Dr Anurag Joshi
email: dranuragjoshi@gmail.com

Dr Murali Varma

Professor Bharat Jasani
email: jasani@cardiff.ac.uk

Sue Wozniac
email: sue.wozniak@cardiffandvale.wales.nhs.uk

FCAP Consultant Frcpath

Histopathologist

Ms Sue Wozniak

Potential unreliability of normal tissue as positive control in diagnostic immunohistochemistry of poorly differentiated carcinoma

Keywords: immunohistochemistry, internal control, tumour differentiation, prostate cancer immunohistochemistry, internal control, tumour differentiation, prostate cancer

HAL is

High-molecular weight cytokeratin (HMWCK) antibody clone 34βE12 is commonly used to help distinguish high-grade urothelial carcinoma from prostatic adenocarcinoma as it is consistently expressed by urothelial carcinoma but negative in prostate cancer [START_REF] Varma | Diagnostic utility of immunohistochemistry in morphologically difficult prostate cancer: review of current literature[END_REF][START_REF] Chuang | Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma[END_REF] . The two cases described below were both referred to us for consultation with a differential diagnosis of urothelial vs. prostatic carcinoma. Tumour cells were judged to be immunonegative for prostatic markers as well as 34βE12 immunostaining (supported by adequately strong positive reactions observed in the internal control tissue elements). Despite this, unequivocal positivity for 34βE12 was observed using a more sensitive assay in our laboratory-indicating a diagnosis of urothelial carcinoma. Prostate needle biopsies performed on a 66 year old man with a 'malignant feeling' prostate and serum PSA <0.1ng/L, revealed a poorly differentiated carcinoma (Fig 1-A) that was negative for immunohistochemical staining with PSA (Prostate Specific Antigen) and HMWCK (clone 34βE12, DAKO, 1:50, Labvision autostainer, pretreatment with Trypsin for 12 minutes, 37°C, pH7.8). The basal cells of adjacent benign prostate glands were strongly 34βE12 positive (Fig 1-B).

The appearances were considered consistent with a high grade prostate carcinoma, however, in the absence of PSA staining, urothelial carcinoma could not be excluded and the case was referred to us.

At our institution, we confirmed the negativity for PSA and PSAP (prostate specific acid phosphatase). However, staining for HMWCK in Two months after the TURP, the patient expired because of unrelated medical causes. There had been no clinical, serological or radiological evidence of prostate cancer.

COMMENT

Positive controls are used to exclude false negative results resulting from suboptimal immunostaining assay performance. In the two cases presented, 34βE12 immunostaining had been considered satisfactory due to strong immunoreactivity in the internal positive control tissue.

However, the original immunohistochemical method used in these two cases has been shown to be suboptimal with respect to the sensitivity of 34βE12 immunostaining as an urothelial marker (a heat induced antigen retrieval method is preferred to enzyme predigestion in this scenario) [START_REF] Varma | High molecular weight cytokeratin antibody (clone 34βE12): a sensitive marker for differentiation of high-grade invasive urothelial carcinoma from prostate cancer[END_REF] .

The cases highlight an important issue regarding reliance upon internal positive controls in routine immunohistochemistry. Internal controls are generally regarded as superior to external controls by virtue of these 'control' cell populations having been subjected to identical fixation as the tumour cells being investigated. In these cases, although the original 34βE12 immunostaining showed strong positivity in the positive internal controls (basal cells of benign prostatic acini in case 1 and benign urothelium in case 2) the urothelial carcinoma cells were falsely negative, as subsequently shown by a more sensitive method. The sensitivity of 34βE12 as an urothelial marker has been shown to be higher with heat retrieval pre-treatment as used in our laboratory as compared to enzyme pre-digestion that had been employed in the referring laboratories [START_REF] Varma | High molecular weight cytokeratin antibody (clone 34βE12): a sensitive marker for differentiation of high-grade invasive urothelial carcinoma from prostate cancer[END_REF] .

Internal positive controls are most reliable when they are similar in their antigen content and distribution to the "tumour cells". Under these circumstances, the quality control of assay sensitivity afforded by them is considered ideal; as is the case when 34βE12 is used as a basal cell marker to support the diagnosis of prostatic adenocarcinoma (where the basal cells of benign prostatic glands serve as the internal control).

It should be noted that the same issues would also apply to external positive controls. The external positive control tissue must be appropriate to the tissue being tested. Thus, when 34βE12 is used as a prostatic basal cell marker, benign prostate would be a suitable internal/external positive control. However, if the same antibody is used as an urothelial marker, the ideal external control would be high-grade urothelial carcinoma.

  positive and negative tissue controls is an important part of the technical quality assurance in immunohistochemistry. Positive controls may be 'external', where known positive tissue is immunostained in parallel to the test slide; or 'internal', with known positive tissue/ cells present within the tested tissue section. Internal controls are generally considered superior to external controls as the control cells have been subjected to identical conditions of fixation and immunostaining as the test cells. We describe 2 cases to demonstrate that even an apparently good quality internal control result is not adequate proof of assay sensitivity.

2

 2 our laboratory (clone 34βE12; DAKO; 1:40; ChemMate/ HRP detection kit; microwave pre-treatment 800W 20min, EDTA pH7.0) revealed diffuse and intense staining of tumour cells (Fig 1-C). A diagnosis of high grade urothelial carcinoma was made and confirmed on a subsequent radical cysto-prostatectomy (Fig 1-D). CASE Transurethral resection of the prostate (TURP) performed on a 73-yearold man for urinary retention (PSA 1.5ng/L) showed a poorly differentiated carcinoma (Fig 2-A) that was negative for PSA, PSAP and HMWCK (clone 34βE12, DAKO, Ventana autostainer, Protease 1 pretreatment) (Fig 2-B). Background benign urothelium was strongly 34βE12 positive (Fig 2-B, inset). Because of this immunohistochemical origin of this tumour was considered uncertain and the case was referred for our further opinion. Immunohistochemistry performed in our laboratory confirmed that the tumour cells were negative for PSA and PSAP, however, HMWCK (clone 34βE12; DAKO; 1:40; ChemMate/ HRP detection kit; microwave pre-treatment 800W 20min, EDTA pH7.0) stained the tumour cells diffusely and intensely (Fig 2-C). The immunohistochemical staining profile was thus interpreted as in keeping with high-grade urothelial carcinoma.

  Fig 1 A) Prostate biopsy with poorly differentiated carcinoma (H&E) B) Tumour cells are negative on 34ßE12 immunostaining after enzyme predigestion, note positive staining in prostatic basal cells (internal control) C) Diffusely positive tumour cells on 34ßE12 immunostaining after heat mediated antigen retrieval. D) Follow-up cystectomy specimen showing invasive urothelial carcinoma and urothelial carcinoma in situ (inset) (H&E) 209x297mm (300 x 300 DPI)

Fig 1 A)

 1 Fig 1

Published on behalf of the British Division of the International Academy of Pathology Histopathology


Sophie E Polo

Stamatios E Theocharis

Laure Grandin

Laetitia Gambotti

Guillemette Antoni

Alexia Savignoni

Bernard Asselain

Efstratios Patsouris

Dr Geneviève Almouzni
email: genevieve.almouzni@curie.fr.

overexpression in human solid tumors

Keywords: chromatin assembly, carcinoma, marker, prognosis, proliferation chromatin assembly, carcinoma, marker, prognosis, proliferation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cancer is a major cause of death in developed countries [START_REF] Jemal | Cancer statistics[END_REF] . Early tumor detection as well as appropriate therapeutic decisions based on predicted clinical outcome and response to treatment are of key importance to treat cancer patients. Prediction of clinical outcome is generally based on the anatomical extent of disease (stage) and the degree of tumor differentiation (grade). In addition, molecular biomarkers 2 including proliferation markers [START_REF] Whitfield | Common markers of proliferation[END_REF] can be used for cancer diagnosis and prognosis. A common feature of cancer pathogenesis is indeed uncontrolled and rapid cell proliferation [START_REF] Hanahan | The hallmarks of cancer[END_REF] . Accordingly, genes regulating cell proliferation have been observed to be overexpressed in the vast majority of tumor cells [START_REF] Whitfield | Common markers of proliferation[END_REF] .

Not only genetic alterations but also abnormalities at the chromatin level can contribute to the aberrant pattern of gene expression that promotes proliferation of cancer cells [START_REF] Esteller | Epigenetics provides a new generation of oncogenes and tumour-suppressor genes[END_REF][START_REF] Iacobuzio-Donahue | Epigenetic Changes in Cancer[END_REF][START_REF] Jones | The epigenomics of cancer[END_REF] . Chromatin abnormalities have even been proposed to be an early and seminal event for cancer progression [START_REF] Baylin | Epigenetic gene silencing in cancer -a mechanism for early oncogenic pathway addiction?[END_REF] , and anti-cancer treatments targeting chromatin components are in development [START_REF] Yoo | Epigenetic therapy of cancer: past, present and future[END_REF] . While active research is aimed at a better understanding of distinct chromatin defects during tumoral progression, Chromatin alterations are certainly considered as valuable markers of malignant transformation 10 11 , which may also provide prognostic information (reviewed in [START_REF] Kurdistani | Histone modifications as markers of cancer prognosis: a cellular view[END_REF] ).

