P = 0.1) or non-responders (3.5 (0 to 11) to 4 (1 to 7), P = 0.79). Likewise, there was no difference in the change in MRI synovitis scores following treatment between the responder and non-responder groups (0 (-3 to 6) and 1 (-7 to 7) respectively, P = 0.83). Individually, neither etanercept nor anakinra treatment led to a significant   difference in MRI synovitis scores in either the responder or non-responder groups ( Table 2). Looking specifically at the knee responders, there was a significant difference in the MRI synovitis scores of the knee responders (n = 15) at Week 0 compared to Week 12 (6 (4 to 12) and 4 (2 to 11), P = 0.049), but not of the knee non-responders (n = 5), (4 (0 to 8) and 4 (1 to 7), P = 1.0).

Associations between ΔCD3, ΔDAS28 and ΔMRI

The primary aim of this study was not to compare the clinical efficacy or specific effects on the synovium of two different biologic agents, but to seek a candidate biomarker of disease response. Change in this biomarker should correlate with change in disease activity and be irrespective of the type of therapeutic intervention used. All patient data were combined, therefore, to determine correlations between change in DAS28 with change in IHC and MRI synovitis scores, as has been done in previous similar studies [12,28].

ΔCD3 expression correlated significantly with ΔDAS28 following treatment (r = 0.49, P = 0.025), (Table 3). No correlations were observed between ΔDAS28 or any of its individual components, and change in expression of the other IHC markers. Figure 2 shows representative images of synovial CD3 expression at baseline and 12 weeks for two patients with differing clinical responses. Patient 1 (etanercept) achieved a ΔDAS28 of 1.22 and ΔCD3 of 19, and Patient 2 (anakinra) achieved a ΔDAS28 of 0.16 and ΔCD3 of -118.

There was a significant correlation between ΔCD3 and ΔMRI synovitis following treatment (r = 0.58, P = 0.009), (Table 3). Furthermore, MRI synovitis and CD3 expression measured at all time points correlated significantly (r = 0.504, P = 0.001), (n = 38). ΔMRI did not correlate with change in expression of the other IHC markers or with ΔDAS28 scores (r = -0.027, P = 0.91). Figure 3 shows representative images of CD3 stained synovium and corresponding MRI scans of a patient who had a ΔDAS28 of 2.54, a ΔCD3 score of 287 and a ΔMRI synovitis score of 4.

Discussion

This study has demonstrated that change in synovial CD3+ T-cell expression correlates with both ΔDAS28 and ΔMRI synovitis scores in a cohort of patients with PsA treated with either anakinra or etanercept.

Over the last 15 years, some fundamental features of the spondyloarthropathy (SpA) synovium have been elucidated. First, the inflamed synovium of SpA appears to differ histologically from that of RA [13][14][15][16]29]. Second, the synovial histology of subtypes of SpA, including oligoarticular versus polyarticular PsA, have been shown to be similar [16,29,30]. Third, there are histological changes in the synovium when patients with SpA Table 3 Correlation of ΔDAS28 and ΔMRI synovitis scores with cell marker expression following biologic treatment

ΔCD3

ΔCD68 sl ΔCD68 ll ΔFVIII   respond to effective treatment. Two studies of anti-TNF therapy in SpA have shown a significant reduction in polymorphonuclear (PMN) cells, CD4+ and CD8+ T cells and macrophage subsets after 12 weeks, plus a trend toward reduced CD3+ T cell numbers [31,32]. Exclusively in PsA, reduction in T cell and sublining macrophage infiltration has been observed as early as 48 hours after an infliximab infusion [33] and also following treatment with alefacept and methotrexate [34,35].

Correlations with clinical outcomes were not performed in these studies. Consistent with our etanercept responders, in a cohort of PsA patients treated with adalimumab, a significant reduction in the number of CD3 positive cells was observed after four weeks [12]. The number of CD68+ cells in the synovial sublining did decrease in the responders of both latter studies, but not significantly, while CD68sl expression in this current study's non-responders significantly increased. Thus, in PsA, change in synovial CD3 cell infiltration, and not CD68, appears to be a superior biomarker of treatment response. Reduction in angiogenesis has been demonstrated in PsA patients after infliximab treatment [36,37], but was not found after etanercept treatment in the present study, and may be related to the difference in mechanism of action between the anti-TNF antibodies compared to etanercept [38].

Only two previously published trials of SpA synovium have measured DAS28 scores. Of 52 SpA patients who may have received infliximab, etanercept or no biologic treatment, DAS28 scores were calculated for 28 patients who had polyarticular disease [28]. These scores correlated only with change in CD163 expression, a macrophage subset marker, in the lining layer, and not with change in CD3 or CD68 expression in the sublining layer. The patients with PsA were not evaluated as a distinct group. Only one other trial, which used adalimumab or placebo, has exclusively enrolled PsA patients and used DAS28 as a primary clinical outcome measure [12]. Consistent with our results, that study also demonstrated a significant correlation between ΔCD3 and ΔDAS28. Taking these two papers together, four different agents have now been used in two PsA cohorts (anakinra/etanercept and adalimumab/placebo) and both studies have found this proportional relationship between ΔCD3 and ΔDAS28.

While not conclusive, the findings in this study are also consistent with the hypothesis that T-cells play an active role in PsA pathogenesis. Large numbers of T cells are present in the PsA synovium, synovial fluid and subchondral bone beneath the entheses [13,29,39], where bone oedema and erosions can occur. Th1 derived cytokines dominate in the PsA synovium [35,40] and the CD8+ cell population contains T cell repertoires which are oligoclonally expanded [41]. The association of PsA with human leukocyte antigen (HLA) Class 1 [42], the development of Ps and PsA as a manifestation of the Acquired Immunodeficiency Syndrome (AIDS) and the transmission of PsA following bone marrow transplantation [43,44] all suggest T-cells take part in disease expression. Lastly, the fact that cyclosporin and ustekinumab, which impair T-cell activation, and alefacept, which specifically targets activated T cells, are effective in PsA, support T-cell involvement further [45][46][47].

The use of MRI in PsA research has been reviewed in detail [48], and an MRI scoring system for hands in PsA has recently been developed by the OMERACT imaging group [49,50]. We opted for our scoring method as it focuses on knee synovitis and is semi-quantitative. MRI synovitis has been shown to reduce significantly following anti-TNF therapy in PsA [51,52], and we found this to be the case in our combined group of clinical knee responders. These former studies assessed mostly hand joints as opposed to exclusively knees and used quantitative analysis. Correlations of MRI findings and histopathology in inflammatory arthritis are emerging in the literature. Bollow et al. compared dynamic MRI and sacroiliac joint immunohistochemistry [53] and found T cells and macrophages to be the most common inflammatory cells in active SpA sacroiliitis, although >95% of the tissue obtained was cartilage and bone. In severe AS, MRI-detected bone oedema has correlated well with histological bone marrow oedema of zygoapophyseal joints, but less so with actual inflammatory cell infiltrates [54]. Other studies correlating MRI findings with synovial and bone oedema histology have been undertaken in RA [55,56], but not yet in PsA. This is the first study, therefore, to demonstrate a relationship between MRI synovitis and CD3 expression in PsA, both at all time points in the study and when comparing the changes with treatment.

No relationship was found in this cohort between ΔMRI synovitis and ΔDAS28. The fact that this MRI data reflects change in a single joint only, in contrast to DAS28, and that improvement in DAS28 may or may not involve the knee, will contribute to this. The strongest correlation being between ΔCD3 expression and ΔMRI synovitis scores is not surprising, as these originate from the same single joint and are objectively measured, excluding any subjectivity of clinical scoring and additional pathologies that could influence single joint clinical scores.

As two patients did not undergo follow-up MRI and three different patients did not have adequate synovial tissue for analysis, our study is limited by some uncoupling of the patient groups included in IHC and MRI analyses. Also, in three pre-treatment scans (two etanercept, one anakinra), insufficient fat suppression may have led to an underestimation of the degree of synovitis.

Conclusions

This study demonstrates a significant correlation between synovial ΔCD3 expression and ΔDAS28, and synovial ΔCD3 expression and ΔMRI synovitis scores in a cohort of 25 patients with PsA treated with either anakinra or etanercept. The establishment of ΔCD3 as a candidate biomarker of treatment response in PsA should prompt other studies using different therapeutic agents to reinforce this concept, and also to determine its ability to predict future clinical outcomes. Further work focusing on changes in peripheral blood T-cell subsets for a more easily accessible biomarker could prove useful.

Figure 1
1ΔCD3 of combined responders and non-responders (A), etanercept responders (B). and ΔCD3 of combined responders versus non-responders (C).

ΔDAS280.49 (0.025*) 0.27 (0.24) -0.07 (0.77) 0.244 (0.30) ΔMRI 0.58 (0.009*) 0.22 (0.378) 0.07 (0.78) 0.33 (0.18) Correlation coefficient (P =). N = 21 for all IHC groups except FVIII (n = 20) and n = 19 for all MRI groups; ll, lining layer; sl, sublining layer.

