J Michael

Dixon Ae

Lorna Renshaw

Ae E Jane Macaskill

A E Oliver

Young Ae

Juliette Murray

A E David

Cameron Ae

Gillian R Kerr

A E Dean

B Evans Ae

William R Miller

J M Dixon

E J Macaskill

Á O Young

Á J Murray

Á D Cameron

Á W R Miller

Increase in response rate by prolonged treatment with neoadjuvant letrozole

Keywords: Breast conserving surgery, Endocrine therapy, Neoadjuvant, Letrozole, Large operable and locally advanced breast cancer

Purpose The aim of this study was to investigate the potential benefits of prolonged treatment with neoadjuvant letrozole. Patients and Methods About 182 consecutive patients have been treated in Edinburgh with neoadjuvant letrozole for 3 months or longer and 63 patients have continued on letrozole beyond 3 months. Outcomes are reported. Results Of the 63 patients who continued on letrozole, 38 patients took letrozole for more than 1 year and 23 took letrozole for more than 24 months. The median reduction in clinical volume in the first 3 months in these 63 patients was 52%. Similar reductions in median clinical volume were seen between three to 6 months (50%), 6-12 months and 12-24 months (medians 37 and 33%, respectively). At 3 months 69.8% of the 182 patients had a partial or complete response. The response rate increased to 83.5% with prolonged letrozole treatment. Continuing letrozole beyond 3 months increased the number of women who initially required mastectomy or had locally advanced breast cancer who were subsequently suitable for breast conserving surgery from 60% (81/134) at 3 months to 72% (96/134). Thirty-three women remain on letrozole alone (man age at diagnosis 83 years) and at 3 years the median time to treatment failure has not been reached. Conclusion Continuing letrozole in responding patients beyond 3-4 months achieves further clinical reduction in tumour size. For elderly women with a short life expectancy letrozole alone may provide long-term disease control.

Introduction

Until recently neoadjuvant therapy of breast cancers consisted predominately of cytotoxic chemotherapy. References [START_REF] Bear | Indications for neoadjuvant therapy for breast cancer[END_REF][START_REF] Perloff | Chemotherapy before and after mastectomy in stage III breast cancer[END_REF][START_REF] Schick | Preoperative chemotherapy followed by mastectomy for locally advanced breast cancer[END_REF][START_REF] Bonadonna | Primary chemotherapy to avoid mastectomy in tumors with diameters of 3 cm or more[END_REF] Studies comparing neoadjuvant with adjuvant chemotherapy have shown that while disease-free, progression-free and overall survival are not changed by the timing of treatment, better outcomes are seen in patients who respond to neoadjuvant therapy [START_REF] Cleator | Good clinical response of breast cancers to neoadjuvant chemoendocrine therapy is associated with improved overall survival[END_REF][START_REF] Van Der Hage | Preoperative chemotherapy in primary operable breast cancer: results from the European Organization for Research and Treatment of Cancer Trial 10902[END_REF][START_REF] Wolmark | Preoperative chemotherapy in patients with operable breast cancer: nine-year results from National Surgical Adjuvant Breast and Bowel Project B-18[END_REF]. These results highlight a key benefit of neoadjuvant therapy, the use of response to predict long-term outcome. Endocrine treatment is now emerging as an attractive alternative in postmenopausal women with large operable or locally advanced hormone receptor positive breast cancers, many of whom cannot tolerate the toxicities of chemotherapy.

