F Didier

D Radice

S Gandini

R Bedolis

N Rotmensz

Ae A Maldifassi

B Santillo

Ae A Luini

V Galimberti

E Scaffidi

Ae D Radice

Ae S Gandini

Ae R Bedolis

Ae N Rotmensz

Ae B Santillo

Ae V Galimberti

Ae E Scaffidi

Ae F Lupo

Ae S Martella

Ae J Y Petit

Á R Bedolis

Á A Maldifassi

Á E Scaffidi

Á F Lupo

Á S Gandini

Á N Rotmensz

Á B Santillo

Á V Galimberti

S Martella

J Y Petit

Does nipple preservation in mastectomy improve satisfaction with cosmetic results, psychological adjustment, body image and sexuality?

Keywords: Satisfaction with cosmetic plastic surgery results, Mastectomy, Nipple areolar complex sparing, Psychological adjustment, Body image, Sexuality

Does nipple preservation in mastectomy improve satisfaction with cosmetic results, psychological

adjustment, body image and sexuality?

Introduction

Nowadays breast cancer surgery has become less and less mutilating and it is now accepted that early-breast cancer patients treated either by mastectomy or by breast-conserving procedures have comparable survival rates [START_REF] Fisher | Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF][START_REF] Veronesi | Conservative treatment of early breast cancer: long term results of 1232 cases treated with quadrantectomy, axillary dissection and radiotherapy[END_REF]. However, a mastectomy is required in case of multifocal, large tumors; extensive local recurrences after conservative treatment; or patient's fear when proposing conservative treatment. The negative psychological impact of breast loss has been frequently studied [START_REF] Andersen | Sexual functioning morbidity among cancer survivors, current status and future research directions[END_REF][4][START_REF] Hopwood | Body image problems in cancer patients[END_REF][START_REF] Sk | Does cosmetic outcomes from treatment of primary breast cancer influence psychosocial morbidity?[END_REF][START_REF] Schain | Breast cancer surgeries and psychosexual sequelae: implications for remediation[END_REF][START_REF] Schover | Partial mastectomy and breast reconstruction. A comparison of their effects on psychosocial adjustment, body image and sexuality[END_REF][START_REF] Schover | The impact of breast cancer on sexuality, body image and intimate relationships[END_REF]; it is now well recognized that cancer treatment can have a negative impact on emotional well being, body image perception, sexuality and breast conservative surgery helps women to cope better with cancer [START_REF] Sk | Comparison of psychological aspects and patient satisfaction following breast conserving surgery, simple mastectomy and breast reconstruction[END_REF][START_REF] Sk | The psychological impact of immediate rather than delayed breast reconstruction[END_REF]. In our clinical experience in contact with patients, as surgeons and as psychologists, we observed that the removal of the NAC when a mastectomy needs to be performed may contribute to increase the sense of mutilation and emotional suffering.

To reduce this negative psychological impact, since 2002 at our institute, women undergoing mastectomy, for cancers located outside the central area of the breast, have been proposed a new type of nipple sparing mastectomy (NSM) associated with intraoperative electron-beam radiotherapy (ELIOT) delivered to the region of the areola. Results were published by Petit et al. [START_REF] Petit | The nipple-sparing mastectomy : early results of a feasibility study of a new application of perioperative radiotherapy (ELIOT) in the treatment of breast cancer when mastectomy is indicated[END_REF][START_REF] Petit | When mastectomy becomes inevitable: the nipple-sparing approach[END_REF][START_REF] Petit | Nipple-sparing mastectomy in association with intra operative radiotherapy (ELIOT): a new type of mastectomy for breast cancer treatment[END_REF]. The effect of removal of the NAC during mastectomy and of nipple reconstruction procedures has not been often studied; Wellisch et al. [START_REF] Wellish | The psychological contribution of nipple addition in breast reconstruction[END_REF] demonstrated the positive psychological impact of NAC reconstruction after mastectomy and breast reconstruction when comparing patients with or without NAC reconstruction. We decided to further investigate the possible advantages of NAC preservation in mastectomy. In our experience, there is also a growing interest regarding patient's satisfaction with the results of plastic surgery, since it is known to influence the coping process and psychological adjustment [START_REF] Sk | Does cosmetic outcomes from treatment of primary breast cancer influence psychosocial morbidity?[END_REF]. For this reason, we decided to develop a new specific questionnaire in order to understand whether NAC preservation could improve patients' satisfaction with the results of breast reconstructive and plastic surgery and psychological well-being.

Aims of the study

The purpose of the present study is to investigate the influence of nipple sparing in mastectomy on patient's satisfaction with cosmetic results, body image, sexuality and psychological adjustment comparing two groups of women who underwent radical mastectomy with IBR and either NAC sparing or NAC reconstruction. Our objectives are [START_REF] Fisher | Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF] to understand whether women who are submitted to the loss of their breast while keeping their NAC are more satisfied with cosmetic results than are women who do not conserve the NAC, (2) to understand whether women who are submitted to the loss of their breast while keeping their NAC are more satisfied with body image perception and sexuality, [START_REF] Andersen | Sexual functioning morbidity among cancer survivors, current status and future research directions[END_REF] to understand if NAC sparing has a positive impact on adjustment helping women to cope better with the psychological consequences of mastectomy and if NAC sparing increases fear of recurrence.

Materials and methods

Study design

Phase I

Between 2002 and 2004, we developed and tested a specific self-administered questionnaire to assess patient's satisfaction with cosmetic results following breast plastic reconstruction; to investigate the influence of NAC sparing on body image, sexuality and psychological adjustment. We also performed interviews with target patients selected for our psychological study.

Phase II

From 2004, we enrolled women who underwent radical mastectomy, with immediate breast plastic reconstruction with or without NAC preservation. The criteria we chose in order to select patients for the study were the following: primary invasive breast cancer (T1, T2, T3) and primary ductal carcinoma in situ; age over 18; no previous chemotherapy; no bilateral surgery for breast cancer. We chose these inclusion criteria in order to have a homogeneous group of women and to exclude factors that may add confounding variables, which could modify perception of body image. We compared two groups of patients: women who preserved their nipple during mastectomy and women who did not preserve their nipple and underwent successive NAC reconstruction, considered as the control group. The self-administered questionnaire was sent by mail. In the NSM group, for those women who had an IBR, questionnaires were sent by mail 1 year after breast surgery, except for those who had an expander to whom questionnaires were sent 1 year after the definitive prosthesis was placed. In the control group questionnaires were sent by mail about 1 year after NAC reconstruction was performed.

Four hundred and fifty-three women (N = 453) who were proposed NAC sparing or removal during mastectomy were enrolled in our study. Eligible patients agreeing to participate in the psychological study were enrolled after giving written informed consent. A small sample of non-responders was interviewed by telephone after 18-24 months from surgery. Two psychologists independently reviewed the interviews and listed eight categories of reasons for missingness. Reasons for missingness were further classified as: not related to surgery or questionnaire (missing completely at random, MCAR), not surgery related (missing at random, MAR) and surgery related (missing not at random, MNAR).

Brief description of the new surgery technique

Since 2002 in the Departments of Breast Surgery and of Plastic Surgery of our institute, when a mastectomy was the treatment chosen for breast cancer treatment, the patient was informed of the possibility to preserve her nipple when the tumor corresponded to inclusion criteria of the surgical study. The new type of mastectomy (NSM) (using a subcutaneous mastectomy technique) was associated with intraoperative electron-beam radiotherapy (ELIOT) delivered to the region of the areola. The NSM is performed leaving 5 mm of glandular tissue behind the nipple areola complex to preserve its blood supply. The reconstruction is immediately performed by prosthesis or by expander then replaced with a definitive prosthesis. At the moment of surgery the inclusion criteria for NAC sparing were: small tumors located at least 1 cm outside the areola margins; absence of nipple retraction or bloody discharge; absence of retroareolar microcalcifications. Multifocality was not a cause of exclusion, provided that all the tumor sites were distant from the areola. Patients were excluded at the time of the operation if the frozen examination of the retroareolar tissue was positive for carcinoma: in these cases ELIOT was not delivered. Patients who could not have NAC sparing and completed surgery with NAC reconstruction were included in the control group. The new surgery technique was explained and discussed with the patient: advantages, disadvantages and possible complications were illustrated. All patients signed a written surgical informed consent before surgery.

Methods

Instruments

Item generation and scale construction

Between April 2002 and August 2004 we developed our specific questionnaire. A MEDLINE search from 1980 to 2002 was made for relevant quality of life issues concerning patient satisfaction with cosmetic results after breast reconstruction surgery, psychological adjustment after mastectomy, body image perception, perception of NAC sparing or NAC reconstruction after mastectomy, sexuality and fear of recurrence after breast cancer. A working group of psycho-oncologists, breast surgeons, plastic surgeons, data-managers and statisticians was formed. The first phase (step I generation of relevant items) was aimed at compiling a list of relevant issues that covered the domains of interest, mainly perception of NAC sparing, satisfaction on cosmetic results, body image and sexuality, and the group decided to include a list of items. The initial draft version was given to 10 target patients, 10 health care professionals and 10 non health care professionals. They were asked to state whether the issues were clear, important or not and if any aspects of interest had been overlooked. The second phase (Step II) consisted in translating the issues into a set of questions; the working group modified the items after analysis and deleted issues that were considered less important. Some items were added. Then (Step III) we performed a pretesting of the provisional questionnaire: this version had 23 items.

Preliminary field testing

Finally, a preliminary field testing (Step IV) of the questionnaire with target patients was performed. We chose to administer the questionnaire (23 items) to three groups of women who underwent three different types of surgery (42 radical mastectomies with nipple preservation, 37 radical mastectomies with nipple suppression and 50 lumpectomies), in order to explore if the items and the rating scale chosen were able to detect differences in opinions or perception among patients undergoing with three different surgical techniques. At this stage, we were also interested in collecting information on (a) ease of understanding and acceptability of items; (b) redundancy of items; (c) missing items; (d) overall ease of completion. Patients completed questionnaires while at our institute for routine scheduled visits or at home (each questionnaire was sent by mail with a pre-paid envelop for restitution) and were encouraged to make additional comments. Thanks to this preliminary field testing, the questionnaire was modified in order to meet our objectives; we deleted, rephrased and added items as necessary. Because the first version of our questionnaire was too general, in order to better understand the impact of surgery and NAC sparing on body image and sexuality and to increase the level of specificity we added different items. Thanks to P. Hopwood study [START_REF] Hopwood | A body image scale for use with cancer patients[END_REF], still not validated in Italian, to our preliminary results and to the patients' interviews we included some specific items that assess the satisfaction with body image before and after the disease.