Figure 2
2Synovial images showing CD3 expression in a DAS28 moderate responder (Patient 1) and non-responder (Patient 2). A and B are baseline and Week 12 images of Patient 1, and C and D are baseline and Week 12 images of Patient 2 respectively.

Figure 3
3Baseline (A) and Week 12 (B) MRI scans with corresponding baseline (C) and Week 12 (D) CD3 stained synovium (red-brown). Thickened enhanced synovium (* in A) has improved following treatment.

Abbreviations Δ: change in; AEC: amino-ethylcarbazole; CASPAR: Classification of psoriatic arthritis; CRP: C-reactive protein; d VAS: disease visual analogue scale; DAS28: disease activity score assessing 28 joints; DIA: digital image analysis; EULAR: European League Against Rheumatism; FVIII: Factor VIII; HAQ: health assessment questionnaire; IHC: immunohistochemistry; IL-1: interleukin 1; IOD: integrated optical density; ll: synovial lining layer; MRI: magnetic resonance imaging; n/a: not applicable; NR: non-responder; OMERACT: outcome measures in rheumatology clinical trials; pVAS: pain visual analogue scale; PsA: psoriatic arthritis; R: responder; RA: rheumatoid arthritis; SJC: swollen joint count; sl: synovial sublining layer; SpA: spondyloarthropathy; TJC: tender joint count; TNF: tumour necrosis factor; VAS: visual analogue scale.

Table 1
1Clinical parameters of patients with PsA at baseline and 12 weeks following treatmentAnakinra n = 10
Etanercept n = 15

Week 0
Week 12
P-value
Week 0
Week 12
P-value

TJC28
9.5 (3 to 19)
7 (1 to 13)
0.033
11 (1 to 25)
2 (0 to 28)
0.004

TJC68
24.5 (9 to 52)
13.5 (3 to 35)
0.015
15 (3 to 57)
3 (0 to 38)
0.002

SJC28
8 (1 to 18)
5 (0 to 8)
0.032
4 (1 to 21)
1 (0 to 8)
0.005

SJC68
20.5 (6 to 27)
8 (0 to 23)
0.028
9 (3 to 43)
2 (0 to 16)
0.007

ESR
18.5 (3 to 79)
7.5 (2 to 46)
0.059
14 (4 to 91)
5 (1 to 26)
0.001

CRP
17 (4 to 70)
7.5 (0 to 29)
0.044
7 (0 to 42)
0 (0 to 18)
0.092

d VAS
66 (27 to 85)
47 (12 to 70)
0.051
44.5 (5 to 93)
15 (2 to 56)
0.002

p VAS
65.5 (21 to 91)
46.5 (20 to 75)
0.169
50 (22 to 93)
12 (2 to 66)
0.001
HAQ
1.25 (0.75 to 2.38)
1.13 (0.25 to 1.88)
0.057
1.13 (0 to 2.5)
0.25 (0 to 2.25)
0.01

DAS28
5.03 (3.77 to 7.16)
4.17 (2.35 to 5.98)
0.022
5.26 (3.08 to 6.95)
2.01 (0.14 to 5.35)
0.001

Median (range). d VAS, disease visual analogue scale; p VAS, pain visual analogue scale.

Table 2
2Change in cell marker expression following treatment in the responder and non-responder groupsWeek 0
Week 12
P-value

Pontifex et al. Arthritis Research & Therapy 2011, 13:R7 http://arthritis-research.com/content/13/1/R7
Pontifex et al. Arthritis Research & Therapy 2011, 13:R7 http://arthritis-research.com/content/13/1/R7 Page 6 of 10
Pontifex et al. Arthritis Research & Therapy 2011, 13:R7 http://arthritis-research.com/content/13/1/R7 Page 10 of 10
AcknowledgementsThe authors wish to acknowledge the assistance of Drs. Ceara Walsh, Ronan Mullan and Tom Smeets. The digital image analysis aspect of this work was supported by the Dutch Arthritis Association and the European Community's F6 (Autocure) funding. The investigator originated protocols were supported by Amgen and Wyeth. Funders had no involvement in the design, production, results or presentation of this research.Competing interests UF had grant research support from GlaxoSmithKline and is a consultant for and received grant research support from Wyeth. PPT is a consultant for Abbott, Amgen, Schering-Plough, and Wyeth. DJV received grant research support from GlaxoSmithKline, is a consultant for and received grant research support from Schering-Plough, is a site primary investigator for Roche, and is a consultant for and received grant research support from Wyeth. OF received grant research support from Abbott, is a primary investigator for Bristol Myers Squibb, and received grant research support from Wyeth. The other authors declare that they have no competing interests.Authors' contributions EKP cut and stored the etanercept slides and performed the initial IHC staining, performed the digital image analysis, collated all data, performed all statistical analysis and wrote the manuscript. DMG participated in the autostainer immunohistochemistry. MG cut and stored all anakinra slides and performed initial IHC staining. MV made a substantial contribution to the DIA part of this work and arranged DIA data ready for analysis. AG was involved in patient recruitment and clinical assessment. IB participated in the autostainer immunohistochemistry. UF made a substantial contribution to the statistical analysis. BB made a substantial contribution to the conception and design of the study. PPT has been involved in the DIA aspect of this work and revising the manuscript critically for content. RG arranged the MRI scanning and performed the MRI synovitis scoring. DV made a substantial contribution to the conception and design of the study and coordinated the arthroscopies. OF conceived of the study, participated in its design and coordination and has been involved in the revision of the manuscript critically for content. All authors have read and approved the final version of this manuscript.
A prospective, clinical and radiological study of early psoriatic arthritis: an early synovitis clinic experience. D Kane, L Stafford, B Bresnihan, O Fitzgerald, Rheumatology (Oxford). 42Kane D, Stafford L, Bresnihan B, FitzGerald O: A prospective, clinical and radiological study of early psoriatic arthritis: an early synovitis clinic experience. Rheumatology (Oxford) 2003, 42:1460-1468.

Consensus on a core set of domains for psoriatic arthritis. D D Gladman, P J Mease, V Strand, P Healy, P S Helliwell, O Fitzgerald, A B Gottlieb, G G Krueger, P Nash, C T Ritchlin, W Taylor, A Adebajo, J Braun, A Cauli, S Carneiro, E Choy, B Dijkmans, L Espinoza, D Van Der Heijde, E Husni, E Lubrano, D Mcgonagle, A Qureshi, E R Soriano, J Zochling, J Rheumatol. 34Gladman DD, Mease PJ, Strand V, Healy P, Helliwell PS, Fitzgerald O, Gottlieb AB, Krueger GG, Nash P, Ritchlin CT, Taylor W, Adebajo A, Braun J, Cauli A, Carneiro S, Choy E, Dijkmans B, Espinoza L, van der Heijde D, Husni E, Lubrano E, McGonagle D, Qureshi A, Soriano ER, Zochling J: Consensus on a core set of domains for psoriatic arthritis. J Rheumatol 2007, 34:1167-1170.

Considerations in the evaluation of surrogate endpoints in clinical trials. summary of a National Institutes of Health workshop. V G De Gruttola, P Clax, D L Demets, G J Downing, S S Ellenberg, L Friedman, M H Gail, R Prentice, J Wittes, S L Zeger, Control Clin Trials. 22De Gruttola VG, Clax P, DeMets DL, Downing GJ, Ellenberg SS, Friedman L, Gail MH, Prentice R, Wittes J, Zeger SL: Considerations in the evaluation of surrogate endpoints in clinical trials. summary of a National Institutes of Health workshop. Control Clin Trials 2001, 22:485-502.

Synovial tissue macrophages: a sensitive biomarker for response to treatment in patients with rheumatoid arthritis. J J Haringman, D M Gerlag, A H Zwinderman, T J Smeets, M C Kraan, D Baeten, I B Mcinnes, B Bresnihan, P P Tak, Ann Rheum Dis. 64Haringman JJ, Gerlag DM, Zwinderman AH, Smeets TJ, Kraan MC, Baeten D, McInnes IB, Bresnihan B, Tak PP: Synovial tissue macrophages: a sensitive biomarker for response to treatment in patients with rheumatoid arthritis. Ann Rheum Dis 2005, 64:834-838.

Synovial macrophages as a biomarker of response to therapeutic intervention in rheumatoid arthritis: standardization and consistency across centers. B Bresnihan, D M Gerlag, T Rooney, T J Smeets, C A Wijbrandts, D Boyle, O Fitzgerald, B W Kirkham, I B Mcinnes, M Smith, A K Ulfgren, D J Veale, P P Tak, J Rheumatol. 34Bresnihan B, Gerlag DM, Rooney T, Smeets TJ, Wijbrandts CA, Boyle D, Fitzgerald O, Kirkham BW, McInnes IB, Smith M, Ulfgren AK, Veale DJ, Tak PP: Synovial macrophages as a biomarker of response to therapeutic intervention in rheumatoid arthritis: standardization and consistency across centers. J Rheumatol 2007, 34:620-622.