Initial studies performed in Edinburgh suggested there may be benefits to using aromatase inhibitors rather than tamoxifen as neoadjuvant therapy in postmenopausal women with ER + cancers [START_REF] Dixon | Letrozole as primary medical therapy for locally advanced and large operable breast cancer[END_REF][START_REF] Dixon | Neoadjuvant tamoxifen and aromatase inhibitors: comparisons and clinical outcomes[END_REF]. This led to a series of randomised trials. The PO24 trial compared 4 months treatment with neoadjuvant letrozole or tamoxifen in postmenopausal women with large hormone positive breast cancers that required mastectomy or were locally advanced [START_REF] Eiermann | Preoperative treatment of postmenopausal breast cancer patients with letrozole: a randomized double-blind multicenter study[END_REF]. Results from this study demonstrated that letrozole achieved a significantly higher clinical response rate (55% vs. 36%) and more patients treated with letrozole than with tamoxifen were suitable for breast-conserving surgery (45% vs. 35%). Letrozole now had a product licence in the UK for neoadjuvant use. Subsequent studies have been performed with anastrozole and exemestane treating patients for between 3 and 4 months prior to surgery and showed that compared with tamoxifen both drugs increased the number of patients becoming eligible for breast conserving surgery [START_REF] Smith | On behalf of the IMPACT Trialists: Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicentre, double-blind randomized trial[END_REF][START_REF] Semiglazov | Neoadjuvant endocrine therapy: exemestane vs tamoxifen in postmenopausal ER + breast cancer patients (T1-4, N1-2, M0)[END_REF].

The 3-4 months treatment duration of neoadjuvant endocrine therapy was based largely on experience with neoadjuvant chemotherapy, but more recent studies with longer durations of neoadjuvant chemotherapy suggest prolonging treatment duration increases the overall response rate [START_REF] Bear | A randomized trial comparing preoperative doxorubicin/cyclophosphamide (AC) to preop AC followed by docetaxel (T) and to preop AC followed by postoperative T in patients with operable carcinoma of the breast: results of NSABP B-27[END_REF]. There are a number of issues that need to be addressed with regard to neoadjuvant hormone therapy: do longer durations of treatment produce prolonged tumour shrinkage, do they increase the numbers achieving a response and do they increase the number of patients who become eligible for breast-conserving surgery? Furthermore, is letrozole alone a satisfactory long-term treatment for some patients with breast cancer? The aims of the present study were to review retrospectively the Edinburgh experience of patients treated with neoadjuvant letrozole to assess first whether patients continue to respond beyond the usual 3-4 months treatment period, and second to identify how many patients treated with long-term letrozole alone become resistant to letrozole and require a change of therapy.

Patients and methods

About 182 patients with large operable or locally advanced oestrogen receptor rich breast cancers have been treated consecutively with neoadjuvant letrozole for 3 months or longer in the Edinburgh Breast Unit. These patients have been treated since the issue of a licence for the use of letrozole in the neoadjuvant setting and do not include the series of patients presented in our early publications [START_REF] Dixon | Letrozole as primary medical therapy for locally advanced and large operable breast cancer[END_REF]. The aim of studying this series of patients was to identify factors associated both with response to therapy and longterm outcome. This study was approved by the local Ethics Committee and the Research and Development Department of the hospital. All women were postmenopausal, defined as amenorrhea for 1 year or with oestradiol, luteinising hormone and follicular stimulating hormone levels in the postmenopausal range. The mean age of the 182 patients was 76.5 years with a median of 78 and a range of 55-95 years. All had ER Allred scores between 5 and 8 with the majority having Allred scores of 8 (121) or 7 (59). All patients were treated with 2.5 mg of letrozole.

Patients were reviewed initially after 2, 6 and 12 weeks of treatment to assess tolerance and to measure response by clinical measurement with callipers and imaging (ultrasound and clinical measurements at 0, 6 and 12 weeks and mammography at 0 and 12 weeks). At 3 months all patients were reviewed by a single surgeon and a treatment decision taken. The decision was either to perform surgery, to continue letrozole, or following discussion at a multidisciplinary meeting to switch to another therapy. This decision was based on an assessment of response, operability, fitness for operation and willingness of the patient to proceed to surgery. Patients who continued letrozole beyond 3 months were reviewed at 6, 9, 12, 18 and 24 months and yearly thereafter.