The structural organization of DNA with histone proteins into chromatin [START_REF] Kornberg | Structure of chromatin[END_REF] is indeed of key importance for the regulation of gene expression, and maintenance of such an organization is thus crucial to preserve genome integrity and cellular identity [START_REF] Groth | Chromatin challenges during DNA replication and repair[END_REF] . Chromatin assembly [START_REF] Polo | Chromatin assembly: a basic recipe with various flavours[END_REF] involves specific factors called histone chaperones that escort histone proteins up to their deposition onto DNA [START_REF] Corpet | Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information[END_REF][START_REF] Koning | Histone chaperones: an escort network regulating histone traffic[END_REF] . Both during replication [START_REF] Stillman | Chromatin assembly during SV40 DNA replication in vitro[END_REF] and repair events [START_REF] Gaillard | Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I[END_REF] , chromatin assembly is coupled to DNA synthesis. The only chaperone know so far that is able to promote this activity is called CAF-1 (Chromatin Assembly Factor-1), a trimeric nuclear [START_REF] Smith | Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro[END_REF] , made of p150, p60 [START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I -a molecular link between newly synthesized histones and DNA replication[END_REF] and p48 subunits 22 in human cells. Recent studies have highlighted CAF-1 intimate relationship with cell proliferation. In particular, CAF-1 expression is massively downregulated in quiescent cells [START_REF] Polo | Chromatin assembly factor-1, a marker of clinical value to distinguish quiescent from proliferating cells[END_REF] and appears essential for human cell proliferation [START_REF] Nabatiyan | Silencing of chromatin assembly factor 1 in human cells leads to cell death and loss of chromatin assembly during DNA synthesis[END_REF][START_REF] Ye | Defective S phase chromatin assembly causes DNA damage, activation of the S phase checkpoint, and S phase arrest[END_REF][START_REF] Hoek | Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo[END_REF] . Furthermore, we previously validated the use of CAF-1 p60 expression as a proliferation marker of clinical value in breast cancer [START_REF] Polo | Chromatin assembly factor-1, a marker of clinical value to distinguish quiescent from proliferating cells[END_REF] . Here, we sought to extend these findings to a series of different human solid tumors and test the validity of CAF-1 p60 expression as a prognostic factor.

F o r P e e r R e v i e w 4 complex

Materials and methods

Selection of cases

Archival formalin-fixed, paraffin-embedded tissues and clinical material of common solid tumors including renal, colon, gastric, thyroid, pancreas, prostate, cervical, endometrial and breast cancer cases were provided by the First Department of Pathology of the University of Athens, which is a referral facility for the four University Hospitals in Athens, Greece.

Selected patients correspond to consecutive cases of primary invasive tumors selected from an electronic medical record and diagnosed between 1994 and 2005, depending on the tumor type. All patients were examined routinely, according to institutional guidelines for each cancer type. The data reviewed, recorded in the hospital files, included baseline clinical and pathological data (available in Supporting information, Supplementary Table S1), the occurrence and the cause of death. Data were extracted in 2006. Immunohistochemical studies were performed on tissues obtained at surgery for cancer diagnosis. None of the patients had received either chemotherapy or radiotherapy before surgery. Patients received appropriate treatment afterwards, following standard procedures based on disease stage. Each patient had given informed consent and the study was approved by the ethical committee of the Medical School of University of Athens.

Immunohistochemical staining

Immunostainings for p60, Ki-67 and MCMs were performed on formalin-fixed paraffin embedded tissue sections (4 µm) as previously described [START_REF] Polo | Chromatin assembly factor-1, a marker of clinical value to distinguish quiescent from proliferating cells[END_REF] using the corresponding antibodies: CAF-1 p60 (mAb8133, Abcam), Ki-67 (MIB1, DAKO) and MCM proteins (MCM-2: MCA1859; MCM-5:MCA1860, Serotec). The percentages of positively stained cells in immunocytochemistry experiments (labeling indexes) were obtained by scoring at least 1000 cells in each case by two independent observers, and the mean values were used for the following statistical analyses (S-Plus 2000 software, MathSoft Inc.). Correlations between continuous variables were studied using Spearman's rank correlation test. For multiple group comparisons of continuous variables the non parametric Kruskal-Wallis test was employed. Comparisons of labeling indexes obtained with two different markers between paired samples were performed by the non parametric Wilcoxon's signed-rank test. Statistical significance was taken as p<0.05. Survival was assessed through medical records and defined as cause-specific survival, calculated from the date of surgical excision of the primary tumor to the date of death due to cancer or last followup. Survival data were systematically collected on the date of data extraction by telephone call to the patient. One case was censored because mortality was due an accident. Losses-tofollow-up were limited to an acceptable percentage of cases except in cervical cancer, where we verified that it did not bias the analysis since baseline characteristics of patients with or without survival data were not statistically different (Table S1). Survival curves were derived from Kaplan-Meier estimates. The association of CAF-1 p60 expression with clinical outcome was determined by univariate and multivariate Cox regression models with forward stepwise inclusion of factors and an inclusion criterion of p<0.20. CAF-1 p60 expression was introduced as continous variable and as categorical variable in the models. Categorical variable was obtained by performing a median split of distribution. Due to the strong correlation between CAF-1 p60 and Ki-67, the latter was excluded from multivariate analyses.

Statistical analyses

Results

CAF-1 is a proliferation marker in various human carcinomas

In order to test the validity of CAF-1 expression as a proliferation marker in solid tumors, we performed immunohistochemical detection of CAF-1 p60 on a series of paraffinembedded tissue sections including breast (n=79), cervical (n=64), endometrial (n=62), renal (n=55), prostate (n=43), gastric (n=64), colon (n=108), pancreatic (n=47) and thyroid (n=52) carcinoma cases (Figure 1A). In each cancer type, we tested the correlation of CAF-1 p60 expression with the expression of the known marker of proliferation Ki-67 [START_REF] Brown | Monoclonal antibody Ki67: its use in histopathology[END_REF] . We also analyzed the association of CAF-1 expression with clinicopathological variables important for patients´ care and their final outcome (listed in Supporting information, Supplementary Table S1), in particular stage and grade.

Strong positive correlations were observed between CAF-1 p60 and Ki-67 labeling indexes in all tumors examined (from r=0.80 in cervical cancer to r=0.99 in pancreatic cancer, Figure 1B), demonstrating that CAF-1 is a proliferation marker of general value in human carcinomas. A consistently weaker correlation, although still significant, was observed with MCM proliferation markers [START_REF] Tachibana | Cell-cycle-dependent regulation of DNA replication and its relevance to cancer pathology[END_REF][START_REF] Freeman | Minichromosome Maintenance Proteins as Biological Markers of Dysplasia and Malignancy[END_REF][START_REF] Gonzalez | Control of DNA replication and its potential clinical exploitation[END_REF] as exemplified in gastric cancer (Figure 2A). Interestingly, MCM markers also commonly display significantly higher labeling indexes than CAF-1 p60 (p<0.001 in colon, gastric and thyroid cancer, Figure 2B) while the fraction of cells detected with CAF-1 p60 and Ki-67 is comparable (based on Wilcoxon's signed-ranked test, data not shown).

Furthermore, CAF-1 p60 expression was significantly associated with histological grade in breast, cervical, endometrial and renal cell carcinoma (p=0.016, p=0.037, p=0.0006, p=0.0006, respectively), and with disease stage in endometrial and renal cell carcinoma Taken together, these data demonstrate that CAF-1 is a proliferation marker of clinical significance in a series of human solid tumors.

CAF-1 expression is associated with adverse clinical outcome in a subset of tumor types

The significant association of CAF-1 p60 expression with tumor stage and grade at least in a subset of tumor types prompted us to analyse the potential prognostic value of CAF-1 marker. We therefore examined the association between CAF-1 p60 expression and causespecific patient survival by Kaplan-Meier analysis and Cox regression models on a series of patients with median follow-up ranging from 8 months for pancreatic cancer to 81 months for cervical cancer (Supporting information, Supplementary Table S1). Interestingly, CAF-1 p60 expression stood out as an independent predictor of survival in renal, endometrial and cervical cancers (Table 1, Figure 3). High levels of CAF-1 p60 immunostaining were indeed associated with poor clinical outcome in these cancer types (hazard ratio for death, 4.4, 5.5 and 3.3, with p=0.006, 0.03 and 0.04 for renal, endometrial and cervical tumors, respectively), based on multivariate Cox regression analyses (Table 1, Figure 3). The most significant differences were observed in renal cancer with 16

Discussion

In this study, we have shown that the expression of the chromatin assembly factor CAF-1 is a clinically relevant indicator of cell proliferation in human carcinomas, thus confirming and extending our previous findings in breast cancer cases [START_REF] Polo | Chromatin assembly factor-1, a marker of clinical value to distinguish quiescent from proliferating cells[END_REF] . This property most likely relates to CAF-1 function coupled to DNA replication. Given the additional role of CAF-1 in chromatin assembly coupled to DNA repair [START_REF] Gaillard | Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I[END_REF][START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF][START_REF] Martini | Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells[END_REF][START_REF] Polo | New Histone Incorporation Marks Sites of UV Repair in Human Cells[END_REF][START_REF] Lan | In situ analysis of repair processes for oxidative DNA damage in mammalian cells[END_REF][START_REF] Okano | Spatial and temporal cellular responses to single-strand breaks in human cells[END_REF] , it would also be worthwhile investigating its potential utility in monitoring/predicting tumor response to therapy. This requires a detailed analysis of CAF-1 expression levels in cells exposed to different sources of DNA damage, an issue that has begun to be considered only in one study [START_REF] Nabatiyan | Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells[END_REF] .

Our analyses also reveal that CAF-1 displays significant but notably weak correlations with MCM proliferation markers, which generally label a larger proportion of cells, suggesting that they detect distinct cell populations. This is consistent with the idea that MCMs mark cells with replicative potential in addition to actively proliferating cells [START_REF] Stoeber | DNA replication licensing and human cell proliferation[END_REF] while CAF-1 only labels the latter. A combination of both MCM and CAF-1 markers would thus be most useful to consider in difficult cases since MCM markers seem more sensitive and CAF-1 more discriminative.

In addition to its clinical utility for refining cancer diagnosis, CAF-1 marker also displays prognostic value, at least in a subset of the tumor types analysed (in particular in renal carcinomas), which can be useful for guiding therapeutic decisions. Interestingly, CAF-1 p60 was also recently put forward as a marker of prognostic value in prostate cancer and squamous cell carcinoma of the tongue [START_REF] Staibano | Overexpression of chromatin assembly factor-1 (CAF-1) p60 is predictive of adverse behaviour of prostatic cancer[END_REF][START_REF] Staibano | Chromatin assembly factor-1 (CAF-1)-mediated regulation of cell proliferation and DNA repair: a link with the biological behaviour of squamous cell carcinoma of the tongue?[END_REF] , which further expands the list of human carcinomas in which high CAF-1 p60 expression is associated with adverse clinical outcome. Elucidation of the biological role of CAF-1 in cell proliferation and tumorigenesis is also of major interest. In particular, whether CAF-1 overexpression has oncogenic potential, as recently described for replication licensing factors [START_REF] Liontos | Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior[END_REF] , remains to be examined. Furthermore, assessment of CAF-1 expression in early pre-cancerous lesions would also help clarifying when CAF-1 overexpression occurs in the course of cancer development.