Effects of oral prednisolone on biomarkers in synovial tissue and clinical improvement in rheumatoid arthritis. D M Gerlag, J J Haringman, T J Smeets, A H Zwinderman, M C Kraan, P J Laud, S Morgan, A F Nash, P P Tak, Arthritis Rheum. 50Gerlag DM, Haringman JJ, Smeets TJ, Zwinderman AH, Kraan MC, Laud PJ, Morgan S, Nash AF, Tak PP: Effects of oral prednisolone on biomarkers in synovial tissue and clinical improvement in rheumatoid arthritis. Arthritis Rheum 2004, 50:3783-3791.

Synovial tissue sublining CD68 expression is a biomarker of therapeutic response in rheumatoid arthritis clinical trials: consistency across centers. B Bresnihan, E Pontifex, R M Thurlings, M Vinkenoog, H El-Gabalawy, U Fearon, O Fitzgerald, D M Gerlag, T Rooney, M G Van De Sande, D Veale, K Vos, P P Tak, J Rheumatol. 36Bresnihan B, Pontifex E, Thurlings RM, Vinkenoog M, El-Gabalawy H, Fearon U, Fitzgerald O, Gerlag DM, Rooney T, van de Sande MG, Veale D, Vos K, Tak PP: Synovial tissue sublining CD68 expression is a biomarker of therapeutic response in rheumatoid arthritis clinical trials: consistency across centers. J Rheumatol 2009, 36:1800-1802.

Composite measures in psoriatic arthritis: GRAPPA. D D Gladman, R Landewe, N J Mchugh, O Fitzgerald, D Thaci, L Coates, P J Mease, A A Qureshi, G G Krueger, C T Ritchlin, A F Kavanaugh, A Garg, J Rheumatol. 37Gladman DD, Landewe R, McHugh NJ, Fitzgerald O, Thaci D, Coates L, Mease PJ, Qureshi AA, Krueger GG, Ritchlin CT, Kavanaugh AF, Garg A: Composite measures in psoriatic arthritis: GRAPPA 2008. J Rheumatol 37:453-461.

Modified disease activity scores that include twenty-eightjoint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. M L Prevoo, M A Van 't Hof, H H Kuper, M A Van Leeuwen, L B Van De Putte, P L Van Riel, Arthritis Rheum. 38Prevoo ML, van 't Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL: Modified disease activity scores that include twenty-eight- joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995, 38:44-48.

Van Riel PL: Performance of response criteria for assessing peripheral arthritis in patients with psoriatic arthritis: analysis of data from randomised controlled trials of two tumour necrosis factor inhibitors. J Fransen, C Antoni, P J Mease, W Uter, A Kavanaugh, J R Kalden, Ann Rheum Dis. 65Fransen J, Antoni C, Mease PJ, Uter W, Kavanaugh A, Kalden JR, Van Riel PL: Performance of response criteria for assessing peripheral arthritis in patients with psoriatic arthritis: analysis of data from randomised controlled trials of two tumour necrosis factor inhibitors. Ann Rheum Dis 2006, 65:1373-1378.

Van den Bosch F: Comparison of different outcome measures for psoriatic arthritis in patients treated with infliximab or placebo. B Vander Cruyssen, De Keyser, F Kruithof, E Mielants, H , Ann Rheum Dis. 66Vander Cruyssen B, De Keyser F, Kruithof E, Mielants H, Van den Bosch F: Comparison of different outcome measures for psoriatic arthritis in patients treated with infliximab or placebo. Ann Rheum Dis 2007, 66:138-140.

A prospective, randomised, placebo-controlled study to identify biomarkers associated with active treatment in psoriatic arthritis: Effects of adalimumab treatment on synovial tissue. A W Van Kuijk, D M Gerlag, K Vos, G Wolbink, M De Groot, M A De Rie, A H Zwinderman, B A Dijkmans, P P Tak, Ann Rheum Dis. 68van Kuijk AW, Gerlag DM, Vos K, Wolbink G, de Groot M, de Rie MA, Zwinderman AH, Dijkmans BA, Tak PP: A prospective, randomised, placebo-controlled study to identify biomarkers associated with active treatment in psoriatic arthritis: Effects of adalimumab treatment on synovial tissue. Ann Rheum Dis 2009, 68:1303-1309.

Reduced synovial membrane macrophage numbers, ELAM-1 expression, and lining layer hyperplasia in psoriatic arthritis as compared with rheumatoid arthritis. D Veale, G Yanni, S Rogers, L Barnes, B Bresnihan, O Fitzgerald, Arthritis Rheum. 36Veale D, Yanni G, Rogers S, Barnes L, Bresnihan B, Fitzgerald O: Reduced synovial membrane macrophage numbers, ELAM-1 expression, and lining layer hyperplasia in psoriatic arthritis as compared with rheumatoid arthritis. Arthritis Rheum 1993, 36:893-900.

study of the synovial histology in rheumatoid arthritis, spondyloarthropathy, and osteoarthritis: influence of disease duration and activity. D Baeten, P Demetter, C Cuvelier, Van Den, F Bosch, E Kruithof, N Van Damme, G Verbruggen, H Mielants, E M Veys, De Keyser, F: Comparative Pontifex, Arthritis Research & Therapy. 13Ann Rheum DisBaeten D, Demetter P, Cuvelier C, Van Den Bosch F, Kruithof E, Van Damme N, Verbruggen G, Mielants H, Veys EM, De Keyser F: Comparative Pontifex et al. Arthritis Research & Therapy 2011, 13:R7 http://arthritis-research.com/content/13/1/R7 study of the synovial histology in rheumatoid arthritis, spondyloarthropathy, and osteoarthritis: influence of disease duration and activity. Ann Rheum Dis 2000, 59:945-953.

McInnes IB: Macrophage-derived cytokine and nuclear factor kappaB p65 expression in synovial membrane and skin of patients with psoriatic arthritis. C L Danning, G G Illei, C Hitchon, M R Greer, D T Boumpas, Arthritis Rheum. 43Danning CL, Illei GG, Hitchon C, Greer MR, Boumpas DT, McInnes IB: Macrophage-derived cytokine and nuclear factor kappaB p65 expression in synovial membrane and skin of patients with psoriatic arthritis. Arthritis Rheum 2000, 43:1244-1256.

Synovial histopathology of psoriatic arthritis, both oligo-and polyarticular, resembles spondyloarthropathy more than it does rheumatoid arthritis. E Kruithof, D Baeten, L De Rycke, B Vandooren, D Foell, J Roth, J D Canete, A M Boots, E M Veys, De Keyser, F , Arthritis Res Ther. 7Kruithof E, Baeten D, De Rycke L, Vandooren B, Foell D, Roth J, Canete JD, Boots AM, Veys EM, De Keyser F: Synovial histopathology of psoriatic arthritis, both oligo-and polyarticular, resembles spondyloarthropathy more than it does rheumatoid arthritis. Arthritis Res Ther 2005, 7: R569-580.

The OMERACT filter for Outcome Measures in Rheumatology. M Boers, P Brooks, C V Strand, P Tugwell, J Rheumatol. 25Boers M, Brooks P, Strand CV, Tugwell P: The OMERACT filter for Outcome Measures in Rheumatology. J Rheumatol 1998, 25:198-199.

Blocking the receptor for C5a in patients with rheumatoid arthritis does not reduce synovial inflammation. C E Vergunst, D M Gerlag, H Dinant, L Schulz, M Vinkenoog, T J Smeets, M E Sanders, K A Reedquist, P P Tak, Rheumatology (Oxford). 46Vergunst CE, Gerlag DM, Dinant H, Schulz L, Vinkenoog M, Smeets TJ, Sanders ME, Reedquist KA, Tak PP: Blocking the receptor for C5a in patients with rheumatoid arthritis does not reduce synovial inflammation. Rheumatology (Oxford) 2007, 46:1773-1778.

Modulation of CCR2 in rheumatoid arthritis: a double-blind, randomized, placebo-controlled clinical trial. C E Vergunst, D M Gerlag, L Lopatinskaya, L Klareskog, M D Smith, F Van Den Bosch, H J Dinant, Y Lee, T Wyant, E W Jacobson, D Baeten, P P Tak, Arthritis Rheum. 58Vergunst CE, Gerlag DM, Lopatinskaya L, Klareskog L, Smith MD, van den Bosch F, Dinant HJ, Lee Y, Wyant T, Jacobson EW, Baeten D, Tak PP: Modulation of CCR2 in rheumatoid arthritis: a double-blind, randomized, placebo-controlled clinical trial. Arthritis Rheum 2008, 58:1931-1939.

