Harriet Salbach-Andrae

Nora Klinkowski

Martin Holzhausen

Katja Frieler

Inga Bohnekamp

Cornelia Thiels

Caroline Bender

Walter Vandereycken

Ae N Klinkowski

Ae K Frieler

Keywords:

Objective To assess the performance of the German version of the Anorectic Behavior Observation Scale (ABOS) as a parent-report screening instrument for eating disorders (ED) in their children. Methods Parents of 101 ED female patients (80 with Anorexia Nervosa; 21 with Bulimia Nervosa) and of 121 age-and socioeconomic status (SES)-matched female controls completed the ABOS. Results Confirmatory factor analysis supported the original three-factor structure model of the ABOS. Cronbach's alpha coefficients indicated good internal consistency for the three factors and the total score in the total sample. The best cut-off point (100% sensitivity and specificity) in the German version was ‡23. Conclusion The ABOS may be a useful additional instrument for assessing ED.

Introduction

Various self-report instruments have been developed to assess symptoms related to eating disorders. The widely used Eating Attitudes Test (EAT-40: [11]; EAT-26: [12]) and the Eating Disorder Inventory (EDI: [13]; EDI-2: [10]; EDI-3: [9]) for example, were designed to explore cognitive and behavioural symptoms of eating disorders (ED). These instruments provide information helpful in understanding the patient. As the psychopathology related to ED is very heterogeneous, such descriptive information is particularly relevant in individual cases.

Anorexia Nervosa (AN) can develop from about 8 years of age, reaching a peak between 15 and 18 years. Bulimia Nervosa (BN) becomes more common in young adulthood [14]. Within these age-groups, gathering data from the patients' parents and including them in the treatment is very important, because patients suffering from ED often deny or minimize their own disorder [4,15,16,18,20].

The Anorectic Behavior Observation Scale (ABOS) developed by Vandereycken and Meermann [19] assesses symptoms of patients suffering from AN and BN based on her or his parents' or carers' observations. The ABOS can be a useful instrument for the evaluation of the course of eating disorders, e.g. before and after treatment. Moreover, it can be applied for screening subjects in whom the eating disorder appears to be in an early stage. So far the ABOS has been used mainly in adolescents from the age of 12 years on. Vandereycken [18] confirmed the validity and reliability of the Dutch version of the ABOS, and Uehara et al. [17] approved its three-factor structure in a Japanese sample.

The aim of this study was (1) to confirm the threefactor structure of the ABOS using data from a German sample; and (2) to assess the validity and clinical usefulness of the German version of the ABOS in a clinical eating disorder sample.

Method j Subjects

Informed consent was obtained from the participants and their parents. The study was approved by the Institutional Review Board. We analysed the ABOSdata (German version) from 101 female ED in-and outpatients recruited from the Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Charite ´-Universita ¨tsmedizin Berlin (Germany). Sixty-three patients suffered from AN restricting subtype (ANR), 17 from AN binge-purge subtype (ANBP), and 21 from BN. All ED patients met the DSM-IV criteria for ED [2] and were aged between 12 and 18 years. AN was diagnosed by a structured interview (SIAB; [6-8]) that assesses the prevalence and severity of specific eating-related pathology over the past 3 months according to DSM-IV diagnostic criteria for people aged between 12 and 65 years. Inter-rater reliability of the SIAB is high, Cronbach's alpha coefficients range between 0.43 and 0.91 [8]. Clinically experienced and trained research assistants under the supervision of the attending child and adolescent psychiatrist conducted the interviews. Two patients had to be excluded due to missing ABOS data. Patients suffering from ED other than AN and BN were not included in the study. All parents knew about the diagnoses of their child. Socioeconomic status (SES) was measured on an eleven-point scale assessing the parents' self-reported occupational status [5]. The scale places each occupation into one of eleven categories (1 = unskilled laborer, 6 = professional employed, 7 = unskilled self-employed, 11 = professional selfemployed). We distinguished three SES groups: High (groups 5, 6, 10, 11; e.g., manager, physician), middle (groups 3, 4, 9; e.g., electrician, nurse) and low (groups 1, 2, 7, 8; e.g., cashier, textile machine operator).

Furthermore, the questionnaire was administered to 134 female controls matched for age and SES drawn from German high schools. Total body height (in cm) and body mass (in kg) were measured using standard techniques, and Body Mass Index (BMI) was calculated. Control subjects with BMI < 18 and >24 or Tscores above 59 on the two broadband scales and the total problem scale of the child behavior checklist (CBCL) were excluded from the study [START_REF] Achenbach | Manual for the child behavior checklist/4-18 and 1991 profile[END_REF]. Sixteen participants had to be excluded due to missing CBCL or ABOS values, and also due to T-scores above 59 on the subscales or on the total problem scale of the CBCL. Table 1 shows the sociodemographic characteristics of all subjects. j Measurement

ABOS

The ABOS, originally developed by Vandereycken and Meermann [19], evaluates a patient's eating behaviour based on information obtained by his or her relatives. It consists of 30 dichotomous items to be answered with Ôyes' (2 points) or Ôno' (0 points) if relatives are certain, and Ô?' (1 point) if relatives are doubtful about the answer. The higher the total score, the more pathological the subject is regarded (highest score possible = 60). Relatives should base their ratings of the 30 items on observations of the patient during the past month at home. The reliability and validity of the ABOS have been confirmed by Vandereycken [18]. The original version showed For the German version, ABOS-items were first translated into German by an independent translator. To assure adequate item-wordings in the German version, the German text was then translated back to English by a second independent translator. The two English versions (original and back-translation) were regarded as similar enough to accept the German translation by an English native speaker. Table 2 presents item examples of the ABOS.

j Statistical analysis

Two analyses were conducted to evaluate the theoretical structure (i.e., three factors) of the scale: First, internal consistency was assessed using Cronbach's alpha. Second, confirmatory factor analysis was conducted to test whether our data fitted the original three-factor model proposed by Vandereycken [18] using the analysis of moment structure (AMOS) version 4 statistical software package. Chi-square values to degrees of freedom ratios (CMIN/df), root mean square residual (RMR), goodness of fit index (GFI), adjusted GFI (AGFI), were calculated as the respective standardised goodness of fit indices in the confirmatory model. The following criteria were used to indicate an appropriate fit of the model with the present data: CMIN/df < 2.0; GFI > 0.9; AGFI > 0.9; RMR < 0.08. Also, the receiver operating characteristics (ROC) curve of the ABOS was calculated to assess the discriminant capacity of the questionnaire; sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were specified.

Results j Descriptives and group comparisons

Table 1 shows sociodemographic characteristics, body mass indexes (BMI (kg/m 2 )) and ABOS total score in the four groups. There were no significant differences regarding age and SES. As expected, there was a statistically significant difference in mean BMI (kg/m 2 ). Scheffe ´post-hoc comparisons showed a significant lower BMI (kg/m 2 ) in ANR patients than ANBP patients (P < 0.05), BN patients (P < 0.001), and healthy controls (P < 0.001). Furthermore, ANBP patients had a significant lower BMI (kg/m 2 ) than BN patients (P < 0.001) and normal controls (P < 0.001). No significant differences in BMI (kg/m 2 ) were found between BN patients and healthy controls (P = 0.99).

j Reliability (internal consistency)

Cronbach's alpha was used to evaluate the reliability of the German version of the ABOS. Internal consistencies of the three factors and the total score were high for the entire sample, but much lower for the two subsamples (see Table 3). readers can contact the authors for original CFA data. The global indices of goodness of fit derived from the original study [18] showed an equally low or slightly lower fit on GFI, AGFI, and CMIN/df. RMR was the only parameter that indicated a better fit in the original Belgian data than in the present study. In the Japanese study [17] slightly better or equal fit on GFI, AGFI, CMIN/df and RMR were found. j Sensitivity, specificity, PPV and NPV

In our sample a score of ‡23 as cut-off yielded the best sensitivity and specificity possible. Table 5 summarizes sensitivity, specificity, PPV and NPV of the ABOS questionnaire for the detection of ED. The ROC curve is presented in Fig. 1.

Discussion

According to our findings, the ABOS can be used as a valid and reliable screening tool for ED in adolescents (in addition to existing self-report instruments). Confirmatory factor analysis using data from a German sample gives support for the three-factor structure proposed in the original Belgian study [18]. It was found that the internal consistency of the 30-item instrument was high for the total sample. However, reliability for the two subsamples was much lower; a fact that can largely be explained by the massively decreased intragroup variance in the subsamples compared to the total sample [START_REF] Bortz | Forschungsmethoden und Evaluation: fu ¨r Human-und Sozialwissenschaftler[END_REF]. Reliability of the German ABOS clearly needs closer scrutiny in a population-based sample (see below). Using a total cut-off score of ‡23, the ABOS offered excellent levels of sensitivity and specificity. A limitation of this study is that based on the current sample (a clinical ED subsample and a non-clinical school-based sample), it is not possible to derive a cut-off score that would be appropriate for early identification of ED in the population that likely come with lower ABOS scores. Rather, the cut-off score ‡23 informs about the test value that best discriminates girls that already have an ED in need for treatment from a school-based sample of girls. Future studies should examine, whether these levels of sensitivity, specificity, and predictive values can be replicated when screening for ED cases within the population.