Our results also need to be considered in light of the emerging concept of cancer stem cells [START_REF] Ward | Cancer stem cells: at the headwaters of tumor development[END_REF] , which refers to a small subpopulation of tumor cells that is required for tumor growth and therefore constitutes the main target of anti-cancer therapies. It would be interesting to compare CAF-1 expression in cancer stem cells relative to other cells within the tumor to determine if this marker is also associated with the cancer stem cell phenotype and if it could contribute to more sensitive detection methods for cancer stem cells. Another population of cells in which assesment of CAF-1 expression would be informative is stromal fibroblasts of the tumor microenvironment, which are known to potentiate tumor development [START_REF] Bhowmick | Stromal fibroblasts in cancer initiation and progression[END_REF] . These cells have indeed been reported to differ from their normal counterparts in terms of proliferation/migration capacities, which relates to differences in gene expression arising from chromatin changes rather than genetic alterations [START_REF] Qiu | No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas[END_REF][START_REF] Hu | Distinct epigenetic changes in the stromal cells of breast cancers[END_REF][START_REF] Allinen | Molecular characterization of the tumor microenvironment in breast cancer[END_REF] Finally, in light of recent genome-wide analyses of chromatin marks that identify specific alterations in human cancer cells (reviewed in 46 ), our study underlines that a better understanding of chromatin-related events associated with tumorigenesis can prove useful for cancer diagnosis and prognosis.

8 (

 8 p=0.012 and p=0.015, respectively). No significant associations with any other clinicopathological data examined were noted in our study.

9 (

 9 deaths in 22 patients with high p60 staining (median survival 8 months, range 2-66 months) compared with 7 deaths in 29 patients with low p60 staining (median survival 17 months, range 3-144 months). Our analyses did not show any significant prognostic value of CAF-1 detection in colon (p=0.64), gastric (p=0.43) and pancreatic (p=0.23) cancer. Univariate Cox regression analyses showed similar trends with CAF-1 p60 expression introduced as continous variable in the models data not shown). Longer follow-up of patients will be required to examine CAF-1 prognostic value in slowly progressing breast, prostate and thyroid cancers.Taken together, our data highlight the tight association of CAF-1 p60 expression with the proliferative state of the cells in various human solid tumors, validating its use as a general proliferation marker. Additionally, CAF-1 p60 level proved of prognostic significance in a subset of tumor types, namely renal, cervical and endometrial. These new data extend the clinical value of CAF-1 to assess cancer progression.

  M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 2007;8(4):286-98.

Figure 1 :

 1 Figure 1 : Comparison between CAF-1 p60 and Ki-67 labeling indexes in various solid

Figure 2 :

 2 Figure 2: Comparison between CAF-1 p60 and MCMs labeling indexes.

Figure 3 :

 3 Figure 3 : Association of CAF-1 p60 expression level with clinical outcome.

Table 1 :

 1 CAF-1 p60 prognostic value (prediction of cause-specific survival) determined by univariate and multivariate Cox regression models in the indicated tumors.

		UNIVARIATE		MULTIVARIATE
	Cancer	N HR 95% C.I.	p	HR	95% C.I.	p
	Renal	51
	p60=<15%	29 1		1
	p60>15%	22 4.9 [1.9 -12.6] <0.001 4.4 [1.4 -14.0] 0.006 *
	Endometrial 52
	p60=<35%	26 1		1
	p60>35%	26 8.7 [1.9 -38.7]	0.005 5.2 [1.1 -25.6]	0.03 †
	Cervical	35
	p60=<40%	17 1		1
	p60>40%	18 2.6 [0.8 -8.1]	0.08 § 3.3 [1.0 -10.6]	0.04 ‡
	Gastric	63		-	-	NS
	p60=<55%	33 1
	p60>55%	30 1.4 [0.6 -2.9]	0.43
	Pancreatic	39		-	-	NS
	p60=<20%	19 1
	p60>20%	20 1.5 [0.8 -2.8]	0.23
	Colon	108		-	-	NS
	p60=<23%	55 1
	p60>23%	53 0.9 [0.5 -1.5]	0.64
	In addition of p60, forward stepwise inclusions were performed with:
	* age (not included in the final model), TNM (p=0.003) and Fuhrman grade (p=0.003)
	† histological grade (not included in the final model) and FIGO stage (p=0.005)
	‡ histological grade and lymph node invasion (global p-value=0.009)
	§ In the cervical cancer cases analysed in our study, squamous histological type that has good
	prognosis is more frequently, although not significantly, associated with high p60 values than
	adenocarcinoma type, an indicator of bad prognosis. This explains why p60 shows a

Page 25 of 25 Published on behalf of the British Division of the International Academy of Pathology Histopathology

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Acknowledgements

This work was supported by la Ligue Nationale contre le Cancer (Equipe labellisée la Ligue), ACI-2007-Cancéropôle IdF 'Breast Cancer and Epigenetics', the European Commission Network of Excellence Epigenome (LSHG-CT-2004-503433), contract RTN (HPRN-CT-2002-00238), ACI-DRAB (no 04393) and a collaborative program 'Paramètres Epigénétiques' between the Curie Institute and the CEA. S. E. P. was supported by an ARC fellowship followed by a post-doctoral fellowship from Human Frontier Science Program Organization. We thank Faculty Members of the Medical School of the University of Athens (E. Agapitos, S. Tseleni, A. Kyroudi, G. Kouraklis, D. Mytropoulos) for samples and clinical data collection.

F o r P e e r R e v i e w


Uta Flucke
email: u.flucke@pathol.umcn.nl

Theo Jm Hulsebos

Han Van Krieken

Thomas Mentzel

Theo J M Hulsebos

J Han

J M Van Krieken

Myxoid epithelioid sarcoma: a diagnostic challenge! A report on six cases

Keywords: epithelioid sarcoma, myxoid, soft tissue tumors, myoepithelioma epithelioid sarcoma, myxoid, soft tissue tumours, myoepithelioma

HAL is

Myxoid epithelioid sarcoma: a diagnostic challenge! A report on six cases

Aims: Epithelioid sarcoma (ES) is a distinct sarcoma-type with a specific morphology and immunophenotype. Whereas focal myxoid change does occur, to our knowledge only two cases of ES with diffuse myxoid stroma were reported until today. To better characterize the myxoid variant of ES, we describe six additional cases and discuss the differential diagnoses.

Methods and results: Cases were retrieved from the authors' files and studied histologically, immunohistochemically and by molecular methods. The age of the patients, four females and two males, ranged from 16 to 74 years (median: 33 years). The neoplasms arose in an extremity (two cases), the abdominal wall, groin, perineum, and shoulder (one case each). Histologically, four cases were of the conventional type, and two were of the proximal variant and the immunophenotype was typical for ES. The tumour stroma however, revealed prominent myxoid changes, ranging from 50 to 90% (median: 75%). Only one of the proximal type ES showed a SMARCB1 mutation, whereas the other tumours had no mutation of this gene.

Conclusions:

The myxoid variant of ES represents a diagnostic challenge and may be confused with other myxoid benign and malignant neoplasms. The main differential diagnosis is myoepithelioma of the skin and soft tissue.

Abbreviations: ES, epithelioid sarcoma; EMC, extraskeletal myxoid chondrosarcoma; CK, pancytokeratin; EMA, epithelial membrane antigen; SMA, alpha smooth muscle actin; EMA, epithelial membrane antigen; GFAP, glial fibrillary acidic protein; NA, not analyzed In the first description of 62 cases of epithelioid sarcoma (ES) in 1970 by Enzinger, extensive extracellular mucinous material was present in 5 of 15 cases, that had been stained for mucopolysaccharides. [START_REF] Enzinger | Epithelioid sarcoma: a sarcoma simulating a granuloma or a carcinoma[END_REF] Nevertheless, myxoid stromal change in ES was described as a rare occurrence in subsequent studies. [START_REF] Evans | Epithelioid sarcoma: a clinicopathologic and prognostic study of 26 cases[END_REF][START_REF] Hasegawa | Proximal-type epithelioid sarcoma: a clinicopathologic study of 20 cases[END_REF][START_REF] Fisher | Epithelioid sarcoma of Enzinger[END_REF] In contrast to ES, benign or malignant myoepithelial neoplasms of skin and soft tissues show often, at least focally, prominent myxoid stroma. [START_REF] Kilpatrick | Mixed tumors and myoepitheliomas of soft tissue: A clinicopathologic study of 19 cases with a unifying concept[END_REF][START_REF] Kutzner | Cutaneous myoepithelioma: an underecognized cutaneous neoplasm composed of myoepithelial cells[END_REF][START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF][START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF] Like ES, cases of myoepithelioma tend to occur on the extremities of adolescents and adults and may show overlapping morphological features with ES such as a nodular proliferation of spindled and epithelioid tumour cells. More rarely, myoepithelioma and ES may occur in children. [START_REF] Enzinger | Epithelioid sarcoma: a sarcoma simulating a granuloma or a carcinoma[END_REF][START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF][START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF][START_REF] Chase | Epithelioid sarcoma: Diagnosis, prognostic indicators and treatment[END_REF][START_REF] Casanova | Epithelioid sarcoma in children and adolescents: A report from the Italian Soft Tissue Sarcoma Committee[END_REF] The immunophenotype of myoepitheliomas and ES shares similarities with simultaneous expression of epithelial markers and vimentin, although actins are more rarely expressed in ES. Discriminating markers for myoepitheliomas are S-100, GFAP and calponin, positive in the majority of cases. [START_REF] Kutzner | Cutaneous myoepithelioma: an underecognized cutaneous neoplasm composed of myoepithelial cells[END_REF][START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF][START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF] Approximately 50 to 67% of ES are positive for CD34 which is not seen in myoepitheliomas. [START_REF] Hasegawa | Proximal-type epithelioid sarcoma: a clinicopathologic study of 20 cases[END_REF][START_REF] Kilpatrick | Mixed tumors and myoepitheliomas of soft tissue: A clinicopathologic study of 19 cases with a unifying concept[END_REF][START_REF] Kutzner | Cutaneous myoepithelioma: an underecognized cutaneous neoplasm composed of myoepithelial cells[END_REF][START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF][START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF][START_REF] Mirra | The fibroma-like variant of epithelioid sarcoma[END_REF][START_REF] Guillou | Proximal-type epithelioid sarcoma, a distinctive aggressive neoplasm showing rhabdoid features: clinicopathologic, immunohistochemical, and ultrastructural study of a series[END_REF][START_REF] Miettinen | Epithelioid sarcoma: an immunohistochemical analysis of 112 classical and variant cases and a discussion of the differential diagnosis[END_REF][START_REF] Laskin | Epithelioid sarcoma. New insights based on an extended immunohistochemical analysis[END_REF][START_REF] Chbani | Epithelioid sarcoma. A clinicopathologic and immunohistochemical analysys of 106 cases from the French Sarcoma Group[END_REF] Most cases of ES (> 80%) show loss of SMARCB1 protein expression, and this phenomenon is also described in a subset of myoepithelial carcinomas. [START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF][START_REF] Chbani | Epithelioid sarcoma. A clinicopathologic and immunohistochemical analysys of 106 cases from the French Sarcoma Group[END_REF][START_REF] Hornick | Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma[END_REF][START_REF] Kohashi | Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor[END_REF] In this study we describe the so far largest series of 6 cases of the myxoid variant of the distal and proximal type ES, and discuss the differential diagnosis, with special emphasis on the distinction from benign and malignant myoepithelioma of skin and soft tissue.