Because a woman's overall psychological health, relationship satisfaction and premorbid sexual life appear to be strong predictors of postcancer sexual satisfaction [START_REF] Schover | The impact of breast cancer on sexuality, body image and intimate relationships[END_REF], we included items to assess the satisfaction with sex before and after the disease. We added one item to evaluate the possible influence of factors not related to surgery on sexuality. We also asked the patient's partner to express their opinion and level of satisfaction with cosmetic plastic results after surgery.

In total, the last version of the questionnaire consisted of 56 items (see Appendix 1 for the whole questionnaire). The questionnaire is composed of four categories: (1) satisfaction with plastic-surgery cosmetic results (26 items), (2) body image (before and after the disease, 13 items), ( 3) sexuality (before and after the disease, 16 items), (4) fear of recurrence (1 item). We chose a 5-likert scale ranging from ''not at all'' to ''very much''. One of the most important items regards the feeling of mutilation (see item 15bis) in the category (2) related to body image.

Ten semi-structured in-depth interviews were performed by psychologists in order to evaluate the comprehensibility and the content of the items in the revised version. All women expressed deep suffering not only for the loss of their breast but also for the loss of their NAC. One patient stated that the nipple areola was perceived as the signature of breast identity, it was, in her words, ''the eyes of the breast''. When we asked women who underwent the new technique (preservation of the NAC) to explain why they agreed to participate in the study, most answered that one of the reasons was ''to decrease their sense of mutilation''. When interviewed, all women who did not conserve their nipple expressed their disappointment for not having preserved their nipples.

Field testing

The final version of the questionnaire was administered to patients enrolled in the psychological study conducted along the surgical study, from September 2004 to September 2006. At the beginning of the study, part of the sample was used to assess compilation time (N = 197 patients): 72 patients (36.5%) needed \10 min to complete the questionnaire, 67 patients (34.0%) needed 10-15 min and only 6 patients (3.0%) needed [30 min. Overall the average time of compilation was 13.4 min. Ninety-two point four percent patients did not receive any help during the compilation. One hundred seventy-five patients (88.4%) considered the questionnaire comprehensible, 92.4% (n = 183) did not find the items embarrassing, 93.4% of patients (n = 185) considered the items pertinent. The second version of the questionnaire under field testing, at the time of the preliminary analysis, was well accepted by patients (response rate = 63% in the NSM group, response rate = 72% in the group with nipple reconstruction) and we received very good feedback from patients on appropriateness of content and comprehensibility. Analysis of missing data on the responses' frequency didn't reveal any problematic items.

Statistical methods

Descriptive statistics and frequencies are given for sociodemographic features and relevant clinical variables, at surgery time, by treatment group (NAC vs. Controls). Comparisons between treatment groups are tested through Mantel-Haenszel tests or Fisher's Exact test. We present histograms by treatment groups for statistically significant items of the questionnaire.

One of the most important questions being the one regarding feeling of mutilation, we analyzed frequency distribution of missing answers to feeling of mutilation question (see item 15bis) by treatment group and reasons of missingness. Cohen's Kappa was used to assess the agreement between the two psychologists, who evaluated the reasons for missingness. We assessed the association between missing values for item 15bis and treatment group, with a logistics regression model to take into account clinically or psychologically relevant variables. Odds Ratios (OR) with their 95% Confidence Intervals (CI) are evaluated for treatment group and clinically or psychologically relevant variables. Frequency distributions for the reasons arising from phone interviews, classified as MCAR, MAR and MNAR are evaluated. Two-sided P values below the conventional 5% threshold were considered statistically significant.

Results

Description of the sample

From September 2004 to September 2006, 310 women with NAC preservation (NSM group), and 143 patients with successive NAC reconstruction (control group) were mailed the questionnaire. At the time of analysis, 51.2% of women with mastectomy, IBR and NAC sparing and 67.8% of women with mastectomy, IBR and successive NAC reconstruction, answered the questionnaires. Thus a total of 256 questionnaires were available from patients who had completed 1 year of follow-up after definitive complete breast reconstruction surgery.

Socio-demographic and relevant clinical variables by treatment group (control group vs. NSM group) at surgery time are summarized in Table 1. Mean age at surgery for NSM group and control group were 46 years (range = 26-73) and 47 (range = 24-80), respectively (Table 1). The comparison between non-responders and responders showed no significant differences between the two groups regarding age, geographic origin (P = 0.74), marital status (P = 0.69). The comparison regarding education showed statistically significant differences (Chi square P value 0.002): education was higher in the NSM group.

Impact of NAC preservation (NSM) on body image perception

In our questionnaire, results showed an important decrease of satisfaction with overall body image, after the disease and surgery, in both groups (Fig. 1a). The women who expressed an increased dissatisfaction (answer ''quite a bit'' or more) were 10% in the NSM group and 9% in the control group. When interviewed regarding the item ''did you feel feminine?'' women felt less feminine after surgery (Fig. 1b), especially in the control group: 16% of the patients in the NMS group and 26% in the control group expressed ''quite a bit'' or more decrease in femininity after surgery. Regarding nakedness, women had more difficulty in looking at themselves naked after surgery (Fig. 1c), especially in the control group; 15% of women in the NSM group had an increased difficulty (''quite a bit'' or more) in looking at themselves naked and 34% in the control group. We also observed an increased difficulty in being seen naked by their partners (Fig. 1d), especially in the control group; 19% of women expressed a ''quite a bit'' or more increased difficulty in the NSM group and 39% in the control group answered ''quite a bit'' ore more. Analysing in details the differences between the two groups after surgery (Figs. 2, 3), we observed that they were significantly different in answering the questions regarding difficulty in looking at themselves naked and being seen naked by their partners (P = 0.001 and P = 0.003, respectively). The score distribution that assessed the feeling of mutilation (Fig. 4, item 15bis) also showed a significant difference between the two groups (P = 0.003).

Impact of NAC preservation (NSM) on feeling of mutilation: the contribution of the analysis of missing values to understand better this issue

The analysis of distribution frequency of missing answers to the question on feelings of mutilation (item 15bis) showed a significantly higher proportion of missingness in the control group compared to the NSM group (17/97 vs. 2/159, in the control and NSM groups, respectively; P \ 0.001). Furthermore, evaluating all demographicdependent variables and clinical data (histology, complications, treatments) to verify if they were associated to missing answers to question 15bis, we observed a significant relationship only with the surgical procedure (OR = 0.313, 95% CI: 0.117-0.837). All Odds Ratios (OR) with their 95% Confidence Intervals (CI) are presented in Table 2.

Impact of NAC preservation (NSM) on sexuality

Ninety-one percent of women in the NSM group felt sexually attractive before the disease versus 54.5% after cancer and treatments, 98% of women in the control group felt sexually attractive before cancer versus 72% after cancer (Fig. 5). There were no significant differences between the two groups regarding sexual life and impact of nipple sparing on sexuality.

Impact of NAC preservation on satisfaction with cosmetic results

A significant difference was observed regarding the level of satisfaction with the appearance of the nipple in the two groups (P = \.0001) (Fig. 6) as well as regarding the level of satisfaction with the sensitivity of the nipple (P = 0.001) (Fig. 7). Satisfaction was higher in the NSM group.

Impact of NAC preservation on the psychological adjustment

Ninety-three percent (143/154) of patients in the NSM group reported that the preservation of their nipple helped them to cope with the disease and its consequences (Item 31). Ninety-two percent (142/155) of them reported that the preservation of their nipple helped them to feel less mutilated (Item 32). Eighty-nine percent (79/89) of the women who did not preserve their nipple (control group) would have preferred to have done so (Item 33, percentages correspond to the score of ''quite a bit'' or more).

Regarding anxiety for the future and fear of recurrence, globally NSM does not seem to increase the level of anxiety. Women in both groups expressed globally a low level of fear of recurrence (respectively 31% in the NSM expressed a high level of fear, vs. 28% in the control group).

Interviews of non-responders

Considering the large number of nipple non-responders (48.2%), it was decided to investigate the reason for this lack of responses to the questionnaire. We telephoned a random sample of non-responders (N = 34 in the NSM group and N = 6 in the control group). The interviews were aimed at evaluating how much surgery-dependent dissatisfaction affects the lack of questionnaire returns. The reasons arising from our phone interviews were grouped into eight categories and further classified as MCAR (Missing Completely at Random), MAR (Random, that is only related to the questionnaire or psychological reasons, but not to the surgical procedure) and MNAR (Non-random or related to surgery). Frequency distribution of MCAR, MAR and MNAR are presented in Table 3. Only 12.5% of patients reported that they did not return their questionnaires for surgery-related reasons. Agreement was evaluated through Cohen's Kappa. Following the interpretation suggested by Landis and Koch [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF] the agreement between two psychologists who conducted a blind evaluation of the reasons for not returning the questionnaires was almost perfect (Kappa = 0.92, 95% CI : 0.70-1.00).

Discussion

It is now recognized that women after mastectomy are more likely to develop psychological adjustment disorders and there are recognized benefits of breast conservation and breast reconstruction over mastectomy [START_REF] Sk | Comparison of psychological aspects and patient satisfaction following breast conserving surgery, simple mastectomy and breast reconstruction[END_REF][START_REF] Sk | The psychological impact of immediate rather than delayed breast reconstruction[END_REF][START_REF] Sanger | Comparison of the psychological effects of breast-saving procedures with the modified radical mastectomy[END_REF][START_REF] Polivy | Psychological effects of mastectomy on a woman's feminine self-concept[END_REF]. Previous studies have investigated the influence of cosmetic outcome on psychosocial morbidity in primary breast cancer [START_REF] Sk | Does cosmetic outcomes from treatment of primary breast cancer influence psychosocial morbidity?[END_REF][START_REF] Nano | Psychological impact and cosmetic outcome of surgical breast cancer strategies[END_REF]. These studies reported a good correlation between satisfaction with cosmetic outcomes and positive body image perception, sexuality and self-esteem and showed that the more conservative was breast surgery the better were quality of life and psychological adjustment.