In conclusion, the German version of the ABOS could be particularly useful in the early stages of an ED, when patients show a tendency to deny or minimize their problems [20]. The parents' viewpoint seems to be a valuable source of information for both the assessment and treatment of ED in children and adolescents.

j

  Confirmatory factor analysis (CFA)Comparisons of the goodness-of-fit indices between the original Belgian model, the Japanese and the German model are shown in Table4. The original three-factor model [18] fits our data relatively well as indicated by CMIN/df, GFI, AGFI, and RMR. Inter-

Table 1

 1 Group means and standard deviations for demographic and BMI data

	Variable	AN-R (n = 63)	AN-BP (n = 17)	BN (n = 21)	Healthy controls (n = 134)	ANOVA or v 2 -test
	Age (years) a SES: high b SES: middle b	15.9 ± 1.4 19 (30.2%) 40 (63.5%)	16.5 ± 1.4 7 (41.2%) 8 (47.1%)	16.5 ± 1.6 2 (9.5%) 18 (85.7%)	16.2 ± 0.8 28 (20.9%) 100 (74.6%)	F = 2.2, P = 0.09 v 2 = 9.8; df = 6, P = 0.13
	SES: low b BMI (kg/m 2 ) a ABOS c	4 (6.3%) 15.0 ± 1.4 35.94 ± 4.35	2 (11.8%) 16.0 ± 0.7 38.88 ± 6.90	1 (4.8%) 20.3 ± 1.6 34.24 ± 5.54	6 (4.5%) 20.1 ± 1.7 4.17 ± 5.06	F = 173.5, P < .001*** F = 758.4, P < 0.001***
	SES socioeconomic status, BMI (kg/m 2 ) body mass index (kg/m 2 ), AN-R Anorexia nervosa restricting type, AN-BP anorexia nervosa bingeing/purging type, BN bulimia
	nervosa a Mean ± SD b Absolute frequency (%) c ABOS total score

Table 4

 4 Goodness-of-fit for the three-factor model in German, Belgian and Japanese samples Chi-square values to degrees of freedom ratios, GFI goodness of fit index, AGFI adjusted GFI, RMR root mean square residual

	Factor model	CMIN	GFI	AGFI	RMR
	Present study (Germany)	1.78	0.70	0.66	0.08
	Original Belgian study (16)	2.38	0.72	0.68	0.02
	Japanese Study (15)	1.55	0.84	0.80	0.08
	CMIN/df

Table 3

 3 Reliability (cronbach's alpha) of the ABOS

		Whole sample	Eating disorder	Healthy controls
	Total score	0.95	0.52	0.76
	Factor I	0.94	0.53	0.67
	Factor II	0.75	0.57	0.42
	Factor III	0.87	0.67	0.51

Table 5

 5 Sensitivity, specificity, positive and negative predictive values for ABOS Gowers SG (2008) Management of eating disorders in children and adolescents. Arch Dis Child 93:331-334 15. le Grange D, Crosby RD, Rathouz PJ, Leventhal BL (2007) A randomized controlled comparison of family-based treatment and supportive psychotherapy for adolescent bulimia nervosa. Arch Gen Psychiatry 64:1049-1056 16. Lock J, Agras WS, Bryson S, Kraemer HC (2005) A comparison of short-and long-term family therapy for adolescent anorexia nervosa. J Am Acad Child Adolesc Psychiatry 44:632-639 17. Uehara T, Takeuchi K, Ohmori I, Kawashima Y, Goto M, Mikuni M, Vandereycken W (2002) Factor-analytic study of the anorectic behavior observation scale in Japan: comparisons with the original Belgian study.

							1,0
	Cut-off Sensitivity Specificity Positive predictive	Negative predictive
				value	value
	‡5	1	0.65	0.74	1		0,8
	‡10	1	0.83	0.85	1
	‡19 ‡20 ‡23	1 1 1	0.99 0.99 1	0.98 0.99 1	1 1 1	Sensitivity	0,6
							0,4
							0,2
							0,0
							0,0	0,2	0,4	0,6	0,8	1,0
								1 -Specificity
						Fig. 1 ROC curve for ABOS

Anorectic Behavior Observation Scale (ABOS)


Massimiliano Cazzaniga

Tarik Gheit

Chiara Casadio

Noureen Khan

Debora Macis

Francesco Valenti

Mara Jo Miller

Bakary S Sylla

Suminori Akiba

Bernardo Bonanni

Ae Tarik Gheit

A E Chiara

Casadio Ae

A E Debora

Macis Ae

A E Mara

Jo Miller

A E Bakary

S Sylla

A E Suminori

Akiba Ae

Bernardo Bonanni Ae

Andrea Decensi

A E Umberto

Veronesi Ae

Massimo Tommasino
email: tommasino@iarc.fr

S Bakary

Andrea Sylla

Umberto Decensi

Bernardo Veronesi

Chiara Bonanni

Massimo Casadio

Tommasino

Á C Casadio

Á D Macis

Á F Valenti

Á B Bonanni

Á U Veronesi

Á M Tommasino

Á S Akiba

Analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis

Keywords: HPV, Breast cancer, Ductal lavage, Breast fluids

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

Introduction

The possible involvement of the epithelio-tropic human papillomavirus (HPV) in breast cancer has been proposed repeatedly in recent decades [START_REF] Lawson | Viruses and human breast cancer[END_REF]. The HPV family comprises approximately 100 different types that have been sub-grouped in different genera according to their genomic DNA sequence [START_REF] De Villiers | Classification of papillomaviruses[END_REF]. In addition, the HPVs can be subdivided into mucosal or cutaneous, based on their tissue tropism. At least 15 different mucosal HPVs of genus alpha, namely [START_REF] De Villiers | Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast[END_REF][START_REF] Hennig | Human papillomavirus 16 in breast cancer of women treated for high grade cervical intraepithelial neoplasia (CIN III)[END_REF][START_REF] Forslund | High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in ''Stripped'' biopsies from the same tumors[END_REF]33,35,39,45,51,52,56,58,59, 68, 73 and 82, have been clearly linked to cervical cancer and classified as high-risk types [START_REF] Munoz | Epidemiologic classification of human papillomavirus types associated with cervical cancer[END_REF]. Three additional HPV types of the same genus are classified as probable high-risk types, 26, 53 and 66, while several others, including HPV6 and 11, are considered low-risk types that are normally associated with benign cervical lesions [START_REF] Munoz | Epidemiologic classification of human papillomavirus types associated with cervical cancer[END_REF]. Emerging lines of evidence indicate that also another group of HPVs that belongs to the genus beta may be involved in human carcinogenesis, i.e., non-melanoma skin cancer (NMSC) [START_REF] Pfister | Chapter 8: human papillomavirus and skin cancer[END_REF]. In vitro studies have shown that the main oncoproteins E6 and E7 from HPV16 are able to immortalise primary mammary epithelial cells, providing additional evidence for a possible role of this virus in breast carcinogenesis [START_REF] Dimri | Mammary epithelial cell transformation: insights from cell culture and mouse models[END_REF].

However, studies aiming to determine the presence of HPV DNAs in breast lesions by different polymerase-chain reaction (PCR)-based methods have produced contrasting results. Low or high prevalence of HPV DNA in breast cancer of women was found in several studies. HPV16 or HPV18 were preferentially found in European women, while other types, e.g., 33, were frequently found in Japanese and Chinese cohorts [START_REF] Kroupis | Presence of high-risk human papillomavirus sequences in breast cancer tissues and association with histopathological characteristics[END_REF][START_REF] Grenier | Differential immunohistochemical and biological profile of squamous cell carcinoma of the breast[END_REF][START_REF] Tsai | Relationship between viral factors, axillary lymph node status and survival in breast cancer[END_REF][START_REF] Kan | Identification of human papillomavirus DNA gene sequences in human breast cancer[END_REF][START_REF] Gumus | HPV DNA frequency and subset analysis in human breast cancer patients' normal and tumoral tissue samples[END_REF][START_REF] Lonardo | Human papillomavirus in breast cancer[END_REF][START_REF] Yu | HPV33 DNA in premalignant and malignant breast lesions in Chinese and Japanese populations[END_REF][START_REF] Yu | Human papillomavirus type 33 DNA in breast cancer in Chinese[END_REF][START_REF] Damin | Evidence for an association of human papillomavirus and breast carcinomas[END_REF][START_REF] Tsai | Association of viral factors with non-familial breast cancer in Taiwan by comparison with non-cancerous, fibroadenoma, and thyroid tumor tissues[END_REF][START_REF] De Villiers | Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast[END_REF]. De Villiers et al. have recently shown that a broad spectrum of mucosal and cutaneous HPV types could be found in breast specimens using several PCR-based methods [START_REF] De Villiers | Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast[END_REF]. In the same study the presence of the low-risk HPV types 6 and 11 in breast tissues was also proved by in situ hybridization [START_REF] De Villiers | Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast[END_REF]. Additional studies have reported a high prevalence of HPV DNA in breast cancer of women with a history of cervical pre-malignant and malignant lesions [START_REF] Widschwendter | Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history[END_REF][START_REF] Hennig | Human papillomavirus 16 in breast cancer of women treated for high grade cervical intraepithelial neoplasia (CIN III)[END_REF][START_REF] Liu | Human papillomavirus DNA is present in a subset of unselected breast cancers[END_REF], suggesting that HPV DNA may migrate through bloodstream from the original site of infection to the breast, or alternatively, may be transmitted by hand from the female perineum to the breast. In contrast to the studies cited above, no DNA from low-risk and high-risk HPV types was detected in several other independent studies using type-specific and/or consensus PCR primers in Paget's diseases, papillomas, papillary carcinomas and infiltrating ductal breast carcinomas [START_REF] Wrede | Absence of HPV 16 and 18 DNA in breast cancer[END_REF][START_REF] Bratthauer | Etiology of breast carcinoma: no apparent role for papillomavirus types 6/11/ 16/18[END_REF][START_REF] Gopalkrishna | Absence of human papillomavirus DNA in breast cancer as revealed by polymerase chain reaction[END_REF][START_REF] Czerwenka | Human papilloma virus DNA: a factor in the pathogenesis of mammary Paget's disease?[END_REF][START_REF] Manavi | Papilloma virus and c-erbB-2 expression in diseases of the mammary nipple[END_REF][START_REF] De Cremoux | No evidence of human papillomavirus DNA sequences in invasive breast carcinoma[END_REF][26]. In particular, Gopalkrishna et al. reported that when breast cancer cells were collected by aspiration with a fine needle to avoid any possible contamination of surrounding tissues, no HPV DNA was detected in the tumour tissue [START_REF] Gopalkrishna | Absence of human papillomavirus DNA in breast cancer as revealed by polymerase chain reaction[END_REF].