Material and methods:

The cases were retrieved from the files of the Department of Pathology, Radboud University Medical Centre Nijmegen (Nijmegen, The Netherlands), of the Dermatopathologie Bodensee (Friedrichshafen, Germany) and from the consultation files of one of the authors (TM). One case was selected from PALGA, the nationwide network and registry of histopathology and cytopathology in the Netherlands.

Slides were reviewed and the diagnosis of ES was based on histological and immunohistochemical criteria according to WHO classification. [START_REF] Guillou | Epithelioid sarcoma[END_REF] Cases in which 50% or more of the tumour area showed myxoid change were included in the study. The specimens had been obtained by surgical excision, fixed in 10% buffered formalin, and processed for paraffin embedding. Four micrometer-thick sections were stained with haematoxylin and eosin, and immunohistochemically by the labeled streptavidinbiotin technique using commercially available antibodies; appropriate positive and negative controls were used in each case. Antibodies, their sources and dilutions are summarized in Table 1. Genomic DNA was extracted from the paraffin embedded specimens of all six cases by use of commercially available kits (Qiagen, Venlo, The Netherlands). SMARCB1 mutation analysis was performed as described previously. [START_REF] Hulsebos | Germline mutation of INI1/SMARCB1 in familial schwannomatosis[END_REF] The clinical information were obtained from the referring clinicians and pathologists.

Results

:

Clinical findings

Details are summarized in Table 2.

The neoplasms arose in four females and two males. The age of the patients ranged from 16 to 74 years (median: 33 years). Anatomic sites included the extremities (two cases), the abdominal wall, perineum, groin, shoulder (one case each). The patients were treated by wide excision or reexcision with tumour free margins in all cases. Local recurrences were seen in three cases, two of the distal and one of the proximal type. The two recurring neoplasm of the distal type showed two and four recurrences respectively and subsequently lymph node metastases developed.

Pathological features

The size of the neoplasms ranged from 1.0 to 4.0 cm (median: 1.8 cm). Sites of involvement were the dermis and subcutis of the abdominal wall, schoulder, and, hand and soft tissue of the lower leg (distal type), and subcutis of the groin, and vulva ( proximal type).

Microscopically, in all tumours a (multi)nodular growth with infiltrative margins was seen (Figure 1). The tumour cells were arranged in nests and cords, with the myxoid stromal background. A reticular growth pattern was observed in three cases (two of the distal type and one of the proximal type) (Figures 2 and3). Classical solid areas were seen in all cases (Figures 1 and4). The epithelioid tumour cells contained abundant eosinophilic cytoplasm and round, vesicular nuclei. Prominent nucleoli were variably seen. In all four cases of the distal type scattered spindle-shaped tumour cells were noted. In two cases plasmocytoid cells were also recognized (Figure 5). Centrally the tumours showed hyalinization (4 cases) and/or necrosis (3 cases).

The two cases of proximal type were composed of larger epithelioid and rhabdoid tumor cells (Figures 3 and4).

Necrosis was seen in one of the proximal type, and in all but one of the distal ES. Inflammatory reaction was more prominent in the cases of the distal type. The mitotic rate ranged from 12 to 24 mitoses per 2mm 2 in the cases of the proximal type and from 3 to 11 mitoses per 2mm 2 in the cases of the distal type. Two cases were 90% myxoid, and one case each showed 80% and 70% myxoid stromal change, whereas in two cases 50% myxoid areas were observed (Table 2).

Immunohistochemically, tumour cells in all cases stained strongly positive for vimentin, pancytokeratin (Figure 6) and EMA. In addition, CD34 was positive in two cases (Figure 7), and expression of SMA was focally present in two cases. A weak and focal positivity of calponin was seen in three cases. SMARCB1 (BAF47) was absent in all six cases (Figure 8). None of the neoplasms expressed S-100 protein, GFAP, and p63 (Table 3).

SMARCB1 gene status

All tumours were investigated for the presence of mutations in the SMARCB1 gene. In one of them a mutation in exon 6 was recognized, which we reported previously. [START_REF] Flucke | Direct evidence of mutational inactivation of SMARCB1/INI1 in epithelioid sarcoma[END_REF] In this case of proximal type, localized in the vulva, we identified a c.769C>T transition resulting in the generation of an in-frame stopcodon (CAG->TAG, p.Q257X) in the transcript and premature termination of SMARCB1INI1 protein synthesis. In accordance with the two-hit model for tumour genesis, we noted considerable loss of the wild-type copy of this gene in the tumour. All other cases had no mutation of SMARCB1.

Discussion:

Myxoid tumours of soft tissue encompass a heterogenous group of lesions characterized by a marked abundance of extracellular mucoid (myxoid) matrix. They demonstrate significant variability in their biological behavior thus including indolent tumours, tumours with a tendency to recur locally but not metastasize, and malignant tumours. [START_REF] Van Roggen | Myxoid tumors of soft tissue[END_REF] Although prominent myxoid stromal changes in classical ES is well known, to date, to our knowledge only two cases of ES with diffuse myxoid changes have been described. [START_REF] Enzinger | Epithelioid sarcoma: a sarcoma simulating a granuloma or a carcinoma[END_REF][START_REF] Fisher | Epithelioid sarcoma of Enzinger[END_REF][START_REF] Miettinen | Epithelioid sarcoma: an immunohistochemical analysis of 112 classical and variant cases and a discussion of the differential diagnosis[END_REF][START_REF] Fadare | Myxoid epithelioid sarcoma: Clinicopathologic analysis of 2 cases[END_REF] We report an additional series of six cases of myxoid ES and discuss their morphological, immunohistochemical and molecular features, and the differential diagnosis to other benign and malignant neoplasms of skin and soft tissues.

The reported neoplasms, two of the proximal and four of the distal type, were localized in the dermis, subcutis and deep soft tissues, and showed classical morphological and immunohistochemical features of ES as established. In addition, prominent myxoid stromal changes were seen in all cases, and tumour cells were often arranged in cords and nests, a rather reticular architecture was seen in three cases.

Our findings are comparable to those described earlier for two distal type myxoid ES. showing loss of SMARCB1 protein expression in more than 80% of cases of ES. [START_REF] Chbani | Epithelioid sarcoma. A clinicopathologic and immunohistochemical analysys of 106 cases from the French Sarcoma Group[END_REF][START_REF] Hornick | Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma[END_REF][START_REF] Kohashi | Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor[END_REF][START_REF] Flucke | Direct evidence of mutational inactivation of SMARCB1/INI1 in epithelioid sarcoma[END_REF] Only one of our proximal type ES showed a SMARCB1 mutation, whereas the other tumours had no mutation. The low frequency of mutations affecting the SMARCB1 gene in our series of ES is comparable to others and suggests that the expression of SMARCB1 in this tumour type is down-regulated by alternative mechanisms, such as epigenetic modification. [START_REF] Kohashi | Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor[END_REF][START_REF] Flucke | Direct evidence of mutational inactivation of SMARCB1/INI1 in epithelioid sarcoma[END_REF] The main differential diagnosis of myxoid ES represents myoepithelioma of skin and soft tissues.

These neoplasms also arise predominantly in the extremities of (young) adults and children. Because of the plasticity of myoepithelial cells, they show heterogeneous morphological features, consisting of spindled, epithelioid, plasmocytoid and clear cells often set in a (chondro)myxoid or collagenous/hyalinized stroma. [START_REF] Kilpatrick | Mixed tumors and myoepitheliomas of soft tissue: A clinicopathologic study of 19 cases with a unifying concept[END_REF][START_REF] Kutzner | Cutaneous myoepithelioma: an underecognized cutaneous neoplasm composed of myoepithelial cells[END_REF][START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF] Hence there is morphological overlap with myxoid ES, showing predominantly epithelioid and spindle shaped cells. Ductal structures in myoepitheliomas are of help in the distinction of both entities. This feature was not observed in our cases. Futhermore all of our cases showed also classical morphology of ES in at least 10% of tumour area. The distal variants in our series had a granulomatous appearance and necrosis. Moreover none of the tumours of our series expressed S-100 protein, which was seen in 87% respective 93% of myoepithelial neoplasms. [START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF] The presence of pankeratin and EMA was 100% in our cases as expected in ES, while AE1/AE3 was positive in only 77% and EMA only in 63% of myoepithelial neoplasms. [START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF] Smaller series showed similar results. [START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF][START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF] The focal staining for SMA in two and calponin among three of our cases was very weak and incomparable to what is seen in myoepithelial neoplasms. [START_REF] Kilpatrick | Mixed tumors and myoepitheliomas of soft tissue: A clinicopathologic study of 19 cases with a unifying concept[END_REF][START_REF] Kutzner | Cutaneous myoepithelioma: an underecognized cutaneous neoplasm composed of myoepithelial cells[END_REF][START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF][START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF] Two of our cases showed discriminating positivity for CD34, which is in line with approximately 50 % of ES. [START_REF] Hasegawa | Proximal-type epithelioid sarcoma: a clinicopathologic study of 20 cases[END_REF][START_REF] Guillou | Proximal-type epithelioid sarcoma, a distinctive aggressive neoplasm showing rhabdoid features: clinicopathologic, immunohistochemical, and ultrastructural study of a series[END_REF][START_REF] Miettinen | Epithelioid sarcoma: an immunohistochemical analysis of 112 classical and variant cases and a discussion of the differential diagnosis[END_REF][START_REF] Chbani | Epithelioid sarcoma. A clinicopathologic and immunohistochemical analysys of 106 cases from the French Sarcoma Group[END_REF] Negative staining results we observed for GFAP, and p63, analogous to the results of others, whereas positivity was described in myoepithelial neoplasms in about 46 -100% and 7 -27% respectively. [START_REF] Kutzner | Cutaneous myoepithelioma: an underecognized cutaneous neoplasm composed of myoepithelial cells[END_REF][START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Hornick | Cutaneous myoepithelioma: A clinicopathologic and immunohistochemical study of 14 cases[END_REF][START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF][START_REF] Laskin | Epithelioid sarcoma. New insights based on an extended immunohistochemical analysis[END_REF] Like in ES, SMARCB1 protein expression in myoepithelial carcinomas may be absent, as it has been shown recently. [START_REF] Gleason | Myoepithelial carcinoma of soft tissue in children: An aggressive neoplasm analysed in a series of 29 cases[END_REF][START_REF] Hornick | Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma[END_REF] The genetic background thereof is still unknown.