Clinical and ultrasound volumes were calculated using the formula pD 3 /6 where 'D' is the mean diameter of two perpendicular measurements on clinical or mammographic assessment and the mean of two diameters and the depth measurement on ultrasound. A patient with no clinical mass palpable after 3 months was considered as having 100% reduction in clinical volume or a complete clinical response. The % change in clinical volume was calculated using the formula 100x (initial volumefinal volume) 7 initial volume. The change in volume between 3 and 6 months was calculated using the following formula: 100x (volume at 6 monthsvolume at 3 months) 7 (volume at 3 months) and other volumes were calculated similarly. Changes in volume were calculated for the first 3 months, between 3 and 6 months, between 6 and 12 months and between 12 and 24 months. These data were entered into an Excel spreadsheet and plotted using Minitab v.14. Time to treatment failure for the group taking letrozole longer than 6 months was calculated using a Kaplan-Meier estimate; the minimum treatment duration was 6 months and the maximum 5 years.

Results

The clinical response rates and outcomes at 3 months for all 182 patients are shown in Tables 1 and2. One hundred and twenty seven patients (69.8%) had a complete or partial response and only four patients had disease progression ([25% increase in tumour volume). Complete clinical responses were only seen in patients with T1 and T2 cancers and no patient with a T3 or T4 cancer had a complete clinical response at 3 months. The four patients with progression of their disease were treated with surgery, three requiring mastectomy and one being successfully treated by breast conserving surgery. Of the 119 patients who had surgery at 3 months, 99 had breast conserving surgery and 19 had mastectomy. The 19 undergoing mastectomy included 15 patients who had locally advanced breast cancer which was not initially considered suitable for surgery but these patients became operable following 3 months of letrozole. Three had progressive disease and the final patient had an invasive lobular cancer that reduced in size but still required mastectomy. All 16 responding patients who had a mastectomy were considered unlikely to ever be candidates for breast conserving surgery even if they had continued to respond.

Conversion to breast conserving surgery

One hundred and thirty-four of the 182 patients had a cancer that either required mastectomy or was locally advanced at the outset of treatment. By 3 months 81 of these 134 (60%) were suitable for and had breast conserving surgery.

Results for patients on prolonged letrozole treatment

Sixty-three patients continued on letrozole beyond 3 months for the following reasons: 26 were responding but had not responded sufficiently to allow breast-conserving surgery, 15 patients were responding but still had inoperable disease, 13 were unfit and considered unsuitable for surgery and nine refused surgery. The mean age of these 63 patients was 77.9 years (median 79) which is not significantly different from the age of the patients who had surgery at 3 months, mean age 75.7 years (median 77)comparison between two groups P = 0.09. The initial T stage of patients who continued on letrozole was significantly different from that of the initial T stage of patients who had surgery at 3 months with significantly more patients having T4 cancers in the group continuing on surgery, P = 0.021 (Table 3). Twenty per cent of patients with T1 cancers, 29% with T2, 35% with T3 cancers and 47% of patients with T4 cancers continued on letrozole for longer than 3 months. Of the 63 patients who continued letrozole beyond 3 months, the duration of letrozole in 25 was less than 1 year; the remaining 38 patients took letrozole for more than 1 year and 23 took letrozole for more than 24 months.

The mean reduction in clinical volume in the first 3 months in these 63 patients was 46.7%, with a median of 52%. There was a similar continued reduction in clinical Changes in clinical volume for individual patients who continued on letrozole for longer than 3 months are shown in Fig. 1 and summarised in Fig. 2. The changes in volume as assessed by ultrasound are shown in Fig. 3 and summarised in Fig. 4. There were four patients from 3 to 6 months where clinical or ultrasound assessment indicated that the tumour volume had increased in volume by [25%.

In three of these patients other assessments including mammography and clinical measurement suggested the lesion was either static or smaller so treatment with letrozole was continued. None of these three showed progression at later assessments. From 6 to 12 months there were three patients whose tumours increased in size on ultrasound. Clinical assessment and mammography suggested there had been no significant change in size in two and both patients were infirm and unfit for surgery, so treatment with letrozole was continued and subsequently their disease has been static. Between 12 and 24 months, three patient's tumours did increase in size, one by 41%, one by 42% and one by 85% on ultrasound. These tumours were increasing on other assessments. Two of these patients underwent surgery and the third had a change in hormonal treatment.