To further assess the possibility that mucosal and/or cutaneous HPV types play a role in breast carcinogenesis, we determined their prevalence in breast ductal lavage (DL) fluids of women at increased risk for breast cancer. We also aimed to determine their prevalence in colostrum and milk in order to verify the prevalence of HPV in physiological fluids.

Materials and methods

Collection of the specimens

Ductal lavage specimens and superficial layers of the nipple epidermis

One hundred fifteen ductal lavage specimens (DLs) were collected from September 2006 to July 2007 at the European Institute of Oncology (Milan, Italy) in a consecutive series of 90 women at increased risk for breast cancer, i.e., history or presence of contralateral (n = 62) or ipsilateral (n = 13) breast cancer, or unaffected women with a BRCA1/2 mutation or having [10% of risk of mutation according to the Berry-Parmigiani model (n = 40) [START_REF] Parmigiani | Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2[END_REF] (age ranging from 24 to 65, average +45.29). The nipple area was washed with a solution containing 96% ethanol (Neoxinal, Milan, Italy). After insertion of the catheter in the duct, the part was anaesthetised by injection of 2 ml 2% lidocaine solution. Ducts were washed with 10-12 ml of physiological solution. Two ml of DL fluid was mixed with 2 ml of fixative (PreservCyt, Cytyc, Boxborough, MA, USA) and sent to the International Agency for Research on Cancer (IARC) for HPV typing.

After the first 70 procedures, the collection of 45 DLs was preceded by removal of the nipple epidermis by tape stripping. This procedure was introduced to reduce the risk of contamination of DLs with HPV DNA presence on the skin. For this purpose, a plaster (Fixomull, BDF Beiersdorf AG, Hamburg, Germany) was attached to the skin and stripped with sterile forceps after approximately 1 min. After the tape stripping, collection of DL fluid was performed as described above. The tapes were transferred in sterile cap tubes and sent to IARC for the detection of HPV DNA.

The ductal lavages were performed approximately 1 month after the surgical intervention and before the beginning of any type of therapy. No clear presence of malignant cells was detected by cytological analysis in all ductal lavages. In only four specimens we observed atypical suspicious malignancy. Of these four women, two had ipsilateral cancer, one had contralateral cancer and one is at [10% risk of mutation. All women were informed of the aim of the program and gave their written consent. This study was approved by the ethical committees of the European Institute of Oncology and IARC.

Colostrum and milk

To broaden our analysis to other breast fluids, we have extended our study to include milk and colostrum. We have chosen an Asian population to evaluate the possibility that the HPV prevalence in breast fluids may be influenced by genetic features. Twenty-five breast milk and ten colostrum samples were collected at the Department of Epidemiology and Preventive Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. This evaluation was done in average risk women in order to determine the possible presence of HPV in physiological mammary fluids. After extensive washing of the nipple area and the hand of lactating mother, colostrum or milk was collected by dropping the liquid directly into sterile tubes. Precautions were taken to avoid any contact between the skin and the sterile tubes. All specimens were sent to IARC for the detection of HPV DNA. The study was approved by the ethical committees of Kagoshima University and IARC.

DNA extraction

Ductal lavage

DNA extraction was performed by using the Qiagen BioRobot EZ1 with the EZ1 DNA tissue kit and EZ1 DNA tissue card or QIAamp Ò UltraSens TM Virus kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). To monitor the possible occurrence of cross-contamination between the different specimens during DNA extraction, tubes containing buffer only were also included.

Tape attached material

DNA purification was performed using the QIAamp DNA Micro Kit according to the instructions provided by the manufacturer (QIAGEN, Hilden, Germany).

Colostrum and milk

DNA was isolated using QIAamp DNA blood mini kit (QIAGEN, Hilden, Germany). For preparation of DNA, 200 ll native colostrum or milk was used. Lysis, digestion with proteinase, precipitation and elution of membranebound DNA were carried out following the instructions of the manufacturer.

DNA quality control

The quality of the DNA extracted from all specimens described above was controlled by amplification of part of the b-globin gene with the primers previously described [START_REF] Saiki | Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase[END_REF]. Only a minority of specimens resulted b-globin negative, i.e., one sample for the first set of ductal lavages (1/70) and three samples after tape stripping (3/45), while all milk and colostrum specimens were b-globin positive.

Detection of HPV DNAs

Multiplex PCR and HPV typing Detection of HPV DNA was performed using two recentlydeveloped highly sensitive and specific assays [START_REF] Gheit | Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types[END_REF][START_REF] Gheit | Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect Betapapillomavirus types[END_REF]. The two assays detected 19 mucosal high-risk and probable high-risk HPV types [START_REF] De Villiers | Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast[END_REF][START_REF] Hennig | Human papillomavirus 16 in breast cancer of women treated for high grade cervical intraepithelial neoplasia (CIN III)[END_REF]26,[START_REF] Forslund | High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in ''Stripped'' biopsies from the same tumors[END_REF]33,35,39,45,51,52,53,56,58,59, 66, 68, 70, 73 and 82) and 25 cutaneous HPV types from genus beta (HPV5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 47, 49, 75, 76, 80, 92, 93 and 96). The assay for the detection of mucosal HPV type was slightly modified with respect to the previous version [START_REF] Gheit | Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types[END_REF], which resulted to be less sensitive than the GP5+/6+ reverse line blot assay in detecting HPV16. We have modified the PCR forward primer for HPV16 E7

(5 0 -TTATGAGCAATTAAATGACAGCTCAG-3 0 ). The new version of the multiplex PCR assay showed a comparable sensitivity to the GP5+/6+ reverse line blot assay in detecting HPV16 DNA (data not shown).

Detection of HPV6 and 11 DNA

The presence of HPV6 and 11 DNA was determined by PCR using HPV type-specific primers (HPV6 E7 forward 5 0 -GTA TTA GAC CTG CAA CCT CCA GAC-3 0 ; HPV6 E7 reverse 5 0 -GTG CGC AGA TGG GAC ACA CTA TG-3 0 ; HPV11 E7 forward 5 0 -TAC TAG ACC TGC AGC CTC CTG AC-3 0 ; HPV11 E7 reverse 5 0 -GAT GGG ACA CAC AAT ATT TAG TGT GC-3 0 ). The size of the PCR products was 252 bp for HPV6 E7 and 244 bp for HPV11 E7.

Results

DNA from mucosal and cutaneous HPV types is detected in ductal lavage fluid

We initially tested the presence of HPV DNA in 70 DLs from women at increased risk for breast cancer (affected women, BRCA1/2 mutation carriers or [10% of risk of mutation according to the Berry-Parmigiani model [START_REF] Parmigiani | Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2[END_REF]). Two different detection assays for 25 cutaneous beta and 19 mucosal high-risk alpha HPV types were used [START_REF] Gheit | Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types[END_REF][START_REF] Gheit | Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect Betapapillomavirus types[END_REF].

Only one specimen of the 70 (1.4%) tested positive for mucosal high-risk HPV DNA (type 51), while cutaneous beta HPV types were detected in ten ductal lavage specimens (10/70, 14%) (Table 1). Of ten cutaneous HPVpositive samples, seven were found to have more than one HPV type. Altogether, 32 beta HPV DNAs were present in the ten breast specimens. HPV38 was the most frequently detected type, followed by types 9 and 22 (Fig. 1). HPV positivity was not preferentially associated with a group of women at specific risk, i.e., genetic predisposition or contralateral breast cancer (data not shown).

A recent study reported the presence of DNA from cutaneous and mucosal HPV types in areolar and cancer tissues from female patients with breast carcinoma [START_REF] De Villiers | Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast[END_REF]. In particular, the mucosal low-risk HPVs were found in breast tissue by PCR and in situ hybridisation. Therefore, we also searched for DNA of the low-risk mucosal HPV types 6 and 11, using E7 type-specific primers. No DNA from HPV6 and 11 was detected in the 70 DLs analysed.