There are very few studies assessing the impact of NAC reconstruction on breast reconstruction: e.g., we found only one specific article [START_REF] Wellish | The psychological contribution of nipple addition in breast reconstruction[END_REF] that showed that NAC reconstruction had a positive impact on patient's satisfaction with cosmetic results.

In our study, we wanted to understand the psychological contribution of nipple conservation versus nipple reconstruction comparing NSM versus mastectomy with breast and NAC reconstruction. Investigating patient's treatment preferences, we clearly found that most of women who did not preserve their nipple would have preferred keeping it P=<.0001 and the majority of women who preserved their nipple expressed a very high level of satisfaction for having done so. Analyzing our results in details, we can see that the new technique of NSM has impacted very positively especially on patient satisfaction with cosmetic results and body image related to nakedness. Results showed that the type of surgery in favour of NSM positively influences patient perception e.g., when the patient looks at herself naked or is seen naked by her partner. Women who have had nipple conservation (NSM) experienced less difficulty than women who did not keep their nipple. Our study also showed that patients who preserved their nipple felt less mutilated regarding their breast with a significant difference between the two groups.

Our results showed an important decrease of overall satisfaction with body image, after the disease and surgery, in both groups. This result could probably be attributed to the impact of the type of surgery on the level of expectations of the patients: women in the NSM group more likely had a higher level of expectations and more likely felt disappointed about overall satisfaction with their body. We also found a higher education level in the NSM group and we can hypothesize that a higher education may have influenced negatively the level of satisfaction because patients could be more demanding and could also have more expectations.

The analysis of frequency distribution of missing answers to the question of feelings of mutilation (item 15bis), one the main domain of our study, showed a significantly higher proportion of missingness in the control group compared to the NSM group. This difference cannot be ascribed to difficulties related to the item itself, because we obtained very good scores on the analysis of comprehensibility. The higher proportion of missingness in the control group may be explained by the painful emotional resonance generated by this item : we can say that women in the control group faced two losses in their body; the loss of the breast and the loss of their nipple and probably they felt more distressed regarding mutilation than women in the NSM group. We decided to analyse more in-depth the missing data of our study, given the importance of this issue. Results obtained interviewing non responders over the phone indicated that our results were not biased because of non-responders. Eighty-eight percent of the patients sampled for this interview did not reply for reasons not related to surgery but for emotional reasons. Filling in the questionnaire put pressure on the patient's psychological denial defences that may be an adaptative coping mechanism. Women may refuse to fill in a questionnaire or a specific item to avoid painful emotions.

In many studies, it appeared that breast conservation protected women's perception of their bodies but did not contribute to a more positive sexual adjustment over time [START_REF] Schain | Mastectomy versus conservative surgery and radiation therapy. Psychosocial consequences[END_REF]. Regarding the negative psychological impact of mastectomy, we wanted to understand if NAC preservation in mastectomy may have a positive impact on sexuality after breast surgery. Overall, a high number of women in both groups expressed many difficulties in their sexuality after the diagnosis and surgery. There is an important decrease of satisfaction in sexual life. It is understandable that sexuality after cancer diagnosis and treatments may present difficulties. Sexual activity, sexual pleasure and well-being need a relaxed frame of mind, dealing with grief, due to a series of losses such as the loss of one's health, the loss of one's breast, with anxiety associated with fear for death and the future does not facilitate a relaxed and pleasant sexual activity.

Rising interest in improved cosmesis has led to the introduction of NSM as an acceptable alternative to traditional mastectomy. From the oncological point of view recent studies [START_REF] Petit | The nipple-sparing mastectomy : early results of a feasibility study of a new application of perioperative radiotherapy (ELIOT) in the treatment of breast cancer when mastectomy is indicated[END_REF][START_REF] Petit | When mastectomy becomes inevitable: the nipple-sparing approach[END_REF][START_REF] Petit | Nipple-sparing mastectomy in association with intra operative radiotherapy (ELIOT): a new type of mastectomy for breast cancer treatment[END_REF][22][START_REF] Banerjee | Preservation of nipple-areola complex in breast cancer-a clinicopathological assessment[END_REF][START_REF] Ueda | Cosmetic outcome and patient satisfaction after skinsparing mastectomy for breast cancer with immediate reconstruction of the breast[END_REF] and a recent review of the literature [START_REF] Chung | Nipple-sparing mastectomy: where are we now?[END_REF] showed that the risks and complications of NSM, with or without ELIOT, were acceptable, when compared to the traditional surgical treatment of breast cancer and NSM can be considered as a viable option in the appropriate setting.

To conclude, our study showed the point of view of women who expressed a very high level of satisfaction for having preserved their nipple and perceived this surgery as helpful to better deal with the traumatic experience of breast cancer and breast loss. Our results clearly showed that most women who could not preserve their nipple would have preferred keeping it. Patients who preserved their nipple felt less mutilated regarding their breast. The new technique of NSM has impacted positively on patient satisfaction with cosmetic results, with femininity and body image especially related to nakedness.

Acknowledgment This study has been supported by AVON Italy.

Appendix 1

Assessment of the patients' satisfaction with cosmetic results, physical and emotional impact of mastectomy In this questionnaire you will be asked how you feel about the physical and emotional impact of mastectomy with or without nipple sparing.

We would like to know from you how you felt about your physical aspect BEFORE your diagnosis:

Fig. 1 a

 1 Fig. 1 a Dissatisfaction with the body b Did you feel feminine? c Did you find it difficult to look at yourself naked? d Did you find difficult being seen naked by your partner?

Fig. 2 Fig. 3 Fig. 4 Table 2 Fig. 5

 23425 Fig.2Did you find it difficult to look at yourself naked?

Fig. 6 Fig. 7 Table 3

 673 Fig.6Have you felt satisfied with the appearance of your nipple?

Table 1

 1

	Summary statistics for relevant socio-demographic and	Group	Nipple (N = 159)	Control (N = 97)	Total (N = 256)
	clinical features at surgery time by treatment group	Mean age years (min-max) Education	46 (26-73)	47 (24-80)	46 (24-80)
		\High school	27 (17.0%)	32 (33.0%)	59 (23.6%)
		CHigh school	130 (81.8%)	61 (62.9%)	191 (76.4%)
		Missing	2 (1.2%)	4 (4.1%)	6 (2.3%)
		Marital status
		Married/living together	125 (78.6%)	66 (68.0%)	191 (74.6%)
		Divorced/single	21 (13.2%)	31 (31.9%)	52 (20.3%)
		Missing	13 (8.2%)	0	13 (5.1%)
		Histology
		Invasive Ca	125 (78.6%)	80 (82.5%)	205 (80.1%)
		In situ Ca	33 (20.8%)	13 (13.4%)	46 (17.9%)
		Missing	1 (0.6%)	4 (4.1%)	5 (1.9%)

  Have you been satisfied with the size of your breast? 17. Have you been satisfied with the shape of your breast? 18. Have you been satisfied with your breast skin sensitivity? 19. Have you been satisfied with the appearance of your scar? 20. Has the other breast been remodeled? YES NO If YES, we would like to know: 21. Have you been satisfied with the size? 22. Have you been satisfied with the shape? 23. Have you been satisfied with your breast skin sensitivity? 24. Have you been satisfied with the appearance of your scar? Now we would like to know from you, regarding cosmetic results of plastic surgery: 25. Do you consider the overall result of the surgery is similar to the natural aspect of your breast? 26. Have you been satisfied with cosmetic result of the plastic surgery overall? 27. Did your surgery results meet with your expectations? 28. Did you regret having had the breast reconstruction? 29. Did you have nipple sparing surgery? YES NO Please, answer only if YES: 30. Have you been satisfied having preserved your nipple? 31. Did the preservation of your nipple help you to cope with the disease and surgical consequences? 32. Did the preservation of your nipple help you to feel less mutilated (disabled)? Please answer only if it has NOT been possible to preserve your nipple: 33. Would you have preferred to keep your nipple? Please indicate, why: ………………………………… During the last four weeks: 34. Have you felt satisfied with the appearance of your nipple? 35. Have you felt satisfied with your nipple sensitivity? Now we would ask you some questions about an aspect of life which could be important for a woman, regarding sexuality. We would like to know, BEFORE the disease: 36. Was sexuality an important part of your life? 37. Did you feel sexually attractive? 38. Was your breast important to your sexual life? 39. Was your nipple important to your sexual life? During the last 4 weeks: 40. Was sexuality an important part of your life? 41. Did you feel interested in sexual activities? 42. Did you feel sexually attractive? 43. Have you been feeling less sexually attractive because of the disease or its treatment? 44. Were you sexually active? Please, answer only of you have been sexually active during the last four weeks. 45. Did you feel enjoyment during sexual activities? 46. Did your breast play an important part in your sexuality? 47. Did your nipple play an important part in your sexuality? 48. Have you noticed a change regarding your partner in your sexual intimacy? 49. Have you noticed a diminished frequency of breast caressing in your partner? 50. Have you noticed that you have a reduced sexual drive? Now we would like to know from you if during the last 12 months: 51. If there has been personal situation, a part from your disease and breast surgery, that would have interfered with your sexuality? YES NO Only if you want, please indicate:

	6. Were you satisfied with the size of your breast?
	7. Were you satisfied with the shape of your breast?
	8. Were you satisfied with the appearance of your nipple?
	During the last four weeks:
	9. Have you felt unsatisfied with your body?
	10. Have you felt physically attractive?
	11. Have you felt less physically attractive because of the
	disease or its treatment?
	12. Have you been feeling feminine?
	13. Have you been feeling less feminine because of the
	disease or its treatment?
	14. Did you find it difficult to look at yourself naked?
	15. Did you find it difficult being seen naked by your
	partner?
	15bis. Did you experience feelings of mutilation?
	Regarding the breast operated and reconstructed:
	16.
	1. Did you feel unsatisfied with your body?
	2. Did you feel physically attractive?
	3. Did you feel feminine?
	4. Did you find it difficult to look at yourself naked?
	5. Did you find it difficult being seen naked by your
	partner?

s Family problems s Grief s Partner problems

Breast Cancer Res Treat (2009) 118:623-633

Other……………………………………………………

We would like to know during the last four weeks:


Kari Hemminki
email: k.hemminki@dkfz.de

Asta Försti

Justo Lorenzo Bermejo

Kari Hemminki Ae

Asta Fo

A E Justo

Lorenzo Bermejo

Á A Fo

Á J Lorenzo Bermejo

Surveying germline genomic landscape of breast cancer

Keywords: Familial risk, Population attributable fraction, Genotype relative risk, Cancer genetics

come

Recent studies on somatic alterations in breast cancer have revealed large numbers of mutated genes, the commonest ones of which have been referred to as 'mountains' and the rarer ones as 'hills' in the genomic landscape [START_REF] Wood | The genomic landscapes of human breast and colorectal cancers[END_REF]. Among germline mutations predisposing to breast cancer several variants have also been described, whereby the germline landscape of breast cancer susceptibility is taking form [START_REF] Stratton | The emerging landscape of breast cancer susceptibility[END_REF]. Along with the previously characterized high-penetrance genes, such as BRCA1 and BRCA2, moderate-penetrance and low-penetrance susceptibility alleles have recently been detected [START_REF] Cox | A common coding variant in CASP8 is associated with breast cancer risk[END_REF][START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF][START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF][START_REF] Hunter | A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer[END_REF][START_REF] Hemminki | Etiologic impact of known cancer susceptibility genes[END_REF]. The level of penetrance is defined by the relative risk that the alleles inflict in mutation carriers. However, when the genomic landscape is being surveyed and the perspective is being widened over the individuals into the population, measures beyond the individual risk will be useful. The familial risk is one of them, but it is useful only if the causative variants are known [START_REF] Hemminki | Estimating risks of common complex diseases: familial and population risks[END_REF], which is not the case with the newly detected low-penetrant variants of unknown function. Thus, the commonly cited low familial risks of the new variants are probably only the minimal estimates. The new variants are likely to be markers of rarer functional variants which would confer a higher familial risk, the magnitude of which can only be estimated once the functional variants have been identified [START_REF] Hemminki | Estimating risks of common complex diseases: familial and population risks[END_REF]. A more robust measure is the population attributable fraction (PAF), which has many useful properties particularly for genomic landscaping. There it defines the proportion (etiological proportion) of the disease occurrence explained by the variant under study. We review some properties of PAF and apply these to the genes/loci that can be grouped by their variable penetrance [START_REF] Stratton | The emerging landscape of breast cancer susceptibility[END_REF].

The PAF of each variant shows its relative etiologic impact. Importantly, the PAF is identical for the marker and its linked (linkage disequilibrium D 0 = 1.0) causative variant, thus making it a more robust tool for genomic landscaping than the familial risk [START_REF] Hemminki | Estimating risks of common complex diseases: familial and population risks[END_REF]. PAFs have been used extensively for environmental risk factors of diseases in order to rank them and to assess the prospective gains in disease prevention. Their use in cancer genetics is relatively new [START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF][START_REF] Hunter | A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer[END_REF][START_REF] Gudmundsson | Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24[END_REF][START_REF] Yeager | Genome-wide association study of prostate cancer identifies a second risk locus at 8q24[END_REF], probably because the mutant variants of the 'classical' high-penetrance cancer genes are so rare that their contribution to the population burden is low compared to the high individual risks [START_REF] Hemminki | Etiologic impact of known cancer susceptibility genes[END_REF]. Some recent studies on low-penetrance genes do cite PAFs, referring to the high population burden conferred by the variants in spite of their low relative risks. The PAF of a gene variant integrates any unmeasured gene-gene and gene-environment interactions for the particular study population, which implies that PAF may vary from one population to the next [START_REF] Hemminki | Constraints for genetic association studies imposed by attributable fraction and familial risk[END_REF]. The variation could be expected to distinguish populations with variable allele frequencies and/or environmental exposures, i.e., those with large differences in cancer incidence. Age structure of the population is important in the presentation of any risk parameters, which has been dealt elsewhere [START_REF] Hemminki | Etiologic impact of known cancer susceptibility genes[END_REF]. Suffice to point out that when more than 10% of the women will develop breast cancer (i.e., the cumulative risk is over 10%) any relative risk even for factors causing breast cancer in all women has to be below 10. Most data on carrier frequencies emanate from relatively young populations, where relative risks may exceed 10 but extrapolation to the whole population remains an enigma.

Population attributable fractions (PAFs) can be calculated based on the genotype relative risks of the variants and on their allele frequencies as shown elsewhere [START_REF] Hemminki | Etiologic impact of known cancer susceptibility genes[END_REF]. Among the high-penetrance genes, the PAFs for BRCA1 and BRCA2 each are at or above 0.5% in European populations aged up to 70 years [START_REF] Hemminki | Etiologic impact of known cancer susceptibility genes[END_REF]; the PAFs would be somewhat lower in the whole population where we could consider around 1.0% a good estimate (Fig. 1). For some of the moderate-penetrance genes, ATM, BRIP1, CHEK2 and PALB2, the data are still limited to single populations [START_REF] Cybulski | CHEK2 is a multiorgan cancer susceptibility gene[END_REF][START_REF] Erkko | A recurrent mutation in PALB2 in Finnish cancer families[END_REF][START_REF] Renwick | ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles[END_REF][START_REF] Seal | Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles[END_REF][START_REF]TheCHEK2BreastCancerCase-ControlConsortium. CHEK2*1100delC and susceptibility to breast cancer: a collaborative analysis involving 10,860 breast cancer cases and 9,065 controls from 10 studies[END_REF]. For CHEK2*del1100delC and ATM variants the PAF estimates are about 0.5% each; adding up the other CHEK2 variants, BRIP1 and PALB2 mutations the sum PAF would be around 1.5% (Fig. 1). Among low-penetrance alleles, PAF for TGFbeta1 and CASP8 may account for 5.7 and 3.2%, respectively [START_REF] Cox | A common coding variant in CASP8 is associated with breast cancer risk[END_REF]; the five loci described by Easton et al. [START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF] (close to genes/loci FGFR2, TNRC9, MAP3K1, LSP1, 8q) can be calculated to confer a joint PAF of 36%, assuming independence and multiplicative epistatic interactions; the two loci described by Stacey et al. on chromosomes 2q35 and 16q12 confer PAFs of 5.3 and 7.1%, assuming that estrogen receptor-positive breast cancers accounted for 50% of all breast cancer [START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF]. Assuming that the low-penetrance loci act independently and multiplicatively, their joint PAF can be estimated at 58% (Fig. 1).

The present calculations are based on the published data on genotype relative risks and frequencies of the variant alleles, both of which may vary according to the populations studied. The exact results depend also on the assumed mode for genotypic and epistatic interactions for which limited data are available. The evidence appears, nevertheless, compelling in pointing to the remarkably high population impacts of the recently described heritable loci compared to the 'classical' high-penetrance genes. As it seems now, the genome scans on breast cancer have opened a wide genomic landscape of flat hills which abruptly break off from the few well known mountain ridges. The lack of intermediary peaks has been considered a paradox, which however may be explained by the applied research tools: linkage studies were used to find the highpenetrance genes and genome scans were used to find the low-penetrance variants. By definition, the former detects rare variants of high risk while the latter finds common variants of low risk [START_REF] Hemminki | Constraints for genetic association studies imposed by attributable fraction and familial risk[END_REF]. We may have missed the intermediary mountains because we have not had the right tools to find them: they are of too low risk to be found in linkage studies and too rare to be found in genome scans. The most feasible way to find them would probably be to focus on relatively rare variants in familial and bilateral breast cancer cases [START_REF] Houlston | The future of association studies of common cancers[END_REF][START_REF] Hemminki | The balance between heritable and environmental aetiology of human disease[END_REF][START_REF] Johnson | Counting potentially functional variants in BRCA1, BRCA2 and ATM predicts breast cancer susceptibility[END_REF].

Fig. 1

 1 Fig. 1 PAFs for high-, moderate-and low-penetrance variants underlying susceptibility to breast cancer

Breast Cancer Res Treat (2009) 113:601-603

Acknowledgements

The study was supported by the Deutsche Krebshilfe, the BMBF for NGNF+, the Swedish Cancer Society, the Swedish Council for Working Life and Social Research, the EU, LSHC-LT-2004-503465 and EU Food-CT-2005-016320.


M J Pepels
email: mpepels@hotmail.com

Ae K A Boomars

Ae R Van Kimmenade

Ae P S Hupperets

P S Hupperets

K A Boomars

R Van Kimmenade

Life-threatening interstitial lung disease associated with trastuzumab: case report

Keywords: Breast cancer, Dyspnoea, Eosinophils, Infiltrates, Trastuzumab

A female patient with HER2 positive, metastatic breast cancer presented with pulmonary infiltrates, and a plural effusion dyspnoea after several months of trastuzumab treatment. She had been treated without complications with six courses of docetaxel and trastuzumab in combination with dexamethasone with partial remission of disease. Malignancy, infection and cardiomyopathy were excluded as causes of dyspnoea. Pleural and broncheoalveolar fluid analyses (BAL) showed eosinophils. A diagnosis of trastuzumab-induced pneumonitis was made. After treatment with steroids there was gradual clinical improvement and disappearance of infiltrates. Although a causative association between trastuzumab and this patient's pulmonary syndrome was not proven, the potential for this toxicity should be considered.

Introduction

The HER-2/neu proto-oncogene is a poor prognostic factor and is amplified in 20-30% of patients with primary breast cancer. Trastuzumab, a monoclonal antibody directed against HER2, improves response, time to progression, and overall survival in both the metastatic and adjuvant settings in patients with HER2 positive breast cancer [START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer[END_REF][START_REF] Piccart | Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer[END_REF][START_REF] Slamon | Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[END_REF]. Trastuzumab is introduced as standard adjuvant treatment to patients with HER-2 positive tumors [START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer[END_REF]. Before, trastuzumab was already introduced as first-line treatment in combination with a taxane after prior use of anthracyclines, for metastasized disease. Patients who express high levels of HER-2/neu are most likely to respond to trastuzumab.

Case description

A 56-year-old non-smoking female underwent neoadjuvant chemotherapy for cT3N1M0 ductal carcinoma of the left breast in December 2003. The chemotherapy consisted of six courses of 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC) administered every 3 weeks. Chemotherapy resulted in a partial remission after which a radical mastectomy was performed in June 2004. Histopathological findings revealed an invasive ductal carcinoma of 2.2 cm, grade III with HER-2(3+) receptor overexpression. With 1 out of 10 axillary lymph nodes involved with metastatic cancer.

Immunohistochemical staining for oestrogen and progesterone receptors was negative. At subsequent follow-up visits no signs of recurrent disease were found.