Epithelioid haemangioendothelioma (EHE) of skin and soft tissues may also enter the differential diagnosis of myxoid ES. This distinct malignant vascular tumour, most commonly occurring in middle-aged patients, has a wide range of localization. Tumours of the skin are very rare and described as both single and multiple lesions; the last is typically in progressive ES. Histologically, EHE shows a (multi)nodular architecture and is composed of short strands, cords, or small clusters of epithelioid to slightly spindled cells with an eosinophil cytoplasm and vesicular nuclei. Intracytoplasmatic vacuoles, usually a prominent feature in EHE, could be in some cases very subtle and are occasionally recognized in ES due to lipid droplets. The typically myxohyaline stroma shows variably haemorrhage and/or haemosiderin deposits. Discriminating immunohistochemical features are positivity for specific F o r P e e r R e v i e w 7 endothelial markers, as CD31 (with a strong membranous reaction) and Fli-1 (with nuclear staining) in EHE, whereas EMA, usually positive in ES, was rarely focally detected in EHE. Cytokeratin and CD34, variably positive in both tumours, are not useful in the differential diagnosis. [START_REF] Quante | Epithelioid hemangioendothelioma presenting in the skin : A clinicopatholologic study of eight cases[END_REF][START_REF] Weiss | Epithelioid hemangioendothelioma and related lesions[END_REF][START_REF] Mentzel | Epithelioid hemangioendothelioma of skin and soft tissues : clinicopathologic and imuunohistochemical study of 30 cases[END_REF][START_REF] Billings | Epithelioid sarcoma like hemangioendothelioma[END_REF] Another differential diagnosis of ES represents extraskeletal myxoid chondrosarcoma (EMC). EMC typically shows a multinodular growth pattern with interlacing cords of round or plump spindled tumour cells set in a chondomyxoid background. In contrast to ES, the cells of EMC are less epithelioid and the architecture is more homogeneous. A cellular high grade variant and rhabdoid features is also described showing greater degree of morphological overlap especially with proximal type ES. [START_REF] Lucas | High-grade extraskeletal myxoid chondrosarcoma: a high-grade epithelioid malignancy[END_REF][START_REF] Kohashi | SMARCB1/INI1 protein expression in round cell soft tissue sarcomas associated with chromosomal translocations involving EWS: A special reference to SMARCB1/INI1 negative variant extraskeletal myxoid chondrsarcoma[END_REF] The rhabdoid features in EMC were associated with loss of SMARCB1/INI1 expression and partly with genetic alteration in SMARCB1/INI. [START_REF] Kohashi | SMARCB1/INI1 protein expression in round cell soft tissue sarcomas associated with chromosomal translocations involving EWS: A special reference to SMARCB1/INI1 negative variant extraskeletal myxoid chondrsarcoma[END_REF] If requiring immunohistochemistry for a definitive diagnosis, S-100 is expressed in a minority of cases, and epithelial and myogenic markers are usually absent. [START_REF] Hornick | Myoepithelial tumors of soft tissue. A clinicopathologic and immunohistochemical study of 101 cases with evaluation of prognostic parameters[END_REF][START_REF] Oliveira | Extraskeletal myxoid chondrosarcoma: a clinicopathologic, immunohistochemical, and ploidy analysis of 23 cases[END_REF] Typical translocations, t(9;22) (q22;q12), t(9;17) (q22;q11), and t(9;15) (q22;q21), furnish proof of this sarcoma. [START_REF] Sciot | T(9;22) (q22-31;q11-12) is a consistent marker of extraskeletal myxoid chondrsarcoma: evaluation of three cases[END_REF][START_REF] Hisaoka | TFG is a novel fusion partner of NOR1 in extraskeletal myxoid chondrosarcoma[END_REF] Recently an epithelioid variant of myxofibrosarcoma has been described composed of mainly epithelioid fibroblastic cells set in a myxoid stroma. In addition pseudolipoblasts and numerous elongated, curvilinear blood vessels are present, mostly seen in the hypocellular myxoid areas. [START_REF] Nascimento | Epithelioid variant of myxofbrosarcoma: expanding the clinicomorphologic spectrum of myxofibrosarcoma in a series of 17 cases[END_REF] This tumour usually arises in elderly patients, lacks the expression of epithelial markers and do not show loss of SMARCB1 protein expression.

In summary myxoid ES represents a rare variant in the spectrum of ES and prominent myxoid stromal changes are seen in the distal and proximal type respectively. Particularly in small biopsies correct diagnosis can be problematic, and cases of benign and malignant myoepithelioma, extraskeletal myxoid chondrosarcoma and epithelioid myxofibrosarcoma have to be distinguished from myxoid ES.

  Hulsebos T J M, van Krieken J H J M & Mentzel T

25

 25

  negative for SMARCB1 protein expression. This confirms recently published studies,

Figure 1 .

 1 Figure 1. Classical features in one of the distal type ES (case 1) with nodular architecture and inflammatory reaction; myxoid background is also seen.

Figure 2 .

 2 Figure 2. Epithelioid and spindle-shaped tumour cells in case 3 are arranged in cords given a reticular growth pattern. Note the prominent myxoid stroma.

Figure 3 .

 3 Figure 3. This neoplasm of the proximal type is composed of nests and cords of enlarged tumour cells with rhabdoid features (case 2).

Figure 4 .

 4 Figure 4. Predominantly solid areas in a proximal type ES (case 6).

Figure 5 .

 5 Figure 5. Epithelioid and spindle tumour cells set in a prominent myxoid stroma; scattered plasmocytoid cells are also present (case 4).

Figure 6 .

 6 Figure 6. Pan-cytokeratin expression was a characteristic feature of all cases (case 6).

Figure 7 .

 7 Figure 7. CD34 positivity was observed in two cases (case 6).

Figure 8 .

 8 Figure 8. All cases showed loss of SMARCB1 protein expression (case 6).

  Classical features in one of the distal type ES (case 1) with nodular architecture and inflammatory reaction; myxoid background is also seen 180x134mm (300 x 300 DPI)Epithelioid and spindle-shaped tumour cells in case 3 are arranged in cords given a reticular growth pattern. Note the prominent myxoid stroma. 180x134mm (300 x 300 DPI)This neoplasm of the proximal type is composed of nests and cords of enlarged tumour cells with rhabdoid features (case 2). 180x134mm (300 x 300 DPI)Predominantly solid areas in a proximal type ES (case 6). 180x134mm (300 x 300 DPI)Epithelioid and spindle tumour cells set in a prominent myxoid stroma; scattered plasmocytoid cells are also present (case 4). 180x134mm (300 x 300 DPI) Pan-cytokeratin expression was a characteristic feature of all cases (case 6).180x134mm (300 x 300 DPI)

Table 1 .

 1 Panel of antibodies used in this study

	Antigen	Clone	Dilution	Source
	Vimentin	V9	1:150	Dako
	Pan-cytokeratin	MNF116	1:100	Dako
	Pan-cytokeratin	AE1/3	1:50	Dako
	CD34	HPCA-1	1:100	Becton & Dickinson
	SMA	1A4	1:300	Dako
	BAF 47	25	1:250	BD Transduction Laboratories
	S-100	Polyclonal	1:2000	Dako
	Calponin	CALP1	1:400	Dako
	EMA	E29	1:3000	Dako
	P63	4A4	1:400	Dako
	GFAP	GA-5	1:200	Biogenix

Table 2 .

 2 Clinical and morphological details of six cases of myxoid ES

	Case	Sex	Age	Location	Type	Size	Amount	Recurrences	Metastases
			(years)			(cm)	of	(n)
							myxoid
							changes
							(%)
	1	M	48	abdominal	distal	1.6	80	2	lymph
				wall					node groin
				(dermis,
				subcutis)
	2	F	22	vulva	proximal 1.5	70	NA	NA
				(subcutis)
	3	F	16	lower leg	distal	2.0	90	NA	NA
				(soft
				tissue)
	4	M	74	shoulder,	distal	4.0	50	no	no
				(dermis,
				subcutis)
	5	F	41	hand	distal	1.0	50	4	lymph
				(subcutis)					nodes
									axilla
	6	F	25	groin	proximal 4.0	90	1	NA
				(subcutis)

Table 3 .

 3 Immunohistochemical results of six cases of myxoid ES

	Case Vimentin CK CD34 EMA SMA BAF47 S100 GFAP P63 Calponin
	1	+	+	+	+	-	-	-	-	-	-

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Acknowledgement:

The authors thank PALGA, the nationwide network and registry of histopathology and cytopathology in the Netherlands, especially Dr. Mariel Casparie, for selecting epithelioid sarcoma cases.