Stripping of the superficial epidermis of the nipple strongly decreases HPV positivity in DLs

The collection of the DL fluids is performed using a catheter. Before insertion in the mammary ducts, the catheter may touch the nipple skin and contact any HPV flora present on the skin. Thus, the HPV DNA detection in the ten out of the 70 DL fluids positive for the beta HPV types may have been due to external contamination. Alternatively, the HPV positivity is associated with a real infection of the ducts. To discriminate between these two possibilities we introduced a modification in the procedure for the collection of the DL fluids. Forslund et al. reported a new method that consists in the removal of the superficial layers of the epidermis by tape stripping and found a strongly decreased HPV DNA prevalence in skin biopsies [START_REF] Forslund | High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in ''Stripped'' biopsies from the same tumors[END_REF]. Based on these findings, the skin superficial layers of the nipple area were removed by tape before the collection of the DL fluids. We collected and analyzed 45 DLs for the presence of the alpha and beta HPV types. No DNA from the alpha HPVs were detected in DLs, while only two specimens contained a single infection with beta HPV types, i.e., 5 and 24. Thus, the HPV-positivity in DLs strongly decreased after stripping of the superficial layers of the nipple skin.

Beta HPV types are highly prevalent in the epidermis of the nipple

The very low prevalence of HPV DNA in DLs after tape stripping suggests that the nipple area is highly colonised by different HPV types. To confirm this hypothesis we extracted total DNA from the tape and determined the presence of the beta HPV types. A b-globin signal was obtained in 41 of the 45 samples (91%); of these 37 were positive for beta HPV DNA. The majority of the specimens contained multiple beta HPV types (Table 2). HPV DNA was also detected in two of the four b-globin negative samples (Table 2). HPV5, 12, 15, 24, 47 and 75 were the most prevalent types, being present in approximately 35% of the tape specimens (Fig. 2). Together, these data show that beta cutaneous HPV types are highly prevalent in the nipple skin area.

DNA of cutaneous and mucosal HPV types were seldom detected in colostrum or milk

To further investigate the presence of HPV DNA in mammary ducts, we analyzed ten and 25 samples of colostrum and milk, respectively. Only one sample (1/10, 10%) of colostrum contained the mucosal high-risk HPV16, while two milk samples resulted positive for the beta HPV type 5 or 23. In line with the data obtained in DL fluids, the analysis of the milk and colostrum indicates that HPV DNA is very rarely present in mammary ducts.

Discussion

The possible involvement of HPV in breast carcinogenesis is still under debate. Many studies have reported the presence of HPV DNA in breast tissues, while others did not confirm the findings [START_REF] Lawson | Viruses and human breast cancer[END_REF]. Damin et al. found 25% of HPV positivity in 101 breast cancer tissues [START_REF] Damin | Evidence for an association of human papillomavirus and breast carcinomas[END_REF]; moreover, Kroupis et al. [START_REF] Kroupis | Presence of high-risk human papillomavirus sequences in breast cancer tissues and association with histopathological characteristics[END_REF] showed a 16% rate of HPV positivity in more than 100 breast cancer tissue specimens, and they suggested a All of these data are derived from histological samples of breast cancer tissue; our study is the first to look for an eventual HPV infection in cytological samples. In fact, we have analysed the presence of the DNA from mucosal and cutaneous HPV types in the lavage fluid of mammary ducts. Cutaneous beta HPV types were found in 14% (10/70) of the breast specimens, while DNA from mucosal high-risk HPV types was detected in only one sample (i.e., HPV51) (1.4%). However, when the collection of DL fluids was preceded by the removal of the superficial layers of the nipple epidermis by tape stripping, the HPV positivity was dramatically reduced, dropping from a total number of HPV infections of 32 to 2. These findings suggest that the majority, if not all, of the HPV DNA detected in DL fluids may have been transported from the epidermis into the ducts by the catheter.

The collection of colostrum and milk samples did not involve any mechanical stress, and HPV positivity was extremely low in these specimens, i.e., three HPV DNAs in 35 samples (%8%).

Surprisingly, the spectrum of the HPV types detected in the 70 DL fluid collected without tape stripping differs from the one detected in the material extracted from the 45 tapes (Figs. 1 and2). Different hypotheses can be envisaged to explain these findings. It is possible that the most frequently detected HPV types in the DL fluids (9, 22, 23 and 38) are present on the skin of the nipple at a higher copy number than the other beta HPVs. Therefore, they have a higher probability than the others to be transported by the catheter into or infect the ducts. Alternatively, the different HPV spectrum detected in DLs and tapes may be simply due to the fact that the two types of specimens were obtained from two distinct groups of women. However, since the women included in the two groups do not show any apparent difference, e.g., race and place of residence, and were randomly selected, the second hypothesis is less probable. In future studies, we intend to determine the copy number of the different beta HPV types present at the nipple epidermis by real-time PCR.

Our current data do not reveal whether the few HPV DNAs detected in the colostrum, milk and DLs collected after skin stripping correspond to real HPV infections. The detection of viral transcripts in cytological material will provide convincing proof for the presence or absence of an active HPV infection in the mammary ducts.

In addition to HPV, several studies have provided evidence for the involvement of other viruses in breast carcinogenesis, e.g., cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus (HHV)-8 and mouse mammary tumour virus (MMTV)-like virus [START_REF] Lawson | Viruses and human breast cancer[END_REF]. The evaluation of the presence of these viruses in DL fluids may provide important insight into their possible role in breast cancer.

In conclusion, our findings show a very limited presence of HPV in ductal lavage fluid, colostrums and milk, and do not support a major role of HPV in breast carcinogenesis. Further methodological approaches are needed to improve our ability to evaluate the presence of HPV infection in cytological samples, but at present a direct role of HPV in breast carcinogenesis appears to be unlikely.

Fig. 1

 1 Fig. 1 Detection frequency of beta HPV types in 70 DL fluids

Fig. 2

 2 Fig. 2 Detection frequency of beta HPV types in 45 samples of the nipple epidermis removed by tape

Table 1

 1 Single or multiple HPV DNAs detected in ten of the 70 DL fluids samples

	Patient	Alpha mucosal	Beta cutaneous HPV types
	number	high-risk
		HPV types
	1	Negative	8
	5	Negative	9, 38
	9	Negative	92
	13	Negative	20, 21, 22, 23, 36, 80
	14	Negative	9, 17, 24, 38
	16	Negative	75, 76, 93
	26	51	5, 22
	27	Negative	5, 9, 93
	32	Negative	15, 17, 20, 22, 23, 24, 36, 38, 80
	42	Negative	38
	Patients not included in the Table tested negative for alpha and beta
	HPV types

Table 2

 2 Single or multiple HPV DNA in each of the 45 tapes containing nipple epidermis

	Patient number	b-globin	Beta cutaneous HPV types
	1	+	12, 15, 17
	2	-	5, 15, 36, 75
	3	+	12, 15
	4	+	5, 9, 12, 15, 17, 21, 23, 47
	5	+	5, 12, 15, 19, 20, 24, 38, 47, 75
	6	+	5, 47
	7	+	-
	8	+	5, 12, 15, 24
	9	+	5, 8, 12, 23, 24, 47, 80
	10	+	5, 15, 20, 23, 24, 75
	11	+	14, 15, 20, 22, 24, 36
	12	+	12, 22, 38, 47, 75
	13	+	5, 12, 20, 24, 38, 75
	14	+	15, 24, 47
	15	-	-
	16	+	8, 14, 20, 23, 24, 36, 38, 47, 75, 80
	17	+	8, 19, 36, 80
	18	+	5, 8, 12, 20, 23, 24, 38, 93
	19	+	5, 14, 23, 24
	20	+	15, 20
	21	+	5, 12, 20, 21, 22, 23, 24, 47, 75, 80
	22	+	15, 47
	23	+	15, 23, 24
	24	+	5, 12, 15, 17, 23, 25
	25	+	5, 15, 17
	26	+	5
	27	+	12, 47
	28	-	93
	29	+	5, 22, 36
	30	+	12, 19, 22, 36, 75, 92
	31	-	-
	32	+	5, 22, 24
	33	+	12
	34	+	19, 23
	35	+	15, 24, 75
	36	+	75
	37	+	5, 12, 15, 23, 47, 75
	38	+	8, 15, 17, 47
	39	+	-
	40	+	-
	41	+	20, 22
	42	+	9, 23, 47, 75
	43	+	8, 20, 24, 47, 75
	44	+	8, 47
	45	+	-

Breast Cancer Res Treat (2009) 114:599-605

Acknowledgement We are grateful to all the members of our laboratory for their cooperation. The study was partially supported by grants from the European Union ''INCA'' (LSHC-2005-018704), the Association for International Cancer Research, La Ligue Contre le Cancer (Comite ´du Rho ˆne and Comite ´de la Dro ˆme).


Valerie Speirs
email: v.speirs@leeds.ac.uk

Abeer M Shaaban

HAL is

LETTER TO THE EDITOR

The rising incidence of male breast cancer Valerie Speirs AE Abeer M. Shaaban Published online: 14 May 2008 Ó Springer Science+Business Media, LLC. 2008 Although breast cancer rates are declining in Western societies, quoted figures are restricted to female breast cancer. Recently in this journal, Stang and Thomssen reversed this trend by analysing male breast cancer data from the SEER Program of the National Cancer Institute, which showed an increased incidence in male breast cancer of 1.0 per 100,000 in the late 1970s to around 1.2 per 100,000 at the start of this decade [START_REF] Stang | Decline in breast cancer incidence in the United States: what about male breast cancer?[END_REF].