Best response for all 182 patients is shown in Table 4. By giving patients letrozole for longer than 3 months, the overall response rate in all 182 patients increased from 69.8% at 3 months to 83.5%. The complete response rate in the 63 patients who continued letrozole was three of 63 Of the 63 patients treated with letrozole for longer than 3 months, 21 patients eventually underwent surgery (15 breast conserving procedures and six mastectomies), two patients continued letrozole treatment but had radiotherapy for responding but locally advanced disease, seven switched to other hormonal therapies and 33 women remain on letrozole and their disease is currently controlled on this treatment alone. The mean age of this group at diagnosis was 83 years with a median of 84 years.

Prolonged treatment with neoadjuvant letrozole treatment resulted in an increase in the number of patients who were converted from requiring a mastectomy or who were inoperable to be suitable for breast conserving surgery from 81 of 134 (60%) at 3 months with 96 (72%) eventually being treated with breast conservation.

Time to treatment failure

The median period of follow up of those on prolonged letrozole was short at 3 years and at this time, the median time to treatment failure had not been reached with over 70% having their disease controlled on letrozole alone.

Discussion

In the late 1970s and early 1980s it was common practice to treat patients over the age of 70 years with tamoxifen alone and not consider them for surgery. A series of randomised trials compared tamoxifen alone versus surgery alone [START_REF] Gazet | Prospective randomized trial of tamoxifen vs surgery in elderly patients with breast cancer[END_REF][START_REF] Kenny | Long-term follow-up of elderly patients randomised to primary tamoxifen or wedge mastectomy as initial therapy for operable breast cancer[END_REF][START_REF] Fentiman | Treatment of operable breast cancer in the elderly: a randomised clinical trial EORTC 10851 comparing tamoxifen alone with modified radical mastectomy[END_REF] and a recent meta-analysis comparing surgery with primary endocrine therapy for operable breast cancer in elderly women found no difference in overall survival [START_REF] Hind | Surgery versus primary endocrine therapy for operable primary breast cancer in elderly women (70 years plus)[END_REF]. Several randomised trials compared tamoxifen alone with surgery followed by tamoxifen and in all but one of these trials there was a significant increase in local events in the tamoxifen alone group [START_REF] Mustacchi | Tamoxifen alone versus adjuvant tamoxifen for operable breast cancer of the elderly: long-term results of the phase III randomized controlled multicenter GRETA trial[END_REF][START_REF] Fennessy | on behalf of the Closed Trials Working Party of the Cancer Research UK Breast Cancer Trials Group: Late follow-up of a randomized trial of surgery plus tamoxifen versus tamoxifen alone in women aged over 70 years with operable breast cancer[END_REF][START_REF] Bates | On behalf of the CRC Breast Cancer Trials Group UK: Breast Cancer in the Elderly: Surgery improves survival. The results of a Cancer Research Campaign Trial[END_REF][START_REF] Willsher | Investigation of primary tamoxifen therapy for elderly patients with operable breast cancer[END_REF][START_REF] Capasso | Surgery and tamoxifen versus tamoxifen as treatment of stage I and II breast cancer in over 70 years old women: Ten years follow-up[END_REF]. One study also showed a significantly higher mortality in patients treated by tamoxifen alone but this increase in mortality was only apparent after 3 years of follow up [START_REF] Smith | On behalf of the IMPACT Trialists: Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicentre, double-blind randomized trial[END_REF]. A meta-analysis found a non-significant trend in favour of combining surgery with tamoxifen versus tamoxifen alone for overall survival (HR 0.86, P = 0.06) and a significant improvement in local disease control in favour of surgery and tamoxifen (HR 0.28, P \ 0.00001) [START_REF] Hind | Surgery versus primary endocrine therapy for operable primary breast cancer in elderly women (70 years plus)[END_REF]. Many of these early studies did not select patients based on oestrogen receptor status and all used tamoxifen.