Characteristic magnetic resonance imaging entheseal changes of knee synovitis in spondylarthropathy. D Mcgonagle, W Gibbon, P O'connor, M Green, C Pease, P Emery, Arthritis Rheum. 41McGonagle D, Gibbon W, O'Connor P, Green M, Pease C, Emery P: Characteristic magnetic resonance imaging entheseal changes of knee synovitis in spondylarthropathy. Arthritis Rheum 1998, 41:694-700.

The relationship between the extensor tendon enthesis and the nail in distal interphalangeal joint disease in psoriatic arthritis-a high-resolution MRI and histological study. A L Tan, M Benjamin, H Toumi, A J Grainger, S F Tanner, P Emery, D Mcgonagle, Rheumatology (Oxford). 46Tan AL, Benjamin M, Toumi H, Grainger AJ, Tanner SF, Emery P, McGonagle D: The relationship between the extensor tendon enthesis and the nail in distal interphalangeal joint disease in psoriatic arthritis-a high-resolution MRI and histological study. Rheumatology (Oxford) 2007, 46:253-256.

Classification criteria for psoriatic arthritis: development of new criteria from a large international study. W Taylor, D Gladman, P Helliwell, A Marchesoni, P Mease, H Mielants, Arthritis Rheum. 54Taylor W, Gladman D, Helliwell P, Marchesoni A, Mease P, Mielants H: Classification criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum 2006, 54:2665-2673.

Development and validation of the European League Against Rheumatism response criteria for rheumatoid arthritis. Comparison with the preliminary American College of Rheumatology and the World Health Organization/International League Against Rheumatism Criteria. A M Van Gestel, M L Prevoo, M A Van 't Hof, M H Van Rijswijk, L B Van De Putte, P L Van Riel, Arthritis Rheum. 39van Gestel AM, Prevoo ML, van 't Hof MA, van Rijswijk MH, van de Putte LB, van Riel PL: Development and validation of the European League Against Rheumatism response criteria for rheumatoid arthritis. Comparison with the preliminary American College of Rheumatology and the World Health Organization/International League Against Rheumatism Criteria. Arthritis Rheum 1996, 39:34-40.

Etanercept in the treatment of psoriatic arthritis and psoriasis: a randomised trial. P J Mease, B S Goffe, J Metz, A Vanderstoep, B Finck, D J Burge, Lancet. 356Mease PJ, Goffe BS, Metz J, VanderStoep A, Finck B, Burge DJ: Etanercept in the treatment of psoriatic arthritis and psoriasis: a randomised trial. Lancet 2000, 356:385-390.

Measurement of cytokine and adhesion molecule expression in synovial tissue by digital image analysis. M C Kraan, M D Smith, H Weedon, M J Ahern, F C Breedveld, P P Tak, Ann Rheum Dis. 60Kraan MC, Smith MD, Weedon H, Ahern MJ, Breedveld FC, Tak PP: Measurement of cytokine and adhesion molecule expression in synovial tissue by digital image analysis. Ann Rheum Dis 2001, 60:296-298.

Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis. J J Haringman, M Vinkenoog, D M Gerlag, T J Smeets, A H Zwinderman, P P Tak, Arthritis Res Ther. 7Haringman JJ, Vinkenoog M, Gerlag DM, Smeets TJ, Zwinderman AH, Tak PP: Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis. Arthritis Res Ther 2005, 7:R862-867.

The validation of simple scoring methods for evaluating compartmentspecific synovitis detected by MRI in knee osteoarthritis. L A Rhodes, A J Grainger, A M Keenan, C Thomas, P Emery, P G Conaghan, Rheumatology (Oxford). 44Rhodes LA, Grainger AJ, Keenan AM, Thomas C, Emery P, Conaghan PG: The validation of simple scoring methods for evaluating compartment- specific synovitis detected by MRI in knee osteoarthritis. Rheumatology (Oxford) 2005, 44:1569-1573.

Identification of synovial biomarkers of response to experimental treatment in early-phase clinical trials in spondylarthritis. E Kruithof, L De Rycke, B Vandooren, De Keyser, F Fitzgerald, O Mcinnes, I Tak, P P Bresnihan, B Veys, E M Baeten, D , Arthritis Rheum. 54Kruithof E, De Rycke L, Vandooren B, De Keyser F, FitzGerald O, McInnes I, Tak PP, Bresnihan B, Veys EM, Baeten D: Identification of synovial biomarkers of response to experimental treatment in early-phase clinical trials in spondylarthritis. Arthritis Rheum 2006, 54:1795-1804.

Detailed analysis of the cell infiltrate and the expression of mediators of synovial inflammation and joint destruction in the synovium of patients with psoriatic arthritis: implications for treatment. A W Van Kuijk, P Reinders-Blankert, T J Smeets, B A Dijkmans, P P Tak, Ann Rheum Dis. 65van Kuijk AW, Reinders-Blankert P, Smeets TJ, Dijkmans BA, Tak PP: Detailed analysis of the cell infiltrate and the expression of mediators of synovial inflammation and joint destruction in the synovium of patients with psoriatic arthritis: implications for treatment. Ann Rheum Dis 2006, 65:1551-1557.

Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy. D Baeten, E Kruithof, L De Rycke, A M Boots, H Mielants, E M Veys, De Keyser, F , Arthritis Res Ther. 7Baeten D, Kruithof E, De Rycke L, Boots AM, Mielants H, Veys EM, De Keyser F: Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy. Arthritis Res Ther 2005, 7:R359-369.

Histological evidence that infliximab treatment leads to downregulation of inflammation and tissue remodelling of the synovial membrane in spondyloarthropathy. E Kruithof, D Baeten, F Van Den Bosch, H Mielants, E M Veys, De Keyser, F , Ann Rheum Dis. 64Kruithof E, Baeten D, Van den Bosch F, Mielants H, Veys EM, De Keyser F: Histological evidence that infliximab treatment leads to downregulation of inflammation and tissue remodelling of the synovial membrane in spondyloarthropathy. Ann Rheum Dis 2005, 64:529-536.

Immunomodulatory effects of etanercept on peripheral joint synovitis in the spondylarthropathies. E Kruithof, L De Rycke, J Roth, H Mielants, F Van Den Bosch, De Keyser, F Veys, E M Baeten, D , Arthritis Rheum. 52Kruithof E, De Rycke L, Roth J, Mielants H, Van den Bosch F, De Keyser F, Veys EM, Baeten D: Immunomodulatory effects of etanercept on peripheral joint synovitis in the spondylarthropathies. Arthritis Rheum 2005, 52:3898-3909.

Early effects of tumour necrosis factor alpha blockade on skin and synovial tissue in patients with active psoriasis and psoriatic arthritis. A Y Goedkoop, M C Kraan, M B Teunissen, D I Picavet, M A De Rie, J D Bos, P P Tak, Ann Rheum Dis. 63Goedkoop AY, Kraan MC, Teunissen MB, Picavet DI, de Rie MA, Bos JD, Tak PP: Early effects of tumour necrosis factor alpha blockade on skin and synovial tissue in patients with active psoriasis and psoriatic arthritis. Ann Rheum Dis 2004, 63:769-773.

Alefacept treatment in psoriatic arthritis: reduction of the effector T cell population in peripheral blood and synovial tissue is associated with improvement of clinical signs of arthritis. M C Kraan, A W Van Kuijk, H J Dinant, A Y Goedkoop, T J Smeets, M A De Rie, B A Dijkmans, A K Vaishnaw, J D Bos, P P Tak, Arthritis Rheum. 46Kraan MC, van Kuijk AW, Dinant HJ, Goedkoop AY, Smeets TJ, de Rie MA, Dijkmans BA, Vaishnaw AK, Bos JD, Tak PP: Alefacept treatment in psoriatic arthritis: reduction of the effector T cell population in peripheral blood and synovial tissue is associated with improvement of clinical signs of arthritis. Arthritis Rheum 2002, 46:2776-2784.

Reduction of synovial sublining layer inflammation and proinflammatory cytokine expression in psoriatic arthritis treated with methotrexate. D Kane, M Gogarty, J O'leary, I Silva, N Bermingham, B Bresnihan, O Fitzgerald, Arthritis Rheum. 50Kane D, Gogarty M, O'Leary J, Silva I, Bermingham N, Bresnihan B, Fitzgerald O: Reduction of synovial sublining layer inflammation and proinflammatory cytokine expression in psoriatic arthritis treated with methotrexate. Arthritis Rheum 2004, 50:3286-3295.

Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective singlecentre study. A Y Goedkoop, M C Kraan, D I Picavet, M A De Rie, M B Teunissen, J D Bos, P P Tak, Arthritis Res Ther. 6Goedkoop AY, Kraan MC, Picavet DI, de Rie MA, Teunissen MB, Bos JD, Tak PP: Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single- centre study. Arthritis Res Ther 2004, 6:R326-334.