In the UK around 350 cases of male breast cancer are diagnosed annually [2]. To determine if this figure is changing, we reviewed data obtained from members of the United Kingdom Association of Cancer Registries (UKACR). In most cases data was provided directly from UKACR members both as absolute numbers and European Age Standardised Rates (EASR;1991[START_REF] Giordano | Breast carcinoma in men: a population-based study[END_REF]. As shown in Fig. 1, male breast cancer incidence is increasing in the UK, paralleling US data which shows a concordant increase over a similar time period [START_REF] Stang | Decline in breast cancer incidence in the United States: what about male breast cancer?[END_REF][START_REF] Fentiman | Male breast cancer[END_REF]. This begs the question of why male rates are rising.

Age is the single biggest risk factor for male breast cancer. We are an ageing population and increased male breast cancer may well parallel increased longevity. However this increase is also reflected in ASR rates, which accounts for this. Nowadays, men are more health conscious and with increased public awareness of breast cancer in general e.g. through pink ribbon campaigns, they may be much more likely to seek medical attention for breast symptoms than ever before, which could contribute to increased diagnosis.

Men who have been repeatedly exposed to radiation from a young age and over a long time period are at greater risk of developing breast cancer [START_REF] Giordano | Breast carcinoma in men: a population-based study[END_REF]. One might expect that males living within the fallout zone from the 1986 Chernobyl nuclear disaster (which included the UK) may be at greater risk. Although reports are sketchy, an article published in Croatian suggests that post-Chernobyl, the ratio of male breast cancer increased from 1:139 to 1:79 [START_REF] Belicza | Change in the occurrence of breast cancer in hospital registries (1980-2000)[END_REF].

Rising levels of obesity resulting from physical inactivity and poor diet is a serious public health issue in both the US and UK and may be contributory to increasing male breast cancer. High estrogen levels are linked to breast cancer and local estrogen biosynthesis by breast tissue adipocytes may be an important factor in obese individuals. Increased use of pesticides, many of which are weakly estrogenic, by the agricultural industry and their subsequent introduction into the food chain could also add to rising trends.

Increased alcohol intake has been associated with male breast cancer [START_REF] Memon | Male breast cancer[END_REF] and this is probably indirectly linked to alcohol-induced liver damage and subsequent hyperestrogenism. Liver cirrhosis is associated with increased estrogen levels which could increase breast cancer risk. There are suggestions that this risk is higher when cirrhotic patients live longer [START_REF] Misra | Cancer of the breast in a male cirrhotic: is there an association between the two?[END_REF]. The so-called 'binge drinking' culture that has developed in recent years, particularly in the UK could also be a casual factor.

Although male breast cancer is rare, for the patient a diagnosis is often unexpected but it should be regarded as no less important than other 'male' illnesses. Its rarity precludes randomised clinical trials but its rising trend, which seems likely to continue, suggests coordinated multi-centre approaches are necessary to accumulate sufficient numbers for meaningful study into the biology of male breast cancer. Ultimately this may help clinicians better understand the disease and thus improve outcome for men with breast cancer.

Fig. 1

 1 Fig. 1 Breast cancer incidence in males in the UK according to data from UKACR. (a) Total numbers from 1981 to 2004. Data not available from: * three, ** two and *** one region. (b) European age-standardised incidence rates per 100,000 person-years at risk from 1991 to 2004 from nine UKACRs and data not available from one region (+)

Acknowledgements Thanks to the following for providing statistics: Northern and Yorkshire Cancer Registration and Information Service, Scottish Cancer Registry, North West Cancer Intelligence Service, Eastern Cancer Registration and Information Centre, Thames Cancer Registry, Trent Cancer Registry, West Midlands Cancer Intelligence Unit, Oxford Cancer Intelligence Unit Welsh Cancer Intelligence and Surveillance Unit, Northern Ireland Cancer Registry. Our male breast cancer programme is supported by the Breast Cancer Campaign (UK).


Noemi Malandrino

Lorenzo Leggio

Giovanni Addolorato

teaching and research institutions in France or abroad, or from public or private research centers.

LETTER TO THE EDITOR

Link between insulin and metabolic disorders in cancer: does leptin play a key role? To the Editor, We read with great interest the recent paper by Goodwin et al. [START_REF] Goodwin | High insulin levels in newly diagnosed breast cancer patients reflect underlying insulin resistance and are associated with components of the insulin resistance syndrome[END_REF] on the association between insulin resistance and other components of the metabolic syndrome (e.g., obesity, high waist-hip ratio, impaired lipid profile) in patients affected by breast cancer. As correctly reported by the authors [START_REF] Goodwin | High insulin levels in newly diagnosed breast cancer patients reflect underlying insulin resistance and are associated with components of the insulin resistance syndrome[END_REF], a state of hyperinsulinemia can act on breast cancer prognosis through several mechanisms, such as: (i) a direct interaction between insulin and insulin/IGF receptors able to stimulate cell proliferation; (ii) some indirect effects of insulin on the circulating levels of both IGF and of insulin/IGF binding proteins; (iii) an effect of insulin on the free circulating estrogen levels.

A further explanation supporting this hypothesis could be related to the ability of insulin to stimulate the expression and secretion of leptin by adipocytes [START_REF] Capristo | Metabolic alterations as a risk factor for cancer[END_REF][START_REF] Malmstro ¨m | Insulin increases plasma leptin concentrations in normal subjects and patients with NIDDM[END_REF][START_REF] Mueller | Evidence that glucose metabolism regulates leptin secretion from cultured rat adipocytes[END_REF][START_REF] Greco | Lowering of circulating free-fatty acids levels and reduced expression of leptin in white adipose tissue in post-obesity status[END_REF]. Therefore, hyperleptinemia is usually present in some states of hyperinsulinemia like in obesity [START_REF] Capristo | Metabolic alterations as a risk factor for cancer[END_REF][START_REF] Malmstro ¨m | Insulin increases plasma leptin concentrations in normal subjects and patients with NIDDM[END_REF][START_REF] Mueller | Evidence that glucose metabolism regulates leptin secretion from cultured rat adipocytes[END_REF][START_REF] Greco | Lowering of circulating free-fatty acids levels and reduced expression of leptin in white adipose tissue in post-obesity status[END_REF]. Consistently, a possible role of leptin in the development and progression of breast cancer has been suggested [START_REF] Capristo | Metabolic alterations as a risk factor for cancer[END_REF]. In fact in addition to its neuroendocrine functions (i.e., regulation of appetite and energy expenditure), leptin can act as a mitogen and angiogenic factor via paracrine and autocrine mechanisms. Due to its mitogen effect, a condition of hyperleptinemia could favour the development of malignancies. In close agreement with this hypothesis, several lines of evidence support the role of leptin in the neoplastic processes [START_REF] Perera | Identification of proteins secreted from leptin stimulated mcf-7 breast cancer cells: a dual proteomic approach[END_REF][START_REF] Slattery | Leptin and leptin receptor genotypes and colon cancer: gene-gene and gene-lifestyle interactions[END_REF][START_REF] Stattin | Leptin is associated with increased prostate cancer risk: a nested case-referent study[END_REF][START_REF] Ramani | Leptin's mitogenic effect in human liver cancer cells requires induction of both methionine adenosyltransferase 2A and 2beta[END_REF]. Moreover, this is further supported by some genetic evidences showing a correlation between serum leptin levels, leptin receptor polymorphisms, and the risk of breast cancer [START_REF] Koda | Expression of leptin and its receptor in female breast cancer in relation with selected apoptotic markers[END_REF][START_REF] Soma | Leptin augments proliferation of breast cancer cells via transactivation of HER2[END_REF]. In sum, although further prospective data are needed, leptin should be mentioned among the key factors in understanding the relationship between hyperinsulinemia and related metabolic factors in subjects with cancer risk. This also gets the basis for future potential therapeutical approaches (i.e., leptin-inhibiting drugs, strict adherence to weight-reducing programs) in the prevention and treatment of malignancies.


Muhammad U Rashid

Diana Torres

Farah Rasheed

Faisal Sultan

Abdul R Shakoori

Asim Amin

Klaus Schlaefer

Ute Hamann
email: u.hamann@dkfz-heidelberg.de

A E Diana

Torres Ae

Sultan AE A E Faisal

A E Asim

Amin Ae

Klaus Schlaefer Ae

Á F Rasheed

Á F Sultan

Á D Torres

Á U Hamann

No association of miscarriage and carrier status in Pakistani breast/ovarian cancer patients with a history of parental consanguinity

To the editor About 80% of breast and ovarian cancer families and about 50% of site-specific breast cancer families are due to germ line mutations in either BRCA1 (MIM 113705) or BRCA2 (MIM 600185) [1][2][3]. Women carrying a pathogenic mutation in BRCA1 or BRCA2 face average lifetime risks of 65% and 45%, respectively, of developing breast cancer, and corresponding ovarian cancer risks of 39% and 11% [4]. In humans, almost all BRCA carriers have been found to be heterozygous [5,6]. The lack of viable homozygous BRCA mutation carriers in humans suggests that homozygous fetuses may be more susceptible to spontaneous abortions. Further evidence for this hypothesis comes from in vivo animal studies showing that mice homozygous for mutations in the BRCA1 or BRCA2 gene die early in embryogenesis [7,8].