With the advent of the new aromatase inhibitors which are superior to tamoxifen in the neoadjuvant setting both in terms of response rates and converting patients to breast conserving surgery [START_REF] Eiermann | Preoperative treatment of postmenopausal breast cancer patients with letrozole: a randomized double-blind multicenter study[END_REF][START_REF] Smith | On behalf of the IMPACT Trialists: Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicentre, double-blind randomized trial[END_REF][START_REF] Semiglazov | Neoadjuvant endocrine therapy: exemestane vs tamoxifen in postmenopausal ER + breast cancer patients (T1-4, N1-2, M0)[END_REF][START_REF] Ellis | Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for erbB-1 and/or erbB-2 positive, estrogen receptor positive primary breast cancer: evidence from a phase III randomised trial[END_REF] there has been a renewed interest in using hormonal therapy to treat elderly patients either prior to surgery or as a sole treatment. The only data available on the use of aromatase inhibitors are from series or from randomised neoadjuvant trials which have treated patients for between three and 4 months [START_REF] Eiermann | Preoperative treatment of postmenopausal breast cancer patients with letrozole: a randomized double-blind multicenter study[END_REF][START_REF] Smith | On behalf of the IMPACT Trialists: Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicentre, double-blind randomized trial[END_REF][START_REF] Semiglazov | Neoadjuvant endocrine therapy: exemestane vs tamoxifen in postmenopausal ER + breast cancer patients (T1-4, N1-2, M0)[END_REF]. Studies which have used longer durations of neoadjuvant chemotherapy have shown that in comparison to the standard 3 months treatment, longer durations increase the rates of complete pathological response [START_REF] Bear | A randomized trial comparing preoperative doxorubicin/cyclophosphamide (AC) to preop AC followed by docetaxel (T) and to preop AC followed by postoperative T in patients with operable carcinoma of the breast: results of NSABP B-27[END_REF]. It was our perspective that longer durations of treatment with aromatase inhibitors might have a similar effect. The current study provides the first data from a sufficiently large group of patients treated for prolonged periods with neoadjuvant letrozole to confirm that prolonged treatment does produce continued tumour shrinkage and improves response rates.

In the current report the majority of patients who continued on letrozole had continuing reductions in tumour volume for up to 2 years. This review has also demonstrated that if there was evidence of tumour shrinkage by 3 months but the tumour remained inoperable or was only suitable for treatment by mastectomy prolonging treatment allowed more tumours to become operable and to become treatable by breast conserving surgery rather than mastectomy. Continued response occurred in the majority, but not in all patients and small numbers of women whose tumours initially responded to letrozole did become resistant and started to increase in size. Based on these observations it is our current practice to advise patients to undergo surgery if the cancer having been locally advanced becomes operable or if a cancer that is best treated initially by mastectomy becomes suitable for breast conserving surgery. Importantly in this study the majority of patients had oestrogen receptor rich tumours having ER Allred scores of 7 and 8 and only occasional patients had ER scores of 5 or 6. Such selection is important because we have previously demonstrated that the highest rates of tumour shrinkage are in patients whose tumours have the highest levels of ER [START_REF] Ellis | Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for erbB-1 and/or erbB-2 positive, estrogen receptor positive primary breast cancer: evidence from a phase III randomised trial[END_REF][START_REF] Macaskill | Neoadjuvant use of endocrine therapy in breast cancer[END_REF]. While aromatase inhibitors do show activity even in tumours with low levels of ER, response rates, tumour shrinkage and reduction in proliferation are less than in tumours with high levels of ER [START_REF] Ellis | Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for erbB-1 and/or erbB-2 positive, estrogen receptor positive primary breast cancer: evidence from a phase III randomised trial[END_REF].

Thirty-three patients out of the 63 who were treated for longer than 3 months remain on letrozole alone. Some of these women have been on letrozole for 5 years. These are an elderly population, median age 84 years, and many have co-morbidities. As with tamoxifen, there is likely to be a group of women whose tumours can be controlled for prolonged periods by letrozole alone [START_REF] Horobin | Long-term follow-up of elderly patients with locoregional breast cancer treated with tamoxifen only[END_REF]. Using microarray we have identified genes at diagnosis and genes which change early during treatment which correlate with response [START_REF] Miller | Phenotypes for endocrine resistance can be indentified by RNA microarray of sequential biopsies and are more variable than those predicting for tumour response[END_REF]. This approach may provide an opportunity to identify groups of women whose disease can be controlled on this treatment alone.