In January 2006 she noticed thickening of the skin under her left mastectomy scar. Histopathologic examination showed an invasive ductal adenocarcinoma, with HER-2(3+) receptor overexpression. A computed tomography of the chest and the abdomen demonstrated local thickening of the skin and abdominal lymphadenopathy. No apparent metastases were present in the lungs, liver or other sites. Before starting treatment she did not suffer from any pulmonary symptoms. Her ECOG performance score was one. In January 2006 treatment was initiated with trastuzumab and docetaxel, every 3 weeks. A gradual decline of the skin thickening and the abdominal lymphadenopathy was demonstrated after six courses of docetaxel/trastuzumab. A chest X-ray taken in March 2006 did still not show any abnormalities. In May 2006 docetaxel was discontinued and trastuzumab was continued as monotherapy.

Eight days after the first administration of trastuzumab (30 days after her last docetaxel) the patient complained of dry cough and progressive dyspnoea. Apart from Oxycontin 20 mg twice daily she was not taking any other medication at that time. Physical examination revealed a temperature of 38°C, a pulse rate of 119/min, blood pressure 125/85 mmHg and an oxygen saturation of 86%. No supplemental oxygen was administered at that time. At the lung bases crackles were noticed. Laboratory tests showed a haemoglobin level 6.2 mmol/l (normal [7.3), leucocytes 7.8 9 10 9 /l, neutrophils 5.0 9 10 9 /l, eosinophils 1,000 9 10 6 /l (normal 40-440), CRP 133 (normal \9 mg/l). Renal and liver biochemistry test results did not show any abnormalities. An NT-proBNP and IgE level were not elevated nor was the ANCA titer.

With 5 l of nasal oxygen, the arterial blood gas analysis showed a hypoxemia with a PO2 of 6.5 kPa (normal [8.7) and an oxygen saturation of 90%.

A chest X-ray showed bilateral patchy consolidations and pleural effusion without cardiomegaly or any other signs of heart failure (Fig. 1).

Blood cultures remained negative. A PCR for CMV was performed and was negative, as was a serological aspergillus antigen test (galactomanantest). A urine sample for Legionella did not show any antigen.

A spiral CT angio of the thorax showed no signs of pulmonary embolism. However, patchy consolidations were observed in both lungs as well as bilateral pleural effusions.

To exclude heart failure a transthoracic echocardiography was performed which showed a normal left ventricular ejection fraction (65%) without any signs of diastolic dysfunction or any other abnormalities.

A diagnostic thoracocentesis was performed. Biochemical analysis of the serous fluid demonstrated an exudate. Microbiological examination revealed many leucocytes, but no bacteria were seen. Ziehl-Neelsen staining was negative. Cytological examination did not reveal any malignant cells. After cultures were taken treatment with amoxicillin with clavulanic acid and levofloxacin was initiated to cover typical and atypical respiratory infections. There was clinical improvement of dyspnoea and oxygen supply was gradually diminished.

During hospitalization, trastuzumab was again administered, 3 weeks after the prior administration. After this dose the patient's temperature rose to 38.7°C. She also complained of progressive dyspnoea and wheezing. Thoracentesis was repeated, and 1,500 ml of pleural fluid was evacuated. Microbiological examination did not show any micro-organisms and cultures remained negative. Cytological examination again revealed no malignant cells; however many eosinophilic granulocytes were observed.

A bronchoalveolar lavage (BAL) was performed, with instillation of 4 9 50 ml NaCl 0.9% in the middle lobe. The cell count showed 257,000 cells/ml. Cell differentiation of 500 cells showed 38.8% alveolar macrophages, 25.2% lymphocytes, 17.8% polymorphonuclear neutrophils, and 18% eosinophils. No malignant cells and no intracellular bacteria were observed. Microbiological cultures of the BAL fluid for bacteria, viruses, fungi and tuberculosis remained negative. An immunofluorescence test for Legionella on BAL fluid was also negative.

An allergic reaction to trastuzumab was highly suspected, based on the presence of eosinophils in BAL and pleural fluid and in the absence of an infectious agent. A lung biopsy via VATS was considered but the risk of ventilatory complications was regarded too high. The patient was treated with prednisolone 40 mg once daily. She gradually recovered and became independent of oxygen. A chest X-ray showed diminishment of the consolidations (Fig. 2).

Discussion

This female patient with HER2 positive breast cancer metastases to skin and abdominal lymph nodes was treated with docetaxel and trastuzumab. After six courses of combined therapy, docetaxel was stopped. Trastuzumab monotherapy was continued. After the first trastuzumab monotherapy infusion our patient was hospitalized because of dry cough and dyspnoea. With supportive therapy she gradually recovered but after a second treatment of trastuzumab there was again deterioration of her symptoms. Eventually, after exclusion of other causes, the pneumonitis appeared to be the consequence of trastuzumab. Analysis of BAL fluid demonstrated a pattern compatible with drug-induced pneumonitis. After treatment with corticosteroids dyspnoea and cough gradually disappeared and the consolidations on the chest X-ray diminished.

In several series, trastuzumab has been well-tolerated, and addition of trastuzumab to chemotherapy does not significantly increase the frequency of side effects [START_REF] Marty | Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group[END_REF]. Infusion-related events are common but rarely severe, and they are more common during the first administration [START_REF] Baselga | Phase II study of efficacy, safety and pharmacokinetics of trastuzumab monotherapy administered on a 3-weekly schedule[END_REF]. In an analysis of the safety of the administration of trastuzumab to 25,000 patients, the only respiratory-associated serious adverse event was bronchospasm [START_REF] Cook-Burns | Retrospective analysis of the safety of Herceptin immunotherapy in metastatic breast cancer[END_REF]. These reactions occurred usually within 2.5 h of administration. However, apart from described infusion-related bronchospasms, rare cases of interstitial pneumonitis have been reported. One patient with an organizing pneumonia and another with a pneumonitis were reported, probably due to trastuzumab administration [START_REF] Vahid | Trastuzumab (Herceptin)-associated lung injury[END_REF][START_REF] Radzikowska | Organising pneumonia caused by trastuzumab (Herceptin) therapy for breast cancer[END_REF]. In trial B-31, four patients in the trastuzumab group had interstitial pneumonitis, one of whom died [START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer[END_REF]. In the N9831 trial, five patients in the trastuzumab group had grade 3+ pneumonitis or pulmonary infiltrates, one of whom died [START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer[END_REF].

Drug-induced infiltrative lung disease (DI-ILD) is the most common form of anti-neoplastic agent-induced respiratory disease. Patterns of DI-ILD are non-specific interstitial pneumonia, eosinophilic pneumonia, hypersensitivity pneumonitis, pulmonary fibrosis, or organizing pneumonia [START_REF] Dimopoulou | Pulmonary toxicity from novel antineoplastic agents[END_REF]. Early withdrawal of the causative drug will often lead to improvement or even cure of the ILD. Corticosteroids may suppress the inflammatory reaction [START_REF] Camus | Interstitial lung disease induced by drugs and radiation[END_REF][START_REF] Camus | Drug-induced infiltrative lung disease[END_REF].

After trastuzumab monotherapy our patient developed symptoms and signs indicative of DI-ILD. Because of the time dependent relationship between the infusion of trastuzumab and the signs and symptoms a causal relationship is likely to exist although it is not proven.

Based on the history, physical examination and the HRCT and the BAL fluid analyses, our patient was diagnosed with drug-induced interstitial lung disease.

A peculiar feature of this case is why this patient did not develop symptoms of trastuzumab-induced pneumonitis earlier in her course? We speculate that before trastuzumab monotherapy was initiated, combination therapy with docetaxel was given with accompanying dexamethasone. Perhaps the concurrent dexamethasone prevented trastuzumab-related complications.

Taken together, the reports of apparent trastuzumabrelated pulmonary interstitial disease in B-31, N9831, and by Vahid, et al., Radzikowska, et al., and ourselves are highly suggestive this syndrome is a rare, but real, complication of this therapy. Clinicians should be aware of it and prepared to respond appropriately, including discontinuation of further treatment or cautious co-administration with corticosteroids.

Fig. 1

 1 Fig. 1 Chest ray at presentation

Breast Cancer Res Treat (2009) 113:609-612


Marc Lacroix

Poor usage of HUGO standard gene nomenclature in breast cancer studies

Dear Editor, Analysis of gene expression in breast cancer cells, notably by reverse transcriptase-polymerase chain reaction (RT-PCR), is widely performed. One promising use of RT-PCR is for the detection of disseminated cancer cells (DTC) in lymph nodes, peripheral blood and bone marrow (for a review, see [START_REF] Lacroix | Significance, detection and markers of disseminated breast cancer cells[END_REF]). Many genes of interest in breast cancer possess multiple names, which may be confusing. However, since 1989, the Human Genome Organisation (HUGO, http://www.hugo-international.org) attempts to ensure that for each gene there is one name and one symbol [START_REF] White | Promoting a standard nomenclature for genes and proteins[END_REF][START_REF] Bruford | The HGNC Database in 2008: a resource for the human genome[END_REF].

To determine to which extent HUGO nomenclature is used, I recently performed a PubMed search for abstracts mentioning DTC detection by RT-PCR in breast cancer patients. The resulting 92 articles were searched for occurrences of standard HUGO gene symbols and names, and of other descriptors («aliases») for the markers targeted by RT-PCR.

As shown in Table 1 for four representative markers, the use of HUGO symbols and names is dramatically low, as compared to that of aliases. The case of SCGB2A2 (gene name: «secretoglobin family 2A, member 2») is probably the most striking among all. This symbol was not found in 34/34 abstracts of papers reporting on the detection of the gene it designates. Instead, the gene was variably symbolized as MAM, hMAM, hMAM-A, MG, MGB1, or MMG, reflecting at least three different names: mammaglobin, mammaglobin A, and mammaglobin 1. Many people performing markers analysis in breast cancer are familiar with the word «mammaglobin». However, there are two different mammaglobins, and a PubMed search using the word «mammoglobin» does not retrieve all papers reporting on SCGB2A2 expression analysis. As reflected by this example, the breast cancer community has not widely adopted the HUGO nomenclature. This impairs the efficient retrieval of information from databases. I suggest that the mention of standard names or symbols in abstracts in place of, or, at least, next to aliases, should be made mandatory. More generally, all actors of research should pay more attention to the usage of standard nomenclature. Indeed, the task of mentioning genes in papers by their official names and symbols will require the collaboration of authors, journals (editors and reviewers). Regarding journals, the pursuit of unique standard gene name and symbol has been encouraged for years by most journals primarily concerned with human genetics. In contrast, the frequency with which HUGO nomenclature is used is still low in clinical journals that publish most of the data concerning tumor cell identification.