Two recent studies have explored the impact of BRCA mutation status on the occurrence of miscarriages. One study included BRCA carriers of Ashkenazi Jewish descent [9], and the other included mutation carriers from United States and Canada [10]. Neither study found a significant difference in the rate of miscarriage between BRCA carriers and non-carriers. We conjectured that miscarriages may occur more frequently in BRCA carriers from consanguineous parents since their offspring are more often homozygous by descent compared to those of unrelated parents, and living homozygous BRCA mutation carriers have not been observed. To explore this question, we studied the rates of miscarriages in Pakistani BRCA mutation carriers and non-carriers with a history of parental consanguinity. Pakistan has one of the highest rates of consanguinity in the world (range 60-70%) [11,12].

Breast and/or ovarian cancer families were recruited at the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan, between June 2001 and November 2004. Of the 165 female index patients, 30 carried a deleterious BRCA mutation, and 135 were without any identifiable mutation. All families, screening methods and mutations have been described previously [13]. Four additional families carrying a deleterious BRCA mutation identified recently were also included in this study. Relevant information regarding potential breast cancer risk factors including family history of cancer, hormone use, reproductive history, medical history and life style factors was collected by interview-administered questionnaires. Detailed information on the outcome of each pregnancy (year of each pregnancy, number of miscarriages including spontaneous and therapeutic abortions, number of still and live born), and parental consanguinity was collected. Spontaneous abortion was defined as loss of a pregnancy before the completion of 20 gestational weeks from the previous menstrual period or fetal weight of \ 500 g according to the recommendation of the World Health Organization [14]. Parental consanguinity was classified into four types: no consanguinity, first cousins, second cousins and more distant relatives. A total of 128 breast/ ovarian cancer patients comprising 29 BRCA carriers and 99 non-carriers who had at least one pregnancy were included in the study. Mean and standard deviation (SD) was calculated for total number of pregnancies, live births, and miscarriages. The Wilcoxon's signed rank test was performed to test if BRCA carriers and non-carriers were independent of the number of pregnancies and its outcome (number of live births and number of miscarriages, respectively). The Student's t-test was used to test for independency of age at diagnosis and carrier status. All other variables were dichotomized, and tested using Fisher's exact test. Two-sided P-values of 0.05 or less were considered as statistically significant. All statistical analyses were performed using SAS, version 9.1.

A comparison of parity-related parameters between BRCA carriers and non-carriers is shown in Table 1. The mean age at diagnosis of breast and ovarian cancer among carriers and non-carriers did not show any significant difference (t-test, P = 0.082 and P = 0.194, respectively). In addition, no difference was found between the two groups in the mean number of pregnancies, live births, miscarriages, and in the proportion of study participants with and without miscarriages.

No association was noted between miscarriage and parental consanguinity, disregarding BRCA carrier status (miscarriage among related couples versus miscarriage among unrelated couples: Fisher's exact test, P = 0.463; miscarriage among married first and second cousins versus miscarriage among unrelated couples: Fisher's exact test, P = 0.435) (data not shown), which is in agreement with previous studies conducted in countries with high rates of consanguinity [15][16][17][18].

When BRCA status was considered, there was no statistically significant difference between the occurrence of miscarriage among related and unrelated study participants (Table 2). Even when the analysis was restricted to marriages between close relatives (first or second cousins), no significant association with miscarriages could be shown.

In the two previous studies the rate of three or more spontaneous miscarriages among Israeli Jewish BRCA carriers with at least one live birth was 4.4% (15/343) and The limitations of this study are that it is not populationbased as the study subjects were recruited based on a personal or family history of breast and/or ovarian cancer in a hospital, and the small study size.

In conclusion, our findings suggest no difference in the occurrence of miscarriage between BRCA carriers and noncarriers when parental consanguinity was taken into consideration. Additional studies are warranted.

1. Narod SA, Ford D, Devilee P, Barkardottir RB, Lynch HT, Smith SA, the Breast Cancer Linkage Consortium (1995) An evaluation of genetic heterogeneity in 145 breast-ovarian cancer families. Am J Hum Genet 56:254-264 2. Narod S, Ford D, Devilee P, Barkardottir RB, Eyfjord J, Lenoir G et al (1995) Genetic heterogeneity of breast-ovarian cancer revisited. Am J Hum Genet 57:957-958 3. Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, the Breast Cancer Linkage Consortium (1998) Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. Am J Hum Genet

Table 1

 1 Comparison of parity-

	related parameters among BRCA carriers and non-carriers	Parameter	BRCA carriers a N (%)	Non-carriers N (%)	P-value
		Total number of patients	29	99
		Age at diagnosis of breast cancer (years) b	24	82
		Mean ± SD	35 ± 8	3 2 ± 8	0.082 c
		Range	22-50	22-70
		Age at diagnosis of ovarian cancer (years)	5	17
		Mean ± SD	39 ± 6	3 5 ± 6	0.194 c
		Range	29-45	26-45
		Year of birth
		Mean ± SD	1965 ± 10	1969 ± 8
		Range	1938-1980	1922-1980
		Total number of pregnancies	116	371
		Mean ± SD	4.0 ± 2.4	3.7 ± 2.2	0.798 d
	SD, standard deviation	Range	1-8	1-12
	a The carrier group includes BRCA1 and BRCA2 carriers b Including three patients who developed ovarian cancer as a	Total number of live births Mean ± SD Range	83 2.9 ± 1.9 0-8	307 3.1 ± 1.8 0-8	0.389 d
	second cancer; exclusion of	Total number of miscarriages	22	60
	these patients yielded mean ages	Mean ± SD	0.8 ± 1.3	0.6 ± 1.0	0.891 d
	at breast cancer diagnoses of 34 years in carriers and 31 years in non-carriers	Range Study participants with miscarriages	0-5	0-4
	(t-test, P = 0.262).	0	19 (66)	63 (64)	1.000 e
		[0	10 (34)	36 (36)
		1	3 (10)	20 (20)
	e No miscarriage versus one or	2	5 (17)	11 (11)
	more miscarriages, Fisher's exact test	3+	2 (7)	5 (5)

c t-test: pooled (equal variance) d Wilcoxon's signed rank test

Table 2

 2 Grant JC, Shami SA (1993) Consanguinity as a determinant of reproductive behaviour and mortality in Pakistan. Int J Epidemiol 22:463-467. doi:10.1093/ije/22.3.463 Comparison of miscarriages between BRCA carriers and non-carriers, and parental consanguinity

	62:676-689. doi:
	10.1086/301749
	4. Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE,
	Hopper JL et al (2003) Average risks of breast and ovarian cancer
	associated with BRCA1 or BRCA2 mutations detected in case
	series unselected for family history: a combined analysis of 22
	studies. Am J Hum Genet 72:1117-1130. doi:10.1086/375033
	5. Friedman E, Bar-Sade Bruchim R, Kruglikova A, Risel S, Levy-
	Lahad E, Halle D et al (1998) Double heterozygotes for the
	Ashkenazi founder mutations in BRCA1 and BRCA2 genes. Am
	J Hum Genet 63:1224-1227. doi:10.1086/302040
	6. Kuschel B, Gayther SA, Easton DF, Ponder BA, Pharoah PD (2001)
	Apparent human BRCA1 knockout caused by mispriming during
	polymerase chain reaction: implications for genetic testing. Genes
	Chromosomes Cancer 31:96-98. doi:10.1002/gcc.1122

a The carrier group includes BRCA1 and BRCA2 carriers b Fisher's exact test

Breast Cancer Res Treat (2009) 116:211-213


Erik Teugels
email: eteugels@uzbrussel.be

Jacques De Grève

Erik Teugels Ae

Jacques De Gre

About the c.156_157insAlu BRCA2 breast cancer predisposing mutation

We followed with interest the articles and commentary notes about the screening of the BRCA2 c.156_157insAlu mutation, a mutation that we have detected for the first in a Portuguese patient residing in Belgium [START_REF] Teugels | De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes[END_REF]. Machado et al. recently described this mutation in 20 Portuguese families [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF], while Peixoto et al. reported a little bit later this same mutation in 14 Portuguese families [START_REF] Peixoto | The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal[END_REF]. All authors agree that this highly recurrent mutation initially occurred in a single patient (founder effect), but with some disagreement about which method to use for the molecular identification of the high breast cancer risk mutation carriers. In a letter to the editor of this journal, Machado and Vaz proposed a three step PCR assay for which they have a patent pending [START_REF] Machado | Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation[END_REF]. The mutant BRCA2 allele is unable to generate wild type full length mRNA, but a shorter messenger missing exon 3 (the target exon for the Alu repeat insertion). A shorter BRCA2 mRNA form missing exon 3 can also be generated by the wt allele, but in (much) smaller amounts when compared to the full length form. The biological significance of the BRCA2 alternative messenger missing exon 3 is actually unknown. According to Machado et al. [START_REF] Machado | Caution should be used when interpreting alterations affecting the exon 3 of the BRCA2 gene in breast/ovarian cancer families[END_REF] the alternative and mutant transcripts missing exon 3 are not identical, but differ at the level of the exon 10 coding sequence, a property that these authors want to exploit in their proprietary assay to detect the c.156_157insAlu associated mutant transcript.

