Christophe Fouqueré
email: christophe.fouquere@lipn.univ-paris13.fr

Virgile Mogbil
email: virgile.mogbil@lipn.univ-paris13.fr

Gabriele Pulcini
email: pulcini@di.ens.fr

Substructural Logic for Orientable and Non-Orientable Surfaces

. Whereas PL is limited to orientable structures, we characterize the whole range of topological surfaces, orientable as well as non-orientable. The system we obtain is a surface calculus that enjoys both cut elimination and focussing properties and comes with a natural phase semantics whenever explicit context is considered.

Among the different viewpoints considered for studying proofs of Linear Logic, let us recall that its graph-theoretical representations may be seen as topological objects and considered as surfaces on which usual proofs are drawn without crossing edges [START_REF] Bellin | Planar and braided proof-nets for multiplicative linear logic with mix[END_REF][START_REF] Métayer | Implicit exchange in multiplicative proofnets[END_REF][START_REF] Melliès | A topological correctness criterion for non-commutative logic[END_REF]. Following that interpretation, Gaubert [START_REF] Gaubert | Two-dimensional proof-structures and the exchange rule[END_REF] provided a way to compute surfaces. Moreover, based on the fact that the exchange rule may model topological operations, non-commutative variants of Multiplicative Linear logic (MLL) were developed: planar logic [START_REF] Melliès | A topological correctness criterion for non-commutative logic[END_REF], the calculus of surfaces [START_REF] Gaubert | Two-dimensional proof-structures and the exchange rule[END_REF] and permutative logic (PL) [START_REF] Andreoli | Permutative logic[END_REF]. In all these cases the conclusions of the proofs are drawn on disjoint oriented circles, more precisely orientable surfaces with boundary. E.g. in PL the underlying structure is that of a permutation, which is a product of disjoint cycles, together with a natural number to express the number of tori, actually a topological invariant of the surface. The shape of such sequents is called a q-permutation and geometrically studied in [START_REF] Pulcini | A geometrical procedure for computing relaxation[END_REF] by one of the present authors. One of the main topological results given in [START_REF] Pulcini | A geometrical procedure for computing relaxation[END_REF] is related to Massey classification theorem [START_REF] Massey | A basic course in algebraic topology[END_REF]: any orientable surface, possibly with boundary, is homeomorphic either to a sphere or to a finite connected sum of tori, possibly with boundary.

We consider in this paper a generalization to surfaces orientable or not. Massey theorem states in that case that it may be homeomorphic also to a finite connected sum of projective planes, possibly with boundary. For that purpose, we consider in our work pq-permutations which are simply obtained from q-permutations by replacing the single index q with an ordered couple (p, q) of positive integers for counting tori handles and projective planes. The shape of our sequents still integrates the topology of a surface and non-trivial exchange rules correspond to surface transformations, following what is done in PL but also in Melliès planar logic [START_REF] Melliès | A topological correctness criterion for non-commutative logic[END_REF]. After presenting the logical system, we prove a few logical properties: it enjoys both cut elimination and the focussing property, as PL does. We give a phase semantics that is sound and complete with respect to the calculus. Though a phase semantics may seem a too elementary result, it allows us to tackle the problem of contextual structures. The aim of our work is to shed new light on the relationship between topology and logic.

sPL: A Sequent Calculus for Surfaces

Formulas of sPL are inductively built from a countable infinite set of atoms A = {a, b, c, . . . , a ⊥ , b ⊥ , c ⊥ , . . .} and the two usual multiplicative connectives and ⊗, together with a unary bar operation ( _) that models the inversion of the orientation:

F ::= F ∈ A | F | F 1 F 2 | F 1 ⊗ F 2 IDENTITY GROUP ax. ⊢ 0 0 (A, A ⊥ ) ⊢ p q Σ, (Γ, A) ⊢ p ′ q ′ Ξ, (∆, A ⊥ ) cut ⊢ p+p ′ q+q ′ Σ, Ξ, (Γ, ∆) ORIENTABLE STRUCTURAL RULES ⊢ p q Σ, (Γ, ∆) cylinder ⊢ p q Σ, (Γ), (∆) ⊢ p q Σ, (Γ), (∆) torus ⊢ p q+1 Σ, (Γ, ∆) ⊢ p q Σ invert ⊢ p q Σ NON-ORIENTABLE STRUCTURAL RULES ⊢ p q Σ, (Γ, ∆) Möbius ⊢ p+1 q Σ, (Γ, ∆) ⊢ p q Σ, (Γ), (∆) Klein ⊢ p+2 q Σ, (Γ, ∆) LOGICAL RULES ⊢ p q Σ, (Γ, A, B) ⊢ p q Σ, (Γ, A B) ⊢ p q Σ, (Γ, A) ⊢ p ′ q ′ Ξ, (∆, B) ⊗ ⊢ p+p ′ q+q ′ Σ, Ξ, (Γ, A ⊗ B, ∆)
Table 1: Sequent calculus for sPL

The negation is defined as usual by de Morgan duality and preserves the bar operation. A sequent is denoted ⊢ p q Γ where Γ is a multiset of cyclic sequences which are formulas separated by ',' within parenthesis, and p and q are integers with the intuition they denote a pq-permutation (p for projective planes). We write () for an empty cycle. Type derivations are built from the rules of table 1.

Remark that the key rules of divide and merge in PL are also in sPL as respectively cylinder and torus acting as orientable rules.

Cut Elimination and Focussing Property

Theorem 1 (cut-elimination). Any proof of a sPL sequent can be rewritten into a cut-free proof of the same sequent.

A standard proof by case analysis need a particular attention for commutative conversions involving the Möbius or the Klein rule. This result can also be obtained as a consequence of focalization.

A focalized sequent calculus, called foc-sPL, may be defined with sequents of the form ⊢ p q Γ|Σ where Γ is the focus -a distinguished cyclic sequence -and Σ is a multiset of cyclic sequences of formulas separated by ';'. The cut rule acts only on focusses. The main ingredients are the following ones: a focus rule is added and Klein and torus rules are changed in the following way:

⊢ p q |(Γ); Σ focus ⊢ p q Γ|Σ ⊢ p q Γ, Λ, ∆|Σ torus' ⊢ p q+1 Γ, ∆, Λ|Σ ⊢ p q Γ, Λ, ∆|Σ Klein' ⊢ p+2 q Γ, ∆, Λ|Σ
In such a presentation the defocus rule is simply a special case of the cylinder rule (with () neutral w.r.t. ';'). As it follows from topological considerations, structures of proofs in foc-sPL may be normalized in such a way that cylinder applications arrive only at the end of a proof construction. Such proofs are called maximally focalized and it is then possible to prove a cut-elimination property on them.

Proposition 2 (Maximal Focalization). A sequent is provable in foc-sPL if and only if there exists a proof such that cylinder rules are applied only at the end. Moreover cuts in a maximally focalized proof in foc-sPL may be eliminated.

Sketch. As the cut rule is applied to focalized formulas and that cut and cylinder rules commute, one may consider that cuts are applied to sequents of the form ⊢ p q Γ|. The rest is done by case analysis.

We are finally able to prove that provability in sPL and foc-sPL are equivalent, hence a cut-elimination theorem for sPL follows.

Proposition 3 (Focussing property). A sequent is provable in sPL if and only if it is provable in foc-sPL.

Phase Semantic

A phase space is provided that is proved to be complete and valid with respect to the calculus. This should be considered as a first step towards a better understanding of the calculus and its relation to geometry. In fact, this is not at all obvious if we notice that there is not yet satisfying proof semantics for non-commutative logic (NL) even though its phase space has been given together with its sequent calculus (by Ruet in his 1997'thesis). What is the main difficulty when turning to a calculus of surfaces? Or equivalently what makes NL an easier situation? The orientation has to be taken into account, more than that the context cannot be neglected. In NL, the non-commutative structure is an order variety. Hence a formula on which an operation is applied may be 'extracted' from its context: the structure of the semantics is close to what is required with Linear Logic. This is no more true in the calculus of surfaces as see-saw structural rules are not valid: one is required to deal explicitly with the context.

For that purpose, a support phase space Supp(M ) interpreting formulas is embedded into a context phase space Con(M ) interpreting sequents. The two phase spaces are defined from an associative monoid and give rise to two closure operations ⊥ and † in such a way that the fundamental proposition is provable:

Proposition 4. Let M = (M, ⋆, 1) be a (not necessarily commutative) associative monoid with neutral element 1, let F, G ⊂ M, (F ⋆ G ⊥⊥ ) ⊥⊥ = (F ⋆ G) ⊥⊥ (F ⋆ G † † ) † † = (F ⋆ G) † †
We consider as usual that a fact is a subset A of the support phase space such that A ⊥⊥ = A. Operations are defined on facts:

A ⊗ B def = (A ⋆ B) ⊥⊥ A B def = (B ⊥ ⋆ A ⊥ ) ⊥ 1 def = ⊥ ⊥ ⊥ is given A ⊕ B def = (A ∪ B) ⊥⊥ A & B def = A ∩ B⊤ def = M 0 def = ⊤ ⊥
Although the general lines for proving soundness and validity of the model are standard, their proofs are more complex as they require to consider explicitly the context.

Principal line of current work: Relaxation

Relaxation is the binary relation induced by structural transformations -divide, merge, Möbius and Klein -on the set of pq-permutations. We write α ≺ β, β relaxes α, for meaning that the pq-permutation α can be rewritten into β through a suitable series of applications of structural rules. Since each structural rule increases the topological genus of the transformed surface, relaxation turns out to impose a partial order on the set of pq-permutations. We pose the problem of providing an algorithm for the decision of relaxation, namely an effective procedure able to answer to the question 'α ≺ β ?' being given two pq-permutations α and β.

Two parallel solutions have been already afforded in case of q-permutations, namely in case of combinatorial structures encoding orientable surfaces. The first one has been considered in [START_REF] Andreoli | Permutative logic[END_REF] and consists in interpreting orientable transformations -divide and merge -as the effect of composing q-permutations with a suitable transposition. Being established such an algebraic correspondence, the solution comes straightforwardly by stressing very standard achievements in theory of permutations. The other solution provides a geometrical and interactive approach. For answering to our question 'α ≺ β ?' we compute the surface S α * S β obtained by composing, through identification of paired edges occurring on the boundaries, the two surfaces S α and S β respectively corresponding to α and β. In [START_REF] Pulcini | A geometrical procedure for computing relaxation[END_REF], it is proved that the topological genus of S α * S β provides information enough to decide relaxation.

The passage from q to pq-permutations turns out to be critical from the point of view of the decision of relaxation. Whereas orientable transformations exclusively act at the level of the combinatorial structure of pq-permutations, Möbius and Klein also affect their supports so as to make impossible any resort to theory of permutations. On the contrary, we guess that the just mentioned geometrical procedure might admit a very natural extension in order to include non-orientable transformations and surfaces. As a matter of fact, it is an often remarked logical phenomenon that genuinely interactive approaches allow to avoid technical problems due to syntactical bureaucracy.

5 At the end...

Our logical system sPL generalizes Permutative logic, and in this way it is an embedding of the multiplicative Cyclic Linear logic (CyLL) and MLL. A completely unexplored field of research is that one of proof-nets for PL and sPL. The starting point should be the criterion for proof-nets of Planar Logic which just consists in requiring, together with the logical correctness, the planarity of the graph [START_REF] Melliès | A topological correctness criterion for non-commutative logic[END_REF]. In this direction the main difficulty is that structural rules are usually 'transparent' with respect to the syntax of proof-nets so as we need to recover this kind of information by stressing the geometrical structure of the net. As far as PL is concerned, some useful results could be borrowed from [START_REF] Métayer | Implicit exchange in multiplicative proofnets[END_REF], whereas the non-orientable side of the question misses at all of contributions.

Finally we developed a framework allowing to characterize the relationship between logic and orientable as well as not orientable surfaces. Semantical issues have not yet been explored and we are peculiarly interested in denotational semantic to give a topological interpretation of formulas and proofs. The existence of a phase semantics for sPL may provide some alternative algebraic tools for studying the geometry of 2-manifolds. A standard application of phase semantics consists in singling out redundant rules, namely rules which are superfluous with respect to the deductive power of the system, typically the cut rule. Now, since the basic topological transformations are embodied into our system, the classical classification theorem might find an interesting alternative proof when addressed in terms of semantics.


PhD Maria Cristina Rapanotti
email: cristinarapanotti@yahoo.it

Roberta Caruso

Emanuele Ammatuna

Serena Zaza

Laura Trotta

Mariadomenica Divona

Daria Funaro

Laura Cicconi

Giorgio Federici

Sergio Amadori

Giulio De Rossi

Francesco Lo-Coco

MOLECULAR CHARACTERIZATION OF PAEDIATRIC IDIOPATHIC HYPEREOSINOPHILIA

Keywords: Hypereosinophilia, CHILDHOOD HAEMATOLOGICAL MALIGNANCIES, CLONALITY, IgH, MOLECULAR

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Summary

Hypereosinophilic syndromes (HES) include a group of heterogeneous diseases characterized by the persistent increase of the number of eosinophils in blood and bone marrow. Few cases of paediatric hypereosinophilia (pHES) have been described in the literature. Early identification of pHES that may evolve towards a lymphomyeloproliferative disease is relevant in light of prognostic and therapeutic implications. Molecular features of 10 pHES patients were analysed at presentation and during their clinical outcome, including analysis of BCR-ABL and FIP1L1/PDGFRA fusion genes, quantitation of Wilm's tumour suppressor gene copy number and clonality of T-cell receptor and immunoglobulin heavy chain (IgH). All patients had normal karyotype and germ line T-cell receptor configuration. Five children showed IgH clonality at presentation: of these, two developed a B non-Hodgkin lymphoma and a B-lineage acute lymphocytic leukaemia at six and 12 months, respectively, two spontaneously reverted to a polyclonal IgH profile during the follow-up, and the last one persisted with pHES without B-clonal evolution after 19 months. One patient had a PDGFRA/FIP1L1 fusion and achieved hematologic and molecular remission after imatinib therapy.

IgH rearrangement was observed to be a frequent molecular feature of pHES in children and may precede B-cell clonal expansion and evolution into B-cell malignancies.

Introduction

Hypereosinophilic syndromes (HES) include a group of heterogeneous diseases characterized by the persistent increase of eosinophil numbers in the peripheral blood and the bone marrow [START_REF] Chusid | The hypereosinophilic syndrome: analysis of fourteen cases with review of the literature[END_REF][START_REF] Brito-Bapapulle | The eosinophilias, including the Idiopathic Hypereosinophilic Syndrome[END_REF][START_REF] Bain | The idiopathic hypereosinophilic syndrome and eosinophilic leukemias[END_REF][START_REF] Gleich | The hypereosinophilic syndromes:current concepts and treatments[END_REF]. HES is predominantly a disease of males (male:female ratio 9:1) and is usually diagnosed between the ages of 20 and 50 years. Tissue infiltration by eosinophils is responsible for the end-organ damage that results from the activity of proteins released by the eosinophil granules. A major change in the new WHO 2008 classification was the reorganization of chronic eosinophilic leukaemia CEL/HES into "CEL not otherwise specified (CEL, NOS)" and "myeloid and lymphoid neoplasms with eosinophilia associated with PDGFRA, PDGFRB, and FGFR1 abnormalities" (Klion et al., 2006;[START_REF] Bain | Chronic eosinophilic leukemia, not otherwise specified. Chapter Myeloproliferative Neoplasms[END_REF].

The relevance of identifying PDGFR-rearranged eosinophilic disorders is related to the efficacy of treatment with imatinib mesylate, [START_REF] Gotlib | The FIP1L1-PDGFRalpha fusion tyrosine kinase in hypereosinophilic syndrome and chronic eosinophilic leukemia: implications for diagnosis, classification, and management[END_REF][START_REF] Gotlib | The FIP1L1-PDGFRalpha fusion tyrosine kinase in hypereosinophilic syndrome and chronic eosinophilic leukemia: implications for diagnosis, classification, and management[END_REF][START_REF] Helbig | A single weekly dose of imatinib is sufficient to induce and maintain remission of chronic eosinophilic leukaemia in FIP1L1-PDGFRA-expressing patients[END_REF].

However, the myeloproliferative CEL, NOS are also frequently responsive to imatinib, suggesting that other as yet unrecognized mutations affecting tyrosine kinases may underlie their pathogenesis [START_REF] Salem | Effective treatment of hypereosinophilic syndrome with imatinib mesylate[END_REF].

A diagnosis of CEL, NOS requires evidence of clonality of the eosinophils. Since in many cases it is impossible to define clonality, the diagnosis of idiopathic HES is still made. The diagnosis of idiopathic HES is based on exclusion criteria and frequently remains doubtful because of the lack of genetic markers [START_REF] Brito-Bapapulle | The eosinophilias, including the Idiopathic Hypereosinophilic Syndrome[END_REF]. Early discrimination between idiopathic HES confined to an unidentified reactive process and hypereosinophilia accompanied by a clonal proliferation eventually evolving into a lymphoproliferative disease is relevant in light of its obvious prognostic and therapeutic implications [START_REF] Smith | Unexplained hypereosinophilia and the need for cytogenetic and molecular genetic analyses[END_REF][START_REF] Roche-Lestienne | Molecular characterization of the idiopathic hypereosinophilic syndrome (HES) in 35 French patients with normal conventional cytogenetics[END_REF].

In the latter case, the hypereosinophilia is the result of a primitive T or B lymphoid disorder and can be considered as an epiphenomenon. Long-term evolution of this form of hypereosinophilia is often complicated by T-cell lymphoma or leukaemia, indicating that T cells are involved in the pathogenesis of these disorders (Raghavar et al., 1987;[START_REF] Cogan | Brief report: clonal proliferation of type 2 helper T cells in a man with the hypereosinophilic syndrome[END_REF][START_REF] Roufosse | Clonal Th2 lymphocytes in patients with the idiopathic hypereosinophilic syndrome[END_REF][START_REF] Bank | The hypereosinophilic syndrome associated with CD4+CD3-helper type 2 (Th2) lymphocytes[END_REF][START_REF] Roumier | Hypereosinophilia with abnormal T cells, trisomy 7 and elevated TARC serum level[END_REF]. Evolution into B-cell malignancies has been occasionally reported both in adults and in childhood (Blatt et al., 1992;[START_REF] Urtasùn | Hipereosinophilia mantenida que precede al diagnostico de una leucemia linfoblastica aguda[END_REF][START_REF] Bernasconi | Hypereosinophilia in acute lymphoblastic leukemia: an association to be remembered[END_REF]Hill et al., 2003;[START_REF] Wynn | Acute lymphoblastic leukemia with hypereosinophilia: report of a case with 5q deletion and review of the literature[END_REF][START_REF] Horigome | Fatal eosinophilic heart disease in a child with neurofibromatosis-1 complicated by acute lymphoblastic leukemia[END_REF][START_REF] Rapanotti | Idiopathic hypereosinophilic syndrome: a case evolving in B-lymphoblastic lymphoma[END_REF][START_REF] Bhatti | Adult B lymphoblastic leukaemia/lymphoma with hypodiploidy (-9) and a novel chromosomal translocation t(7;12)(q22;p13) presenting with severe eosinophilia -case report and review of literature[END_REF]. Finally, the expression level of WT1 has been recently investigated as an additional marker to the standard parameters so far considered in the diagnosis of CEL, NOS in the adult population, and it seems to be particularly useful in cases where standard molecular and cytogenetic analyses are not informative [START_REF] Cilloni | WT1 transcript amount discriminates secondary or reactive eosinophilia from idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia[END_REF].

Few cases of pHES have been described in the literature (Blatt et al., 1992;[START_REF] Urtasùn | Hipereosinophilia mantenida que precede al diagnostico de una leucemia linfoblastica aguda[END_REF][START_REF] Bernasconi | Hypereosinophilia in acute lymphoblastic leukemia: an association to be remembered[END_REF]Hill et al., 2003;[START_REF] Scheinfeld | Fatal fulminant hypereosinophilic syndrome with a petechial rash in a 16year-old girl[END_REF][START_REF] Wynn | Acute lymphoblastic leukemia with hypereosinophilia: report of a case with 5q deletion and review of the literature[END_REF][START_REF] Chien | Acute lymphoblastic leukemia presenting with urticarial plaques and hypereosinophilia in a child[END_REF][START_REF] Horigome | Fatal eosinophilic heart disease in a child with neurofibromatosis-1 complicated by acute lymphoblastic leukemia[END_REF][START_REF] Rapanotti | Idiopathic hypereosinophilic syndrome: a case evolving in B-lymphoblastic lymphoma[END_REF][START_REF] Rives | Idiopathic hypereosinophilic syndrome in children: report of a 7-year-old boy with FIP1L1-PDGFRA rearrangement[END_REF][START_REF] Farruggia | Hypereosinophilic syndrome in childhood: clinical and molecular features of two cases[END_REF]. pHES has a slight male predominance and shows a distinct biologic and clinical course compared with the adult forms. The disease in children is commonly associated with chromosomal abnormalities, including trisomy 8 and gene rearrangements that may involve ABL [START_REF] Brito-Bapapulle | The eosinophilias, including the Idiopathic Hypereosinophilic Syndrome[END_REF][START_REF] Bain | The idiopathic hypereosinophilic syndrome and eosinophilic leukemias[END_REF]. In addition, pHES has also been described in association with acute lymphoblastic leukaemia, while the abnormal fusion FIP1L1/PDGFRA has rarely been found [START_REF] Rives | Idiopathic hypereosinophilic syndrome in children: report of a 7-year-old boy with FIP1L1-PDGFRA rearrangement[END_REF], and evolution of idiopathic HES into a B-cell neoplasia has been only occasionally described (Blatt et al., 1992;[START_REF] Jain | Approaches to the treatment of hypereosinophilic syndromes: a workshop summary report[END_REF][START_REF] Urtasùn | Hipereosinophilia mantenida que precede al diagnostico de una leucemia linfoblastica aguda[END_REF][START_REF] Bernasconi | Hypereosinophilia in acute lymphoblastic leukemia: an association to be remembered[END_REF]Hill et al., 2003;[START_REF] Scheinfeld | Fatal fulminant hypereosinophilic syndrome with a petechial rash in a 16year-old girl[END_REF][START_REF] Wynn | Acute lymphoblastic leukemia with hypereosinophilia: report of a case with 5q deletion and review of the literature[END_REF][START_REF] Chien | Acute lymphoblastic leukemia presenting with urticarial plaques and hypereosinophilia in a child[END_REF][START_REF] Horigome | Fatal eosinophilic heart disease in a child with neurofibromatosis-1 complicated by acute lymphoblastic leukemia[END_REF][START_REF] Rapanotti | Idiopathic hypereosinophilic syndrome: a case evolving in B-lymphoblastic lymphoma[END_REF][START_REF] D'angelo | Acute lymphoblastic leukemia with hypereosinophilia and 9p21 deletion: case report and review of the literature[END_REF]. Here, we report the molecular and clinical features of 10 pHES studied at diagnosis and during follow up.

Patients and methods

Patients and healthy donors

Between November 2001 and October 2007, 10 children (six males and four females) with marked hypereosinophilia lasting for at least six months were observed at the Division of Haematology, Bambino Gesù Children's Hospital of Rome. The main clinical and laboratory features at the time of presentation are summarized in Table 1. The median absolute number of eosinophils was 10.1/mm³ (range 1.5-70.8/mm³). Five patients presented with anaemia, while the platelet count was normal in all but one patient (UPN6). In three cases (UPN2, UPN4 and UPN5), there were no signs of organ involvement at diagnosis. Eosinophil infiltration in the bone marrow at presentation ranged from 21.5% to 80% (median 35%). Cardiac involvement was documented by echocardiography and electrocardiography (EKG) in four patients who presented with chronic cardiac tamponade and pleural effusion, sinus tachycardia, restrictive cardiomyopathy and pancarditis with mitral valvular insufficiency, respectively (UPN3, UPN6, UPN7 and UPN8). Three patients (UPN3, UPN7 and UPN9) presented with pulmonary involvement with interstitiopathy, pleural effusion and asthmatic bronchitis. All but two patients (UPN1 and UPN10) started therapy with steroids (prednisone 1 mg/kg/day) followed

Clinical and laboratory exams

Bone marrow (BM) aspirate and complete clinical chemistry, including serum immunofixation and IgE count assessed by the PRIST method, and allergic tests (RAST method), were performed in all patients together with blood culture, virological investigations and immunophenotype. Instrumental examinations including chest radiography, abdomen ultrasound, total body computed tomography scan, EKG and echocardiography were carried out to exclude the causes of secondary hypereosinophilia and to evaluate end-organ damage. Immunophenotype was performed by flow cytometry at onset in all cases. The following monoclonal antibodies were used for the characterization of potential abnormal T or B clonal population: CD19, CD10, CD20, CD22, CD24, HLADR, CD3, CD7, CD5, CD4, CD8, CD79a, CD13, CD33, CD34 and TdT.

Genetic studies included conventional karyotyping on bone-marrow-derived G-banded metaphases.

Molecular biology analysis was performed by RT-PCR to identify the presence of the BCR-ABL and FIP1L1/PDGFRA fusion genes (vanDongen et al., 1999;[START_REF] Bacher | A combination of cytomorphology, cytogenetic analysis, fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction for establishing clonality in cases of persisting hypereosinophilia[END_REF]. In order to assess the clonality of IgH and TCR repertoires, BM genomic DNA was extracted from bone marrow mononuclear cells. TCR clonality was performed by DHPLC sequencing of the TCRβ genomic region [START_REF] Cave | Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes[END_REF]. IgH clonality analysis was performed on BM genomic DNA by amplifying CDRI, CDRII and CDRIII regions of the heavy chain immunoglobulin genes (IgH-PCR). Amplification of the CDRIII region was performed using CA1 and CA2 primers, respectively, the "consensus primers" for variable and joining regions of the heavy chain [START_REF] Nizet | Follow-up of residual disease (MRD) in B lineage acute leukaemias using a simplified PCR strategy: evolution of MRD rather than its detection is correlated with clinical outcome[END_REF]. Amplifications of CDRI and CDR II were performed using oligo-degenerate sequence primers mapping the frameworks FR2 and FR3, as previously described [START_REF] Diss | Detection of monoclonality in low-grade B-cell lymphomas using the polymerase chain reaction is dependent on primer selection and lymphoma type[END_REF].

Quantitative WT1 assessment (RQ-PCR) and fluorescence measurement were performed on the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystem, Epster City, CA, USA) as described by Cilloni and co-workers (2007).

Control populations

Blood samples from 30 healthy donors were taken from the Transfusion Centre as a negative control population. Bone marrow aspirates from 25 B-ALL and peripheral blood from 25 chronic lymphocytic leukaemia (CLL) patients were used as positive controls, all of which had a monoclonal IgH profile and a polyclonal TCRβ repertoire. To assess the prevalence of IgH rearrangement in chronic myeloid neoplasms, we analysed a cohort of 10 patients affected by

Results

Diagnostic findings

Biological and clinical features at onset are summarized in Table 1. Immunophenotype analysis was normal in all but one patient (UPN9), who showed a very low proportion of T-helper lymphocytes, with a CD4/CD8 ratio of 0.5%. The CD4/CD8 ratio was normal in all other patients. All patients expressed CD33/CD13 antigens in their myeloid compartments, whereas the CD34 marker was never expressed. All patients had a normal karyotype.

Molecular analysis documented a B-clonal disorder showing a monoclonality of the IgH-CDRII region (framework FR2) in 5/10 patients (UPN1, UPN2, UPN7, UPN8 and UPN9).

FIP1L1/PDGFRA transcript was found in one patient (UPN6) who was diagnosed as having a myeloid neoplasm with eosinophilia and abnormalities of PDGFRA. TCR rearrangements were not detected in any of the patients. Quantitative WT1 analysis was performed in 6/10 patients at presentation (UPN4, UPN5, UPN6, UPN8, UPN9 and UPN10). Over-expression of WT1 (cut-off > 90 copies) was detected in 3/6 patients (UPN 4, UPN5 and UPN8).

Patient follow-up

A spontaneous reduction of eosinophils with normalization of total blood cell count was observed in two patients (UPN1 and UPN10). Two patients (UPN 4 and UPN 5) were treated with steroid therapy that resulted in a reduction of the eosinophil count within 15 and 20 days, respectively, from treatment initiation, while in another two patients reversion was achieved after the withdrawal of a neurological drug (Clobazam) and surgical correction of a congenital anomaly of the thoracic duct, respectively (UPN2 and UPN3, 7 and 2 months, respectively). B-cell clonality was detected at onset in patients UPN1 and UPN2 and converted spontaneously to a polyclonal profile in subsequent follow-up bone marrow aspirates.

Patient UPN6 started therapy with Imatinib (400 mg/day) and is now in continuous complete

Discussion

According to the scarce existing literature on pHES, most patients with persistent hypereosinophilia do not show known molecular lesions at presentation. Hence, diagnostic refinement of the disease remains difficult, and frequently malignancy of hypereosinophilia may be defined only after a careful clinical and biologic follow-up (Blatt et al., 1992;[START_REF] Jain | Approaches to the treatment of hypereosinophilic syndromes: a workshop summary report[END_REF][START_REF] Urtasùn | Hipereosinophilia mantenida que precede al diagnostico de una leucemia linfoblastica aguda[END_REF][START_REF] Bernasconi | Hypereosinophilia in acute lymphoblastic leukemia: an association to be remembered[END_REF]Hill et al., 2003;[START_REF] Scheinfeld | Fatal fulminant hypereosinophilic syndrome with a petechial rash in a 16year-old girl[END_REF][START_REF] Wynn | Acute lymphoblastic leukemia with hypereosinophilia: report of a case with 5q deletion and review of the literature[END_REF][START_REF] Chien | Acute lymphoblastic leukemia presenting with urticarial plaques and hypereosinophilia in a child[END_REF][START_REF] Horigome | Fatal eosinophilic heart disease in a child with neurofibromatosis-1 complicated by acute lymphoblastic leukemia[END_REF][START_REF] Rapanotti | Idiopathic hypereosinophilic syndrome: a case evolving in B-lymphoblastic lymphoma[END_REF][START_REF] Rives | Idiopathic hypereosinophilic syndrome in children: report of a 7-year-old boy with FIP1L1-PDGFRA rearrangement[END_REF][START_REF] D'angelo | Acute lymphoblastic leukemia with hypereosinophilia and 9p21 deletion: case report and review of the literature[END_REF]. Moreover, to the best of our knowledge, no studies have systematically analysed TCR and IgH clonality in a sizable number of pHES.

In the present series, 6/10 patients with hypereosinophilia fulfilling the criteria of persistent (> 6 mos) and elevated (> 1.5 × 10 9 /L) eosinophil count had molecular evidence of WBC clonality, including monoclonal IgH rearrangement in five patients and FIP1L1/PDGFRA fusion in one. The remaining four patients showed polyclonal B-and T-cell repertoires and had no detectable cytogenetic and molecular aberrations. We note however that lesions other than the FIP1L1/PDGFRA fusion have been reported in adults with hypereosinophilia. These may involve PDGFRA, PDGFRB and FGFR1 through other fusions and point mutations [START_REF] Jain | Approaches to the treatment of hypereosinophilic syndromes: a workshop summary report[END_REF]Teffery et al., 2006;[START_REF] Bain | Chronic eosinophilic leukemia, not otherwise specified. Chapter Myeloproliferative Neoplasms[END_REF]. Unfortunately, we were not able to search for these alterations in the present series because of the lack of adequate material. B-cell clonality was the most frequently detected alteration in our pHES patients (50%). In particular, in two cases (UPN7 and UPN8) a monoclonal rearrangement of the IgH-CDRII was already detectable in the marrow at six and 12 months, respectively, prior to the development of a B-cell lymphoproliferative disorder, clearly indicating clonal evolution. While the origin of the neoplastic proliferation from the same clone detected at diagnosis is documented by the same IgH rearrangement, it remains unclear whether a second undetected alteration might have occurred that contributed to successive clonal expansion and overt lymphoproliferative disease. However, our In an attempt to interpret these latter findings, we can hypothesize that B-cell clonality might, in these patients, remain under the control of the immune system and be extinguished after the stimulus has ceased. Interestingly, as shown in UPN2, IgH clonality might accompany a clearly reactive and transient pHES, whereby a drug used for the underlying neurological disease was most likely responsible for the prolonged hypereosinophilia. Precursor B-acute lymphoblastic leukaemia/lymphoma with exaggerated eosinophilia is a rare entity, with less than 50 cases including adult and paediatric populations reported since 1973, when it was first described by [START_REF] Spitzer | Lymphoblastic leukemia with marked eosinophilia: a report of two cases[END_REF][START_REF] D'angelo | Acute lymphoblastic leukemia with hypereosinophilia and 9p21 deletion: case report and review of the literature[END_REF]. The most common cytogenetic abnormality encountered in ALL with eosinophilia is the t(5;14), which is characterized by overproduction of interleukin 3 (IL-3) [START_REF] Meeker | Activation of the interleukin-3 gene by chromosome translocation in acute lymphocytic leukemia with eosinophilia[END_REF]. The latter entity is now included as "B lymphoblastic leukaemia/lymphoma with t(5;14); IL-3-IgH" in the new WHO classification of lymphoid neoplasms published in 2008 [START_REF] Meeker | Activation of the interleukin-3 gene by chromosome translocation in acute lymphocytic leukemia with eosinophilia[END_REF], Swerdlow et al., 2008). In this case, eosinophilia is secondary to overproduction of IL-3 by the lymphoblasts, resulting from activation of the IL-3 gene on chromosome 5 after its translocation adjacent to the IgH gene on chromosome 14 [START_REF] Meeker | Activation of the interleukin-3 gene by chromosome translocation in acute lymphocytic leukemia with eosinophilia[END_REF].

IgH rearrangement was present in five of our cases at the time of diagnosis of hypereosinophilia, was associated with a normal karyotype and antedated the development of a B-cell disease by several months in the two cases (UPN7 and UPN8) where this occurred.

In our study, we were not able to find a correlation between the level of hypereosinophilia at presentation and its possible aetiology. In addition, even if the WHO classification does not include WT1 analysis in the diagnostic screening of hypereosinophilic syndrome, a supplementary role of this assay has recently been reported for the adult population [START_REF] Cilloni | WT1 transcript amount discriminates secondary or reactive eosinophilia from idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia[END_REF]. With respect to the question of whether WT1 expression in children is different from that in adults, we did not find any correlation between WT1 expression levels and the differential diagnosis of HES, CEL, NOS or reactive disease.

In conclusion, although though the study of a limited series, our results suggest that HES in children has distinct biologic and clinical features compared with adult cases, indicating a different underlying disease and molecular pathogenesis. In addition, we suggest that, besides the identification of T-cell clonality, IgH rearrangement should be taken into consideration during the diagnostic characterization of paediatric hypereosinophilia.

  interferon up to 1 MIU three times a week. All patients were followed up at regular intervals with periodic clinical controls and standard laboratory and instrumental evaluation.

  (CML) (five patients), essential thrombocytemia (three patients) and chronic myelomonocytic leukaemia (two patients), all of which demonstrated polyclonal IgH.

  molecular and haematological remission with a follow-up of 36 months. Six months after diagnosis, patient UPN7 developed a B-NHL showing the same monoclonal rearrangement IgH-CDRII (framework FR2) documented at the onset.Patient UPN8 was treated for one year with steroids and α-interferon, achieving a decrease of protocol, eventually obtaining complete remission, which is currently maintained at a follow-up of 24 months. Karyotype and T-cell configuration were normal and polyclonal, respectively, in both UPN7 and UPN8. Patient UPN9, who showed monoclonality in the IgH-CDRII region both at onset and in follow-up marrow samples without any evidence of a Blymphoid malignancy, is still under treatment with α-interferon because of skin and lung involvement at onset. She had complete regression of organ involvement after a follow-up of 19 months.

  that detection of B-cell clonality at presentation does not invariably imply subsequent development of a neoplastic lymphoproliferative disease, as shown by the two patients with clonal IgH that did not show any progression in successive clinical and molecular evaluations.

Table 1 . Patient characteristics and biological features of bone marrow at presentation and clinical evolution.

 1

	U P N	Sex/Age (years)	Hb (g/dL)	WBC 10 3 /mm 3	Eos. count 10 3 /mm 3	PLT count 10 3 /mm 3	Eos. (%)	Signs of organ at diagnosis involvement	Karyotype	BCR-ABL and analysis FIP1L1/PDGFRA	TCRβ β β β clonality	WT1 (cut-off < expression	IgH clonality	Final diagnosis	Clinical evolution and treatment	IgH status at last follow up
												90)
	1	M/2	10	63.2	45,496	248	80	Mild hepato-splenomegaly	46, XY	Negative	Polyclonal	N.A.	(FR2/JH) CDRII-IgH	associated with IgH Hypereosinophilia	No treatment Normalization	Polyclonal
														clonality
														Hypereosinophilia	Normalization
	2	M/6	12.4	26	15,340	288	60	Absent	46, XY	Negative	Polyclonal	N.A.	(FR2/JH) CDRII-IgH	clonality associated with IgH	drug/steroid α-interferon Stop neurological	Polyclonal
															treatment
								Chronic cardiac							Normalization
	3	M/10	12.7	4.5	1,512	274	30	tamponade and	46, XY	Negative	Polyclonal	N.A.	Polyclonal	Idiopathic HES	Surgical correction/steroid	Polyclonal
								pleural effusion							α-interferon treatment
	4	M/5	14.1	94.9	70,830	281	70	Absent	46, XY	Negative	Polyclonal	503	Polyclonal	Idiopathic HES	steroid α-interferon Normalization	Polyclonal
															treatment
	5	F/15	12.8	13.9	6,950	130	80	Absent	46, XX	Negative	Polyclonal	240	Polyclonal	Idiopathic HES	steroid α-interferon Normalization	Polyclonal
															treatment
	6	M/16	8.5	13.5	5,284	171	34	Lympho-adenomegaly splenomegaly, cardiomyopathy restrictive	46, XY	PDGFRΑ Α Α Α/FIP1L1 positive	Polyclonal	< 30	Polyclonal	ement PDGFRΑ Α Α Α rearrang with MPN associated	Myeloid neoplasm Imatinib therapy + 36 mos Complete remission ↓	Polyclonal
	7	M/1.4	7.8	52.6	31,022	18	30	Hepato-splenomegaly, sinus tachycardia, asthmatic bronchitis	46, XY	Negative	Polyclonal	N.A.	CDRII-IgH (FR2/JH)	clonality associated with IgH Hypereosinophilia	NHL-B + 60 mos High-grade NHL treatment Complete remission ↓	Polyclonal
															ALL-B
	8	F/9	12.8	19.6	10,100	401	35	Pancarditis with mitral valvular insufficiency	46, XX	Negative	Polyclonal	1876	CDRII-IgH (FR2/JH)	associated with IgH Hypereosinophilia	AIEOP2006 ↓ treatment	Polyclonal
														clonality	Complete remission 24 mos
																CDRII-IgH
	9	F/7	10.3	8,31	4,603	834	40	Interstitial pneumonia	46, XX	Negative	Polyclonal	< 30	CDRII-IgH (FR2/JH)	Hypereosinophilia associated with IgH	Persistent HES α-interferon therapy	(FR2/JH) α-interferon
														clonality	+ 19 mos	therapy
	10	F/1	10.3	67.6	49,920	301	22	Hepatomegaly	46, XX	Negative	Polyclonal	< 30	Polyclonal	Idiopathic HES	Normalization
															No treatment

British Journal of Haematology

Acknowledgements

The authors would like to thank Ms Pamela Paoletti and Ms Tara Mayte Suarez Viguria for their excellent and dedicated technical assistance. This study was partially supported by Associazione Italiana contro la Ricerca sul Cancro (AIRC), Associazione Italiana contro le Leucemie (AIL) and Progetto Integrato Oncologia (PIO), Ministero della Salute.


Catharina H M J Van Elssen
email: j.vanelssen@immuno.unimaas.nl

Peter W H Frings

Freek J Bot

Koen K Van De Vijver

Mariska B Huls

Bob Meek

Pierre Hupperets

Wilfred T V Germeraad

Gerard M J Bos

Expression of aberrantly glycosylated Mucin-1 in ovarian cancer

Keywords: Tn/STn-epitopes, glycosylation, ovarian adenocarcinoma, Tumour associated antigen, glycosylation Tn, STn, ovarian adenocarcinoma, tumour associated antigen, MUC1 and glycosylation

Aims: Mucin 1 (MUC1) is an important tumour-associated antigen (TAA), both overexpressed and aberrantly glycosylated in adenocarcinomas. The objective of this study was to examine the MUC1-glycosylation status in primary ovarian adenocarcinomas and metastatic lesions.

Methods and results: Paraffin-embedded tissue sections of 37 primary ovarian adenocarcinomas representing all histotypes (22 serous, 5 mucinous, 2 clear cell, 8 endometrioid), 4 serous borderline tumours with intraepithelial carcinoma, 7 sections of ovarian endometriosis and 13 metastatic lesions were analysed by immuno-histochemistry.

Non-neoplastic ovarian surface epithelium and serous cystadenomas were used as controls.

All epithelia expressed MUC1 protein. Of primary tumours, 76% expressed the differentiation-dependent glycoform and 84% the cancer-associated glycoform (Tn/Sialyl-Tnepitopes). In metastatic lesions this was 77% and 85%, respectively. Notably, in 57% of ovarian endometriosis and 75% of intraepithelial lesions, the cancer-associated MUC1 epitopes were expressed, whereas normal ovarian surface epithelium and serous cystadenomas did not express these epitopes.

Conclusions:

The underglycosylated MUC1 epitopes are expressed by all histotypes of primary ovarian adenocarcinomas, by the vast majority of metastatic lesions and by ovarian cancer precursor lesions, whereas not by normal ovarian tissue. These results indicate that MUC1-associated Tn/STn-epitopes are important targets for immunotherapy and diagnostic imaging in ovarian cancer patients.

Introduction

Ovarian cancer is the second most common gynaecological malignancy in developed countries, and the most common cause of death among women developing gynaecological cancer [START_REF] Jemal | Cancer statistics[END_REF][START_REF] Parkin | Global cancer statistics[END_REF] . The high mortality rate is ascribed to the fact that more than 60% of ovarian cancer patients already have advanced disease at diagnosis. Therefore, ovarian cancer is also referred to as "silent killer". Metastasis as well as intra-abdominal growth are frequent, because the malignantly transformed epithelium of the ovarian surface is continuous with the peritoneal mesothelium [START_REF] Cannistra | Cancer of the ovary[END_REF] .

The current treatment of ovarian cancer is surgical resection, followed by platinumbased chemotherapy. Despite advances in chemotherapy treatment, which have led to improved survival, relapse is still frequent in the majority of patients with advanced disease 4 .

To improve overall survival of ovarian cancer patients there is an urgent need for new treatment options. Immunotherapy has been demonstrated to be successful in the treatment of cancer [START_REF] Iannello | Role of antibody-dependent cell-mediated cytotoxicity in the efficacy of therapeutic anti-cancer monoclonal antibodies[END_REF] . For antibody-mediated immunotherapy or cancer vaccines to be successful, identification of tumour-associated antigens (TAA), against which an immune response is induced, is of utmost importance. Up to this moment there have been few candidate-TAAs identified for ovarian cancer and clinical effects are limited [START_REF] Frederick | Preclinical studies and clinical utilization of monoclonal antibodies in epithelial ovarian cancer[END_REF] .

An interesting TAA is Mucin 1 (MUC1), because it is overexpressed in most adenocarcinomas [START_REF] Singh | Clinical potential of mucins in diagnosis, prognosis, and therapy of ovarian cancer[END_REF][START_REF] Gendler | MUC1, the renaissance molecule[END_REF][START_REF] Taylor-Papadimitriou | MUC1 and cancer[END_REF] . MUC1 is a protein expressed on the apical surface of most simple, secretory epithelia and on a variety of hematopoietic cells [START_REF] Ho | Heterogeneity of mucin gene expression in normal and neoplastic tissues[END_REF][START_REF] Brayman | MUC1: a multifunctional cell surface component of reproductive tissue epithelia[END_REF] . MUC1 is a transmembrane mucin with an extracellular domain made up largely of 20 amino acid tandem repeats. After translation, the MUC1 protein becomes modified by extensive O-glycosylation. Within each tandem repeat, two serines and three threonines represent five potential O-glycosylation sites.

The extent of glycosylation mainly depends on the expression of tissue-specific glycosyltransferases [START_REF] Gendler | MUC1, the renaissance molecule[END_REF] . Importantly, it has been demonstrated that in most adenocarcinomas and some haematological malignancies MUC1 is aberrantly glycosylated, has lost its apical expression and is secreted into the circulation [START_REF] Cloosen | Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma[END_REF][START_REF] Ichige | Expression of the DF3-P epitope in human ovarian carcinomas[END_REF][START_REF] Burchell | O-linked glycosylation in the mammary gland: changes that occur during malignancy[END_REF][START_REF] Von Mensdorff-Pouilly | Humoral immune response to polymorphic epithelial mucin (MUC-1) in patients with benign and malignant breast tumours[END_REF][START_REF] Richards | Antibodies reactive with the protein core of MUC1 mucin are present in ovarian cancer patients and healthy women[END_REF] . Due to aberrant glycosylation of MUC1 on tumour cells, new glycosylation-specific peptides are revealed, including the MUC1associated Tn/STn-epitopes. These epitopes are generated when O-glycosylation is terminated immediately after the first N-acetylgalactosamine (GalNAc) addition (Figure 1) [START_REF] Springer | T and Tn, general carcinoma autoantigens[END_REF][START_REF] Van Leeuwen | Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector[END_REF] . The mechanisms responsible for Tn-and STn formation are still ill understood. It has been shown that alterations in ST6GalNAc-I transferase induces premature termination of glycosylation and subsequently enhances STn formation [START_REF] Marcos | Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen[END_REF] . Another documented mechanism involves somatic mutation of the Cosmc gene, a molecular chaperone regulating the enzyme responsible for the addition of galactose (Gal) to the first GalNAC 20 . MUC1-associated Tnand STn-epitopes, both recognized by MAb 5E5 [START_REF] Van Leeuwen | Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector[END_REF][START_REF] Sorensen | Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancerspecific anti-MUC1 antibody responses and override tolerance[END_REF][START_REF] Tarp | Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat[END_REF] , are potential TAAs, which can be targeted in immunotherapy but are also of importance in diagnostic imaging [START_REF] Springer | T and Tn, general carcinoma autoantigens[END_REF][START_REF] Karsten | What makes MUC1 a tumor antigen?[END_REF] .

Over-expression of MUC1 has been reported in ovarian cancer [START_REF] Giuntoli | Mucin gene expression in ovarian cancers[END_REF][START_REF] Feng | Expression of MUC1 and MUC2 mucin gene products in human ovarian carcinomas[END_REF] . The expression of MUC1-associated Tn/STn-epitopes has been suggested [START_REF] Tashiro | Immunohistochemical study of mucin carbohydrates and core proteins in human ovarian tumors[END_REF] , based on a similar expression pattern of MUC1 epitopes as well as Tn/STn-epitopes. However, the expression of MUC1associated Tn-and STn-epitopes has never been proven.

In this study, we determined whether primary ovarian tumours and metastatic lesions express aberrantly glycosylated MUC1. Since the most widely accepted candidate for the origin of ovarian cancer is the ovarian surface epithelium (OSE) [START_REF] Mandai | Ovarian cancer in endometriosis: molecular biology, pathology, and clinical management[END_REF] , we examined the MUC1 glycosylation status of OSE adjacent to the tumour in neoplasic ovaria and the epithelium of serous cystadenomas. Additionally, we analysed MUC1 expression of ovarian endometriosis and serous borderline tumours with intraepithelial carcinoma / micropapillary growth, because both have been reported to be precursor lesions of invasive ovarian adenocarcinomas [START_REF] Scully | Classification of human ovarian tumors[END_REF] . We demonstrate that the MUC1-associated Tn/STn-epitopes are expressed on all different ovarian The MAb SM3 (supernatant [START_REF] Burchell | Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin[END_REF] ) was contributed by Prof. Dr. J. Taylor-Papadimitriou (Guy's Hospital, London, United Kingdom). MAb 5E5 (supernatant [START_REF] Sorensen | Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancerspecific anti-MUC1 antibody responses and override tolerance[END_REF][START_REF] Tarp | Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat[END_REF] ) was donated by Prof. Dr.

H. Clausen (Department of Glycobiology, University of Copenhagen, Denmark). A detailed description of the specificities of the MUC1 antibodies used in this study has been published previously [START_REF] Van Leeuwen | Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector[END_REF] . Briefly, the MAb 214D4 recognizes human MUC1 irrespective of its glycosylation pattern, MAb SM3 recognizes the differentiation-dependent glycoforms and MAb 5E5 exclusively recognizes cancer-associated glycoforms of MUC1 (MUC1-associated Tn and STn) (Figure 1) [START_REF] Sorensen | Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancerspecific anti-MUC1 antibody responses and override tolerance[END_REF] . Although the MAbs SM3 and 5E5 both display cancer-associated reactivity [START_REF] Burchell | Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin[END_REF] , MAb SM3 also stains healthy human tissue as has been shown previously in the kidney and thymus [START_REF] Girling | A core protein epitope of the polymorphic epithelial mucin detected by the monoclonal antibody SM-3 is selectively exposed in a range of primary carcinomas[END_REF][START_REF] Cloosen | Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy[END_REF] . IMMUNOHISTOCHEMISTRY Paraffin-embedded tissue sections were deparaffinised by xylol (Merck, Darmstadt, Germany) treatment and rehydrated in decreasing concentrations of methanol (Merck). After rehydration, endogenous peroxidase was blocked (0.5% H 2 O 2 in methanol) for 30 minutes and the slides were washed in PBS (Sigma-Aldrich, St. Louis, USA) supplemented with 0.05% Tween 20 (Merck). Specific staining of MUC1 was determined by incubation with primary anti-MUC1 MAbs or matched isotype controls overnight at 4°C. Afterwards, sections were rinsed with PBS / Tween 20 and incubated with Envision-HRP (DAKO, Glostrup, Denmark) for 20 minutes at room temperature. The colour was developed by incubation for 3 minutes at room temperature using a substrate buffer containing 3,3'-diaminobenzidine (5 µg/mL; DAKO) and 0.5% H 2 O 2 (Sigma, Zwijndrecht, the Netherlands) in PBS / Tween 20.

Cytospins of the breast carcinoma cell line ZR75-1 (Department of Pathology, Maastricht

University Medical Center + ) proven to stain positive for all anti-MUC1 MAbs served as intraexperimental positive control for all antibodies [START_REF] Cloosen | Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy[END_REF] . The histological images were analysed by microscope (Carl Zeiss, Sliedrecht, the Netherlands) with an objective magnification of 10-20x. Digital pictures were taken by a Jenoptic camera (Carl Zeiss). The ovarian tumor slides were analysed independently by five investigators (C.V.E.; F.B.; M.H.; W.G.; K.V.d.V.).

Staining of cytoplasm and cell membranes was evaluated and cells were considered positive when at least one of these components showed positive staining.

Results

EXPRESSION OF MUC1-ASSOCIATED TN/STN-EPITOPES IN OVARIAN CANCER AND ADJACENT HEALTHY TISSUE

To determine the MUC1 glycosylation status, we stained normal and malignantly transformed ovarian epithelium with different MUC1 glycosylation-specific antibodies. Figure 2 shows a tissue section, containing normal OSE and malignantly transformed epithelium. Clearly, the MUC1 protein was expressed on the apical surface of normal ovarian epithelium, whereas the differentiation-dependent and cancer-associated glycoforms were not detected (Figure 2). In all 9 neoplastic ovarian tissue samples containing OSE, MUC1 expression of the normal epithelium was limited to the apical surface.

Strikingly, the malignantly transformed epithelium of the same ovary showed abundant expression of the MUC1 protein (Figure 2B), as well as the differentiationdependent and cancer-associated glycoforms of MUC1, detected by MAb SM3 and 5E5, respectively (Figure 2C,D). Furthermore, in some malignant cells apical expression of MUC1 was lost, and MUC1 was detected on the entire cell surface (Figure 2D, arrows).

As an additional control of benign ovarian epithelium, serous cystadenomas were analysed for expression of MUC1. All of these cysts (7/7) expressed the MUC1 protein (MAb 214D4) and also 29% (2/7) expressed the differentiation-dependent glycoform, (MAb SM3) but none of them (0/7) the cancer-associated MUC1 glycoform (MAb 5E5).

EXPRESSION OF MUC1 AND CANCER-ASSOCIATED MUC1 ON DIFFERENT OVARIAN CANCER HISTOTYPES

Since there are four different epithelial ovarian cancer histotypes, all were examined using the panel of MUC1 glycosylation-specific antibodies. The prevalence differs for each histotype, with the serous adenocarcinoma being the most common type of epithelial ovarian cancer, and the clear cell and endometrioid adenocarcinoma being rare [START_REF] Cannistra | Cancer of the ovary[END_REF] . This was reflected in the number of tumours that was available for examination for each different histotype (22 serous adenocarcinomas, 5 mucinous adenocarcinomas, 2 clear cell carcinomas and 8 endometrioid adenocarcinomas). All ovarian cancer histotypes expressed MUC1 (37/37) (Table 1). The differentiation-dependent glycoform (MAb SM3) was found on 82% (18/22) of the serous, 60% (3/5) of the mucinous, 63% (5/8) of the endometrioid and on all of the clear cell (2/2) adenocarcinomas. The cancer-associated glycoform (MAb 5E5) was expressed on 86% (19/22) of the serous (Figure 3A), 80% (4/5) of the mucinous (Figure 3B), all of the clear cell (2/2) (Figure 3C) and on 88% (7/8) of the endometrioid adenocarcinomas (Figure 3D).

In contrast to serous cystadenomas, ovarian endometriosis can serve as a precursor lesion of ovarian cancer, especially of the endometrioid and clear cell subtypes [START_REF] Mandai | Ovarian cancer in endometriosis: molecular biology, pathology, and clinical management[END_REF] . Since most of the endometrioid adenocarcinomas express the MUC1 cancer-associated glycoform, we investigated the MUC1 glycosylation pattern of ovarian endometriosis. All endometriosis lesions expressed MUC1 (7/7) (Figure 4A), the differentiation-dependent glycoform (MAb SM3) was expressed by 57 % (4/7) and the cancer-associated glycoform (MAb 5E5) by 71%

(5/7) of the endometriosis lesions (Figure 4B,C).

Additionally, serous borderline tumours with intraepithelial ovarian carcinoma, another precursor lesion of invasive ovarian adenocarcinoma, was examined for its MUC1 expression. All of these lesions expressed the MUC1 protein (4/4) (Figure 5A), 50% (2/4) the differentiation-dependent glycoform (Figure 5B) and 75% (3/4) the cancer-associated glycoform (Figure 5C) In summary, the MUC1-associated Tn/STn-epitopes could be detected in tissue sections of all different ovarian cancer histotypes. Importantly, these epitopes were expressed on 86% of the serous adenocarcinomas, the most frequently diagnosed ovarian malignancy.

For the other histotypes the number of tumour sections analysed was too low to draw firm

EXPRESSION OF MUC1 AND CANCER-ASSOCIATED MUC1 IN OVARIAN CANCER

METASTASES

As already indicated, ovarian tumours are prone to metastasize, therefore metastatic lesions were examined for their MUC1 expression. In 8 cases, it was possible to analyze both the primary tumour as well as the metastases. In the metastatic lesions, expression of MUC1 was detected in all (13/13) of the samples (Figure 6B). The differentiation-dependent MUC1 glycoform was detected in 77% (10/13) and the cancer-associated MUC1 glycoform in 85% (11/13) of metastatic lesions (Figure 6C,D). When the MUC1 expression profile of the metastases was compared with the profile of the primary tumour, the expression of MUC1 (MAb 214D4) was similar (100%). Additionally, in 63% (5/8) there was a similar expression profile of the differentiation-dependent MUC1 glycoform (MAb SM3) and even better results were obtained for the expression of MUC1-associated Tn/STn-epitopes (MAb 5E5), which showed comparable expression in all cases.

Discussion

We demonstrate that aberrantly glycosylated, cancer-associated MUC1 is expressed in situ in most ovarian cancer tumours, but not in OSE and serous cystadenomas. Moreover, we show that the cancer-associated MUC1 epitopes are not only present in primary tumours, but also in ovarian cancer precursor lesions and in metastatic lesions. The expression of cancerassociated MUC1 epitopes in breast cancer has already been identified [START_REF] Sorensen | Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancerspecific anti-MUC1 antibody responses and override tolerance[END_REF] . Until now, the presence of cancer-associated MUC1 specific Tn-and STn-epitopes in ovarian cancer has been suggested, but not yet proven [START_REF] Tashiro | Immunohistochemical study of mucin carbohydrates and core proteins in human ovarian tumors[END_REF] . Here we show that the cancer-associated MUC1 Tn/STn-epitopes, recognized by MAb 5E5, are indeed expressed in ovarian cancer and not in healthy ovarian tissue. In accordance with previous reports, the MAb SM3 also reacts with ovarian cancer tissue [START_REF] Burchell | Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin[END_REF] . Even though the MAbs SM3 and 5E5 both display this cancerassociated reactivity in ovarian cancer, MAb SM3 also reacts with healthy tissue as has been shown previously in thymus, salivary glands, sebaceous glands and the distal tubulus of the kidney [START_REF] Girling | A core protein epitope of the polymorphic epithelial mucin detected by the monoclonal antibody SM-3 is selectively exposed in a range of primary carcinomas[END_REF][START_REF] Cloosen | Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy[END_REF] . This indicates that antibodies recognizing MUC1-associated Tn/STn-epitopes might have more potential to be used in ovarian cancer diagnostics and especially in immunotherapy.

Several studies have reported on the monoclonality of most epithelial cells in endometriotic cysts [START_REF] Jimbo | Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts[END_REF][START_REF] Nilbert | Monoclonal origin of endometriotic cysts[END_REF] and therefore endometriosis is classified as a tumour-like lesion [START_REF] Scully | Classification of human ovarian tumors[END_REF] .

The fact that the epithelium of ovarian endometriosis also expresses the MUC1-associated Tn/STn-epitopes, further supports the theory that endometriosis is actually a precursor lesion of ovarian cancer [START_REF] Mandai | Ovarian cancer in endometriosis: molecular biology, pathology, and clinical management[END_REF] . Molecular events involved in the transformation of endometriosis have been studied extensively [START_REF] Mandai | Ovarian cancer in endometriosis: molecular biology, pathology, and clinical management[END_REF][START_REF] Gilks | Ovarian carcinoma pathology and genetics: recent advances[END_REF] and decreased glycosylation of MUC1 may be a marker of this process. This is supported by the expression of MUC1-associated Tn/STn-epitopes by serous borderline tumours with intraepithelial carcinoma, which is confirmed to be a precursor of invasive serous adenocarcinomas. If antibodies recognizing the MUC1-associated Tn/STn-F o r P e e r R e v i e w 12 epitopes are suitable for discrimination between high and low risk endometriosis lesions, this will improve risk assessment and possibly therapy of younger women who want to preserve fertility. However, before being used in routine diagnostics, larger series of endometriosis lesions and ovarian tumours have to be analysed and related to clinical outcome in a comparative study.

MUC1 is immunogenic, both humoral and cellular immune responses against normal and underglycosylated MUC1 have been identified [START_REF] Von Mensdorff-Pouilly | Humoral immune response to polymorphic epithelial mucin (MUC-1) in patients with benign and malignant breast tumours[END_REF] . Humoral immune responses have been confirmed by the detection of anti-MUC1 antibodies in the serum of healthy women and in women with ovarian cancer [START_REF] Richards | Antibodies reactive with the protein core of MUC1 mucin are present in ovarian cancer patients and healthy women[END_REF][START_REF] Croce | Humoral immune response induced by the protein core of MUC1 mucin in pregnant and healthy women[END_REF] . Anti-MUC1 antibody levels tend to be lower in cancer patients, which is possibly caused by the formation of immune-complexes with serum-MUC1 [START_REF] Kotera | Humoral immunity against a tandem repeat epitope of human mucin MUC-1 in sera from breast, pancreatic, and colon cancer patients[END_REF] . These anti-MUC1 antibodies have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) of MUC1 positive cancer cells and to be protective in cancer development [START_REF] Moreno | High level of MUC1 in serum of ovarian and breast cancer patients inhibits huHMFG-1 dependent cell-mediated cytotoxicity (ADCC)[END_REF][START_REF] Terry | Incessant ovulation, mucin 1 immunity, and risk for ovarian cancer[END_REF] .

Despite its immunogenicity [START_REF] Karsten | What makes MUC1 a tumor antigen?[END_REF][START_REF] Wesseling | Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components[END_REF][START_REF] Nassar | Pathogenesis of invasive micropapillary carcinoma: role of MUC1 glycoprotein[END_REF] , MUC1 also contributes to escape from tumour immuno-surveillance and metastasis formation. In tumour cells, MUC1 has an anti-adhesive function, which is not only of importance in cancer development [START_REF] Nassar | Pathogenesis of invasive micropapillary carcinoma: role of MUC1 glycoprotein[END_REF] , but might be responsible for a diminished adherence of immune effector cells to the malignant cells [START_REF] Van De Wiel-Van Kemenade | Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction[END_REF] .

Immunotherapy will be essential to enhance immune responses to MUC1. MUC1 peptide vaccination or peptide-pulsed DC vaccination have shown to increase the amount of anti-MUC1 antibodies and induce proliferation of MUC1-specific cytotoxic T cells [START_REF] Von Mensdorff-Pouilly | Humoral immune response to polymorphic epithelial mucin (MUC-1) in patients with benign and malignant breast tumours[END_REF][START_REF] Akporiaye | Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas[END_REF][START_REF] Apostolopoulos | Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRCTN71711835][END_REF][START_REF] Loveland | Mannan-MUC1-pulsed dendritic cell immunotherapy: a phase I trial in patients with adenocarcinoma[END_REF] .

However, induction of a clinical effective cellular or humoral immune response to MUC1 has not yet been achieved. One of the reasons might be central tolerance to the mature MUC1 protein [START_REF] Sorensen | Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancerspecific anti-MUC1 antibody responses and override tolerance[END_REF] . Thymic medullary epithelial cells express the MUC1 protein, thereby most likely inducing negative selection of MUC1-specific T cells. However, the underglycosylated MUC1 isoforms are not present in the thymus, and therefore it was suggested by our group, 13 that tolerance is induced only to the mature MUC1 and not the MUC1-associated Tn/STnepitopes [START_REF] Cloosen | Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy[END_REF] .

In this study, the underglycosylated MUC1-associated Tn/STn-epitopes were identified as potential TAAs, which could be used as targets for immunotherapy and immunodiagnostics in ovarian cancer patients and patients who are diagnosed with ovarian cancer precursor lesions. The expression of these epitopes by ovarian cancer and precursor lesions indicates that there are alterations in MUC1 processing and elucidation of the mechanism behind these alterations might increase our knowledge on ovarian tumour biology.

Cancer-specific MUC1 is expressed in 86% of all different ovarian cancer histotypes and in 85% of metastatic lesions, but not in normal ovarian tissue. Moreover, in the most frequent histotype, the serous adenocarcinoma, the percentage of tumours expressing the cancerassociated MUC1 is high (86%). Additionally, antibodies recognizing MUC1-associated Tn/STn-epitopes represent an effective diagnostic tool for the detection of ovarian cancer cells in tissue and fluids. Finally, since the MUC1-associated Tn/STn antibodies exclusively detect tumour cells, these antibodies could be effectively used for antibody-mediated immunotherapy and additionally radio-labelling could be very useful for in vivo imaging and therapy.

F

  their metastases, while they are absent in normal ovarian tissue, rendering these epitopes potential targets for immunotherapy and immunodiagnostic imaging.

  tissue samples were obtained from files of the Department of Pathology, (Maastricht University Medical Center + ) from 1990 to 2006. The study population consisted of 43 patients diagnosed with epithelial ovarian cancer. For 37 patients, primary tumours were analysed. For 8 of these patients, matched metastases were analysed. Of 5 other patients, only metastatic lesions could be analyed. As a control, normal OSE adjacent to the tumour of 9 MUC1 positive tumours and 7 serous cystadenomas were analysed.The 37 primary ovarian tumours originated from different histotypes and comprised of 22 serous adenocarcinomas, 5 mucinous adenocarcinomas, 2 clear cell carcinomas, 8 endometrioid carcinomas. Additionally, 4 serous borderline tumours with intraepithelial carcinoma and 7 endometriosis lesions were analysed. The 8 matched metastatic lesions were all typed as serous adenocarcinomas and the 5 non-matched metastatic lesions consisted of 4 serous adenocarcinomas and 1 mucinous adenocarcinoma. ANTIBODIES MUC1 antibody 214D4 (purified from the supernatant of the 214D4 cell line[START_REF] Wesseling | Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components[END_REF] ) was kindly provided by Dr. J. Hilkens (the Netherlands Cancer Institute, Amsterdam, the Netherlands).

  all tumours and ovarian cancer precursor lesions examined, expressed the cancer-associated MUC1 epitopes.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 123456 Figure 1. Schematic representation of carbohydrate antigens, MAb and glycosyltransferases assayed in this paper. O-linked glycosylation is initiated by polypeptide N-acetylgalactosaminyltransferases (a), adding N-acetylgalactosamine (GalNAc) to serine (Ser) or threonine (Thr) of the polypeptide backbone, resulting in the Tn antigen, recognised by MAb 5E5. The Tn antigen may be sialylated by sialyltransferase ST6GalNAc-I (b), adding sialic acid (NeuAc) to the GalNAc moiety of Tn, resulting in the sialyl-Tn (STn) antigen, also recognized by MAb 5E5. If not sialylated, core 1 structures may be synthesized by galactosyltransferase Core 1 ß3-GalT (c), adding galactose (Gal) to the GalNAc moiety of Tn, resulting in the T antigen. Addition of further sugar moieties will result in the formation of Core 2 antigen formation, recognised by MAb SM3. 215x128mm (300 x 300 DPI)

Table 1 .

 1 Expression of the different MUC1glycoforms on the different ovarian carcinoma histotypes.

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

Acknowledgements

We thank Prof. Dr. R. Kruitwagen (gynecological oncology) for critical reading of the manuscript. This work was supported by SenterNovem (Project: IS055002). The authors have no conflicting financial interests.