Clinical assessment unless carried out by the same individual is open to interobserver error. The combination of clinical examination, ultrasound and mammography was invaluable in assessing response in the present series. In individual patients during prolonged treatment with letrozole on occasions one method of assessment indicated disease progression, whereas the two other assessments indicated that the tumour was adequately controlled. In such patients treatment with letrozole was continued and in all such patients the next series of assessments indicated that the disease remained controlled by the drug. Multimodality assessment in patients treated by endocrine therapy alone is therefore necessary.

This study has demonstrated that in carefully selected patients, neoadjuvant endocrine therapy with letrozole produces prolonged tumour shrinkage over many months and years. The optimal duration of letrozole is not clear and the decision on the duration of treatment should be individualised. Unless a patient refuses surgery or is considered to have significant morbidities which limit surgical options or there is limited life expectancy, then following a satisfactory response to neoadjuvant letrozole therapy surgery should be performed followed by postoperative radiotherapy in appropriate patients. Treatment by neoadjuvant letrozole to allow breast conserving surgery is associated with good long-term disease control rates providing postoperative radiotherapy is given [START_REF] Dixon | Surgical issues surrounding the use of aromatase inhibitors[END_REF]. In patients who are not suitable for or who refuse surgery, neoadjuvant letrozole given for prolonged periods appears safe and appears to provide this small group of women with longterm disease control.

Fig. 2 Fig. 3

 23 Fig. 2 Mean (circles) and median (squares) values with 95% confidence intervals of the mean for change in clinical volume between 0 and 3 months, 3 and 6 months, 6 and 12 months and 12 and 24 months

Fig. 4

 4 Fig. 4 Mean (circles) and median (squares) values with 95% confidence intervals of the mean for change in ultrasound volume between 0 and 3 months, 3 and 6 months, 6 and 12 months and 12 and 24 months

Table 1

 1 Tumour response after 3 months of letrozole according to WHO criteria

	Response	Number of patients	Percentage
	Complete response	21	11.5
	Partial response	106	58.3
	Static disease	51	28
	Progressive disease	4	2.2
	Response rate 69.8%

Table 2

 2 Outcomes at 3 months for all patients treated with neoadjuvant letrozole

		Neoadjuvant
		letrozole 3 months
		n-182
	Breast conserving	Mastectomy	Continued on	Disease
	surgery		letrozole	progression
	n=99	n=16	n=63	n=4
	(54%)	( 9 %)	(35%)	( 2 %)
			Mastectomy	Breast conserving
				surgery
			n=3	n=1

Table 3 T

 3 -stage of overall group and patients who underwent surgery at 3 months or continued letrozole

		Overall	Patients	Patients having
		group	continuing	surgery at
		N (%)	on letrozole	3 months
			N (%)	N (%)
	T 1	10 (5.5)	3 (4.8)	7 (5.9)
	T 2	91 (50.0)	28 (44.4)	63 (52.9)
	T 3	23 (12.6)	5 (7.9)	18 (15.1)
	T 4	58 (31.9)	27 (42.9)	31 (26.1)
	Total	182 (100)	63 (100)	119 (100)
	N represents the numbers of patients with T 1 , T 2 , T 3 and T 4 cancers in
	each of the groups. T 4 includes direct skin infiltration
	There were significantly less T 4 cancers in those who proceeded with
	surgery compared to those continuing on letrozole, P = 0.021,
	v 2 = 5.359

Table 4

 4 Best response for all 182 patients treated with 3 months or longer of neoadjuvant letrozole

		Number of patients	Percentage
	Complete response	34	18.7
	Partial response	118	64.8
	Static disease	26	14.3
	Progression	4	2.2

Breast Cancer Res Treat (2009) 113:145-151

Acknowledgements Thanks to the Dunhill Medical Trust who provided research Fellowships to Juliette Murray and Oliver Young. This work was supported in part by an unlimited educational grant from Novartis Pharma.