Antiangiogenic effects of anti-tumor necrosis factor alpha therapy with infliximab in psoriatic arthritis. J D Canete, J L Pablos, R Sanmarti, C Mallofre, S Marsal, J Maymo, J Gratacos, J Mezquita, C Mezquita, M C Cid, Arthritis Rheum. 50Canete JD, Pablos JL, Sanmarti R, Mallofre C, Marsal S, Maymo J, Gratacos J, Mezquita J, Mezquita C, Cid MC: Antiangiogenic effects of anti-tumor necrosis factor alpha therapy with infliximab in psoriatic arthritis. Arthritis Rheum 2004, 50:1636-1641.

Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. D Tracey, L Klareskog, E H Sasso, J G Salfeld, P P Tak, Pharmacol Ther. 117Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP: Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. Pharmacol Ther 2008, 117:244-279.

Predominance of CD8+ T lymphocytes in psoriatic arthritis. P Costello, B Bresnihan, C O'farrelly, O Fitzgerald, J Rheumatol. 26Costello P, Bresnihan B, O'Farrelly C, FitzGerald O: Predominance of CD8+ T lymphocytes in psoriatic arthritis. J Rheumatol 1999, 26:1117-1124.

Patterns of cytokine production in psoriatic synovium. C Ritchlin, S A Haas-Smith, D Hicks, J Cappuccio, C K Osterland, R J Looney, J Rheumatol. 25Ritchlin C, Haas-Smith SA, Hicks D, Cappuccio J, Osterland CK, Looney RJ: Patterns of cytokine production in psoriatic synovium. J Rheumatol 1998, 25:1544-1552.

Clonal characteristics of T cell infiltrates in skin and synovium of patients with psoriatic arthritis. I Tassiulas, S R Duncan, M Centola, A N Theofilopoulos, D T Boumpas, Hum Immunol. 60Tassiulas I, Duncan SR, Centola M, Theofilopoulos AN, Boumpas DT: Clonal characteristics of T cell infiltrates in skin and synovium of patients with psoriatic arthritis. Hum Immunol 1999, 60:479-491.

HLA antigens in psoriatic arthritis. D D Gladman, K A Anhorn, R K Schachter, H Mervart, J Rheumatol. 13Gladman DD, Anhorn KA, Schachter RK, Mervart H: HLA antigens in psoriatic arthritis. J Rheumatol 1986, 13:586-592.

Transmission of psoriatic arthritis by allogeneic bone marrow transplantation for chronic myelogenous leukaemia from an HLA-identical donor. T Daikeler, I Gunaydin, H Einsele, L Kanz, I Kotter, Rheumatology (Oxford). 38Daikeler T, Gunaydin I, Einsele H, Kanz L, Kotter I: Transmission of psoriatic arthritis by allogeneic bone marrow transplantation for chronic myelogenous leukaemia from an HLA-identical donor. Rheumatology (Oxford) 1999, 38:89-90.

Development of psoriasis after syngeneic bone marrow transplant from psoriatic donor: further evidence for adoptive autoimmunity. J A Snowden, D C Heaton, Br J Dermatol. 137Snowden JA, Heaton DC: Development of psoriasis after syngeneic bone marrow transplant from psoriatic donor: further evidence for adoptive autoimmunity. Br J Dermatol 1997, 137:130-132.

Comparison of cyclosporin A and methotrexate in the treatment of psoriatic arthritis: a one-year prospective study. A Spadaro, V Riccieri, A Sili-Scavalli, F Sensi, E Taccari, A Zoppini, Clin Exp Rheumatol. 13Spadaro A, Riccieri V, Sili-Scavalli A, Sensi F, Taccari E, Zoppini A: Comparison of cyclosporin A and methotrexate in the treatment of psoriatic arthritis: a one-year prospective study. Clin Exp Rheumatol 1995, 13:589-593.

Ustekinumab, a human interleukin 12/23 monoclonal antibody, for psoriatic arthritis: randomised, double-blind, placebo-controlled, crossover trial. A Gottlieb, A Menter, A Mendelsohn, Y K Shen, S Li, C Guzzo, S Fretzin, R Kunynetz, A Kavanaugh, Lancet. 373Gottlieb A, Menter A, Mendelsohn A, Shen YK, Li S, Guzzo C, Fretzin S, Kunynetz R, Kavanaugh A: Ustekinumab, a human interleukin 12/23 monoclonal antibody, for psoriatic arthritis: randomised, double-blind, placebo-controlled, crossover trial. Lancet 2009, 373:633-640.

Alefacept in combination with methotrexate for the treatment of psoriatic arthritis: results of a randomized, double-blind, placebo-controlled study. P J Mease, D D Gladman, E C Keystone, Arthritis Rheum. 54Mease PJ, Gladman DD, Keystone EC: Alefacept in combination with methotrexate for the treatment of psoriatic arthritis: results of a randomized, double-blind, placebo-controlled study. Arthritis Rheum 2006, 54:1638-1645.

Magnetic resonance imaging in psoriatic arthritis: a review of the literature. F Mcqueen, M Lassere, M Ostergaard, Arthritis Res Ther. 8207McQueen F, Lassere M, Ostergaard M: Magnetic resonance imaging in psoriatic arthritis: a review of the literature. Arthritis Res Ther 2006, 8:207.

The OMERACT Magnetic Resonance Imaging Inflammatory Arthritis Groupadvances and priorities. M Ostergaard, F Mcqueen, P Bird, C Peterfy, E Haavardsholm, B Ejbjerg, M Lassere, O' Connor, P Emery, P Edmonds, J Genant, H Conaghan, P G , J Rheumatol. 34Ostergaard M, McQueen F, Bird P, Peterfy C, Haavardsholm E, Ejbjerg B, Lassere M, O'Connor P, Emery P, Edmonds J, Genant H, Conaghan PG: The OMERACT Magnetic Resonance Imaging Inflammatory Arthritis Group - advances and priorities. J Rheumatol 2007, 34:852-853.

The OMERACT psoriatic arthritis magnetic resonance imaging scoring system (PsAMRIS): definitions of key pathologies, suggested MRI sequences, and. M Ostergaard, F Mcqueen, C Wiell, P Bird, P Boyesen, B Ejbjerg, C Peterfy, F Gandjbakhch, A Duer-Jensen, L Coates, E A Haavardsholm, K G Hermann, M Lassere, O' Connor, P Emery, P Genant, H Conaghan, Pg ; Pontifex, Arthritis Research & Therapy. 13J RheumatolOstergaard M, McQueen F, Wiell C, Bird P, Boyesen P, Ejbjerg B, Peterfy C, Gandjbakhch F, Duer-Jensen A, Coates L, Haavardsholm EA, Hermann KG, Lassere M, O'Connor P, Emery P, Genant H, Conaghan PG: The OMERACT psoriatic arthritis magnetic resonance imaging scoring system (PsAMRIS): definitions of key pathologies, suggested MRI sequences, and Pontifex et al. Arthritis Research & Therapy 2011, 13:R7 http://arthritis-research.com/content/13/1/R7 preliminary scoring system for PsA Hands. J Rheumatol 2009, 36:1816-1824.

Open-label study of infliximab treatment for psoriatic arthritis: clinical and magnetic resonance imaging measurements of reduction of inflammation. C Antoni, C Dechant, Hanns-Martin Lorenz, P D Wendler, J Ogilvie, A Lueftl, M Kalden-Nemeth, D Kalden, J R Manger, B , Arthritis Rheum. 47Antoni C, Dechant C, Hanns-Martin Lorenz PD, Wendler J, Ogilvie A, Lueftl M, Kalden-Nemeth D, Kalden JR, Manger B: Open-label study of infliximab treatment for psoriatic arthritis: clinical and magnetic resonance imaging measurements of reduction of inflammation. Arthritis Rheum 2002, 47:506-512.

Efficacy of infliximab on MRI-determined bone oedema in psoriatic arthritis. H Marzo-Ortega, D Mcgonagle, L A Rhodes, A L Tan, P G Conaghan, P O'connor, S F Tanner, A Fraser, D Veale, P Emery, Ann Rheum Dis. 66Marzo-Ortega H, McGonagle D, Rhodes LA, Tan AL, Conaghan PG, O'Connor P, Tanner SF, Fraser A, Veale D, Emery P: Efficacy of infliximab on MRI-determined bone oedema in psoriatic arthritis. Ann Rheum Dis 2007, 66:778-781.

Quantitative analyses of sacroiliac biopsies in spondyloarthropathies: T cells and macrophages predominate in early and active sacroiliitiscellularity correlates with the degree of enhancement detected by magnetic resonance imaging. M Bollow, T Fischer, H Reisshauer, M Backhaus, J Sieper, B Hamm, J Braun, Ann Rheum Dis. 59Bollow M, Fischer T, Reisshauer H, Backhaus M, Sieper J, Hamm B, Braun J: Quantitative analyses of sacroiliac biopsies in spondyloarthropathies: T cells and macrophages predominate in early and active sacroiliitis- cellularity correlates with the degree of enhancement detected by magnetic resonance imaging. Ann Rheum Dis 2000, 59:135-140.