To agree on a particular gene name not only will make one's own research easier, but will also be helpful to the present generation, as well as future generations, of researchers who are about to enter molecular clinical research.

Table 1

 1 Four markers widely used to detect DTC from breast cancer patients by RT-PCR

	HUGO gene symbol (number of	HUGO gene name (number of abstracts	Frequently-used non-standard names
	abstracts citing this symbol/total	citing this name/total number of abstracts	and symbols (''aliases'')
	number of abstracts citing the marker)	citing the marker)
	KRT19 (0/46)	Keratin 19 (0/46)	Cytokeratin 19; cytokeratin-19; CK19; CK-19
	SCGB2A2 (0/34)	Secretoglobin family 2A, member 2 (0/34)	Mammaglobin; mammglobin A; mammaglobin
			1; MAM; mam; hMAM; hMAM-A; MG;
			MGB1; MMG
	SERPINB5 (0/12)	Serine (or cysteine) proteinase inhibitor,	Maspin; MAS
		clade B (ovalbumin), member 5 (0/12)
	TACSTD1 (1/9)	Tumor-associated calcium signal	Epithelial glycoprotein 2; epithelial
		transducer 1 (1/9)	glycoprotein 40; EGP40; EGP2; KS1/4;
			GA733-2


Eugenio Maiorano
email: emaiorano@anatopat.uniba.it

Meredith M Regan
email: mregan@jimmy.harvard.edu

MD, FRCPath Giuseppe Viale
email: giuseppe.viale@ieo.it

Mauro G Mastropasqua
email: mauro.mastropasqua@ieo.it

Marco Colleoni
email: marco.colleoni@ieo.it

Monica Castiglione-Gertsch

Karen N Price
email: price@jimmy.harvard.edu

Richard D Gelber
email: gelber@jimmy.harvard.edu

Aron Goldhirsch
email: aron.goldhirsch@ibcsg.org

Alan S Coates
email: alan.coates@ibcsg.org

Eugenio Maiorano

Prognostic and predictive impact of central necrosis and fibrosis in early breast cancer: Results from two International Breast Cancer Study Group randomized trials of chemoendocrine adjuvant therapy

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

Results: CNF was present in 84 of 1850 trial patients (4.5%). It was associated with tumor characteristics suggesting poor outcome, but was an independent adverse factor for diseasefree survival. In the presence of CNF outcome was worse regardless of tumor grade, whereas in the absence of CNF, patients with grade 3 tumors had poorer outcome than those with grade 1-2 tumors. Among patients with estrogen receptor-absent tumors chemoendocrine therapy was superior to endocrine therapy alone only in the absence of CNF [HR (chemoendocrine:endocrine)=0.46 in CNF-absent, 0.90 in CNF-present], while among those with receptor-positive disease chemoendocrine therapy was beneficial only in the presence of CNF [HR=0.34 CNF-present, 0.96 CNF-absent].

INTRODUCTION

Extensive central (or geographic) necrosis has long been recognized as a pathological feature in some cases of primary breast cancer. Analysis of patients participating in an early trial of the National Surgical Adjuvant Breast Project (NSABP-04) found that necrosis was likely to be marked in tumors classified as medullary, in larger tumors, in those exhibiting squamous metaplasia or vessel invasion and in those occurring in younger patients [START_REF] Fisher | The pathology of invasive breast cancer. A syllabus derived from findings of the National Surgical Adjuvant Breast Project (protocol no. 4)[END_REF]. Necrosis was seen also in a smaller percentage of cases of infiltrating ductal carcinoma without specific features, especially those of higher histological grade, and in some other histologic types. Recently, several studies have reported on the clinico-pathological characteristics of a distinct subset of breast cancer, showing central acellular zones occupied by necrotic or fibrotic tissues (CNF) [START_REF] Hasebe | Fibrotic focus in invasive ductal carcinoma of the breast: a histopathological prognostic parameter for tumor recurrence and tumor death within three years after the initial operation[END_REF][START_REF] Hasebe | Fibrotic focus in infiltrating ductal carcinoma of the breast: a significant histopathological prognostic parameter for predicting the long-term survival of the patients[END_REF][START_REF] Hasebe | New prognostic parameter of invasive ductal carcinoma of the breast: clinico-pathological significance of fibrotic focus[END_REF][START_REF] Tsuda | Large, central acellular zones indicating myoepithelial tumor differentiation in high-grade invasive ductal carcinomas as markers of predisposition to lung and brain metastases[END_REF][START_REF] Jiminez | Centrally necrotizing carcinomas of the breast. A distinct histologic subtype with aggressive clinical behavior[END_REF][START_REF] Colpaert | Early distant relapse in 'node-negative' breast cancer patients is not predicted by occult axillary lymph node metastases, but by the features of the primary tumour[END_REF][START_REF] Hasebe | Highly proliferative fibroblasts forming fibrotic focus govern metastasis of invasive ductal carcinoma of the breast[END_REF][START_REF] Colpaert | The presence of a fibrotic focus in invasive breast carcinoma correlates with the expression of carbonic anhydrase IX and is a marker of hypoxia and poor prognosis[END_REF][START_REF] Van Den Eynden | Gene expression profiles associated with the presence of a fibrotic focus and the growth pattern in lymph node-negative breast cancer[END_REF]. These studies emphasized that patients harboring invasive ductal breast carcinomas (IDC) with large acellular areas were at higher risk for developing lung and brain metastases and showed poorer survival, in comparison with conventional IDC of comparable tumor grade.

Different criteria were employed by the cited authors to assess CNF, such as extension for 30% or more of the tumor area [START_REF] Tsuda | Large, central acellular zones indicating myoepithelial tumor differentiation in high-grade invasive ductal carcinomas as markers of predisposition to lung and brain metastases[END_REF], for 70% or more of the tumor cross-section (6) or detection at low power view [START_REF] Colpaert | Early distant relapse in 'node-negative' breast cancer patients is not predicted by occult axillary lymph node metastases, but by the features of the primary tumour[END_REF]. Additional features detected in IDC with CNF were: the absence of coagulative necrosis, of squamous metaplasia or keratinization, of osseous or cartilaginous metaplasia, and of matrix-producing features (5); abrupt transition from necrosis/fibrosis to viable tumor cells; high proliferation [START_REF] Hasebe | New prognostic parameter of invasive ductal carcinoma of the breast: clinico-pathological significance of fibrotic focus[END_REF][START_REF] Colpaert | The presence of a fibrotic focus in invasive breast carcinoma correlates with the expression of carbonic anhydrase IX and is a marker of hypoxia and poor prognosis[END_REF][START_REF] Colpaert | Intratumoral hypoxia resulting in the presence of a fibrotic focus is an independent predictor of early distant relapse in lymph node-negative breast cancer patients[END_REF], especially in the tumor fibroblasts; [START_REF] Hasebe | Highly proliferative fibroblasts forming fibrotic focus govern metastasis of invasive ductal carcinoma of the breast[END_REF] and pushing tumor-host interface [START_REF] Jiminez | Centrally necrotizing carcinomas of the breast. A distinct histologic subtype with aggressive clinical behavior[END_REF].

Linking powered by eXtyles More recently, CNF has been reported as a common morphological feature of the tumors belonging to the molecular class of basal-like breast cancers [START_REF] Van Den Eynden | Gene expression profiles associated with the presence of a fibrotic focus and the growth pattern in lymph node-negative breast cancer[END_REF][START_REF] Fulford | Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast[END_REF][START_REF] Livasy | Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma[END_REF] and is associated with activation of Ras oncogene signaling, an activated wound healing signature and a poor prognosis 76-gene signature [START_REF] Van Den Eynden | Gene expression profiles associated with the presence of a fibrotic focus and the growth pattern in lymph node-negative breast cancer[END_REF].

The present study was designed to investigate the impact of CNF on patient outcome, in particular the interrelation of tumor grade and CNF on outcome, and investigate the impact of CNF on the relative effects of chemoendocrine and endocrine therapies in two randomized controlled trials conducted by the International Breast Cancer Study Group for women with lymph node-negative breast cancer.

PATIENTS AND METHODS

Patients were enrolled in one of two randomized clinical trials conducted by the International Breast Cancer Study Group (IBCSG) which have been reported elsewhere [START_REF]International Breast Cancer Study Group (2002) Endocrine responsiveness and tailoring adjuvant therapy for postmenopausal lymph node-negative breast cancer: A randomized trial[END_REF][START_REF]International Breast Cancer Study Group (2003) Adjuvant chemotherapy followed by goserelin versus either modality alone for premenopausal lymph node-negative breast cancer: A randomized trial[END_REF]. Briefly, Trial VIII enrolled pre/peri-menopausal women with lymph node-negative breast cancer. The trial evaluated whether sequential treatment with six 28-day courses of "classical" cyclophosphamide, methotrexate, fluorouracil (CMF) chemotherapy followed by 18 monthly subcutaneous implants of goserelin significantly improved disease-free survival (DFS) as compared with either six 28-day courses of classical CMF alone, or 24 monthly implants of goserelin alone. From 1990 through 1999, a total of 1063 assessable patients were randomized.

Trial IX enrolled postmenopausal women with lymph node-negative breast cancer and evaluated whether sequential treatment with three 28-day courses of classical CMF chemotherapy followed by tamoxifen for 57 months significantly improved DFS as compared Linking powered by eXtyles with tamoxifen alone for 5 years. From 1988 through 1999, a total of 1669 eligible and assessable patients were randomized. Institutional review boards reviewed and approved the protocols, and informed consent was required according to the criteria established within the individual countries. Toward the end of enrollment, on the basis of evidence from other trials [START_REF]Early Breast Cancer Trialists' Collaborative Group (1996) Tamoxifen for early breast cancer: an overview of the randomised trials[END_REF][START_REF]Early Breast Cancer Trialists' Collaborative Group (▪▪▪) Ovarian ablation in early breast cancer: overview of the randomised trials[END_REF] and after some 94% of patients had been entered, the protocols were amended to restrict enrollment to patients whose tumors were estrogen receptor (ER)-positive.