P Sganga

Lens

Cecilia Tamara Avellán

D Rousseau
email: d.rousseau@unesco-ihe.org

J Sganga

P Lens
email: p.lens@uensco-ihe.org

Community based approach towards water quality restoration and pollution prevention of the Flores Creek (Uruguay)

Keywords: water quality, constructed wetlands, SWAT, dairy farms, Santa Lucia River

The micro-river basin of the Flores Creek is an important source of aquifer recharge, which is the drinking water supply of the mid-sized town of Libertad in Southwestern Uruguay as well as the irrigation source of the surrounding rural area, However, it is strongly contaminated by municipal wastewater and dairy sewage. To mitigate this issue, an integrated river basin project based on technical and community approaches was initiated in 2008, raising awareness of the problems to relevant stakeholders and identifying the practically possible solutions. So far, some of the implemented measures are: a) Design and construction of a constructed wetland for one of the larger dairy farms, b) Active participation of the local rural school children in the water quality/level monitoring, c) Integration of the data into the GIS based SWAT model (first time use in Uruguay) that will allow the prediction of the improvement of water quality and quantity following better management practices d) Increased monitoring of the water quality, the flow volume, the biodiversity and the water absorption capacity of the characteristic soils. Therefore, the initial foundation for a successful IRBM has been laid, some relevant changes have occurred (such as the CW) and future steps to be taken are being addressed (such as the cleanup of the municipal sewage treatment facility).

INTRODUCTION General Background

Uruguay is one of the smaller countries in South America covering an area of 176.000 km2 and a population of approximately 3 million people. It has a temperate climate where temperatures vary between 6.5 and 30.9 °C and where the annual precipitations reaches up to 1000 mm of about 90 days of rain a year, being the months between January and March the ones with the most amount of rainfall [START_REF] Rla | Informe final sobre el proyecto de cooperación técnica "Manejo integrado y sostenible de las aguas subterráneas en América Latina" -"Gestión sostenible del Acuífero Raigón, Uruguay[END_REF]. Uruguay is fortunate to have relatively large amounts of surface and subsurface waters. Sadly, drastic changes in land use, such as augmented herd sizes and the implementation of artificial pastures that go hand in hand with the uncontrolled use of chemical fertilizers and pesticides and increased irrigation activities as well as the lack of proper 9th World Wide Workshop for Young Environmental Scientists WWW-YES-Brazil-2009: Urban waters: resource or risks? 26-30 October 2009

2 / 11 sewage disposal systems of dairy farms, have provoked severe deteriorations of aquatic environments.

The dairy sector (3600 dairy farms) plays an important economic role in Uruguay and a trend of increased milk production and thus, the number of cows in milking and sewage, has been occurring for the past decade (from 600000 cows in 1980 to more than 750000 in 2008, MGAP 2009) as is common to other parts of the world as shown by [START_REF] Martinez | Livestock waste treatment systems for environmental quality, food safety and sustainability[END_REF]. The main challenges for the implementation of sewage treatment facilities for the milking parlor effluents lie within the costs of construction and the lack of direct benefit from this investment for the farmers. No economic incentives either by the government or the industries are being offered so far, although a clear link between eutrophication of surface and subsurface waterways and improper sewage disposal has been detected by the Ministry of Environmental Affairs (DINAMA) in a small scale study, where 5 out of 37 surveyed farms complied with national standards (Conaprole, DI-NAMA, 2007).

The Flores Creek watershed is a typical example of the ones found within the dairy belt and is situated in the Southwestern part of Uruguay within the department of San José, 60 km north of Uruguay's capital Montevideo (see figure 1). It drains into the Río San José which flows into the Río Santa Lucía which itself ends up in the Río de la Plata and the Atlantic Ocean. The Flores Creek has two small tributaries the Cañada Clara and the Cañada Fea and its watershed comprises ca. 80 km 2 . Moreover, this creek is to become part of the Santa Lucia Wetlands National Park (HSL 2009), where water and ecosystem quality will become a priority (Figure 2), and where the downstream portion of the Flores Creek would enter into both the core zone (Zona 1) as well as the buffer zone (Zona 2). Water quality and ecosystem problems Through water quality sampling since 2003 we have been able to show the detrimental effects of the outflow of • the primary sewage treatment facility of the city of Libertad and • the raw sewage discharge of several dairy farms, on the water quality, leaving the upstream portion entirely anoxic (see figure 3) [START_REF] Avellan | Developmental instability in Polygonum punctatum and Sagittaria montevidensis in a sewage impacted stream[END_REF]) and rendering some stretches of the tributaries not suitable for aquatic life. We also clearly showed a link between sewage effluent pollution in the Flores Creek and species diversity (see figure 4), being the least polluted sites the more biodiverse ones (from Avellan 2004). Moreover, the creek is an important recharge element for the aquifer since the infiltration rates through the surrounding limestone soil is slow and the aquifer is largely rainwater fed (RLA 2005). High nitrogen rates have already been confirmed in the aquifer (RLA 2005) and correlate strongly with anthropogenic pollution sources.

Wetlands are known to be the most prone sites for aquifer recharge and therefore need special attention, such as a 20 ha Typha sp. wetland between the Bidegain and the Gonzalo site (see figure 5) that might be largely responsible for the enhanced water quality at the latter site.

MATERIALS & METHODS

Research and community based problem identification and solution finding

Following APHA standard procedures, we have been studying the water and ecosystem quality of the Flores Creek since 2003/2004 (for detailed description see [START_REF] Avellan | Developmental instability in Polygonum punctatum and Sagittaria montevidensis in a sewage impacted stream[END_REF]). Further, several reports about the water quality of the region exist, such as the work on the Raigon Aquifer and the ongoing study of the Santa Lucia River Basin by the Ministry of Environmental Affairs [START_REF] Rla | Informe final sobre el proyecto de cooperación técnica "Manejo integrado y sostenible de las aguas subterráneas en América Latina" -"Gestión sostenible del Acuífero Raigón, Uruguay[END_REF], JICA 2008), in which part of the diagnostic of the water and ecosystem quality has been based. From November 2007 onwards, we actively searched for the interaction with potential interest groups. First, we invited all the dairy farmers of the creek for a meeting where their main con-cerns in view of the environmental degradation were addressed and potential solutions they had thought of, heard of or implemented, were discussed. Subsequent to this first meeting, we started to address the public agencies to sense their interest for the matters of integrated catchment management and water pollution at the rural-urban interface.

Several other meetings that were open to all public followed, in which the proposed solutions were presented, explained and discussed and the importance of the natural riverine habitats were underlined.

In coordination with the farmers and the other involved interest groups the following actions to undertake were distinguished:

• innovative and sustainable solution for the dairy farm waste waters Constructed wetlands • integration of the locals environmental education with the school children • use of IT tools to aid in the visualization of the problems and model future scenarios use of SWAT Monitoring of the process to show the changes in the water quality were requested to justify the implementation of the solutions and to potentially pinpoint other sources of pollution.

RESULTS & DISCUSSION

Community based problem identification

The farmers identified soil erosion, water quality and invasive exotic species as well as useful and easy solutions for their sewage treatment facilities as their main concerns. With the public agencies three working agreements were signed.

1. One with the local municipality (Intendencia de San Jose) in particular the hygiene section, who have committed to ongoing water quality monitoring of the creek as well as to exercise pressure on the local water company to enhance the municipal sewage treatment facility. Further, it is likely that the Intendencia will lend their heavy machinery to the farmers for the construction of further constructed wetlands for the dairy farm effluent and set up a plan for the regular maintenance of the systems. 2. The Ministry for Agriculture, especially the Water and Soil section and their laboratory, is performing water analyses on a regular basis to test if the water quality complies with irrigation standards and are also testing the soil humidity and water absorption capacity of the surrounding soils. 3. The Universidad de la Republica is undertaking studies on macro-invertebrates in the creek to see how the species composition has changed in the past 5 years and to monitor further changes which may arise due to the implementation of measures that shall enhance the water quality.

Proposed integrated solutions

Constructed wetland for one of the larger dairy farms

The Bidegain family is one of the six mid sized dairy farms of the Flores Creek catchment (there are also 4 small farms) and has traditionally milked cows for the past 100 years here. In their dairy parlor they milk approx. 200 cows twice a day and use roughly 16 m2 of freshwater a day for washing the cows and rinsing the parlor. About ten years ago they had constructed a double lagoon system as a means of treating the waste water before it entered the Cañada Clara, a tributary to the Flores Creek. However, this system had filled up recently due to the lack of maintenance and they therefore decided to build a new system. Using an online calculation tool developed by local civil engineers and agronomists we were able to calculate the dimensions needed for a sediment trap as well as a double lagoon system (MGAP PPR 2009). Following this, we designed a free water surface constructed wetland of 736 m2 planted with Scirpus americanus (see figure 6) (according to methods proposed in [START_REF] Kadlec | Treatment Wetlands[END_REF]. To optimize future constructed wetlands we undertook root oxygen loss laboratory experiments with four plant species (Typha latifolia, Phragmites australis, Cortedeira selloiana, Juncus acutus), which were carried out at UNESCO-IHE, The Netherlands, sponsored by the UNESCO Keizo Obuchi Research Fellowship. These experiments showed increased oxygen release into the water column of two native species, C. selloiana and J. acutus (Figure 7).

Integration of the data into the GIS based SWAT model

The data has been included and integrated into GIS (ArcView and MapWindows) which is helping in the visualization of the problem zones (figure 10), both in space and time and will allow the prediction of the improvement of water quality and quantity following better management practices (such as constructed wetlands for dairy farm effluents). Figure 10 clearly shows the deterioration of the water quality in the basin and the strong influence that the insufficient sewage treatment facility at the headwaters has. Moreover, preliminary intents to build the model (Soil and Water Assessment Tool SWAT) have been undertaken and the missing data has been identified as well as the sources to fill in the gaps. Thus within the coming months a full model should be built and ready to use, enabling us to predict changes in water quality and levels according to the management practices, the infiltration into the aquifer and the enhancement of the overall system. The Ministry of Agriculture, in particular, will use the outcome of the model to better allocate the irrigation cuotas to the farmers and make them dynamic and in relation to rainfall, in order to not strain the ecological base flow of the creek in drought periods. This model could further form the basis for future uses of SWAT in Uruguay, such as for the large Santa Lucia River Basin.