Correlation of histopathological findings and magnetic resonance imaging in the spine of patients with ankylosing spondylitis. H Appel, C Loddenkemper, Z Grozdanovic, H Ebhardt, M Dreimann, A Hempfing, H Stein, P Metz-Stavenhagen, M Rudwaleit, J Sieper, Arthritis Res Ther. 8143Appel H, Loddenkemper C, Grozdanovic Z, Ebhardt H, Dreimann M, Hempfing A, Stein H, Metz-Stavenhagen P, Rudwaleit M, Sieper J: Correlation of histopathological findings and magnetic resonance imaging in the spine of patients with ankylosing spondylitis. Arthritis Res Ther 2006, 8:R143.

Mode of action of abatacept in rheumatoid arthritis patients having failed TNF blockade: a histological, gene expression and dynamic MRI pilot study. M H Buch, D L Boyle, S Rosengren, B Saleem, R J Reece, L A Rhodes, A Radjenovic, A English, H Tang, G Vratsanos, P O'connor, G S Firestein, P Emery, Ann Rheum Dis. 68Buch MH, Boyle DL, Rosengren S, Saleem B, Reece RJ, Rhodes LA, Radjenovic A, English A, Tang H, Vratsanos G, O'Connor P, Firestein GS, Emery P: Mode of action of abatacept in rheumatoid arthritis patients having failed TNF blockade: a histological, gene expression and dynamic MRI pilot study. Ann Rheum Dis 2009, 68:1220-1227.

Cellular characterization of magnetic resonance imaging (MRI) bone edema in rheumatoid arthritis; implications for pathogenesis of erosive disease. N Dalbeth, T Smith, S Gray, A Doyle, P Antill, M Lobo, Robinson E King, A Cornish, J Shalley, G Gao, A Mcqueen, F M , 10.1186/ar3228Ann Rheum Dis. 62Dalbeth N, Smith T, Gray S, Doyle A, Antill P, Lobo M, Robinson E, King A, Cornish J, Shalley G, Gao A, McQueen FM: Cellular characterization of magnetic resonance imaging (MRI) bone edema in rheumatoid arthritis; implications for pathogenesis of erosive disease. Ann Rheum Dis 2009, 62:279-282. doi:10.1186/ar3228

Change in CD3 positive T-cell expression in psoriatic arthritis synovium correlates with change in DAS28 and magnetic resonance imaging synovitis scores following initiation of biologic therapy-a single centre, open-label study. Pontifex, Arthritis Research & Therapy. 137Cite this article asCite this article as: Pontifex et al.: Change in CD3 positive T-cell expression in psoriatic arthritis synovium correlates with change in DAS28 and magnetic resonance imaging synovitis scores following initiation of biologic therapy-a single centre, open-label study. Arthritis Research & Therapy 2011 13:R7.


Challenges to diagnose metaplastic carcinoma of the breast through cytologic methods: an eight-case series

Seema Lale
Kiyoe Kure
Daniel Lingamfelter
Challenges to diagnose metaplastic carcinoma of the breast through cytologic methods: an eight-case series
C A S E R E P O R T Open Access
Because metaplastic carcinoma of the breast encompasses a great variety of histopathology, diagnostic challenges abound, especially within the realm of cytology. The authors compiled and studied an eight-case series comprised of metaplastic breast carcinomas and lesions initially suspicious cytologically for metaplastic carcinoma in order to assess the degree of cytologic-histologic correlation and to identify recurring problematic themes surrounding the cytology-based diagnosis of this neoplasm. The cytologic and histologic slides from eight cases suspicious for metaplastic breast carcinoma diagnosed by fine needle aspiration (FNA) were collected and analyzed through a seven-year retrospective search of case files at our institution. Based on cytologic characteristics, the cases were separated into three groups. Group 1 consisted of three cases presenting with poorly differentiated adenocarcinoma and squamoid components on FNA. Group 2 was composed of two cases that featured a monophasic, malignant ductal cell population on cytology, while the cytologic specimens for the third group of cases presented with a mesenchymal component with or without a malignant glandular constituent. Cytologic-histologic correlation was present in two of three cases demonstrating a mesenchymal component, and there was 100% sensitivity in the cytologic detection of those mesenchymal elements. However, in only one of three cases was there an accurate cytologic diagnosis of metaplastic carcinoma when squamoid changes were present on FNA. Both cases demonstrating only malignant glandular elements on cytologic specimens revealed an additional component of malignant squamous differentiation upon the examination of mastectomy-derived tissue. These results indicate that squamous-like changes identified on FNA should be interpreted with caution and that sampling error remains a problematic recurrence in cytology. Regardless, there appears to be promise concerning the accurate cytologic diagnosis of metaplastic carcinoma when the lesion is characterized by a mesenchymal component. A study implementing a larger case number is essential in determining the significance of these findings.

Background

Metaplastic carcinoma refers to a highly heterogeneous group of neoplasms characterized by an admixture of adenocarcinoma with "metaplastic" areas typically of spindle, squamous, osseous, or chondroid differentiation. These lesions are rare, with a reported incidence of less than 1% of all breast tumors in some series [1]. Wargotz et al. suggested five variants of metaplastic carcinomamatrix-producing carcinoma, squamous cell carcinoma, spindle cell carcinoma, carcinosarcoma, and metaplastic carcinoma with osteoclastic giant cells [2][3][4][5][6][7]. This entity known as "metaplastic carcinoma" can pose a diagnostic challenge, in part, because it encompasses a wealth of histopathologic variation and, consequently, can be mimicked by a wide array of other disease entities. We report a series of eight cases of metaplastic breast carcinomas or lesions initially suspicious cytologically for metaplastic carcinoma of the breast, describe their cytologic and subsequent histologic features, and discuss various challenges that we encountered in the cytologicbased diagnosis of such an entity.

* Correspondence: firstname.lastname@example.com 3 El Paso County Coroner's Office, Colorado Springs, Colorado, USA Full list of author information is available at the end of the article

Case Presentations

Eight cases suspicious for metaplastic carcinoma of the breast diagnosed on fine needle aspiration (FNA) cytology were selected from the pathology files of Truman Medical Center, Kansas City, Missouri, from 2001 through 2008. Clinically, the lesions presented as localized masses and/or abnormal mammograms. Biopsy and subsequent mastectomy specimens were reviewed for correlation (or lack thereof) with the FNA results. All specimens were reviewed by two pathologists for uniformity of reporting. Clinical data was obtained by chart review.

Materials and methods

The fine needle aspirations were performed using 22-gauge needles and three passes. Half of the smears were air-dried and half were fixed in alcohol. Air-dried smears were stained with Diff-Quik while alcohol-fixed smears underwent Papanicolaou staining. The smears were assessed for adequacy by immediately evaluating the Diff-Quik stained preparations.

The resected tissue was fixed in formalin, processed in a routine fashion, then sectioned and stained with hematoxylin and eosin. For the immunohistochemical analysis, 4-μm-thick paraffin-embedded tissue sections were deparaffinized and endogenous peroxidase quenched using 3% hydrogen peroxide in methanol. The sections were then hydrated through phosphate-buffered saline. For antigen retrieval, slides were placed in "target antigen retrieval" solution (DAKO Cytomation, Carpinteria, CA) and submerged into pre-heated antigen retrieval solution for 30 minutes in a Black and Decker steamer, after which slides were left sitting for ten minutes at room temperature, washed in phosphate buffered saline, and then exposed to the following antibodies and conditions for one hour: S-100 (1:100 dilution), cytokeratin (1:100 dilution) and CEA (1:2000 dilution). Blocking solution was used as a negative control on duplicate slides. Secondary antibody was biotinylated goat anti-immunoglobulin of mouse, rabbit, guinea pig, and rat primary antibodies (Supersensitive Immunodetection System, Biogenex, San Ramon, CA). An avidin-biotin-peroxidase complex with DAB as a chromogen was used for detecting antibody binding.

Results

A total of 8 cases suspicious for metaplastic carcinoma of the breast diagnosed on fine needle aspiration were studied by a retrospective search of files over a period of 7 years. A retrospective review of the biopsies and mastectomies performed in each case was performed. The series consisted of 8 female patients ranging from 41 to 77 years of age, with a mean age of 59 years.

Grossly, tumor sizes ranged from 2.0 cm to 13 cm. Most tumors were described as gray, white and firm. One case contained areas of obvious necrosis.