Central Pathology Review

Retrospective tissue collection was carried out in accordance with institutional guidelines and national laws. More than 80% of patients randomized in Trials VIII and IX had archival tumor material available for review in the IBCSG Central Pathology Laboratory. Central pathology review was conducted without knowledge of patient treatment assignment or outcome. CNF was assessed during central pathology review according to the following criteria:

-extension of necrosis/fibrosis for 1mm or more of the cross section of tumors -absence of coagulative necrosis -abrupt transition from necrosis/fibrosis to viable tumor cells -pushing tumor borders -lack of squamous metaplasia or keratinization -no evidence of osseous or cartilaginous metaplasia and of matrix-producing features Linking powered by eXtyles Expression of ER, progesterone receptors (PgR), HER2 and Ki-67 labeling index (LI) in the primary tumors were determined by immunohistochemistry (IHC) [START_REF] Viale | Chemoendocrine compared with endocrine adjuvant therapies for node-negative breast cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors--International Breast Cancer Study Group[END_REF][START_REF] Viale | Predictive value of tumor Ki-67 expression in two randomized trials of adjuvant chemoendocrine therapy for node-negative breast cancer[END_REF].

Analytic Cohort

Tumor blocks and/or slides were available and assessable for CNF for 776 of 1063 patients (73%) in Trial VIII and 1427 of 1669 patients (86%) in Trial IX. Because of the low prevalence of CNF, the two trial cohorts were analyzed together. The present study compared endocrine therapy alone (goserelin alone in Trial VIII or tamoxifen alone in Trial IX) with chemoendocrine therapy (sequential treatment with CMF followed by goserelin in Trial VIII, or CMF followed by tamoxifen in Trial IX). Patients in Trial VIII assigned to chemotherapy alone were not included in the analytic cohort. A further 81 patients whose tumors could not be assessed for any of ER, PgR and HER2 were also excluded. Thus a total of 1850 patients randomly assigned to treatment groups comparing endocrine with chemoendocrine therapy were included in the analytic cohort. The median follow up of the analytic cohort was 10 years.

Statistical Methods

Patient age at randomization, treatment assignment, tumor size and tumor grade were obtained from trial case report forms. Centrally-determined ER and PgR status were classified as present (≥1% immunoreactive cells) or absent (0%), HER2 was considered as over-expressed if the intensity was scored 3+ vs. (0, 1+, 2+), and Ki-67 was classified as high for ≥19% immunoreactive cells by dichotomizing expression at the median value, as in previous analyses [START_REF] Viale | Chemoendocrine compared with endocrine adjuvant therapies for node-negative breast cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors--International Breast Cancer Study Group[END_REF][START_REF] Viale | Predictive value of tumor Ki-67 expression in two randomized trials of adjuvant chemoendocrine therapy for node-negative breast cancer[END_REF]. A triple-negative tumor was defined as one that was absent of ER and PgR expression and did not overexpress HER2.

Linking powered by eXtyles Logistic regression modeling was used to examine the association of patient and tumor features with presence of CNF. The association of the presence of CNF with DFS, accounting for other patient and tumor features, was evaluated using Cox proportional hazards modeling. DFS was defined as in the trials as the length of time from the date of randomization to any relapse (including ipsilateral breast recurrence), the appearance of a second primary cancer (including contralateral breast cancer), or death, whichever occurred first. In all modeling, dummy categories were used to accommodate unavailable values; pairwise comparisons of interest were made using model contrasts which produced likelihood-ratio tests (logistic) or Wald tests (proportional hazards), and all p-values were obtained from two-sided tests. Hazard ratios (HRs) and 95% confidence intervals (CIs) for the treatment comparisons were estimated from the proportional hazards models. The analysis used SAS version 9.1 (SAS Institute Inc., Cary, NC).

RESULTS

CNF was identified in tumors from 84 of 1850 trial patients (4.5%). Patient and tumor characteristics of the analytic cohort are summarized in Table 1 according to presence or absence of CNF.

Associations of CNF with patient and tumor features

The CNF-positive tumors were equally distributed across the two treatment groups. CNF was observed among 3.6% and 4.9% of the Trial VIII (premenopausal) and Trial IX (postmenopausal) patients' tumors respectively. There was no association between CNF and trial cohort (i.e., menopausal status) or patient age at randomization. Presence of CNF was associated with the following other poor prognostic features (each P<0.001): larger and higher grade tumors; Linking powered by eXtyles absence of ER and PgR; high Ki-67 LI. Although CNF was not significantly associated with HER2 overexpression per se (P=0.88), it was markedly associated with triple-negative tumors lacking ER, PgR and HER2 expression (17.9% of triple negative vs. 2.8% of other tumors had CNF; P<0.001). Much of this association between CNF and triple-negative status was contributed by the absence of ER. In multivariable logistic regression modeling that included trial cohort and selected among tumor features, the tumor triple-negative status, Ki-67 LI status and size were most strongly associated with presence of CNF (each P<0.01; Table 2). In this model, there was a marginal association of trial cohort and CNF (P=0.09; Table 2). Tumor grade was not independently associated with CNF (OR=1.40, 95% CI 0.80 to 2.44; grade 3 vs. grade 1-2) in the multivariable model.

Disease-free survival

CNF was associated with poorer DFS (HR=1.6, 95% CI 1.1 to 2.3; P<0.01; Figure 1) in univariate analysis. Estimated 5-year DFS was 70% ± 5.0 and 84% ± 0.9 among patients having tumors with and without CNF respectively (Table 3). There was evidence of an interaction of tumor grade and CNF (P=0.03) with DFS, such that the presence of CNF was associated with poorer outcome among patients with grade 1 or 2 tumors (HR=2.5, 95% CI 1.4 to 4.3), but this was not seen among patients with grade 3 tumors (HR=1.1, 95% CI 0.7 to 1.8) (Figure 2A). Conversely, the interaction suggests that in the presence of CNF there was poor outcome regardless of tumor grade whereas in the absence of CNF, patients with grade 3 tumors had poorer outcome than those with grade 1-2 tumors. DFS according to the combination of CNF and tumor grade -as Linking powered by eXtyles grade 1, grade 2, grade 3 (each without CNF) and any grade with CNF -is summarized in Figure 2B. These results persisted in multivariable analysis.

The relative benefit of chemoendocrine vs. endocrine therapy alone on DFS in the presence and absence of CNF was examined separately for patients whose tumors did or did not express ER, because of both biological and statistical considerations (P=0.03 for 3-way interaction of ER-status, treatment and CNF). The observed patterns appeared strikingly different (Figure 3). In patients with ER-absent tumors, for whom the benefit of chemoendocrine therapy over endocrine therapy alone might be most expected, this benefit was seen in the majority whose tumors did not show CNF (HR=0.46, 95% CI 0.31 to 0.70), but was less apparent in the minority with CNF present (HR=0.90, 95% CI 0.33 to 2.41). The outcomes for the three patient groups (CNF-present with either treatment, and CNF-absent treated with endocrine therapy alone) were similar and markedly inferior to that for patients with CNF-absent tumors allocated chemoendocrine therapy (Figure 3A). By contrast, among patients with ER-expressing tumors, there was benefit of chemoendocrine therapy over endocrine therapy alone in the minority of patients whose tumors did show CNF (HR=0.34, 95% CI 0.11 to 1.04) but not in the majority whose tumors did not show CNF (HR=0.96, 95% CI 0.78 to 1.18). Outcome was similar and relatively good, except for patients whose tumors showed CNF and who were allocated endocrine therapy alone (Figure 3B).

DISCUSSION

The strength of examining the prognostic and predictive value of tumor markers in the context of prospective clinical trials has been noted by previous authors (1): the treatments and follow-up are more likely to be standardized than would be true of a convenience sample. CNF is a relatively uncommon finding in early invasive breast cancer, and our observation of a prevalence of 4.5% among patients with node-negative breast cancer is consistent with previous reports [START_REF] Fisher | The pathology of invasive breast cancer. A syllabus derived from findings of the National Surgical Adjuvant Breast Project (protocol no. 4)[END_REF][START_REF] Jiminez | Centrally necrotizing carcinomas of the breast. A distinct histologic subtype with aggressive clinical behavior[END_REF]. We found associations between CNF and larger tumors, those lacking hormone receptors and those with a high proliferative fraction, findings again consistent with earlier reports [START_REF] Hasebe | Fibrotic focus in infiltrating ductal carcinoma of the breast: a significant histopathological prognostic parameter for predicting the long-term survival of the patients[END_REF][START_REF] Jiminez | Centrally necrotizing carcinomas of the breast. A distinct histologic subtype with aggressive clinical behavior[END_REF][START_REF] Hasebe | Highly proliferative fibroblasts forming fibrotic focus govern metastasis of invasive ductal carcinoma of the breast[END_REF][START_REF] Colpaert | The presence of a fibrotic focus in invasive breast carcinoma correlates with the expression of carbonic anhydrase IX and is a marker of hypoxia and poor prognosis[END_REF][START_REF] Colpaert | Intratumoral hypoxia resulting in the presence of a fibrotic focus is an independent predictor of early distant relapse in lymph node-negative breast cancer patients[END_REF].

It appears from the present study that the presence of CNF provides information in addition to the prognostic and predictive factors normally recorded in such trials. CNF was associated with a poorer outcome irrespective of histologic grade, and indeed all grades had similarly poor outcomes in the presence of CNF.