We want to comment shortly on two points. First, because of the frequent occurrence of the c.156_157insAlu mutation in Portuguese breast cancer families (while absent in a corresponding control population) and a clear genotype/phenotype correlation, we are convinced that performing the molecular diagnosis on RNA material in addition to genomic DNA is without significant use and much more cumbersome. Moreover, results obtained on RNA from leucocytes are not necessarily representative for what would occur in the breast epithelial cells. A well designed mutation specific single step gDNA PCR/sequence analysis should be sufficient for the correct characterization of high risk patients or families.

Second, the mutation was first reported by us in a publication dating from 2005. As we mentioned in that article, we collected a large number of blood samples from family members residing in Portugal. Then, the team in Lisbon (Dr Vaz and colleagues) accepted to perform for us the genetic counseling for the interested family members living in Portugal. In July 2002 we provided them the clinical phenotypes and genotypes of 24 family members, eight being mutation carriers. We communicated that the mutation corresponded to an insertion of about 300 bp within exon 3 of BRCA2, resulting in the synthesis of mRNA molecules that lack exon 3. This information made it possible to easily characterize the mutation.

For these reasons and aside from ethical considerations, we question the patentability of the assay for specific identification of the c.156_157insAlu mutation.

We hope this specific mutation, together with all other BRCA1/2 mutations, will remain 'public domain' in the interest of all families concerned.


A Peixoto

Á C Santos

Á P Rocha

Á P Pinto

Á S Bizarro

Á M R Teixeira

We wish to reply to the Letter to the Editor written by Machado and Vaz [START_REF] Machado | Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation[END_REF] concerning our recently published article on the c.156_157insAlu BRCA2 founder mutation [START_REF] Peixoto | The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal[END_REF], first discovered by Teugels et al. [START_REF] Teugels | De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes[END_REF] in a Portuguese patient living in Belgium. That letter has several misstatements about our and others' work that must not remain unchallenged.

Machado and Vaz suggest in their letter [START_REF] Machado | Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation[END_REF] that we diagnose the c.156_157insAlu BRCA2 mutation by subjective evaluation of band intensities after RT-PCR. We think that it is clear in our paper [START_REF] Peixoto | The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal[END_REF], under the subheading ''Screening for the c.156_157insAlu BRCA2 mutation'', that we screen for this mutation by using two independent PCRs, one for exon 3 amplification and another specific for the Alu rearrangement. Using this strategy, in positive cases we expect two bands in the first PCR (one band if negative) and one band in the second PCR (none if negative). The second PCR helps to control the first PCR for eventual problems with preferential amplification of the shorter fragment (the normal), whereas the first PCR controls for eventual absence of amplification in the second PCR. This strategy of two independent PCRs (which differs from the nested PCR approach used by Machado et al. [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF]), followed by sequencing of the genomic fragments in positive cases, allows the unambiguous detection of the c.156_157insAlu BRCA2 mutation. In the Results section of our article [START_REF] Peixoto | The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal[END_REF], the molecular diagnosis of families with the c.156_157insAlu BRCA2 mutation is also described under the separate subheading ''Detection of the c.156_157insAlu BRCA2 mutation'', whereas any mention to RT-PCR data appears under the subheading ''Analysis of RNA transcripts''. Therefore, the reason why we performed RT-PCR with primers located in exons 1 and 6 was not to confirm the mutation, but to deliberately be able to compare the physiologic alternative splice lacking exon 3 in controls with the effect of the exon 3 Alu insertion in carriers. Taking advantage of a known polymorphism in BRCA2 exon 2 (previously reported as c.-25G[A [START_REF] Peixoto | The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal[END_REF][START_REF] Dı ´ez | Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects[END_REF][START_REF] Van Der Hout | A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting[END_REF], but described as c.-26G[A according to the recommendations of the Human Genome Variation Society [START_REF]Human Genome Variation Society[END_REF], we clearly demonstrated in Fig. 1 of our paper [START_REF] Peixoto | The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal[END_REF] the preferential production of the transcript without exon 3 by the allele with the Alu insertion and its (presumably residual) production by both alleles in non-carriers.

Machado and Vaz [START_REF] Machado | Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation[END_REF] state that RT-PCR must be used to confirm the c.156_157insAlu BRCA2 mutation in each patient and that a reliable confirmation of this rearrangement must be done using the primers in exons 2 and 10 first described by Nordling et al. [START_REF] Nordling | A large deletion disrupts the exon 3 transcription activation domain of the BRCA2 gene in a breast/ovarian cancer family[END_REF]. Both these claims are unfounded for the following reasons. First, it is not necessary to confirm one given mutation at the RNA level in every new patient once its pathogenic nature is well established, as it is now the case for this BRCA2 rearrangement. Second, Machado and Vaz claim [START_REF] Machado | Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation[END_REF] that these primers amplify specifically the transcript produced from the allele with the Alu insertion, and not the ubiquitous alternative splice transcript, allegedly because only the former transcript includes at least the first part of BRCA2 exon 10 [START_REF] Machado | Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation[END_REF]. They present no data to support this assumption nor do they exist in the literature. In fact, Zou et al. [START_REF] Zou | Structure and expression of variant BRCA2a lacking the transactivation domain[END_REF] have shown that the full length transcript and the ubiquitous alternative splice differ only by the lack of exon 3 in the latter, so exon 10 does not distinguish them. Additionally, the forward primer described by Nordling et al. [START_REF] Nordling | A large deletion disrupts the exon 3 transcription activation domain of the BRCA2 gene in a breast/ovarian cancer family[END_REF] (5 0 -GCTTATTTACCAAGCATTGGA) has two features that might interfere with the amplification process: first, it contains a mismatch in the first base (A[G); second, it is positioned on the SNP c.-26G[A in exon 2, which has an allelic frequency of 28% [START_REF] Dı ´ez | Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects[END_REF]. Indeed, Bonnet et al. [START_REF] Bonnet | Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene[END_REF] have recently used a monoallelic RT-PCR protocol based on the SNP c.-26G[A to demonstrate the complete skipping of exon 3 induced by the presence of the c.316?5G[C BRCA2 mutation. In addition to these methodological flaws, it is likely that Machado et al. [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF] were not able to amplify the transcript lacking exon 3 in controls because they try to amplify a large transcript produced at very low level in non carriers, whereas it can more easily be detected in carriers as that transcript is produced at high levels from the mutated allele. To address this possibility, we have analyzed freshly collected blood samples from five negative controls (confirmed not to carry the c.156_157insAlu BRCA2 mutation). Total RNA was isolated using 1 ml of Tripure isolation reagent (Roche Diagnostics, Indianapolis, USA) and One-Step RT-PCR was performed (QIAGEN GmbH, Germany), as suggested by the manufacturers. Using a 5 0 UTR BRCA2 primer that does not include the SNP c.-26G[A (5 0 -GCTTACTCCGGCCAAAAAAGA) and a reverse primer located in exon 10 (5 0 -TGGTCACATGAAG AAATATGCAATAG) that allows the detection of its first 469 bp, we were able to amplify both the wild type and the ubiquitous transcript lacking exon 3 in all five negative controls (Fig. 1). These data contradict the assumption made by Machado and Vaz [START_REF] Machado | Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation[END_REF][START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF] that the alternative splice transcript in negative controls lacks part of exon 10, as we here show using optimized methods for RNA extraction and analysis. We therefore conclude that RT-PCR studies aiming to detect exon 3 skipping should not be performed for molecular diagnosis of the c.156_157insAlu BRCA2 mutation; instead, this Portuguese founder mutation can reliably be detected by genomic PCR followed by sequencing as we demonstrated in our article [START_REF] Peixoto | The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal[END_REF].

Fig. 1

 1 Fig. 1 RT-PCR analyses from exon 1 to exon 10 of the BRCA2 gene in three normal controls and in one c.156_157insAlu BRCA2 mutation carrier. a Lanes M, 1 Kb DNA standard; Lanes 1, 2 and 3, controls without the c.156_157insAlu BRCA2 mutation; Lane 4, c.156_ 157insAlu BRCA2 mutation carrier; A1 represents the full length transcript and A2 the BRCA2-Dex3 transcript. b Sequencing of the RT-PCR products (A1 band above and A2 band below) from a control individual showing that both the wild type and the ubiquitous transcript lacking exon 3 can be detected in negative controls

  LETTER TO THE EDITOR Molecular diagnosis of the Portuguese founder mutation BRCA2 c.156_157insAlu Ana Peixoto AE Catarina Santos AE Patrı ´cia Rocha AE Pedro Pinto AE Susana Bizarro AE Manuel R. Teixeira