Based on the fine needle aspiration findings, we divided the eight cases into three groups. Group 1 included three cases characterized by poorly differentiated adenocarcinoma and squamoid components on fine needle aspiration. In the first case, fine needle aspiration cytology revealed poorly differentiated ductal carcinoma with focal malignant squamous differentiation. Necrotic debris was also present. Subsequent biopsy and mastectomy findings coincided with the FNA findings, showing poorly differentiated ductal carcinoma with focal malignant squamous differentiation, consistent with metaplastic carcinoma of the breast. Estrogen receptor, progesterone receptor and Her2/neu receptor status were negative.

Cytology in the second case in this group revealed multiple clusters of malignant ductal cells in a background of necrotic debris. One slide also revealed multiple clusters of large cell pearls. Single bizarre cells consistent with squamous cell carcinoma, mixed with ductal cells, were noted (Figures 1, 2). Additionally, clusters of ductal cells with apocrine metaplasia were identified. Interestingly, a subsequent mastectomy specimen revealed microinvasive and in situ ductal carcinoma ( Figure 3). A comedo portion was identified and displayed abundant areas of necrosis lined by large hyperchromatic cells with thick eosinophilic cytoplasm. Squamous elements were not seen. The nipple areola complex was positive for Paget's disease.

The third case in this group revealed numerous groups of poorly differentiated malignant cells showing ductal and squamous differentiation, and subsequent biopsy revealed poorly differentiated invasive adenosquamous carcinoma. The tumor cells were focally positive for EMA but did not stain positively for synaptophysin, chromogranin, CK7, CK20, TTF-1, GCDFP, P63 or type IV collagen. Immunoperoxidase stains for ER, PR and Her-2/neu were negative. This immunohistochemical pattern ruled out a primary breast tumor. Instead, metastatic adenocarcinoma of unknown primary origin was diagnosed.

Group 2 included two cases that were cytologically monophasic. In the first case, touch preparations were performed and revealed poorly differentiated adenocarcinoma. Subsequent mastectomy revealed invasive, poorly differentiated adenocarcinoma with a malignant squamous population. In case 2, fine needle aspiration cytology also revealed only poorly differentiated adenocarcinoma. The subsequent mastectomy, like case 1, revealed invasive poorly differentiated adenocarcinoma admixed with malignant squamous cells. In both of these cases, squamous cells were not detected on touch preparations or fine needle aspiration, and diagnoses of metaplastic carcinoma were made only after examination of the mastectomy tissue specimens.

Group 3 included three cases, all of which cytologically revealed a prominent mesenchymal component (spindle or sarcomatoid population) with or without an adenocarcinoma component.

Fine needle aspiration cytology in the first case revealed three-dimensional clusters and single malignant cells consistent with adenocarcinoma. A prominent component of malignant spindle cells was also identified. Tissue fragments of dense fibrous stroma admixed with malignant cells were observed (Figures 4, 5). The diagnostic impression was poorly differentiated adenocarcinoma, most likely of ductal origin, with possibly another poorly differentiated spindle cell component. Subsequent modified radical mastectomy revealed features of poorly differentiated invasive ductal carcinoma with sarcomatous changes (Figure 6), consistent with metaplastic carcinoma. Extensive areas of necrosis with focal chondroid differentiation were noted. Vascular invasion was present. This tumor was negative for progesterone and Her2-neu receptors but positive for estrogen.

During the FNA in case 2, 60 ml of cystic fluid was drained. Smears from the cyst wall showed atypical epithelioid and spindle cells in a background of fat necrosis, raising the suspicion for metaplastic carcinoma of the breast. The smears had high overall cellularity including multiple, scattered islands within a varying background of fat necrosis and a fibrillary, metachromatic stroma. Several mitotic figures were identified, but necrosis was absent. The case was diagnosed as suspicious for malignancy, with metaplastic carcinoma of the breast in the differential diagnosis. Subsequent biopsy and mastectomy revealed irregular, dense proliferations of plump spindle cells with moderate cytologic and nuclear pleomorphism. The majority of the nuclei showed prominent nucleoli. Mitotic figures were frequent. Additionally, tissue from the biopsy cavity demonstrated scattered, spotty foci of necrosis as well as both perivascular and perineural invasion by the tumor cells.

The proliferating spindle cells revealed diffuse positivity for the myoepithelial markers smooth muscle actin, CD10, and p63, and focal positivity for S-100. Vimentin positivity was strong and diffuse. Additionally, increased positivity for Ki-67 provided evidence for a high proliferation index among the neoplastic cells. The immunohistochemical markers glial fibrillary acid protein (GFAP), desmin, estrogen receptor (ER), and progesterone receptor (PR) did not highlight cells from the lesion. Last, positive staining with the pancytokeratin marker [AE1/AE3 + 8/18] ruled out the possibility of a sarcoma with myoepithelial differentiation. The histology and accompanying immunohistochemical staining patterns were consistent with an infiltrating myoepithelial carcinoma (i.e., sarcomatoid metaplastic carcinoma) of the breast.

Case 3 of this group showed large, pleomorphic sarcomatoid cells with bluish cytoplasm and few multinucleated giant cells and lymphocytes on cytologic smear specimens (Figures 7, 8). The case was diagnosed as favoring metaplastic carcinoma. Subsequent biopsy revealed chondrosarcomatous and pleomorphic spindle cell components with multinucleated giant cells and a brisk mitotic rate. Vimentin was positive in the chondrocytic cells while smooth muscle actin was positive in the spindle cells. S-100 and high molecular weight keratin (HMWK) were both negative. No epithelial component was identified. The differential diagnosis included chondrosarcoma and metaplastic breast carcinoma. A subsequent mastectomy specimen revealed a welldelineated tumor with a distinct chondromyxoid matrix composed of a cartilaginous component and a high grade noncartilaginous sarcoma (Figure 9). The tumor entrapped breast ductal structures. The noncartilaginous sarcoma was composed of highly anaplastic spindle cells with a slightly epithelioid appearance, hyperchromatic nuclei, prominent nucleoli, brisk mitoses (many abnormal) of approximately 11 per 10 high power fields, multinucleated tumor giant cells and osteoclast-type multinucleated giant cells. Interspersed among the high grade sarcomatous areas were nodular and lobulated islands of well-differentiated cartilage showing lacunar spaces occupied by pleomorphic chondrocytes with hyperchromatic nuclei, some of which were binucleated. The transition between the cartilaginous and sarcomatous components was sharp and abrupt without morphologic continuity. In addition there were strips of woven bone formation. S-100 immunostaining highlighted a few chondrocytes, and pancytokeratin (CK) was positive in breast ductal structures. Extraskeletal dedifferentiated chondrosarcoma served as the final diagnosis.

Discussion

Metaplastic carcinomas of the breast comprise a rare, heterogeneous group of neoplasms with varying patterns of metaplasia and differentiation. The common feature of these tumors is the presence of a predominant component other than a glandular cell population. The "metaplastic" terminology applied to this entity refers to the transformation of the glandular component into another cell type, whether it be squamous cells or mesenchymal elements such as spindle cells and osteoclastic-like giant cells. These transformed cells may appear either benign or malignant. Among our 8 cases, three (Group 1) showed squamous-like cells in addition to malignant glandular elements on FNA. Of the three, only case 1 had a perfect cytologic-histologic correlation. The cytology in case 2 showed large cells with bizarre nuclei and thick "keratinizing" cytoplasm, as well as what was originally thought to be keratin pearls. Surprisingly, the tissue from the mastectomy specimen revealed only microinvasive and in situ ductal carcinoma but also showed a comedo component containing cells characterized by dense, eosinophilic cytoplasm that strongly mimicked squamous cells on cytology. In our third case, poorly differentiated adenocarcinoma and squamous cells were revealed on fine needle aspiration, suggesting a possible metaplastic carcinoma. Biopsy confirmed the presence of both malignant glandular and squamous populations, consistent with adenosquamous carcinoma. However, immunohistochemistry ultimately ruled out the possibility of a primary breast tumor, leading to a diagnosis of metastasis.

Needless to say, care must be taken when squamoid changes are seen on FNA.

If we see suspected squamous cells on fine needle cytology, a broad differential diagnosis should be considered in addition to metaplastic carcinoma, including but not limited to such lesions as sarcoma with radiationinduced atypia, adenosquamous carcinoma, and any entity with metaplastic changes such as phylloides tumor or, as we encountered, ductal carcinoma in situ [8]. And, as case 3 shows, there is always the possibility of a metastatic lesion.

In the two cases comprising Group 2, only one component (malignant glandular) was found in the aspirates and touch preparations. However, subsequent histology revealed invasive adenocarcinoma with malignant squamous elements (i.e., metaplastic carcinoma). In our experience, 'sampling' impacts the fine needle aspiration diagnosis. Multiple needle passes and sampling of all areas of the lesion are ideal to ensure an accurate cytologic diagnosis, but in reality compromises oftentimes must be made in response to patient comfort. Therefore, the pathologist commonly must settle for suboptimal samplingtoo few passes, or perhaps sampling areas representing less than 50% or even 25% of the lesion. Also, metaplastic carcinomas not uncommonly reveal prominent cystic or necrotic areas, contributing to further complications in the identification of an additional cell population, especially when less than optimal sampling has occurred [9].