Because the treatment effect of chemoendocrine versus endocrine therapy depends on hormone receptor status as observed in the overall trial results [START_REF]International Breast Cancer Study Group (2002) Endocrine responsiveness and tailoring adjuvant therapy for postmenopausal lymph node-negative breast cancer: A randomized trial[END_REF][START_REF]International Breast Cancer Study Group (2003) Adjuvant chemotherapy followed by goserelin versus either modality alone for premenopausal lymph node-negative breast cancer: A randomized trial[END_REF][START_REF] Viale | Predictive value of tumor Ki-67 expression in two randomized trials of adjuvant chemoendocrine therapy for node-negative breast cancer[END_REF], and because each of the trials was prospectively stratified by estrogen receptor status, we chose to primarily examine the relationship between CNF and treatment effect separately in receptor-absent and receptor-present cohorts. In the receptor-absent cohort, where the benefit of chemoendocrine therapy over endocrine therapy alone had been observed in the trials overall, we indeed found this benefit among the majority of patients without CNF, but the benefit appeared to be completely abrogated by the presence of CNF (Figure 3A). The reason is not clear. It may reflect poorer penetration of chemotherapeutic agents into micro-metastatic tumor deposits in tumors prone to show CNF, perhaps due to associated hypoxia [START_REF] Colpaert | The presence of a fibrotic focus in invasive breast carcinoma correlates with the expression of carbonic anhydrase IX and is a marker of hypoxia and poor prognosis[END_REF][START_REF] Colpaert | Intratumoral hypoxia resulting in the presence of a fibrotic focus is an independent predictor of early distant relapse in lymph node-negative breast cancer patients[END_REF][START_REF] Van Den Eynden | A fibrotic focus is a prognostic factor and a surrogate marker for hypoxia and (lymph)angiogenesis in breast cancer: review of the literature and proposal on the criteria of evaluation[END_REF][START_REF] Shannon | Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies[END_REF][START_REF] Yasuda | Solid tumor physiology and hypoxia-induced chemo/radio-resistance: novel strategy for cancer therapy: nitric oxide donor as a therapeutic enhancer[END_REF][START_REF] Cosse | Tumour hypoxia affects the responsiveness of cancer cells to chemotherapy and promotes cancer progression[END_REF][START_REF] Tan | The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy[END_REF] or some undefined cellular characteristic which reduces the efficacy of the chemotherapeutic agents Linking powered by eXtyles used. The effect of CNF in receptor-positive disease was equally dramatic: whereas endocrine therapy alone appeared adequate for the majority of patients, the outcome was inferior among patients whose tumors showed CNF and who were allocated endocrine therapy alone (Figure 3B). Here the more efficacious therapy may be thought to be the tamoxifen, so it is again possible that the mechanism reflects poorer drug access to the tumor, though an intrinsic characteristic of tumor cells which compromises endocrine efficacy cannot be excluded.

Whatever the mechanism, these results suggest that the presence of CNF identifies a biologically distinct subgroup within early breast cancer and that the difference is important in determining the efficacy of standard chemotherapy and endocrine therapy. We recommend that the presence or absence of CNF, as defined in previous reports (3, 6-9, 11, 20) and in the current paper become a standard part of the pathological reporting of early breast cancer.

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Disease-free survival according to tumor grade and central necrosis fibrosis (CNF),

Figure 3 .

 3 Figure 3. Disease-free survival according to treatment and presence or absence of central

Table 1 . Association of patient and tumor features with presence of CNF 1 .

 1 Percentages sum across the rows. P-values from univariate logistic regression models.

			CNF asbsent	CNF present	Odds Ratio
			N (%)	N (%)	(95% CI)	P-value
	Number of patients	1766 (95.5)	84 (4.5)
	Treatment			0.91
	Endocrine-only	882 (95.6)	41 (4.4)
	CMF	endocrine	884 (95.4)	43 (4.6)
	Trial				0.31
	VIII (pre/perimenopausal)	480 (96.4)	18 (3.6)
	IX (postmenopausal)	1286 (95.1)	66 (4.9)
	Age (y), median (IQR)	57 (50-63)	59 (50-63)	0.69
	Tumor size (cm)			<0.001
	2.0		1069 (97.3)	30 (2.7)	1 (ref)
	> 2.0		662 (92.6)	53 (7.4)	2.9 (1.8, 4.5)
	Unknown	35	1
	Tumor grade			<0.001
	1,2		1075 (97.7)	25 (2.3)	1 (ref)
	3		628 (91.8)	56 (8.2)	3.8 (2.4, 6.2)
	Unknown	63	3
	ER				<0.001
	Absent		292 (85.9)	48 (14.1)	6.4 (4.0, 10.0)
	Present (≥1%)	1354 (97.5)	35 (2.5)	1 (ref)

16

Linking powered by eXtyles

Table 2 . Multivariable logistic regression model for presence of central necrosis/fibrosis

 2

		Estimate ± SE Odds Ratio (95% CI)	P-value
	Trial IX postmenopausal (vs. Trial VIII premenopausal)	0.48 ± 0.28	1.61 (0.93, 2.81)	0.09
	Triple-negative (vs. not)	1.54 ± 0.25	4.65 (2.88, 7.52)	<0.0001
	High Ki-67 LI (vs. low Ki-67 LI)	1.53 ± 0.37	4.62 (2.23, 9.57)	<0.0001
	Tumor size >2cm (vs. ≤ 2cm)	0.65 ± 0.24	1.92 (1.19, 3.10)	<0.01

ACKNOWLEDGEMENT

We thank the local pathologists who submitted tumor blocks and slides, Rosita Kammler and the pathology team in Bern, Stefania Andrighetto at the pathology office in Milan, and Barry Gusterson and Elizabeth Mallon for central pathology review. We thank the patients, physicians, nurses and data managers who participate in the International Breast Cancer Study Group trials. The International Breast Cancer Study Group is supported by Swiss Group for Clinical Cancer Research, Frontier Science and Technology Research Foundation, The Cancer Council Australia, Australian New Zealand Breast Cancer Trials Group (National Health Medical Research Council Grants No. 920876, 950328, 980379, and 100925), National Cancer Institute (Grant No. CA-75362), Swedish Cancer Society, Foundation for Clinical Cancer Research of Eastern Switzerland (OSKK), Cancer Association of South Africa (for South African participation), and Oncosuisse/Cancer Research Switzerland (for collection of tumor blocks within Switzerland).

Conclusion:

The results suggest that the presence of CNF reflects a biological difference in early breast cancer that is important in modulating the efficacy of standard therapies. Accordingly we believe that its presence should be routinely reported.


Josefa Salgado
email: jsalgadog@unav.es

José M Aramendía

Cristina Gutierrez

Carmen Gil

Maitane Robles

Jesús García-Foncillas

Running head: New germline mutation in BRCA2

Keywords: BRCA2, novel mutation, breast cancer, prostatic cancer

Linking powered by eXtyles Mutations in BRCA1 and BRCA2 genes have been shown to be disease-associated with inherited susceptibility to breast and/or ovarian cancer [START_REF] Wooster | Identification of the breast cancer susceptibility gene BRCA2[END_REF][START_REF] Miki | A Strong Candidate for the Breast and Ovarian Cancer Susceptibility Gene BRCA1[END_REF]. A large number of germ-line mutations in these genes have been reported (Breast Cancer Information Core Database, http://www.nchgr.nih.gov/bic). This report concerns a Spanish woman with a family history of breast, ovarian, and prostate cancer, in whom a novel germline mutation was identified. The patient, who is indicated by an arrow in pedigree (Fig. 1a), was a 45 years old multiparous, non-Ashkenazi woman. Fifteen years ago, she was diagnosed and treated for a bilateral breast cancer, and mastectomy was performed. The genealogical tree of the patient revealed a history of grade I prostate cancer, grade II breast cancer and grade III ovarian cancer. After giving the informmed consent of the patient, genomic DNA was automatically extracted from a peripheral blood sample (MagNapure, Roche). Direct sequencing of BRCA1 and BRCA2 genes was done on an automated sequencer ABI PRISM ® 377 (Applied Biosystems) and compared to the consensus wild-type sequences (Genebank No U14680, BRCA1 and Genebank NM_000059, BRCA2). A 5369delATTT frame-shift mutation in exon 11, not yet reported in the BIC database, was detected in BRCA2 (Fig. 1b). This mutation, that generates an aberrant stop codon at position 1723, could affect important BRCA2 functions since it causes absence of BRCA2 C-terminal conserved region, necessary for DSS1 and DNA binding process. Moreover, it affects the BRCA2-BRC globular structural motifs that mediate binding to RAD51, involved in directing strand invasion during recombination [START_REF] Jasin | Homologous repair of DNA damage and tumorogenesis: the BRCA connection[END_REF]. The genetic analysis was offered to other family members being II.4, II.9, III.3, III.4, III.6 and IV.1 to IV.4 recruited in this study (Fig. 1a). Four mutation carriers were detected, two of them already diagnosed with disease: the patient´s father (II.9, prostatic cancer) and the patient´s sister (III.6, breast Linking powered by eXtyles cancer). The other two carriers, IV.2 and IV.3, are not affected by the disease at the moment. A twice per year clinical breast examinations and annual mammography has been recommended for the female mutation carrier (IV.3). In addition, the American Cancer Society recently published guidelines recommending that BRCA carriers and first-degree relatives undergo annual breast magnetic resonance imaging screening [START_REF] Saslow | American Cancer Society guidelines for breast screening with MRI as an adjunct to mammography[END_REF]. For the men (IV.2) an annual clinical breast examination was proposed. The National Comprehensive Cancer Network guidelines recommend that male BRCA mutation carriers should begin prostate screening (digital rectal examination and PSA) beginning at age 50, or earlier, based on the youngest age of diagnosis in the family [START_REF] Daly | National Comprehensive Cancer Network: Genetic/Familial High-Risk Assessment: Breast and Ovarian Clinical Practice Guidelines[END_REF]. Family members II.4, III.3, III.4, IV.1, and IV.4 were not carriers. Frequent truncations found in the Spanish population are 187delAG and 5385insC in BRCA1 and 3036del4, 3492insT, 5374del4, 9254delTCAT and 9538del2 in BRCA2. Geographical variations in the mutations distribution may be due to founder effects [START_REF] Diez | 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] Diez | 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] Duran | Mutational analysis of BRCA2 in Spanish breast cancer patients from Castilla-León: identification of four novel truncating mutations[END_REF][START_REF] Llort | Low frequency of recurrent BRCA1 and BRCA2 mutations in Spain[END_REF][START_REF] Vega | Analysis of BRCA1 and BRCA2 in breast and breast/ovarian cancer families shows population substructure in the Iberian peninsula[END_REF]. We have analyzed the 5369delATTT mutation in 147 families from various Spanish regions with no positive results. Further in low represented Spanish areas will make possible to describe genetic influences and phenotype features associated. At the same time, a study conducted on a population with the same mutation might be of help in determining disease penetrance.

Acknowledgments

We are grateful to the patients, and their families, for their participation in our research project. We thank clinicians and genetic counselors of the Department of Oncology and the Clinical Genetics Unit for their support and encouragement. Cristina Gutierrez has an ADA fellowship from the University of Navarra, Navarra, Spain.