Increased monitoring of the water quality, the flow volume, the biodiversity and the water absorption capacity of the characteristic soils Since 2008, through the working agreements with the public institutions water quality measurements and benthic species diversity as well as soil water absorption capacities monitoring were commenced. Thus a decrease in dissolved oxygen and an increase in nutrients was noted since the last measurements in 2003/2004 (see figure 11) which is due to the lack of maintenance of the municipal sewage treatment facility. Preliminary classification of benthos diversity into classes, within the samples taken in November 2008, confirms the trend revealed in previous years of a gradual increase in diversity downstream of the sewage effluent (Figure 12). This is true even if the sampling season were different, showing the highest species diversity in July 2003 -in winter -whereas the highest species diversity should be expected in spring (Sept-Nov). Further, in 2008 not even the characteristic Tubifex sp. worms were found at the sewage location. As oxygen depletion becomes less of a problem due to sewage input reduction, we expect an increase in species diversity in all sites which will be a clear sign of ecosystem rehabilitation and the justification of the initial investment in the sewage treatment facilities.

CONCLUSIONS

This project is one of the first intents in Uruguay to apply integrated river basin management techniques in order to stop the pollution of the surface and subsurface waters and safeguard the livelihood of the affected rural and urban population. Starting on a sound scientific basis as well as community based approaches, relevant problems, stakeholders and adequate solutions were identified and carried out. Validation of these processes is still occurring and will determine the efficiency of the applied measures. Many problems still need to be tackled such as 1. the pollution caused by the insufficient municipal sewage treatment facility (a claim has already been entered to the Ministry of Environmental Affairs DINAMA in March 2009) 2. the pollution caused by other dairy farms -further CW are in planning 3. data collection (ongoing) 4. fine-tuning of the model -will be aided by the results of some of the studies such as the water absorption and moisture retention analysis 5. further integration of the community -such as the city schools Nevertheless, the most important obstacle of gaining the trust of the involved parties has been overcome.

Figure 1 :

 1 Figure 1: Location of the Flores Creek in South Western Uruguay (see pin) (Google Earth) and sampling locations within the Flores Creek Watershed (produced through ArcView 3.1).

Figure 2 :

 2 Figure 2: Map of the geographical zonation of the future National Park (Flores Creek area only) (HSL 2009)

Figure 3 :

 3 Figure 3: Influence of untreated dairy farm sewage on the ammonium concentration of the Canada Clara (July 2008) and Dissolved Oxygen concentrations along the Flores Creek in 2003 and 2004

9th 11 Figure 4 :

 114 Figure 4: Benthos species diversity of samples taken in July 2003 (winter) and January 2004 (summer)

Figure 5 :

 5 Figure 5: Part of the Typha wetland at about 1/3 of the creek.

9th

  World Wide Workshop for Young Environmental Scientists WWW-YES-Brazil-2009: Urban waters: resource or risks? 26-30 October 2009 6 / 11

Figure 6 :

 6 Figure 6: Images of the construction of the wetland and the vegetated areas.

Figure 7 :

 7 Figure 7: Changes in Dissolved Oxygen Concentration in artificial sewage water according to the used plant

Figure 8 :

 8 Figure 8: Left: Workshop on waste management with the children of 3rd grade of a school in Libertad in cowork with REDES-Friends of the Earth; Right: Water quality monitoring with the rural school children using the donated kits of the WWMD campaign

Figure 9 :

 9 Figure 9: Daily flow rates of the Flores Creek at Paso Flores.

9th 11 Figure 10 :

 1110 Figure 10: Comparative images of dissolved oxygen concentrations in the Flores Creek Watershed that show the clear deterioration of the water quality in time and space.

Figure 11 :

 11 Figure 11: Increase in NH 4 + concentration in 2008 compared to the previous years.

Figure 12 :

 12 Figure 12: Benthos species diversity in the Flores Creek of samples taken in 2003/2004/2008

  9th World Wide Workshop for Young Environmental Scientists WWW-YES-Brazil-2009: Urban waters: resource or risks? 26-30 October 2009

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ACKNOWLEDGEMENTS

This research was made possible by the financial and moral support of the farmers of the creek (Pedro and Roberto Bidegain, Alvaro Boix, Gonzalo Vilaro, Ilse&Juan Hampe), the Intendencia Municpal de San Jose (Dr. Atilio and Dr. Bizensans), the Universidad de la Republica (Dr. Conde and Dr. Arocena, Soledad and Natalia), REDES-Amigos de la Tierra (Carlos Santos), Rotary Club Libertad, Aves del Uruguay and many more. Special thanks to Lorien Walsh and her team of the World Water Monitoring Day Campaign by WEF and IWA for the donation of two water sampling kits and other supplies. The stay in Holland was made possible by the UNESCO Keizo Obuchi Research Fellowship. In Holland, I would like to acknowledge the help of Ann van Griensven and Getnet in matters regarding SWAT; Frank van Dien and Hans van Bruggen regarding constructed wetlands, design and things to consider; Frank Wiegman and Peter Heerings for their aid in the lab; and my office partner Marteen Siebel. I also want to thank my MSc advisor Prof. Emeritus Hough and the staff at Wayne State University for all their help and support in my years with them.


Thai V Hoang

Elisa H Barney Smith

Salvatore Tabbone

EDGE NOISE REMOVAL IN BILEVEL GRAPHICAL DOCUMENT IMAGES USING SPARSE REPRESENTATION

Keywords: Edge noise removal, noise spread, sparse representation, basis pursuit denoising, curvelet transform

A new parametric method for edge noise removal in graphical document images is presented using geometrical regularities of the graphics contours that exists in the images. Denoising is understood as a recovery problem and is done by employing a sparse representation framework with a basis pursuit denoising algorithm for denoising and curvelet frames for encoding directional information of the graphics contours. The optimal precision parameter used in this framework is shown to have linear relationship with the level of the noise. Experimental results show the superiority of the proposed method over existing ones in terms of image recovery and contour raggedness.

INTRODUCTION

The scanning and binarization processes that produce binary document images introduce noise concentrated on the edges of the image objects [START_REF] Mcgillivary | Edge noise in document images[END_REF]. This edge noise has influence on later steps in the chain of automatic document processing. It could affect feature measurement in recognition, reduce image redundancy in compression, and distort skeletons in vectorization. For accurate analysis and recognition of document images, edge noise needs to be removed.

Many methods exist for noise removal in bilevel document images. The most famous and frequently used are median filtering [START_REF]Handbook of Image and Video Processing[END_REF], contour smoothing using chain codes [START_REF] Yu | An efficient algorithm for smoothing, linearization and detection of structural feature points of binary image contours[END_REF] or morphological operators [START_REF]Handbook of Image and Video Processing[END_REF], and kFill filtering [START_REF] O'gorman | Image and document processing techniques for the RightPages electronic library system[END_REF]. The median filter replaces the value of a pixel by the median of the values in the neighborhood of that pixel. It is known to perform well on binary images to remove salt-and-pepper noise, however, it unintentionally removes thread lines and corners. The kFill algorithm is designed to remove salt-and-pepper noise iteratively while maintaining readability of text by using a filter that retains text corners. The value of the parameter k of the filter can be chosen adaptively based on text size and image resolution. Its disadvantage is that the picky points of the contour like the end-points of thin lines are also removed. Contour smoothing based on chain codes replaces a sequence of a chain code by another simpler sequence, usually a shorter one corresponding to the shortest path. Even though this method can produce a smooth contour from a jagged one, it cannot be performed on a noisy contour such as that shown in Fig. 1(b). Morphological operators like erosion, dilation and their combinations opening and closing have their root in set theory. They can be used to remove salt-and-pepper noise but cannot guarantee smooth contours at the output. Thus, most of the existing denoising methods for binary document images are general-purpose, they are not designed specifically for edge noise and do not exploit the directional information in their operation. Contours denoised by these methods are jagged and sometimes shifted from their original positions. Another shortcoming of existing methods is that they do not take into account the information about the level of noise that exists in the binarized document images, denoising is performed in a "blind", non-adaptive way.

Recent research in image degradation modeling [START_REF] Barney | Modeling image degradations for improving OCR[END_REF] shows that edge noise, which results from a combination of the optical system, the additive noise, and the thresholding, can be measured quantitatively and represented by a parameter called Noise Spread (NS), paving the way for the design of a new method to remove edge noise. This paper describes a new parametric method for edge noise removal in bilevel graphical document images exploiting the directional information of graphics contours. Information about the level of edge noise, represented by NS, is used as the input to optimize the method. Directional denoising is facilitated under a sparse representation framework by promoting sparse representation of the document image in an overcomplete dictionary using a basis pursuit denoising algorithm with curvelets as the dictionary. The denoised image reconstructed from this sparse representation is a grayscale one and it can be simply thresholded to obtain the final denoised bilevel image. Our contributions are twofold: directional denoising for edge noise in bilevel graphical document images using curvelet transform in a sparse representation framework and the use of the optimal linear relationship between the framework's parameter and NS, which leads to a superior performance over existing methods.

The remainder of this paper is organized as follows. Section 2 gives some background on edge noise modeling using NS. The edge noise removal algorithm using a sparse representation is presented in Section 3. Experimental results are given in Section 4, and conclusions are drawn in Section 5.

NOISE SPREAD

In document and graphical images the image content contains large regions of white (0) background, with foreground image features displayed in black [START_REF] Mcgillivary | Edge noise in document images[END_REF]. When these are scanned in gray level, the edges change from a step function to ones sloped in the shape of the Edge Spread Function (ESF ). Noise is added, which is generally assumed to be Additive White Gaussian Noise (AW GN ). Document and graphical images are usually processed in bilevel, so this image is then thresholded, usually with a global threshold. While the AW GN is evenly distributed over the whole gray level image, after the thresholding it is concentrated along the edges. This will turn a smooth edge into a rough one.

In [START_REF] Mcgillivary | Edge noise in document images[END_REF] the noise was analyzed and the breadth of the region around the edge where the noise affects the image was defined as the Noise Spread (NS). The NS is not just dependent on the standard deviation of the AW GN , σ noise , but also on the width w of the optics Point Spread Function (P SF ), which determines the slope of the gray level edges, and the level of the binarization threshold, Θ, through the relation

NS = √ 2π • σ noise • w LSF (ESF -1 (Θ)) , (1)
where LSF is the Line Spread Function, or one dimensional P SF . NS provides a measure that can numerically quantify the amount of edge noise, and this measure relates also to the noise visually observed on bilevel images. NS has been shown to be linearly related to the Hamming distance between the original and noisy images, as well as to be correlated with the ability to fit a line to a noisy edge. This gives the potential that the NS could be used as an input to a denoising algorithm that works on bilevel images, in a similar fashion to how the standard deviation of the additive noise is often used as an input to denoising algorithms that work on gray level images.