Vassilis Golfinopouloss

George Pentheroudakis

Smaragda Kamakari

Vasiliki Metaxa-Mariatou

Nicholas Pavlidis

A E George

Pentheroudakis Ae

A E Vasiliki

Metaxa-Mariatou Ae

Á N Pavlidis

Donor-derived breast cancer in a bone marrow transplantation recipient

Keywords:

published or not. The documents may come

We describe the case history of a female patient born in 1978 and diagnosed with acute myelocytic leukemia at the age of 14. After idarubicin/cytarabine induction chemotherapy she underwent conditioning with cyclophosphamide, alemtuzumab, and total body irradiation and received an allogeneic bone marrow graft from her HLA-matched sister. Repeated hematologic and bone-marrow examinations showed complete remission of the disease, sustained to date. In 2006, at the age of 28 years, she palpated a lump at the upper-external quadrant of the right breast. Following diagnostic work-up she underwent modified radical mastectomy and axillary node dissection. Histological examination of the specimen revealed a grade 2 adenocarcinoma of 7.5 cm maximal diameter. Eleven out of 13 resected axillary lymph nodes were involved, while estrogen/progesterone receptors and HER-2 were positive by immunohistochemistry. Staging for distant metastases was negative (AJCC stage pT3pN3M0, IIIC). The patient's sister underwent diagnostic work-up with physical examination, blood tests, chest X-ray and mammary ultrasound and was found to be healthy. Considering the history of allogeneic bone marrow transplantation, we decided to perform an appropriate DNA identity testing in order to define the genetic origin of our patient's tumor.

Genetic analysis

We compared the genetic pattern of the patient's oral epithelial cells, peripheral blood mononuclear cells and formalin-fixed paraffin-embedded neoplastic mammary cells as well as the donor's and both parents' peripheral blood mononuclear cells. For this purpose, DNA was extracted from the above samples, and an ABI AmpFlSTR commercial kit (Applied Biosystems, CA, USA) was used to simultaneously amplify 15 four-nucleotide short tandem repeat (STR) loci as well as one marker for the XY chromosomes.

The analysis of all 15 STR loci showed no major inconsistencies. As expected, the patient's peripheral blood mononuclear cell genetic pattern matched her sister's (donor). The results of the genetic analysis are presented in Table 1 and Fig. 1. All alleles were successfully traced to the paternal and maternal blood (germ-line) samples and were informative for most STR loci (10/15). Based on the inheritance pattern of the variant alleles, two distinct genetic patterns were observed in both the breast tumour and oral mucosa of the patient. These two patterns made up a chimeric population consisting predominantly from the donor graft and to a lesser extent, as estimated by the fluorescence intensity of each peak on the electropherogram, from the patient's germ-line lineage. For example, in the breast cancer sample and oral mucosa of the patient, alleles 13 and 14 for locus D5S818 represent the patient's germ-line lineage of paternal and maternal origin, respectively, whereas alleles 11 and 12 are derived from the donor graft. The presence of a chimeric population suggests repopulation of the patient's epithelia from the transplanted bone marrow-derived stem cells of her sister, as well as host epithelial stem cells.

Discussion

We showed that the breast tumor affecting the patient quite probably originated from donor-derived bone marrow stem cells. Consequently, one is led to hypothesize that donor hemopoietic stem cells repopulated the patient's mammary duct epithelium during allogeneic bone marrow transplantation. There is evidence that such epithelial repopulation is indeed possible and that bone marrow-derived stem cells can produce cancer in the host tissue given adecuate stimulation [START_REF] Jiang | Pluripotency of mesenchymal stem cells derived from adult marrow[END_REF][START_REF] Houghton | Gastric cancer originating from bone marrow-derived cells[END_REF]. A number of similar observations have given rise to speculation concerning a central role of bonemarrow-derived stem cells in the initiation of most epithelial cancers [START_REF] Li | Stem cells and cancer: evidence for bone marrow stem cells in epithelial cancers[END_REF]. In fact, most studies to date concerning the role of donor-derived bone marrow stem cells in transplant recipients have relied on identification of the sex chromosomes in sex-mismatched transplants. These studies have failed to reach consistent conclusions for the origin of tumors observed in such cases [START_REF] Au | Nonhematologic malignancies after allogeneic hematopoietic stem cell transplantation: incidence and molecular monitoring[END_REF][START_REF] