Andrea Luca

Mariagabriella Lotta

Dario Mariani

Ilaria Consonni

Roberta Mancini

Alberto Palla

Dragica Maino

Michele Vucelic

Piermannuccio Pizzuti

Flora Mannucci

Peyvandi

Angelo Bianchi Bonomi

Lotta

Mariagabriella Mariani

Dario Consonni

Ilaria Mancini

Roberta Palla

Alberto Maino

Dragica Vucelic

Michele Pizzuti

Pier Mannuccio Mannucci

Flora Peyvandi
email: flora.peyvandi@unimi.it.

Angelo Bianchi Bonomi

DIFFERENT CLINICAL SEVERITY OF FIRST EPISODES AND RECURRENCES OF THROMBOTIC THROMBOCYTOPENIC PURPURA

Keywords: THROMBOTIC HAEMOLYTIC ANAEMIAS, thrombotic thrombocytopenic purpura, recurrence, relapse, ADAMTS13

published or not. The documents may come

SUMMARY

The clinical course of thrombotic thrombocytopenic purpura (TTP) is characterized by recurrent disease episodes in up to 50% of the cases. The clinical presentation and severity of different TTP episodes have not been systematically compared. Laboratory and clinical information from 51 patients with recurrent disease, stemming from 136 patients with TTP included in the Milan TTP registry (URL: http://www.ttpdatabase.org), were used to compare mortality, symptoms and disease-related laboratory measurements in different disease episodes. The prevalence of severe neurological symptoms (coma, seizures, and focal neurological defects) was significantly lower in recurrences than in the first episode. Platelet counts and haemoglobin levels at presentation were higher in recurrences than in the first disease episode, and lactate dehydrogenase levels were lower.

Also mortality tended to be lower in the second and third disease episodes than in the first.

Recurrences of TTP are generally milder than first episodes. These differences in severity should be taken into account in clinical research on TTP and in patient management. KEY WORDS: thrombotic thrombocytopenic purpura, recurrence, relapse, ADAMTS13.

INTRODUCTION

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease characterized by acute episodes of thrombocytopenia and microangiopathic hemolytic anemia due to disseminated microvascular thrombosis [START_REF] Moake | Thrombotic microangiopathies[END_REF]. The clinical presentation of TTP is variable in terms of type and severity of clinical symptoms [START_REF] Peyvandi | von Willebrand factor cleaving protease (ADAMTS-13) and ADAMTS-13 neutralizing autoantibodies in 100 patients with thrombotic thrombocytopenic purpura[END_REF][START_REF] Scully | Regional UK TTP registry: correlation with laboratory ADAMTS 13 analysis and clinical features[END_REF]= Vesely SK et al, 2003;Zheng XL et al, 2004). Before the introduction of plasma exchange (PEX) for the treatment of acute TTP episodes mortality reached 90% [START_REF] Amorosi | Thrombotic thrombocytopenic purpura: report of 16 cases and review of the literature[END_REF], but decreased to 8-30% following the adoption of PEX [START_REF] Hovinga | Survival and relapse in patients with thrombotic thrombocytopenic purpura[END_REF][START_REF] Rock | Comparison of plasma exchange with plasma infusion in the treatment of thrombotic thrombocytopenic purpura. Canadian Apheresis Study Group[END_REF][START_REF] Scully | Regional UK TTP registry: correlation with laboratory ADAMTS 13 analysis and clinical features[END_REF]). Patients who survive the first acute episode of the disease may remain asymptomatic for the rest of their life or develop one or more recurrence. It is estimated that approximately one third of TTP patients recur, with a higher risk of recurrence in those with severe deficiency of the von Willebrand factor cleaving-protease ADAMTS13 during acute episodes (;Vesely SK et al, 2003;Veyradier A et al, 2001;[START_REF] Hovinga | Survival and relapse in patients with thrombotic thrombocytopenic purpura[END_REF] and remission [START_REF] Ferrari | Prognostic value of anti-ADAMTS 13 antibody features (Ig isotype, titer, and inhibitory effect) in a cohort of 35 adult French patients undergoing a first episode of thrombotic microangiopathy with undetectable ADAMTS 13 activity[END_REF][START_REF] Jin | Relationship between ADAMTS13 activity in clinical remission and the risk of TTP relapse[END_REF][START_REF] Peyvandi | ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission[END_REF]. Few data are available on the clinical presentation and severity of recurrences. It is not uncommon in clinical practice to observe that TTP patients, aware of their condition and under close medical monitoring after the first episode, are diagnosed with recurrence before the development of such severe symptoms as coma or other signs of severe neurological involvement. Accordingly, Scully et al. observed that patients from the UK TTP registry needed less PEX procedures to reach remission during recurrences than during the first episode [START_REF] Scully | Regional UK TTP registry: correlation with laboratory ADAMTS 13 analysis and clinical features[END_REF]. However, the different severity of recurrent TTP episodes has never been the object of a systematic investigation. In addition, it is possible that a number of patients who die during recurrences are not referred for recurrence to expert clinical centers which are the major source of studies on this disease, biasing the perception of the real severity of recurrent episodes. If confirmed, the knowledge that recurrences are clinically milder than first disease episodes would be useful for those who design and carry out observational studies or clinical trials in TTP, in order to avoid lumping together patients with first episodes and recurrences. It would

PATIENTS AND METHODS

Patients and definition of clinical categories

Between 1999 and 2009 physicians from Italy and 10 additional countries (Hungary, Serbia, Canada, Germany, Iran, Lebanon, Romania, Russia, Slovenia and Turkey) referred patients who had a suspected diagnosis of TTP to the registry established at the Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre and provided detailed clinical information and, when available, plasma samples. Information was collected using a standardized clinical questionnaire (available upon request). When necessary to fill gaps of information, clinicians were asked to provide additional clinical documentation and samples. Physicians in Milan (L.A.L. or F.P.) reviewed the cases and confirmed or excluded the diagnosis of TTP. Criteria for TTP diagnosis were the documentation of at least one episode of: (a) thrombocytopenia, (b) microangiopathic haemolytic anemia (c) exclusion of alternative explanations for thrombocytopenia (such as the enterohemorrhagic form of hemolytic uremic syndrome, catastrophic anti-phospholipid antibodies syndrome, pre-eclampsia and related syndromes, sepsis, systemic inflammatory response syndrome, disseminated intravascular coagulation, disseminated malignancy and/or bone-marrow transplantation associated TTP-like syndrome). Remission was defined as persistence of normal platelet counts and hemoglobin levels for at least 30 days after the most recent acute episode and freedom from new disease symptoms. The study was approved by the Institutional Review Board of the Fondazione IRCCS Ca' Granda -Ospedale Maggiore Policlinico and all patients or their tutors gave informed consent. All cases were reviewed for a second time by a single physician (L.A.L.) in 2009 and patients judged to have insufficient information to establish the diagnosis were excluded.

The first TTP episode was classified as secondary when associated to one or more of the following conditions: (1) pregnancy/post-partum, (2) use of drugs reported to be associated with TTP, (3) additional disease associated with TTP (autoimmune diseases and/or HIV infection). Patients who did not fit the aforementioned categories were classified as having idiopathic disease. Presence of symptoms in each TTP episode was defined as prevalence of symptoms at presentation and/or during the acute phase of the disease before remission or death. Symptoms related to thrombocytopenia included petechiae (purpura), ecchymoses and superficial hematomas. Renal involvement included the increase in the serum level of creatinine (serum levels above the normal reference value of each laboratory) and/or presence of alterations of urinalysis such as proteinuria or hematuria. Severe neurological symptoms were coma, seizure and focal neurological signs (motor/sensory deficit or aphasia). Less severe symptoms (headache, amaurosis fugax, mild gait alterations) were included in the category "other neurological symptoms". Cardiovascular involvement included acute myocardial infarction, other acute coronary syndromes, increase in cardiac troponin-T, T wave electrocardiographic alterations and severe hypertension.

Gastrointestinal symptoms included abdominal pain, vomiting and diarrhoea, whereas jaundice was excluded from this category owing to the confounding effect of hemolysis. The category "other symptoms" included fatigue, dyspnoea and other symptoms not included in the aforementioned categories. PEX procedures for each episode were calculated as the number of 1 volume PEX sessions carried out since diagnosis until remission or death, double volume exchanges were considered equivalent to 2 single volume PEX sessions. According to Vesely et al. (Vesely SK et al, 2003), laboratory data recorded as 'at presentation' were the most abnormal findings recorded on the day of diagnosis ±7 days. To compare data from different laboratories, LDH values were normalized to an upper normal limit of 480 IU/L. The plasmatic activity of ADAMTS13 was measured centrally at the Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre using collagen binding assay on citrated plasma as previously described [START_REF] Peyvandi | ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission[END_REF]. Activity of ADAMTS13 below 10% was considered severely deficient. The normal range for ADAMTS13 activity was 46-160%. Data of all patients are included in a digital registry (URL: www.ttpdatabase.org). To assess the severity of different episodes, mortality rates were compared. In addition, each disease episode was compared to the others in patients who had more than one episode. Indicators of disease severity used for comparison were the differences in the prevalence of symptoms, in laboratory values (platelet counts, hemoglobin, LDH, and creatinine), and in the number of PEX procedures carried out in the time span between diagnosis and remission/death. In this matched-pair analysis each individual served as a control for him/herself in different episodes. Patients referred for only one episode were followed-up for vital status to assess how many of them had died of recurrent disease without notification to the caregiver physician and to our registry, because these events might bias the selection of patients with recurrent disease towards non-fatal, less severe cases. This check was carried out only for patients referred from Italian centres. Information on their vital status and (when applicable) cause of death were searched for with different methods: (a) direct contact of the patient, (b) check against mortality registries, (c) postal follow-up through the vital statistic offices of the city of birth or last residence of the patient.

Statistical analysis

Wilcoxon signed-rank test was used to compare continuous variables in the two disease episodes.

RESULTS

After exclusion of patients who did not match the diagnostic criteria and those with insufficient information to establish the diagnosis (n=167), 136 patients with TTP were included in the cohort (number updated at July 2009). The general features of the cohort and clinical information on the first disease episode are in Table I. Twelve patients died of the disease (9% of all patients), 10 during the first disease episode (7.3% of 136 patients with a first disease episode), 2 during the second episode (3.5% of 57 patients referred for more than one episode). None of the 33 patients who had 3 or more disease episodes died of the disease. Of the 12 patients who died, 9 had severe deficiency of ADAMTS13 (activity below 10%). Plasma samples for the centralized measurement of ADAMTS13 activity were available for 135 patients (99%): for 36 (27%) patients only acute disease samples were available, for 53 (39%) only remission samples and for 46 (34%) both acute and remission samples. The prevalence of severe ADAMTS13 deficiency was higher in the group of patients with availability of acute disease samples (67%, 55/82), whereas it was lower in patients with only remission samples available (36%, 19/53) (chi-square test, p<0.001). Among the symptom categories analyzed, cardiovascular symptoms had the lowest prevalence (Table I): an acute coronary syndrome was diagnosed in only 5 patients, 3 of whom had an acute myocardial infarction. Eight of the 136 patients had an acute renal failure according to the RIFLE (Risk of renal dysfunction, Injury to the kidney, Failure of kidney function, Loss of kidney function, End-stage renal disease) criteria for the definition of renal failure in critically ill patients [START_REF] Bellomo | Acute renal failure -definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group[END_REF].

Two of these 8 patients had severe ADAMTS13 activity, three (one with severe ADAMTS13 deficiency) died of the disease at the first episode and only one, who had relapsing episodes, was included in the paired analysis of disease severity.

Of 57 patients who had two (or more) disease episodes, clinical information on the first two episodes of the disease was available for 51 (90%). Their general features and clinical information pertaining to the two episodes are summarized in Table II. The general characteristics and the clinical features at first episode in patients with recurrence were not different from those in patients without recurrence (not shown), but a severe deficiency of ADAMTS13 activity was more prevalent among patients who had recurrences (65%, 37/57) than those without (43%, 29/68) (chi-square test, p=0.02). In patients with multiple disease episodes, the prevalence of severe neurological symptoms and fever was significantly lower during the second episode (Table II). Among disease-related laboratory measurements, platelet counts and hemoglobin levels were higher in the second episode, whereas serum LDH was lower (Table II). Creatinine levels did not significantly differ between the two episodes. Finally, the number of PEX procedures carried out between diagnosis and hospital discharge was smaller for the second episode. Subgroup analyses were performed in groups of recurrent patients with (n=36) or without (n=15) severe deficiency of ADAMTS13. In the group of patients with severe deficiency of ADAMTS13 statistically significant differences were found for prevalence of severe neurological symptoms, fever, hemoglobin levels, platelet counts, LDH values and number of PEX until remission/death. In the group of patients in whom severe deficiency of ADAMTS13 was not found in the available samples (n=15) significant differences in the two episodes were found for platelet counts, hemoglobin and LDH levels. All statistically significant differences found in subgroup analyses were in the same direction of those observed in the primary analysis.

Thirty-three (65%) of the 51 patients included in the paired analysis of disease severity comparing the first and second episode in patients with recurrent disease had at least a third disease episode that developed at a median time of 1.3 years (range: 0.3-8 years) after the second episode. The third episode generally displayed milder clinical and laboratory features in comparison to the first and even to the second episode (Figs 2 and3). The prevalence of purpura (p=0.004, McNemar's test, prevalence at first vs third episode), of severe (p=0.001) and other (p=0.007) neurological symptoms, and of fever (p=0.002) were lower in the third than in the first episode (Fig 2 ).

Hemoglobin was largely influenced by episode number (p<0.001, Wilcoxon signed rank test, levels at first vs third episode) and smaller but statistically significant effects were also observed for platelets (p=0.004) and LDH (p=0.03), whereas creatinine levels were not influenced by recurrence

DISCUSSION

The results of this study show that TTP recurrences usually have a milder clinical course than first episodes of the disease. Neurological involvement, the degree of anemia and thrombocytopenia, which were previously shown to be predictors of 6-month mortality in TTP patients (Wyllie BF et al, 2006), were less severe in the second and third disease episodes than in the first. PEX sessions employed during second episodes were significantly less than those of the first, corroborating the other study findings. The third disease episode displayed even milder clinical course than the first two episodes. A plausible explanation of the fact that TTP recurrences are less severe than first episodes is that recurrences are more rapidly diagnosed and treated. Indeed, many recurrences were diagnosed on the occasion of complete blood counts, in the presence of the laboratory features of TTP (microangiopathic hemolytic anemia and thrombocytopenia), but with mild or no clinical manifestations. A low prevalence of neurological symptoms, relatively high platelet counts and low number of PEX needed to achieve remission in TTP recurrences were previously reported by Bohm et al. in a study evaluating the course of ADAMTS13 activity during acute TTP, including 11 patients with relapsing TTP and 14 with a first disease episode [START_REF] Böhm | The course of ADAMTS-13 activity and inhibitor titre in the treatment of thrombotic thrombocytopenic purpura with plasma exchange and vincristine[END_REF]. However, only two of the relapsing patients of that study were also evaluated during the first episode, allowing a direct comparison. An advantage of this study is that 51 patients with 2 or more TTP episodes could be directly compared during their different episodes. This paired-comparison design allowed excluding confounding due to an influence of individual determinants of disease severity, which might arise when different individuals are compared in different episodes. The design of this study also allowed minimizing confounding due to heterogeneity in the ascertainment of clinical characteristics of different episodes in the same patient, because patients were usually referred to the same centre for first episode and recurrences. Limitations of this study were missing information on some patients and the small absolute number of patients studied. However, given the rarity of TTP and the difficulties of gathering detailed clinical information on clinical episodes we believe that the number and quality of data available for analysis were sufficient to enable the comparison of disease severity described in the study.

The overall prevalence of fatal TTP in the TTP cohort of this study was 9%, with lower mortality (even if not statistically significant) in relapsing episodes. This proportion of fatal disease is consistent with the rate of mortality reported for other cohorts. However, because ours is a tertiary care centre for TTP patients offering free ADAMTS13 testing and medical assistance and counseling, it is possible that recruitment of our patients is biased towards survivors of TTP episodes. Patients who died during the first episode without being referred to our registry do not affect the findings of this study, which is focused on survivors of the first episode who developed recurrences. Instead, we reasoned that deaths for TTP occurring during recurrences, not reported to our registry, might have biased this study towards the recruitment of survivors of TTP recurrence, i. e., a less severe subpopulation of recurrent patients. To exclude the presence of this potential distortion of the results, patients referred for a first TTP episode were followed-up to assess whether or not they had died of the disease without notice to the Hemophilia and Thrombosis Centre. The result of this evaluation showed no evidence of this bias, with the only death that occurred not being causally related to TTP.

Immunosuppressive drugs are used in some instances to prevent recurrent episodes in acquired TTP. In particular, Rituximab, a B-lymphocyte depleting drug, is increasingly used for this purpose.

The use of immunosuppressive drugs and specifically Rituximab might in principle influence the results of our study, if the drugs reduced disease severity in patients that recur while under treatment. However, Rituximab was infrequently used in patients referred to our registry (n=23) and none of the treated patients recurred after treatment, so that the use of this potent immunosuppressive drug cannot have influenced our data on the severity of recurrences.

The knowledge that recurrences are generally milder than first disease episodes should be considered in the design and analysis of observational studies and clinical trials in TTP. An unbalance in the prevalence of patients with recurrent episodes in groups with a risk factor under

  clinical practice to better balance the risk of recurrent disease with that of side effects from potentially harmful preventive therapies such as the use of immunosuppressive agents.

  Fig 1 provides a flow-chart of the study design. To assess the severity of different episodes,

  Categorical variables were compared by McNemar test. P values <0.05 were considered significant. STATA 11 software was used for the analysis (StataCorp. 2009. Stata Statistical Software: Release 11. College Station, TX: StataCorp LP)

  85)(Fig 3). Vital status information were requested for Italian patients reported to have survived the first TTP episode and not known to have had additional disease episodes (n=52, 75% of the 69 survivors of the first TTP episode not referred for recurrence) (Fig1). Vital status information through October 2009 was collected for all 52 patients: by phone calls or e-mail contact with the patients (n=13, 25%), through mortality registries (n= 16, 31%), or from the vital statistic office of the birth or residence city (n=23, 44%). Only one of the 52 patients had died, from a cause that was deemed unrelated to TTP (rupture of mechanic aortic valve prosthesis).

  two different treatment groups might indeed confound the ascertainment of the effect of an exposure or a treatment on acute disease clinical outcomes. The results of this study also have clinical relevance. Although recurrent episodes of TTP should always be regarded as a potentially fatal condition, the relatively less severe clinical course of recurrences should be taken into account when potentially harmful preventive therapies, like long-term immunosuppression, are being planned in patients considered at risk for TTP recurrence.

  Vesely SK, George JN, Lämmle B, Studt JD, Alberio L, El-Harake MA & Raskob GE. (2003) ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients. Blood, 102, 60-8. Veyradier A, Obert B, Houllier A, Meyer D & Girma JP. (2001) Specific von Willebrand factorcleaving protease in thrombotic microangiopathies: a study of 111 cases. Blood, 98, 1765-72. Wyllie BF, Garg AX, Macnab J, Rock GA & Clark WF. (2006) Thrombotic thrombocytopenic purpura/haemolytic uraemic syndrome: a new index predicting response to plasma exchange. British Journal of Haematology, 132, 204-9.Zheng XL, Kaufman RM, Goodnough LT & Sadler JE. (2004) Effect of plasma exchange on plasma ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and nonidiopathic thrombotic thrombocytopenic purpura. Blood, 103, 4043-9.

Table I . General, clinical and laboratory features at first episode of the patients included in the study.

 I

			PATIENTS
	n=		136
	GEOGRAPHIC ORIGIN
	Italy	107 (79%)
	Other country	a	29 (21%)
	REFERRED DURING:
	F o r
	P
	e
	e r
	R
	e
		v i e
			w

Table II . General and clinical features at their first two episodes of the 51 recurrent patients included in paired analysis of disease severity.

 II Continuous variables were expressed as median (interquartile range). A dark gray shadow is used to highlight statistically significant p values (<0.05). a Measured during acute disease or during remission. b Information available on n=37 patients for both episodes; information was not available for the first episode in n=6 patients, for the second in n=2 patients, n=6 patients were not treated with PEX in one or both episodes.

		PATIENTS WITH TTP RECURRENCE
	n=		51
	GEOGRAPHIC ORIGIN
	Italy	41 (80%)
	Other country	10 (20%)
	FEMALE GENDER	11 (21%)
	AGE AT FIRST EPISODE, years	35 (26-44)
	AGE AT SECOND EPISODE, years	38 (30-48)
	TIME TO FIRST RELAPSE IDIOPATHIC DISEASE SEVERE DEFICIENCY OF ADAMTS13 ACTIVITY a F o F o F o REFERRED FOR MORE THAN TWO EPISODES r r r	1.7 (0.5-3.7) 37 (73%) 36 (70%) 33 (64%)
	FIRST EPISODE 25 (49%) 35 (68%) 33 (65%) e P P P Severe neurological Other neurological Cutaneous manifestations of thrombocytopenia CLINICAL SYMPTOMS (n=51) e r e e r e e r	SECOND EPISODE 9 (18%) 25 (49%) 29 (56%)	p= <0.001 0.06 0.48
	Bleeding Renal involvement Cardiovascular symptoms Gastrointestinal symptoms Fever Other symptoms LABORATORY FEATURES (n=47) Platelet counts (x1000/mm3) 80x71mm (600 x 600 DPI) 9 (18%) 15 (41%) 2 (4%) 18 (35%) 21 (41%) 17 (33%) 15 (8-20) R e v i 3 (6%) 18 (35%) 2 (4%) 10 (20%) 8 (16%) 11 (22%) 20 (10-34) e w R e v i e w R e v i e w	0.09 0.58 1.00 0.13 0.003 0.23 0.009
	Hemoglobin (g/dL) 80x88mm (600 x 600 DPI) 7.7 (6.2-9.7)	10 (8.1-11.7)	<0.001
	Lactate dehydrogenase (IU/mL) 1691 (940-2379)	1110 (563-1900) 0.004
	Creatinine (mg/dL)	0.8 (0.8-1.1)	0.9 (0.6-1.3)	0.24
	NUMBER OF PLASMA EXCHANGES (n=37) b	12 (7-19)	7 (5-12)	0.01

80x76mm (600 x 600 DPI)

British Journal of Haematology

Acknowledgement

(92%)

Continuous variables were expressed as median (interquartile range). a Hungary (n=11), Serbia (n=10), Canada, Germany, Iran, Lebanon, Romania, Russia, Slovenia and Turkey (n=1 each) b Autoimmune thyroid disease (n=5), systemic lupus eritematosus (n=3), myasthenia gravis (n=1). c Ticlopidine (n=4), clopidogrel (n=1). d Pregnancy and autoimmune thyroid disease (n=1), pregnancy and systemic lupus eritematosus (n=1). e Measured during acute disease or during remission.


Piotr Jerzy Sek

Antoni Zawrocki

Wojciech Biernat

Janusz Henryk Piekarski
email: janusz.piekarski@umed.lodz.pl

Piotr ; Sek

Piotr Sek

HER2 molecular subtype

Keywords: Mammary Paget's disease, tissue microarrays, molecular subtype, CK5/6, HER2 Mammary Paget's disease, tissue microarrays, molecular subtype, CK5/6, HER2

Introduction

Numerous studies proved that the clinical outcome in the invasive breast carcinoma is significantly associated with its genetic subtype [START_REF] Perou | Molecular portraits of human breast tumors[END_REF][START_REF] Sørlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implication[END_REF][START_REF] Sørlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF][START_REF] Van De Vijver | A gene expression signature as a predictor of survival in breast cancer[END_REF][START_REF] Huang | Gene expression predictors of breast cancer outcomes[END_REF][START_REF] Sortiriou | Breast cancer classification and prognosis based on gene expression profiles from a population based study[END_REF] . Recently, the distinction of the specific molecular subtypes was also applied for in situ breast carcinomas [START_REF] Livasy | Identification of a basal-like subtype of breast carcinoma in situ[END_REF][START_REF] Bryan | Ductal carcinoma in situ with a basal-like phenotype: a possible precursor to invasive basal-like breast cancer[END_REF][START_REF] Tamimi | Comparison of molecular phenotypes of ductal carcinoma in situ and invasive breast cancer[END_REF] . The criteria for molecular classification remained the same as in case of invasive carcinomas. The markers used for defining molecular subtypes were the following: estrogen receptor (ER), human epidermal growth factor receptor type 2 (HER2), epidermal growth factor receptor (EGFR) and cytokeratin 5/6 (CK5/6). This classic panel of molecular profiling markers was proposed by Nielsen et al. for invasive breast carcinomas [START_REF] Nielsen | Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma[END_REF] and later extended to classify carcinomas in situ. Although numerous studies were performed on the molecular subtypes of breast carcinoma, mammary Paget's cells are still a set of specific neoplastic cells that remains to be defined on the molecular grounds. The association between the lesions located in the nipple and the underlying breast carcinoma was described originally in 1874 by Sir James Paget [START_REF] Paget | On the disease of the mammary areola preceding cancer of the mammary gland[END_REF] . Nowadays, the coexistence of parenchymal breast carcinoma is observed in 67%-100% of patients with Paget's disease of the nipple [START_REF] Stomper | Sensitivity of mammography on patients with Paget's disease of the nipple[END_REF][13][START_REF] Sheen-Chen | Paget disease of the breast -an easily overlooked disease?[END_REF] . On the other hand, Paget's disease of the nipple is diagnosed in 0.5%-5% of all breast carcinoma patients [START_REF] Sakorafas | Paget's disease of the breast[END_REF][START_REF] Kollmorgen | Paget's disease of the breast: a 33-year experience[END_REF][START_REF] Dixon | Paget's disease of the nipple[END_REF][START_REF] Ashikari | Paget's disease of the breast[END_REF][START_REF] Nance | Paget's disease of the breast[END_REF] . The expanding oozing and eczematous appearance of the nipple is the most common clinical presentation of the Paget's disease of nipple. The diagnosis of the disease is based on the identification of intraepidermal large, atypical cells with pale cytoplasm and these cells are referred to as 'the Paget's cells' [START_REF] Chaudary | Paget's disease of the nipple: a ten year review including clinical, pathological and immunohistochemical findings[END_REF] . They are dispersed among the basal layer but may be also identified in the upper portions of the epidermis ('pagetoid spread'). Their origin is not clear. There are two hypotheses explaining their derivation. 'Epidermotropic theory' suggests that they derive from the underlying breast carcinoma as they migrate along the mammary duct system into the nipple. The other hypothesis, 'intraepidermal transformation theory', states that the Paget's cells develop in the epidermis as a result of the morphological (and biological) change of the epidermal keratinocytes [START_REF] Paone | Pathogenesis and treatment of Paget's disease of the breast[END_REF][START_REF] Mai | Morphological evidence for field effect as mechanism of tumour spread in mammary Paget disease[END_REF][START_REF] Mai | Mammary Paget's disease: evidence of diverse origin of the disease with subgroup of Paget's disease developing from the superficial portion of lactiferous duct and a discontinuous pattern of tumor spread[END_REF] . The more popular, epidermotropic theory is supported by the observation of frequent coexistence of the invasive breast carcinoma with Paget's disease of the nipple 13,[START_REF] Jamali | Paget disease of the nipple-areola complex[END_REF] . On the other hand, the minority of cases without diagnosable breast cancer 13 . The third theory trying to combine both theories suggests that Paget's cells may develop on either way depending on the local circumstances [START_REF] Lloyd | Mammary and extramammary Paget's disease[END_REF] .

We hypothesise that similarity of the molecular subtypes of Paget's cells to molecular subtypes of underlying breast carcinomas would put in favour the epidermotropic theory. Our analysis of the medical literature concerning Paget disease of the nipple revealed that Paget's cells were not specifically classified in terms of molecular subtyping. To the best of our knowledge there was only one study defining the molecular subtype of breast carcinomas associated with the Paget's disease of the nipple 26 . However, the authors did not determine the molecular profile of the Paget's cells. Therefore, we decided to analyse the expression of the markers defining molecular subtypes of breast carcinomas: ER, HER2, EGFR, and CK5/6. The phenotypic profile was established both in the Paget's cells and in the underlying breast carcinoma. The molecular subtypes were defined as: luminal A (ER+, HER2-, EGFR+/-, CK5/6+/-), luminal B (ER+, HER2+, EGFR+/-, CK5/6+/-), HER2 positive (ER-, HER2+, EGFR+/-, CK5/6+/-), and basal-like (ER-, HER2-, EGFR+ or CK5/6+). Cases negative for all four analysed markers were referred to as 'unclassified'. In order to make our analysis of protein expression in Paget's cells more complete, we also included progesterone receptor (PR) and cytokeratin 17 (CK17). The whole analysis was performed by means of tissue microarray. We tried to determine whether this technique will appear suitable for such a demanding histologic material as the breast nipple with Paget cells. To the best of our knowledge, it is the first attempt of using tissue microarray technique for study on Paget disease of the breast. The analysis was performed on the tissues obtained from the surgery; they were fixed in 10% buffered formalin and embedded in the paraffin blocks. For each patient we retrieved formalin-fixed, paraffin-embedded tissues from the nipple (with Paget's cells) and/or underlying parenchymal DCIS and/or underlying invasive carcinoma. From selected tissue blocks a single section was cut and stained with haematoxylin and eosin. The stained section was used to assess the quality of tissue material and to determine the site of the punch biopsy for the microarray study. Due to poor quality of the archival tissue, a number of cases was rejected from the study. Good quality archival tissue was found in 49 tissue blocks containing Paget's cells, 28 tissue blocks containing associated DCIS and 15 tissue blocks containing associated invasive carcinoma. In these 'good quality' archival tissues complete immunophenotyping was performed. All these tissue blocks were derived from 57 patients.

These patients composed the study group.

Characteristics of study group

Deleted: Finally, 57 cases were included into the study group. In 13 out of 57 cases the Paget disease of the nipple was the sole manifestation of the disease (23%). Paget's disease of the nipple coexisted with an underlying DCIS and invasive ductal carcinoma in 23 (23/57; 40%) and 14 cases (14/57; 25%), respectively. In 7 patients with Paget disease, simultaneous DCIS and invasive ductal carcinoma were identified (7/57; 12%). Althogether, DCIS was diagnosed in 28 cases (28/57; 49%). In this 28 DCIS cases, nuclear grade 3 was diagnosed in 22 cases; nuclear grade 2 was diagnosed in 6 cases. The presence of necrosis was found in 16 DCIS cases. Altogether, invasive ductal carcinoma was diagnosed in 21 cases (21/57; 37%). In these 21 invasive ductal carcinoma cases, histologic grade of carcinoma was determined by means of Nottingham scale [START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF] .

Grade 3 was diagnosed in 16 cases; grade 2 was found in the remaining 5 cases.

Immunohistochemical staining

The sections stained with haematoxylin and eosin (H&E) were used to identify morphologically representative area of interest within the blocks. The original tissue blocks formed the "donor" blocks for creating the Tissue Microarray (TMA). The tissue cores were removed from the "donor" and inserted into a "recipient" paraffin blocks using a Manual Tissue Arrayer MTA-I (Beecher Instruments) with 0,6 mm diameter needles (punches) and 1 mm space between the cores. From every representative area of interest within the block we sampled 4 cores. Therefore, we sampled up to 12 cores from one case (4 cores of Paget's cells, 4 cores of in situ carcinoma and 4 cores of invasive carcinoma). In total, we made four TMAs including tissue cores from the study blocks and cores from the control tissues. As the internal controls we used fragments of the breast carcinomas with known receptor profile (ER+, PR+, HER2+) and the skin biopsies. Sections from TMA blocks were 4 µm thick and were placed onto the silanised slides (Super Frost). The first slide from each TMAs was routinely stained with H&E as a control of morphology. The following cuts were used for immunohistochemical stainings which was performed as follows. After deparaffinization and rehydration the slides containing TMA sections were rinsed and immersed in Target Retrieval Solution (pH 6,0; DakoCytomation, Denmark). They were heated for 8 minutes (2 minutes of max pressure) in a pressure cooker and then cooled slowly for 30 minutes. For detection of immunostaining we used Novolink Polymer Detection System (Novocastra Ltd., UK). After rinsing, the slides were incubated in 3% hydrogen peroxide for 5 minutes to inactivate endogenous peroxidase activity and were incubated in protein blocking agent for 5 minutes to stop nonspecific binding of the antibodies. The next step included incubation with the primary antibodies to ER, PR, EGFR, CK5/6 and CK17 (Table 1). Incubation lasted 90 minutes in the room temperature. Next, the post-primary blocker agent was applied for 30 minutes and then the slides were treated with Polymer, a polyvalent secondary antibody, for 30 minutes. That was followed by incubation with cerium-diaminobenzidine (DAB) solution for maximum 4 minutes and counterstaining with haematoxylin. The last step included dehydration and sealing of the slides. Every assay included tissue samples marked as positive and negative control. For HER2 assessment (Figure 1) we used the HercepTest (DakoCytomation, Denmark) and the whole test was performed according to the instructions of the manufacturer.

Evaluation of immunohistochemical staining

Expression of CK 5/6 and CK 17 were considered positive if any degree of cytoplasmic staining was present in the tumour cells. EGFR were considered positive if any degree of distinct membranous staining was present. Expression of HER2 was considered positive, if strong complete membrane staining was identified in at least 30% of Paget's cells or breast carcinoma cells. Expression of ER and PR was regarded positive if the nuclear staining was present in at least 10% of Paget's cells or breast carcinoma cells.

Criteria of classification of Paget's cells and underlying carcinomas to specific molecular subtypes

The molecular subtypes were defined as: luminal A (ER+, HER2-, EGFR+/-, CK5/6+/-), luminal B (ER+, HER2+, EGFR+/-, CK5/6+/-), HER2 positive (ER-, HER2+, EGFR+/-, CK5/6+/-), and basal-like (ER-, HER2-, EGFR+ or CK5/6+). Cases negative for all four

Results

Immunohistochemical stainings

Due to some loss of studied material during preparation of tissue microarray, final evaluation of immunohistochemical stainings of Paget's cells was feasible in 43 to 47 out of the initial 49 sampled cases depending on the antibody used. Evaluation of immunohistochemical stainings of DCIS was possible in 18-20 out of 28 initially sampled cases. Evaluation of immunohistochemical stainings of invasive ductal carcinoma was made in 12 to 14 out of 15 initially sampled cases. All results of immunohistochemical stainings are presented in Table 2.

Classification of Paget's cells and underlying carcinomas to specific molecular subtypes

Paget's cells

Complete set of results of stainings (ER, HER2, EGFR, CK5/6) necessary for classification to specific molecular subtypes was achieved in 43 cases. HER2 subtype (ER-; HER2+; EGFR+/-;CK5/6+/-) was a dominant molecular subtype of Paget's cells. The diagnostic criteria for this subtype were met in 37 out of 43 cases (86%). In 5 out of 43 cases (12%) the phenotypic profile was indicative of luminal B subtype (ER+; HER2+; EGFR+/-; CK5/6+/-). One case (1/43; 2%) was classified as luminal A subtype (ER+; HER-; EGFR+/-; CK5/6+/-). None of the studied cases could be classified as basal-like nor an unclassified subtype (negative for all four defining markers). Complete set of results of stainings necessary for classification of cases to specific molecular subtypes was achieved in 17 cases. HER2 subtype was a dominant molecular subtype of ductal carcinoma in situ occurring in 14 out of 17 (82%) cases. Two cases were classified as luminal subtypes: one (1/17; 6%) was classified as luminal A subtype, the other (1/17; 6%) was classified as luminal B subtype. One case (1/17; 6%) was classified as basallike subtype (ER-; HER-; EGFR+ and or CK5/6+).

Ductal carcinoma in situ

Invasive ductal carcinoma

Complete set of results of stainings necessary for classification of cases to specific molecular subtypes was achieved in 12 cases HER2-overexpression subtype was a dominant molecular subtype of invasive ductal carcinoma. The criteria for diagnosis of that subtype were met in 10 out of 12 cases (84%). Two cases were classified as luminal subtypes: one each (1/12; 8%) was classified as luminal A and luminal B subtype.

Comparison of distributions of molecular subtypes

The distributions of molecular subtypes in Paget's cells, in the underlying in situ and invasive breast carcinomas were similar (Fisher exact test with Freeman-Halton correction, P=0.93).

Discussion

Paget's cells of the nipple presented predominantly with HER2 molecular subtype. This subtype was identified in 86% of cases. However, the molecular profiles of studied cases were not homogenous in this respect. Luminal B (in 12% of cases) and luminal A (in 2% of cases) were also identified, albeit rarely, and neither basal-like molecular subtype nor unclassified cases were diagnosed. Interestingly, the distribution of molecular subtypes in Paget's cells was very similar to those presented by the underlying in situ and invasive breast carcinomas. In studied groups of carcinoma cells, HER2-positive subtype was a dominant one (86%, 82% and 84% of cases, respectively). Luminal A subtype was F o r P e e r R e v i e w 10 diagnosed in 2%, 6% and 7% of cases, respectively. Luminal B subtype was diagnosed in 12%, 6% and 7% of cases, respectively. Basal-like subtype was diagnosed in only one case (6%) of underlying in situ carcinoma. Very similar distribution of molecular subtypes in Paget's cells and in the underlying in situ and invasive breast carcinomas strongly support common origin of Paget's disease and coexisting invasive and in situ carcinomas. This indicates that Paget's cells are epidermotropic cellular counterpart of parenchymal malignancy. There is a small portion of cases that show discrepant phenotype, that may support independent origin of Paget's disease from underlying breast carcinoma. This observation is supported by another analytical approach. The molecular study of matched mammary Paget's disease and underlying breast carcinoma using loss of heterozygosity and mitochondrial DNA displacement loop sequence analysis revealed differences of molecular alterations in two out of 10 analysed pairs [START_REF] Morandi | Intraepidermal cells of Paget's carcinoma of the breast can be genetically different from those of the underlying carcinoma[END_REF] .

Molecular subtypes in the breast carcinoma were distinguished basing on genomic profiling of mRNA expression, whereas our methodology relies on the identification of protein expression within the tumour cells. We found that the dominant immunohistochemical profile of Paget's cells was characterised by HER2 overexpression and lack of expression of ER, PR, EGFR, CK5/6 and CK17. Overexpression of HER2 in Paget's cells has been already reported in other studies that showed its frequency exceeding 80% [START_REF] Brummer | HER-2/neu expression in Paget disease of the vulve and the female breast[END_REF][START_REF] Anderson | Assessment of HER-2/neu status by immunohistochemistry and fluorescence in situ hybridization in mammary Paget disease and underlying carcinoma[END_REF][START_REF] Schelfhout | Pathogenesis of Paget's disease: epidermal heregulin-α, motility factor, and the HER receptor family[END_REF][START_REF] Fu | Molecular markers in Paget disease of the breast[END_REF][START_REF] Liegl | Androgen receptors are frequently expressed in mammary and extramammary Paget's disease[END_REF] . In our study, the percentage of cases with overexpression of HER2 in Paget's cells was even higher and amounted to the level of 96%. In this respect, our results are concordant with those of other authors. Expression of steroid receptors (ER, PR) and EGFR in our study was also similar to results reported by other authors. The expression of ER in Paget's cells was described in 10-28.6% of studied cases [START_REF] Anderson | Assessment of HER-2/neu status by immunohistochemistry and fluorescence in situ hybridization in mammary Paget disease and underlying carcinoma[END_REF][START_REF] Fu | Molecular markers in Paget disease of the breast[END_REF][START_REF] Liegl | Androgen receptors are frequently expressed in mammary and extramammary Paget's disease[END_REF] , and the expression of PR was described in 0-28.6% [START_REF] Fu | Molecular markers in Paget disease of the breast[END_REF][START_REF] Liegl | Androgen receptors are frequently expressed in mammary and extramammary Paget's disease[END_REF] . We have identified the frequency of these receptors in 14% and 7% of cases of Paget disease, respectively. EGFR expression in Paget's cells was studied by Schelfhout et al. [START_REF] Schelfhout | Pathogenesis of Paget's disease: epidermal heregulin-α, motility factor, and the HER receptor family[END_REF] and revealed in 13% of cases. This generally remains in the similar rank in our study (9%). We did not find the results of any analysis of basal cytokeratin expression in the mammary There is only one report concerning the molecular subtypes of underlying breast carcinomas in patients with Paget's disease of the nipple as defined by protein expression. Lester et al. 26 performed an extensive study of expression of ER, PR, HER2, EGFR, androgen receptor and five types of cytokeratins (CK5/6, CK14, CK17, CK8, CK18). The study was performed in the group of 28 cases (DCIS, 18 cases; invasive carcinoma, 10 cases). They found that in patients with Paget's disease of the nipple, distribution of the molecular profile of underlying DCIS is significantly different from that of invasive carcinomas. In 88% of underlying DCIS HER2 subtype was identified, whereas only 40% of underlying invasive carcinomas presented that profile. Luminal B subtype constituted 6% of DCIS cases and 30% of invasive carcinoma cases in that study. Lester et al. concluded that different molecular subtypes of parenchymal breast carcinomas were most predictive of mammary Paget's disease. Our observations are similar to that by Lester et al. in regard to the molecular profile of DCIS underlying Paget's disease of the nipple. We found that 82% of underlying DCIS belonged to the HER2 subtype whereas other molecular subtypes (luminal A, luminal B and basal-like) were uncommon (6% of cases). However, we found the molecular subtype of the underlying invasive carcinoma similar to that of DCIS coexisting with Paget's disease. In fact, in both studies the common feature was high frequency of HER2 overexpression as determined by immunohistochemistry in DCIS and invasive carcinoma. The discrepancy in molecular profile of these lesions between these studies stems in vast extent from the difference in estrogen receptor expression in the invasive carcinoma component. As the phenotypic profile (ER/EGFR/HER2/CK) identified by immunohistochemistry is 'translated' into molecularly-based classification, expression of HER2 and ER are basic determinants of designation a specific tumour into one of three subtypes: luminal A (ER+, HER2-), luminal B (ER+, HER2+) or HER2-positive (ER-, HER2+).

In the study by Lester et al., ER-positivity was found in 30% of invasive carcinomas whereas in our study only 14% of cases expressed ER. The frequency of HER2 expression in both studies was 80% and 93%. In our view, the main weakness of both studies comparing Paget's disease and underlying invasive carcinoma is low number of analysed cases (10 and 12). As the criteria of assessment of ER and HER2 expression are identical in our study and that by Lester et al., we suspect that this discrepancy in results have incidental character.

These results should be verified on the larger group of cases with invasive breast carcinomas coexisting with Paget's disease of the nipple.

Other authors who undertook immunohistochemical studies on Paget's cells of the nipple, generally analysed smaller number of cases (from 5 to 30) than we did [START_REF] Chaudary | Paget's disease of the nipple: a ten year review including clinical, pathological and immunohistochemical findings[END_REF][START_REF] Brummer | HER-2/neu expression in Paget disease of the vulve and the female breast[END_REF][START_REF] Anderson | Assessment of HER-2/neu status by immunohistochemistry and fluorescence in situ hybridization in mammary Paget disease and underlying carcinoma[END_REF][START_REF] Schelfhout | Pathogenesis of Paget's disease: epidermal heregulin-α, motility factor, and the HER receptor family[END_REF][START_REF] Fu | Molecular markers in Paget disease of the breast[END_REF][START_REF] Liegl | Androgen receptors are frequently expressed in mammary and extramammary Paget's disease[END_REF] . We performed our study in almost fifty cases. This number is similar to number of cases analysed by Liegl et al. [START_REF] Liegl | Androgen receptors are frequently expressed in mammary and extramammary Paget's disease[END_REF] , however, they confined their analysis to Paget's cells whereas we also studied underlying carcinomas. Although other authors studied the expression of ER, PR, EGFR and HER2 in their studies, the panel of molecular subtype defining markers: ER, HER2, EGFR and CK5/6 was not used in studies of Paget's cells of the nipple. To study the expression of proteins in Paget's cells we chose tissue microarray analysis (TMA). The TMA technique has high (90-98%) concordance with the results of classic immunohistochemistry [START_REF] Ginestier | Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers[END_REF][START_REF] Bucher | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF][START_REF] Gillett | Multiple tissue core arrays in histopathology research: a validation study[END_REF][START_REF] Camp | Validation of tissue microarray technology in breast carcinoma[END_REF][START_REF] Zhang | Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer[END_REF] . During the construction of the TMAs we took four cores from each selected portion of the tumour tissue and used 0,6 mm punches. According to several authors three cores per sample are fully representative of the whole tumour [START_REF] Ginestier | Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers[END_REF][START_REF] Camp | Validation of tissue microarray technology in breast carcinoma[END_REF][START_REF] Hoos | Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations[END_REF][START_REF] Nocito | Tissue microarrays (TMAs) for highthroughput molecular pathology research[END_REF] . Taking into account random localization and scarcity of Paget's cells in the epidermis we decided to perform four cores from each case. Likewise, we considered using larger size of punches (1.5 mm), but as described by Kallioniemi et al. multiple 0.6 mm cores are sufficient to acquire an adequate amount of tissue [START_REF] Kallioniemi | Tissue microarray technology for highthroughput molecular profiling of cancer[END_REF] . Before we started the study we were fully aware of difficulties which could be encountered during construction of TMA in such a peculiar material as that occurring in Paget's disease. Indeed, precise sampling of clusters of Paget's cells from nipple epidermis was a difficult part of our study. However, despite difficulties, we were able to study expression of all the analysed proteins in 43 to 47 out of 49 initially sampled cases. Surprisingly, the major problems were encountered when foci of DCIS were sampled. It appeared that a substantial number of DCIS foci were too small to obtain the Deleted: , respectively, and this added less to the discrepant results 13 meaningful material for TMA. This was the reason of relatively inadequate gain of appreciable DCIS cases for immunohistochemical analysis. In our opinion we proved that technique of tissue microarrays can be efficiently used for study of Paget's disease, however some loss of studied material seems to be inevitable. Fortunately, in each of almost fifty sampled cases, the number of Paget's cells in specimens was sufficient to use proposed thresholds of positive cells criteria for evaluation of ER, PR and HER2. This aspect is very important in our study, because we were able to use the same criteria for evaluation of Paget's cells as those for underlying in situ breast carcinoma and invasive carcinoma. In consequence, we could make direct comparisons of protein expression and distribution of molecular subtypes between Paget's cells and breast carcinomas. For the remaining proteins the criteria of positivity were less demanding, as any degree of immunoreactivity was regarded as positive in regard to basal cytokeratins (CK5/6 and CK17) and EGFR. These criteria were shared by other authors [START_REF] Livasy | Identification of a basal-like subtype of breast carcinoma in situ[END_REF][START_REF] Tamimi | Comparison of molecular phenotypes of ductal carcinoma in situ and invasive breast cancer[END_REF][START_REF] Nielsen | Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma[END_REF] . Although a single cell with cytoplasmic staining was sufficient for diagnosis of cytokeratine-positive case, we found only one such case in the whole series. We found only one CK5/6-positive case of DCIS. There was no CK17-positive cases. However, we are convinced about good quality of our IHC staining as CK5/6 (Figure 2) and CK17 were demonstrated in the cells of the nipple epidermis.

Deleted:

In conclusion, our study revealed that HER2-positive molecular subtype was a

  the intraepidermal transformation theory. However, the inability of detection of intraparenchymal breast cancer coexisting with the Paget disease may depend from the insufficient sensitivity of the diagnostic methods

  The search of the database of the Department of Surgical Oncology and the Department of Pathology, Medical University of Lodz, Poland, identified 44 patients with Paget's disease of the nipple who were treated surgically from January 1977 to December 2005. Similarly, we looked for patients with Paget's disease of the nipple who were diagnosed in the Department of Neuropathology and Molecular Pathology, Medical University of Gdansk, Poland, identifying 32 patients in the period from January 1998 to December 2006. Inclusion criteria for the present study were as follows: (i) histologically confirmed Paget's disease of the nipple, (ii) no neoadjuvant chemotherapy applied, (iii) the amount of biological material from both parenchymal carcinoma and Paget disease was sufficient for immunohistochemical analysis, (iv) female gender of the patients. Sixty-four cases fulfilled these inclusion criteria.

Deleted:

  In this group, Deleted: Deleted: for 49 cases of Paget's disease, 28 cases of DCIS and 15 cases of invasive carcinoma.

  to as 'unclassified'. The distribution of molecular subtypes in Paget's cells and in the underlying in situ and invasive breast carcinomas were compared with use of Fisher exact test with Freeman-Halton correction. P-value <0.05 was considered significant.

  Paget's cells. Strangely enough, Lester et al.26 who made such immunostainings did not their manuscript. Therefore, we were not able to compare our results with those of others.

  dominant, but not the sole subtype of Paget's cells of the nipple. The distribution of molecular subtypes in Paget's cells was very similar to distribution of molecular subtypes of underlying in situ and invasive breast carcinomas. Similar distribution of molecular subtypes in Paget's cells and in underlying in situ and invasive breast carcinomas strongly suggest common origin of Paget's cells and underlying carcinomas.

Figure 1 .Figure 2 .

 12 Figure 1. Overexpression of HER2 protein in Paget's cells in nipple epidermis. Magnification x200 Figure 2. Lack of expression of cytokeratin 5/6 in Paget's cells in nipple epidermis. Magnification x200

Figure 1 .Figure 2 .

 12 Figure 1. Overexpression of HER2 protein in Paget's cells in nipple epidermis. Magnification x200 914x685mm (72 x 72 DPI)

Table 1 .

 1 Antibodies used for immunoassays

Table 2 .

 2 The results of the immunohistochemical analysis of Paget's disease and underlying in situ and invasive breast carcinomas

Table 2 .

 2 The results of the immunohistochemical analysis of Paget's disease and underlying in situ and invasive breast carcinomas Number of cases with staining / number of finally evaluated

			cases (%)
		Paget's cells in	Underlying in situ	Underlying
		nipple epidermis	breast carcinoma	invasive breast
				carcinoma
	Estrogen receptor	6/43 (14%)	2/18 (11%)	2/14 (14%)
	Progesterone receptor	3/44 (7%)	1/20 (5%)	1/13 (8%)
	Human epidermal growth	45/47 (96%)	18/20 (90%)	13/14 (93%)
	factor receptor 2
	Epidermal growth factor	4/45 (9%)	3/18 (17%)	3/13 (23%)
	receptor
	Cytokeratin 5/6	1/46 (2%)	0/19 (0%)	0/12 (0%)
	Cytokeratin 17	0/44 (0%)	0/19 (0%)	0/12 (0%)

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


Maria Benevolo
email: benevolo@ifo.it

Irene Terrenato

Marcella Mottolese

Ferdinando Marandino

Mariantonia Carosi

Paola Muti

Francesca Rollo

Livia ; Ronchetti

Luciano ; Mariani

Giuseppe Vocaturo

Amina Vocaturo

Comparative evaluation of nm23 and p16 expression as biomarkers of HR-HPV infection and CIN2+ lesions of the uterine cervix

Keywords: nm23, p16INK4a, Uterine cervix, Human Papillomavirus, diagnostic biomarkers nm23, p16 INK4a, Uterine cervix, Human Papillomavirus, Cervical Intraepithelial Neoplasia, HPV testing, diagnostic biomarkers

Summary

Aims: to investigate the clinical role of nm23 expression in identifying both HR-HPV and high-grade cervical lesions or carcinomas (CIN2+), and compare it with p16 overexpression, as this latter biomarker has already been widely reported in HR-HPV infected cervical lesions.

Methods: Immunohistochemical evaluation of nm23 and p16 in 143 cervical biopsies including negative, low-and high-grade lesions and squamous carcinomas (SC). HR-HPV testing by Digene HC2 and PCR, on the cervico-vaginal samples of the same patients.

Results: In detecting CIN2+, p16 was significantly more sensitive and specific than nm23 (96.3% vs 81.8% and 66% vs 36.4% respectively, both p<0.0001). Concerning HR-HPV detection by HC2, p16 showed a significantly higher specificity than nm23 (82% vs 47%, p<0.0001), although the sensitivities were comparable (71% vs 76%). We found a significantly direct correlation between nm23 and HC2 findings. However, nm23 expression did not correlate with HPV16/18 infection. On the contrary, we observed a significant association between p16 overexpression and HPV 16/18 genotypes. Conclusions: We confirm the promising clinical role of p16 overexpression. Moreover, despite in vitro data regarding the interaction with the HPV-E7 protein, nm23 does not appear to be a more useful biomarker than p16 in identifying CIN2+ or HR-HPV infection.

Introduction

Persistent infection with human papillomavirus (HPV) is generally accepted as a primary cause of cervical cancer. However, neoplastic transformation is a rare complication of a HPV infection which, in the majority of cases, is a transient event [START_REF] Dalstein | Persistance and viral load of high-risk HPV are predictors for development of incident disease[END_REF]. In recent years, considerable time and effort has been spent in order to identify biomarkers which could increase the specificity of DNA-based HPV assays, and which are able to detect those HPV infections significantly involved in cervical tumor progression. Among the emerging risk-progression biomarkers, the p16 protein, a cyclin-dependent kinase inhibitor involved in cell cycle control, is one of the most promising. As widely reported, p16 expression is affected by the HR-HPV E7 protein and its up-regulation in the uterine cervix significantly correlates with the increasing severity of the lesions [START_REF] Klaes | Overexpression of p16 INK4a as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri[END_REF][START_REF] Von | New markers for cervical dysplasia to visualise the genomic chaos created by aberrant oncogenic papillomavirus infections[END_REF][START_REF] Benevolo | Immunohistochemical expression of p16 INK4a is predictive of HR-HPV infection in cervical low-grade lesions[END_REF]. Nevertheless, the immunohistochemical (IHC) evaluation of p16 on histological specimens usually presents a limited reproducibility due to the lack of standardized criteria for the interpretation of immunostaining [START_REF] Tsoumpou | p16 INK4a immunostaining in cytological and histological specimens from the uterine cervix: a systemetic review and metaanalysis[END_REF]. This limitation may be overcome by the use of a panel of wellvalidated biomarkers, which can both increase clinical sensitivity and specificity and provide an internal validation control for each single marker. In the continuing search for more robust biomarkers of risk progression in human cervical lesions, the nm23 protein could represent an emerging and interesting candidate. This protein, which works as a nucleoside diphosphate kinase, displaying antimetastatic potential in human malignancies, is also involved in the control of normal development and differentiation [START_REF] Lombardi | m23: Unraveling its biological function in cell differentiation[END_REF][START_REF] Steeg | Metastasis Suppressor genes[END_REF][START_REF] Hsu | High expression of human nonmetastatic clone 23 type 1 in cancer of uterine cervix and its association with poor cell differentiation and worse overall survival[END_REF]. Preliminary in vitro studies suggest that the physical interaction between the E7 protein from HR-HPV types and the nm23 affected protein stability and function thus favouring the HPV tumorigenic potential [START_REF] Mileo | Multiple interference of the human papillomavirus-16 E7 oncoprotein with the functional role of the metastasis suppressor Nm23-H1 protein[END_REF]. Nevertheless, a limited and conflicting number of clinical studies have been performed to investigate and validate the role of nm23 as a marker of cervical HPV infection, disease onset and progression [START_REF] Wang | Nm23-H1 immunohistochemical expression in multisteps of cervical carcinogenesis[END_REF][START_REF] Wang | Clinical significance of high nm23-H1 expression in intraepithelial neoplasia and early-stage squamous cell carcinoma of the uterine cervix[END_REF][START_REF] Lee | Nm23-H1 protein immunoreactivity in intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix[END_REF][START_REF] Ravazoula | Immunohistochemical analysis of nm23-H1 expression in human cervical lesions[END_REF][START_REF] Hsu | Concurrent high expression of human telomerase reverse transcriptase and human nonmetastatic clone 23 in high grade[END_REF]. In particular, only one study has analysed nm23 expression together with HPV testing (15). By means of IHC, the aims of the present study were: 1) to confirm the in vitro data evaluating whether nm23 may be a useful biomarker in the identification of high grade lesions in a series of human preneoplastic and neoplastic cervical lesions; 2) to verify the potential association between nm23 expression and HR-HPV infection, focusing on HPV 16 and 18; 3) to investigate the relationships between p16 and nm23 proteins evaluating their relative impact in clinical practice.

nm23 and p16 immunostaining

The nm23 and p16 immunostaining was carried out on 5 µm thick sections from formalin fixed paraffin embedded blocks. nm23 IHC was performed using the monoclonal antibody 37.6 directed against the nucleoside diphosphate kinase A, codified by the nm23-H1 gene. Immunoreactivity was revealed by means of a super sensitive streptavidin-biotin immunoperoxidase system (Biogenex, Menarini, Florence, Italy), using 3-amino-9-ethylcarbazole as a chromogenic substrate. All cytoplasmic stainings, independent of occasional nuclear staining, were considered positive. On the basis of staining intensity and localization throughout the epithelium, we distinguished 3 categories of reactivity: negative when any form of staining was observed; positive heterogeneous when only the basal and parabasal epithelial layers presented a weak to moderate immunostaining; positive homogeneous when a strong immunoreactivity was evidenced throughout all the squamous epithelium. Nevertheless, because the heterogeneous and homogeneous positive samples were randomly distributed in the different histological categories and were not related to HPV infection, the staining findings were treated in results and data analysis as dichotomous categorical variables, so that all the positive cases were grouped together and compared with the negatives. p16 expression was revealed by means of a commercially available kit (CINTec Histology Kit, Mtm, Italy), which includes the monoclonal antibody E6H4 (16), following the manufacturer's instructions. Nuclear, with or without cytoplasmic reactivity, was considered positive and, according to the interpretation score proposed by Klaes (2), we considered the diffuse staining of the basal and parabasal cell layers as a positive cut-off. Staining intensity was not graded to avoid subjective interpretation.

HPV testing

The HC2 test was performed on cervicovaginal specimens taken at the same time of colposcopic examination and collected in 20 mL of PreservCyt solution (Cytyc, Italy). For each specimen, a Papanicolaou stained slide was prepared by means of ThinPrep 2000 System (Cytic), to ensure the quality and cellularity of the sample. The HR-HPV DNA detection was performed by means of the Digene HC2 system (Qiagen, Italy), following the manufacturer's recommendations, using 4 mL of each cervico-vaginal samples. The HR-HPV HC2 assay recognizes the 13 most common HR-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Before the HC2 testing, the samples were processed using the Digene HC2 Sample Conversion Kit (Qiagen).

The HPV genotyping test was performed by means of the Linear Array kit (Roche, Italy), utilizing 250 µL of the residual liquid sample. This PCR assay is able to recognize 37 high, intermediate and low risk HPV types [START_REF] Lombardi | m23: Unraveling its biological function in cell differentiation[END_REF][START_REF] Wang | Clinical significance of high nm23-H1 expression in intraepithelial neoplasia and early-stage squamous cell carcinoma of the uterine cervix[END_REF]16,18,26,31,33,35 CP6180). Because of the higher oncogenic potential displayed by HPV 16 and 18 types, in the PCR interpretation we divided the positive results into two categories: 1) presence of HPV 16 and/or 18 sequences with or without other genotypes (16/18+), 2) revelation of HR-HPV genotypes other than 16 and 18, as single or multiple infections, comprising at least one of the genotypes recognized by the HC2 system (HR-HPV+).

Statistical analyses

The Pearson's Chi-Square test was applied for all comparisons between variables of interest and when the p-value was < 0.05 it was considered to be statistically significant.

Results

nm23 and p16 distribution according to histological diagnoses

We evaluated IHC nm23 expression, in parallel with p16, in our series of 143 cervical biopsies. Figure 1A shows nm23 distribution according to histological diagnoses. Among the 32 negative cervical tissues, 17 cases demonstrated nm23 immunoreactivity (53%).

The CIN1, as well as the CIN2 and the CIN3 lesions, demonstrated nm23 positivity in 39 out of 56 (70%), 13 out of 19 (68.5%), and 15 out of 20 (75%) samples respectively, while all the analysed squamous carcinomas displayed a homogeneous cytoplasmic staining (Figure 2). Starting from the negative tissues through to carcinomas, a trend of constant and significant increase of protein overexpression was observed (pχ 2 trend =0.002).

As shown in figure 1B, p16 immunoreactivity was detected in 19% of the normal tissues, in 43% of the CIN1, in 100% of the CIN2, in 95% of the CIN3 and in 94% of the SC cases.

Also in the case of p16 immunoreactivity, we observed a significant increase of p16 overexpression starting from the negative specimens through to the carcinomas (pχ 2 trend < 0.0001), confirming data which was previously obtained by our group (4).

Analysis of HR-HPV distribution

The cervico-vaginal samples from all the 143 patients included in our study were analysed for HR-HPV infection by the HC2 system. HR-HPV infection was found in 109 samples (76%). In particular, as shown in Table 1, HR-HPV infection was found in 14 out of the 32 histologically negative tissues (44%), in 41 out of the 56 CIN1 cases (73%), in 18 out of the 19 CIN2 (95%), and in all the CIN3 lesions as well as in the squamous carcinomas.

The proportion of HR-HPV infected samples significantly increased during progression from negative, CIN1, CIN2 and CIN3 to invasive cancer, with statistical significance (pχ 2 trend <0.0001). In order to verify whether a relationship between nm23 and p16 immunostaining and HR-HPV infection occurred in our series of samples, we compared the IHC findings with HC2 results (Table 2). Concerning HC2 negative patients, 47% were also nm23 negative (16 out of 34), against the 82% (28 out of 34) evidenced in the p16 negative group. Of the 109 HR-HPV positive patients, 83 were nm23 (76%) and 77 p16 positive (71%). We observed a statistically significant correlation between nm23 expression and HR-HPV infection (p(χ 2 ) =0.009), as well as between p16 upregulation and HR-HPV infection (p(χ 2 )<0.0001). This latter finding further confirmed the usefulness of p16 overexpression in HR-HPV detection, as we have already demonstrated in a different series [START_REF] Benevolo | Immunohistochemical expression of p16 INK4a is predictive of HR-HPV infection in cervical low-grade lesions[END_REF].

Relationship between nm23 and p16 immunostaining and HPV genotyping by PCR

A genotyping test was performed in 84 out of the 109 HC2 positive cases. Overall, 45 cases presented the 16 and/or 18 genotypes (HPV 16/18+, 54%) whereas 39 displayed other high risk genotypes (46%), as described in the Materials and Methods Section.

Analysing the distribution of nm23 and p16 immunostaining compared to PCR genotyping (Table 3), we evidenced that nm23 immunoreactivity did not correlate with HPV16/18+ infection (p= 0.251), whereas p16 overexpression showed a significant association with the presence of HPV16/18 types (p=0.008). In fact, even if 38 out of the 45 16/18+ cases showed nm23 immunostaining (84%), 29 out of the 39 HR-HPV+ infected samples were also nm23 immunoreactive (74%). In contrast, among the 45 16/18+ cases, 39 displayed p16 overexpression (87%), while among the 39 HR-HPV+ specimens, only 24 were p16 positive (62%).

HR-HPV infection

To better understand the diagnostic value of the two biomarkers being studied, i.e. nm23 and p16 expression, we evaluated their performance in terms of sensitivity, specificity and predictive values for detection of CIN2+ and/or identification of HR-HPV infection.

In detecting CIN2+ (Figure 3), we found that p16 was significantly more sensitive and specific than nm23 (sensitivity: 96.3% vs 81.8%; specificity: 66% vs 36.4%, both p<0.0001). Positive predictive value (PPV) of p16 was higher than that found for nm23, the difference being statistically significant (63.8% vs 44.5%, p=0.0013). Regarding negative predictive value (NPV), both the markers showed a high value, p16 displaying the highest (96.6% vs 76.2%, p<0.0001).

Concerning HR-HPV infection (Figure 4), we found that the two biomarkers had Comparable sensitivity and NPV (sensitivity: 71% vs 76%, p= 0.41; NPV: 47% vs 38%, p= 0.156). However, p16 showed a significantly higher specificity compared to nm23 (82% vs [START_REF] Benevolo | Immunohistochemical expression of p16 INK4a is predictive of HR-HPV infection in cervical low-grade lesions[END_REF]. Nevertheless, p16 immunostaining lacks standardized interpretation criteria and internal validation controls [START_REF] Tsoumpou | p16 INK4a immunostaining in cytological and histological specimens from the uterine cervix: a systemetic review and metaanalysis[END_REF]. Recently, experimental data have indicated that nm23-H1 could represent a novel, intriguing biomarker of cervical carcinogenesis. In fact, it has been shown that nm23 stability and function is affected by the HR-HPV E7 protein [START_REF] Mileo | Multiple interference of the human papillomavirus-16 E7 oncoprotein with the functional role of the metastasis suppressor Nm23-H1 protein[END_REF].

Nevertheless, the possible molecular links of nm23 to oncogenic HPV have yet to be fully elucidated.

In this context, the aim of our study was to verify whether IHC detection of nm23 could be a useful tool in the identification of CIN2+ lesions and/or HR-HPV infection either as a single marker or used in combination with p16. Concerning p16, we confirmed our previous findings (4) further demonstrating the elevated specificity and sensitivity of this marker in identifying CIN2+ or HR-HPV infected samples, particularly HPV 16/18 infections. In contrast, nm23 did not appear to be a more useful biomarker than p16, showing a lower sensitivity and specificity in CIN2+ detection. Moreover, in identyfing HR-HPV infection, p16 specificity was significantly higher than nm23, although the two markers displayed comparable sensitivities. Nevertheless, in our series, we observed a 12 direct relationship between increasing IHC expression of nm23, high grade cervical lesions and HR-HPV presence. This is a particularly important point. In fact, experimental data showed that in vitro physical interaction between HPV-16 E7 and nm23-H1 and -H2 proteins promotes transcriptional down-regulation and degradation of intracellular content of nm23, favouring the acquisition of invasiveness and resistance to apoptosis [START_REF] Mileo | Multiple interference of the human papillomavirus-16 E7 oncoprotein with the functional role of the metastasis suppressor Nm23-H1 protein[END_REF].

Consistent with this data, loss of Nm23-H1 might allow HPV-16 E7 to promote cell transformation and tumor progression. In contrast, to the best of our knowledge, studies on the expression of nm23 in normal and pathologic human cervical tissues, are limited and conflicting (10-15). Only two in vivo studies have observed a statistically significant decrease of nm23 expression starting from CIN1 to cancer [START_REF] Lee | Nm23-H1 protein immunoreactivity in intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix[END_REF]15). The majority of the in vivo studies, including the present research, have reported an increase in nm23 expression with increased CIN grade [START_REF] Wang | Nm23-H1 immunohistochemical expression in multisteps of cervical carcinogenesis[END_REF][START_REF] Wang | Clinical significance of high nm23-H1 expression in intraepithelial neoplasia and early-stage squamous cell carcinoma of the uterine cervix[END_REF][START_REF] Ravazoula | Immunohistochemical analysis of nm23-H1 expression in human cervical lesions[END_REF][START_REF] Hsu | Concurrent high expression of human telomerase reverse transcriptase and human nonmetastatic clone 23 in high grade[END_REF]. We could hypothesize that in vivo nm23 expression could also be affected by different control mechanisms which are HPVindependent, or that the amount of the E7 protein present in the HR-HPV infected cells might not be sufficient to inactivate the nm23 protein.

Concerning the relationship between HR-HPV and nm23, only one published study has compared nm23 expression with HPV DNA testing in a series of CIN and cervical cancers (15). The Branca study showed no relationship between nm23 and HR-HPV distinguishing HR-HPV positive cases from negatives, without typing. To our knowledge, our study represents the first in which nm23 IHC findings are also compared with HPV genotyping.

In our series of patients, we found a significant direct correlation between nm23 immunoreactivity and HC2 findings. However, when we compared nm23 IHC findings with HPV genotyping, we observed no association between protein expression and HPV16 and 18 genotypes, which are known to have the highest oncogenic potential. This is an important point to be considered. In fact, nm23 expression may also depend on the HR- HPV genotype present and it has been shown that the E6 and E7 oncoproteins from different HPV types have a different efficiency in their binding with the host proteins.

On the contrary, we found a significant association between p16 overexpression and HPV 16/18 infection. These data, further confirm the promising clinical role of p16 as a marker of progression risk, particularly evidencing the correlation between p16 overexpression and presence of HPV 16/18 genotypes.

In conclusion, in our series of patients, the comparative evaluation of the clinical impact of nm23 and p16 proteins, as biomarkers of high grade lesions and HR-HPV detection, showed that nm23 IHC does not display a better performance compared to p16.

Therefore, the introduction of nm23 in clinical practice does not seem reccomendable at present, as it provides no further useful information. Probably additional research must be carried out to study the relationship between this protein and oncogenic HPV proteins before it can be taken into consideration.

F

  formalin fixed cervical specimens from the Regina Elena Cancer Institute archives, collected between July 2004 and October 2007. All cases had a cervicovaginal sample for HPV testing taken at the same time of colposcopic examination and biopsy. Patient median age was 34 years (range 18 to 78 years). The study was reviewed by the Ethics Committee of the Regina Elena Cancer Institute. Histological diagnoses Cervical tissue samples were fixed in 10% buffered formalin, paraffin embedded, and haematoxylin-eosin stained according to routine histological practice. All slides were examined independently by two investigators (FM, MC) without any pathological or clinical information concerning the cases under study. Histological diagnosis revealed 32 tissues without any relevant morphologic lesions (hereinafter referred as negative), 56 CIN (Cervical Intraepithelial Neoplasia) 1, 19 CIN2, 20 CIN3 and 16 invasive squamous carcinomas (SC).

  The nm23 and p16 immunoreactivities were evaluated in terms of sensitivity, specificity and predictive values of the test, considering both histological CIN2 or worse diagnosis (CIN2+), and HR-HPV infection by HC2 test, as the endpoint.

9

 9 Relationship between nm23 and p16 immunostaining and HR-HPV testing

  , PPV and NPV of nm23 and p16 expression in detecting CIN2+ and

Figure 2 :Figure 3 :

 23 Figure 2: nm23 immunohistochemical staining of squamous cell carcinoma of the uterine

Figure 4 :

 4 Figure 4: Representative images of the relationship between sensitivity, specificity,

  of the British Division of the International Academy of Pathology Histopathology

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Table 1 .

 1 HR-HPV distribution according to histological diagnoses in 143 cervical biopsies

	HR-HPV	NEG	CIN1	CIN2	CIN3	SC	Total
	Positive	14 (44%)	41 (73%)	18 (95%) 20 (100%) 16 (100%)	109 (76%)
	Negative	18 (56%)	15 (27%)	1 (5%)	0	0	34 (24%)
	Total	32 (100%) 56 (100%) 19 (100%) 20 (100%) 16 (100%) 143 (100%)
	p(X 2 TREND ) < 0.0001

Table 2 .

 2 nm23 and p16 distribution according to the presence of HR-HPV in 143 cervical biopsies

	HC2
	Negative	Positive	Total

Table 3 .

 3 nm23 and p16 distribution according to HPV genotyping in 84 HC2 positive samples

			PCR
			HPV 16/18 +	HR-HPV +	Total
	nm23*	Positive	38 (84%)	29 (74%)	67
		Negative	7 (16%)	10 (26%)	17
		Total	45 (100%)	39 (100%)	84
	p16°	Positive	39 (87%)	24 (62%)	63
		Negative	6 (13%)	15 (38%)	21
		Total	45 (100%)	39 (100%)	84
	* p(X 2 ) = 0.251
	° p(X 2 ) = 0.008

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

Acknowledgements

This study was supported by the Italian Ministry of Health, Italian Cancer Research Association (AIRC), Lega Italiana per la Lotta contro I Tumori (LILT). We also would like to thank Michael Kenyon for his formal revision of the manuscript and Maria Assunta Fonsi for her secretarial assistance.