EDGE NOISE REMOVAL USING SPARSE REPRESENTATION

Results in computational harmonic analysis [START_REF] Candès | New tight frames of curvelets and optimal representations of objects with piecewise C 2 singularities[END_REF] show that curvelet frames which contain multi-scale, multi-directional, and elongated curvelets (Fig. 1(a)) can provide an optimal representation of objects with edges. Most of the energy of the objects is localized in just a few coefficients of curvelets which overlap and are nearly tangent to the object contours.

In other words, the directional information of object contours is encoded in a small number of curvelet coefficients. Combining this code with the basis pursuit denoising algorithm [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF] will constitute a sparse representation framework allowing directional denoising along object contours.

Curvelet transform

The radial and angular windows, W (r) and V (t), are two smooth, non-negative, and real-valued functions which are supported on [1/2, 2] and [-1, 1] respectively and satisfy the admissibility conditions ∀ r > 0 and t ∈ R:

∞ j=-∞ W 2 2 j r = 1, ∞ l=-∞ V 2 (t -l) = 1. (2)
At each scale j, the mother curvelet ϕ j is defined as:

φj (r, θ) = 2 -3j/4 W 2 -j r V 2 j/2 θ 2π . (3)
A curvelet at scale j, orientation θ l , and position

x j,l k = R -1 θ l 2 -j k 1 , 2 -j/2 k 2 is defined as: ϕ j,l,k (x) = ϕ j R θ l x -x j,l k , (4)
where R θ l is the rotation operator by θ l = 2π2 j/2 l radians with l ∈ Z + such that 0 ≤ l < 2π. The corresponding curvelet coefficients of f ∈ L 2 R 2 is defined as:

c j,l,k = f, ϕ j,l,k = R 2 f (x)ϕ j,l,k (x)dx. (5)
Curvelets constructed in this way define a tight frame in L 2 R 2 , obey the parabolic scaling relation (width = length 2 ), and exhibit an oscillating behavior in the direction perpendicular to their orientation. They can be used as an overcomplete dictionary in a sparse promoting problem of graphical documents. The curvelet transform has a property that the coefficients of those curvelets whose essential support does not overlap with or overlap with but are not tangent to the edge are small and negligible. For example, in Fig. 1(b), coefficients of curvelets of type A and B are negligible, most of the energy of the graphics is localized in just a few coefficients of type C which overlap and are nearly tangent to the edge.

Basis pursuit denoising

The noisy image I is the result of scanning, followed by global thresholding, a noise-free input image I 0 of size w×h, it will contain edge noise of a certain NS. Let y 0 , y ∈ R n (n = wh) be the vectors generated by stacking the columns of I 0 , I respectively, A ∈ R n×K with n K be an overcomplete dictionary allowing sparse representation of y 0 . Let x be the representation of y in A satisfying y = Ax + z, where z ∈ R n stands for the unknown additive edge noise, then finding the sparse representation x of y in A is equivalent to solving the following 0 -optimization problem with precision parameter depending on z:

x = argmin x x 0 subject to Ax -y 2 ≤ . (6)
Finding the solution of an underdetermined system of linear equations like ( 6) is N P -hard [START_REF] Davis | Adaptive greedy approximations[END_REF]. However, recent results [START_REF] Donoho | For most large underdetermined systems of linear equations the minimal 1-norm solution is also the sparsest solution[END_REF] show that if the solution of ( 6) is sufficiently sparse, it is equal to the solution of the following 1 -optimization problem:

x = argmin x x 1 subject to Ax -y 2 ≤ . (7)
This is a convex optimization problem and it can be solved using methods like the interior-point algorithm [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF]. For the edge noise removal problem, the curvelet frame is selected as A. The denoised grayscale image can be obtained from the sparse reconstruction ŷ = Ax, which is then converted to a binary image by simple thresholding ỹ = T (ŷ) where T is the thresholding operator with the threshold value of 0.5.

The precision parameter

In [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF], is a non-zero precision parameter that describes the desired fidelity of the reconstructed ŷ to the noisy y. It is the only parameter that controls the performance of the denoising algorithm. When = 0, y = ŷ = ỹ and as the value of increases, the measure of sparsity of the solution of the problem in [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF] must monotonically increase, since the feasible set is wider, meaning a better alignment of curvelets with graphics contour, which consequently increases denoising performance. However, due to the thresholding operation, deformation in the thresholded ỹ will appear from a certain value of . The selected value of should thus depend on the level of noise that exists in the images. To our knowledge, there exists no work discussing the dependance of on the noise level in image denoising. For zero-mean white and homogeneous Gaussian noise with a known standard-deviation σ, the value of is usually chosen as cnσ 2 , with 0.5 ≤ c ≤ 1.5 [START_REF] Elad | Sparse and Redundant Representations: From Theory to Applications in Signal and Image Processing[END_REF]Chapter 14]. For edge noise, the relation (NS) will be derived to have the form kNS from experiment in the next section.

EXPERIMENTAL RESULTS

Two types of experiments have been carried out, one for the derivation of the relation (NS) and the other for the demonstration of the superiority of the proposed method over existing ones in removing edge noise.

The relation (NS)

A dataset (SetA) containing noisy images has been generated from a ground-truth image "symbol017" which is a 256 × 256 pixel graphical symbol image taken from the GREC2005 database [START_REF] Dosch | Report on the second symbol recognition contest[END_REF], its noisy version is given in Fig. 3(b). The testing database is divided into four subsets, each contains 10 noisy images corresponding to 10 values of NS ranging from 0.2 to 2.0 with increments of 0.2. In order to have best performance, at a specific value of NS, has the value * that corresponds to the peak in a measure of fidelity between ŷ and y 0 , denoted by γ ỹ,y 0 ( ). The measure employed in this work is the normalized cross-correlation. Fig. 2(a the determination of * employing γ ỹ,y 0 ( ). The noisy image in Fig. 3(b) with NS = 2.0 is taken as the input image. At each possible value of , the 1 -optimization problem in [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF] is solved for x and the value of γ ỹ,y 0 ( ) can be calculated thereafter. The plot of γ ỹ,y 0 ( ) in this case has its maximum value of 0.94389 at * = 49. This means that if the input noisy image I has NS = 2.0, in [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF] should have the value 49.

The relation * (NS) obtained from these four subsets is plotted in Fig. 2(b). It is realized that * has a nearly linear relationship with NS for all four subsets, or * kNS. The deviation of * from kNS is small and, moreover, due to the blunt maxima in γ ỹ,y 0 ( ) this deviation has almost no effect on the performance of the proposed method.

Comparison with existing methods

The proposed method has been evaluated on two datasets, the first one is SetA as described in the previous subsection and the second one (SetB) is generated from four ground-truth graphical symbol images "symbol016", "symbol017", "sym-bol024", "symbol081" from the GREC2005 database (their noisy version is given in Fig. 3(a)-3(d)). These images are selected for experiment due to the existence of all possible graphics contour directions. For each ground-truth image, 5 noisy images have been generated corresponding to 5 values of NS = 0.2, 0.6, 1.0, 1.5, 2.0. Fig. 3 provides examples of denoising graphical symbols. The original noisy images with NS = 2.0 are given in the first row. Evidence of directional denoising along noisy contours exists in the denoised images in grayscale in the second row. Edge noise is smoothed out in the direction that is perpendicular to the noisy contour. The final denoised images in binary are given in the third row.

To demonstrate the effectiveness of the proposed method, it has been compared with four frequently used methods: median filtering using a 3×3 neighborhood, kFill filtering with the parameter k = 3, closing then opening using a 3×3 structuring element, and opening then closing using a 3×3 structuring element. The criteria used for comparison are the ability to recover the original image (measured by the normalized crosscorrelation between the denoised and the ground-truth images), and the raggedness of the graphics contours (a moving average based on [12]). The proposed method and four other denoising methods are each applied to datasets SetA and SetB. Normalized cross-correlation and raggedness are measured for each resulting image. The performance of each method per noise level on one dataset is the average performance over all the noisy images in that dataset. The results are shown in Fig. 4 for these two criteria over a range of noise levels. It is observed that as NS increases, the ability to recover the original image decreases and the contour raggedness of the denoised images increases for all methods. However, the decrease in the image recovery and increase in the contour raggedness of the proposed method are nearly constant and are the smallest among all methods for both datasets. The proposed method thus results in a denoised image of best recovery with smoothest contours at all values of NS. It should also be noted from the comparison results that for noisier images, in terms of image recovery and contour raggedness the proposed method produces most significant improvements.

CONCLUSION

A new parametric method for edge noise removal from graphical document images has been presented in this paper using sparse representation. Curvelet transform and basis pursuit denoising are employed for directional denoising along graphics contours. Noise Spread is used as the input parameter to select the precision parameter * of the method, which turns out to have a nearly linear relationship with NS. Experimental results show the superiority of the proposed method over existing ones in terms of image restoration and contour raggedness.

Comparison using the recovery criterion is only feasible when the ground-truth images are available. Thus in this work, as a proof of concept and for the ease of evaluation, the experimental noisy images are generated from ground-truth images. To make the method applicable, in real applications, the value of NS can be estimated directly from the input images [START_REF] Mcgillivary | Edge noise in document images[END_REF]. Performance evaluation of the proposed method using the estimated NS is left as future work.

Fig. 1 .

 1 Fig. 1. Directional denoising using curvelets.

  A plot of γ ỹ,y 0 ( at NS = 2.0 * (NS) relation

Fig. 2 .

 2 Fig. 2. Determination of the precision parameter * using noise level information represented by NS.

Fig. 3 .

 3 Fig. 3. Noisy and denoised images from the dataset SetB at NS = 2.0. First row: original noisy symbol images, y (from left to right: 016, 017, 024, 081). Second row: denoised images, ŷ. Third row: binarized denoised images, ỹ.

Fig. 4 .

 4 Fig. 4. Comparison results with four other denoising methods in terms of image recovery and contour raggedness.