Two of the three cases from the third "mesenchymal" group showed perfect cytologic-histologic correlation, and the mesenchymal components in all three cases were detected on cytology. FNA specimens from case 1 contained both ductal and sarcomatous components of the tumor, while the cytologic examination in case 2 was positive for both epithelioid and spindle cell constituents of the myoepithelial carcinoma. An immunohistochemical panel for keratins was essential to the diagnostic workup of the latter case. Currently, no specific myoepithelial marker is available, so a battery of myogenic markers including basal cell type cytokeratins, p63, and S-100 was performed on subsequent biopsy specimens to establish a myoepithelial differentiation [10]. The cytology in the third case demonstrated large, sarcomatous cells and some multinucleated cells, thereby allowing for the detection of the neoplasm's mesenchymal derivation. Although no distinct epithelioid component was detected, a suspicion for metaplastic carcinoma nonetheless was raised. Primary chondrosarcoma, the eventual diagnosis for this lesion, is a very rare entity but should be kept in mind in the differential diagnosis in such cases, and this diagnosis should be confirmed by cytokeratin and EMA staining of the spindle cell component. In general, the differential diagnosis of spindle cell breast lesions includes metaplastic carcinoma, fibromatosis, pseudoangiomatous stromal hyperplasia, nodular fasciitis, inflammatory myofibroblastic tumor, phyllodes tumor with stromal overgrowth, and metastases [11]. Primary, pure sarcomas of the breast are very rare, the most common of which is malignant fibrous histocytoma, but these lesions still must be considered as possibilities.

Conclusion

In conclusion, our eight-case series identified several cytologic themes that surfaced in the course of diagnosis. Cytologic specimens were 100% sensitive (3/3) in the detection of mesenchymal elements, regardless of the fact that one of these cases ultimately was not diagnosed as metaplastic carcinoma. On the contrary, only in one of our cases was metaplastic carcinoma accurately diagnosed when squamoid changes were present, apparently as a result of a squamous cell mimicker and an unusual metastasis. Lastly, the two cases that were cytologically monophasic for malignant glandular elements both revealed malignant squamous components on subsequent mastectomy-derived tissue, raising speculation that sampling error remains a common culprit in cytology. This series presents intriguing findings, but our case number is quite limited. A significantly larger case series therefore would be beneficial to determine if such diagnostic themes persist on a grander scale.

It is prudent that pathologists and clinicians understand not only the benefits but also the limitations of cytologic diagnoses from FNA specimens. Immediate evaluation of specimen adequacy is useful to eliminate equivocal diagnoses from technical factors. Combining findings derived from both cytology and histology best allows for the proper management of each patient.

Consent

Consent was obtained for publication of this case report and accompanying images. A copy of the consent is available for review by the Editor-in-Chief of this journal.

Figure 1
1Clusters of malignant cells with eosinophilic cytoplasm and dark, angulated nuclei resembling squamous cells (Papanicolaou, ×20).

Figure 2
2A single, atypical squamoid cell displaying eosinophilic cytoplasm and a dark nucleus, next to a cluster of malignant glandular cells (Papanicolaou, ×20).

Figure 3
3Mastectomy specimen reveals microinvasive and in situ ductal carcinoma; no squamous component is seen (H&E, ×20).

Figure 4
4Clusters of stromal cells admixed with malignant cells (Papanicolaou, ×20).

Figure 5 Figure 6
56Singled spindle cells and atypical cells with striped cytoplasm (Papanicolaou, ×20). Poorly differentiated invasive ductal carcinoma with sarcomatous changes (H&E, ×20).

Figure 7 Figure 8
78Multinucleated giant cell (Diff-Quick, ×40). Loosely arranged cluster of pleomorphic sarcomatoid cells (Papanicolaou, ×20).

Figure 9
9Histologic features of the chondrosarcomatous and pleomorphic cell components (H&E, ×20).
Authors' contributions SL and DL constructed the majority of the manuscript. SL and KK were responsible for cytology and histology interpretations. DL and KK contributed significantly to manuscript proofreading and revisions. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
Classification of metaplastic carcinomas of the breast. F A Tavassoli, Pathol Annu. 272Tavassoli FA: Classification of metaplastic carcinomas of the breast. Pathol Annu 1992, 27(Pt 2):89-119.

Metaplastic carcinoma of the breast: clinical, mammographic, and sonographic findings with histopathologic correlation. I Günhan-Bilgen, A Memiş, E E Ustün, O Zekioglu, N Ozdemir, AJR Am J Roentgenol. 1786Günhan-Bilgen I, Memiş A, Ustün EE, Zekioglu O, Ozdemir N: Metaplastic carcinoma of the breast: clinical, mammographic, and sonographic findings with histopathologic correlation. AJR Am J Roentgenol 2002, 178(6):1421-1425.

Metaplastic carcinoma of the breast: I. Matrix producing carcinoma. E S Wargotz, H J Norris, Human Pathol. 207Wargotz ES, Norris HJ: Metaplastic carcinoma of the breast: I. Matrix producing carcinoma. Human Pathol 1989, 20(7):628-635.

Metaplastic carcinomas of the breast. II. Spindle cell carcinoma. E S Wargotz, P H Deos, H J Norris, Hum Pathol. 208Wargotz ES, Deos PH, Norris HJ: Metaplastic carcinomas of the breast. II. Spindle cell carcinoma. Hum Pathol 1989, 20(8):732-40.

Metaplastic carcinomas of the breast. III. Carcinosarcoma. Cancer. E S Wargotz, H J Norris, 64Wargotz ES, Norris HJ: Metaplastic carcinomas of the breast. III. Carcinosarcoma. Cancer 1989, 64(7):1490-9.

Metaplastic carcinomas of the breast. IV. Squamous cell carcinoma of ductal origin. E S Wargotz, H J Norris, Cancer. 652Wargotz ES, Norris HJ: Metaplastic carcinomas of the breast. IV. Squamous cell carcinoma of ductal origin. Cancer 1990, 65(2):272-6.

Metaplastic carcinomas of the breast: V. Metaplastic carcinoma with osteoclastic giant cells. E S Wargotz, H J Norris, Hum Pathol. 2111Wargotz ES, Norris HJ: Metaplastic carcinomas of the breast: V. Metaplastic carcinoma with osteoclastic giant cells. Hum Pathol 1990, 21(11):1142-50.

Khin AT: Fine-needle aspiration cytology of metaplastic carcinoma of the breast. P C Lui, G M Tse, P H Tan, G Jayaram, T C Putti, B Chaiwun, N H Chan, P P Lau, K L Mak, J Clin Pathol. 605Lui PC, Tse GM, Tan PH, Jayaram G, Putti TC, Chaiwun B, Chan NH, Lau PP, Mak KL, Khin AT: Fine-needle aspiration cytology of metaplastic carcinoma of the breast. J Clin Pathol 2007, 60(5):529-33.

Metaplastic breast carcinoma on fine-needle cytology samples: a report of three cases. F Fulciniti, G Mansueto, A Vetrani, A Accurso, A Fortunato, L Palombini, Diagn Cytopathol. 333Fulciniti F, Mansueto G, Vetrani A, Accurso A, Fortunato A, Palombini L: Metaplastic breast carcinoma on fine-needle cytology samples: a report of three cases. Diagn Cytopathol 2005, 33(3):205-9.

Metaplastic breast carcinomas: are they of myoepithelial differentiation?: immunohistochemical profile of the sarcomatoid subtype using novel myoepithelial markers. S Leibl, M Gogg-Kammerer, A Sommersacher, H Denk, F Moinfar, Am J Surg Pathol. 293Leibl S, Gogg-Kammerer M, Sommersacher A, Denk H, Moinfar F: Metaplastic breast carcinomas: are they of myoepithelial differentiation?: immunohistochemical profile of the sarcomatoid subtype using novel myoepithelial markers. Am J Surg Pathol 2005, 29(3):347-53.

Spindle cell (sarcomatoid) carcinoma of the breast: a clinicopathologic and immunohistochemical analysis of 29 cases. M R Carter, J L Hornick, S Lester, C D Fletcher, 10.1186/1746-1596-6-7Am J Surg Pathol. 303Carter MR, Hornick JL, Lester S, Fletcher CD: Spindle cell (sarcomatoid) carcinoma of the breast: a clinicopathologic and immunohistochemical analysis of 29 cases. Am J Surg Pathol 2006, 30(3):300-9. doi:10.1186/1746-1596-6-7

Challenges to diagnose metaplastic carcinoma of the breast through cytologic methods: an eight-case series. Lale, Diagnostic Pathology. 67Cite this article asCite this article as: Lale et al.: Challenges to diagnose metaplastic carcinoma of the breast through cytologic methods: an eight-case series. Diagnostic Pathology 2011 6:7.