	Published online: 14 October 2008
	Ó Springer Science+Business Media, LLC. 2008


R U Ja
email: janicke@uni-duesseldorf.de

LETTER TO THE EDITOR

MCF-7 breast carcinoma cells do not express caspase-3

Reiner U. Ja ¨nicke Published online: 14 October 2008 Ó Springer Science+Business Media, LLC. 2008 Apoptosis, a fundamental process essential for normal tissue homeostasis and development, is closely associated with the activation of a class of aspartate-specific cysteinedependent proteases, called caspases, that lead to the demise of the cell via limited proteolysis of a multitude of cellular substrates [START_REF] Fischer | Many cuts to ruin: a comprehensive update of caspase substrates[END_REF][START_REF] Fuentes-Prior | The protein structures that shape caspase activity, specificity, activation and inhibition[END_REF]. Caspases are expressed as inactive zymogens that become activated upon cleavage by other caspases in a so-called caspase activation cascade, or by mere oligomerization instigated by the formation of large multi-protein complexes such as the death-inducing signaling complex (DISC) or the apoptosome [START_REF] Kroemer | Mitochondrial control of apoptosis: an introduction[END_REF][START_REF] Lavrik | Death receptor signaling[END_REF]. Whereas DISC formation occurs via the so-called extrinsic death pathway that is instigated by activation of members of the death receptor family such as CD95, tumor necrosis factor receptor (TNF-R) or the receptors of the TNF-Rrelated apoptosis-inducing ligand (TRAIL), the apoptosome is formed following activation of mitochondria and is hence termed the intrinsic or mitochondrial death pathway. Based on their order in cell death pathways, caspases can be divided into initiator (caspase-2, -8, -9, and -10) and effector (caspase-3, -6, and -7) caspases. Among them, caspase-3, a member of the latter group, is absolutely crucial for apoptosis induction, as this enzyme is not only activated downstream of both the extrinsic and intrinsic death pathway, it is also responsible for the cleavage of the majority of substrates known so far [START_REF] Fischer | Many cuts to ruin: a comprehensive update of caspase substrates[END_REF][START_REF] Porter | Emerging roles of caspase-3 in apoptosis[END_REF]. More importantly, with the proteolysis of discrete substrates, caspase-3 evokes some of the typical morphological and biochemical alterations associated with apoptosis. For instance, whereas the caspase-3-mediated cleavages of a-fodrin, gelsolin, rho-associated kinase-1 (ROCK-1) and p21-activated kinase 2 (PAK2) contribute to membrane blebbing, cleavage of the inhibitor of the caspase-activated DNase (ICAD) leads to the typical DNA fragmentation pattern observed in apoptosis [START_REF] Fischer | Many cuts to ruin: a comprehensive update of caspase substrates[END_REF]. Furthermore, with the cleavagemediated activation of the calcium-independent phospholipase A2 and subsequent production of the chemotactic phospholipid lysophosphatidylcholine, caspase-3 appears to be also responsible for the generation of so-called ''eatme'' signals that induce migration of phagocytes to the site of apoptotic cell death [START_REF] Lauber | Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal[END_REF]. Thus, caspase-3 not only instigates and pursues the demise of a cell, but, in addition, makes sure that the corpse is properly disposed, a function crucial for avoiding inflammatory processes.

Hence, determination of the processing and activation of caspase-3 are common means to assess apoptotic signal transduction pathways in numerous cell lines of varying origin. Curiously, several reports still claim the presence of caspase-3 in the breast carcinoma cell line MCF-7 in which this enzyme is supposed to contribute to apoptosis signaling [START_REF] Wang | Tumor-specific adenovirus-mediated Puma gene transfer using the survivin promoter enhances radiosensitivity of breast cancer cells in vitro and in vivo[END_REF][START_REF] Yeruva | Methyl jasmonate decreases membrane fluidity and induces apoptosis through tumor necrosis factor receptor 1 in breast cancer cells[END_REF][START_REF] Nizamutdinova | Tanshione I effectively induces apoptosis in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells[END_REF][START_REF] Kumar | Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by a new series of substituted-1,3,4-oxadiazole derivatives[END_REF][START_REF] Cui | p53-mediated cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells[END_REF][START_REF] Hsuuw | Epigallocatechin gallate dosedependently induces apoptosis or necrosis in human MCF-7 cells[END_REF][START_REF] Zhang | Effect of ursolic acid on caspase-3 and PARP expression of human MCF-7 cells. Zhongguo[END_REF][START_REF] Yang | Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata[END_REF][START_REF] Chen | Drug-resistant breast carcinoma (MCF-7) cells are paradoxically sensitive to apoptosis[END_REF]. This was not only demonstrated indirectly via fluorometric assay systems measuring caspase-3-like activities-that might be also elicited by the closely related caspase-7-but by Western blotting analyses demonstrating directly the presence of this protease in MCF-7 cells. However, in addition to the lack of caspase-10 [START_REF] Engels | Caspase-10 sensitizes breast carcinoma cells to TRAILinduced but not tumor necrosis factor-induced apoptosis in a caspase-3-dependent manner[END_REF], an initiator caspase in the extrinsic death pathway, MCF-7 cells do also not express caspase-3 [START_REF] Ja ¨nicke | Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis[END_REF]. Ten years ago, we demonstrated unambiguously that the lack of caspase-3 in these cells is caused by a 47-base pair deletion within exon 3 of the CASP-3 gene resulting in the skipping of this exon during pre-mRNA splicing and introduction of a premature stop codon at position 42 that completely abrogates translation of the CASP-3 mRNA [START_REF] Ja ¨nicke | Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis[END_REF]. Although caspase-3-deficient MCF-7 cells are still sensitive to cell death induction by several stimuli including TNF, staurosporine [START_REF] Ja ¨nicke | Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis[END_REF][START_REF] Ja ¨nicke | Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis[END_REF] and various DNA damaging agents [START_REF] Ja ¨nicke | Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells[END_REF][START_REF] Essmann | Apoptosis resistance of MCF-7 breast carcinoma cells to ionizing radiation is independent of p53 and cell cycle control but caused by the lack of caspase-3 and a caffeine-inhibitable event[END_REF], death of these cells occurs in the absence of DNA fragmentation. In addition, the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing are also not evident in caspase-3deficient MCF-7 cells. As introduction of caspase-3 restored all of these events, our results-and also those of many other laboratories-clearly showed that this protease is crucial for these typical biochemical and morphological changes of cells undergoing apoptosis.

Although caspase-7 might compensate under certain circumstances for the lack of caspase-3, it is well appreciated that the executioner caspases 3, 6 and 7 perform distinct, non-redundant roles during the demolition phase of apoptosis [START_REF] Slee | Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis[END_REF]. In addition, it was shown that caspase-3 is the primary activator of apoptotic (low molecular) DNA fragmentation via cleavage-mediated inhibition of ICAD [START_REF] Wolf | Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation[END_REF]. Thus, it is puzzling that caspase-3-deficient MCF-7 cells exhibit DNA fragmentation and other apoptotic characteristics following cell death induction as described [START_REF] Wang | Tumor-specific adenovirus-mediated Puma gene transfer using the survivin promoter enhances radiosensitivity of breast cancer cells in vitro and in vivo[END_REF][START_REF] Yeruva | Methyl jasmonate decreases membrane fluidity and induces apoptosis through tumor necrosis factor receptor 1 in breast cancer cells[END_REF][START_REF] Nizamutdinova | Tanshione I effectively induces apoptosis in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells[END_REF][START_REF] Kumar | Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by a new series of substituted-1,3,4-oxadiazole derivatives[END_REF][START_REF] Cui | p53-mediated cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells[END_REF][START_REF] Hsuuw | Epigallocatechin gallate dosedependently induces apoptosis or necrosis in human MCF-7 cells[END_REF][START_REF] Zhang | Effect of ursolic acid on caspase-3 and PARP expression of human MCF-7 cells. Zhongguo[END_REF][START_REF] Yang | Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata[END_REF][START_REF] Chen | Drug-resistant breast carcinoma (MCF-7) cells are paradoxically sensitive to apoptosis[END_REF]. While it is still possible that caspase-3-deficient MCF-7 cells display high molecular weight DNA fragmentation that might be occasionally mistaken for the typical apoptotic DNA fragmentation, the detection of a caspase-3 protein in MCF-7 cells [START_REF] Wang | Tumor-specific adenovirus-mediated Puma gene transfer using the survivin promoter enhances radiosensitivity of breast cancer cells in vitro and in vivo[END_REF][START_REF] Yeruva | Methyl jasmonate decreases membrane fluidity and induces apoptosis through tumor necrosis factor receptor 1 in breast cancer cells[END_REF][START_REF] Cui | p53-mediated cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells[END_REF][START_REF] Zhang | Effect of ursolic acid on caspase-3 and PARP expression of human MCF-7 cells. Zhongguo[END_REF][START_REF] Yang | Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata[END_REF][START_REF] Chen | Drug-resistant breast carcinoma (MCF-7) cells are paradoxically sensitive to apoptosis[END_REF] with a partially deleted CASP-3 gene is cause for concern. Such discrepant findings may be partially explained by the use of inappropriate antibodies that cross-react with other caspase-3-unrelated proteins in cellular extracts, a phenomenon we have also observed previously (unpublished data). Alternatively, as there are several variants of MCF-7 cells around, the examined cultures might not contain the original MCF-7 cell line, and a karyotyp analysis to clarify this issue would be appropriate. Nevertheless, similar to our recent note of caution with regard to the presence of caspase-10 in the mouse [START_REF] Ja ¨nicke | Caspase-10 in mouse or not?[END_REF], this letter should make a general readership aware of the fact that the original MCF-7 breast cancer line that can be obtained from the American Tissue Type Culture Collection (ATCC) does not express the caspase-3 protein. Hopefully, this misconception can be avoided in the future.

Breast Cancer Res Treat (2009) 117:219-221

Acknowledgment This work was supported by the Collaborative Research Center ''Extrinsic Ageing'' funded by the Deutsche Forschungsgemeinschaft (SFB 728/B1).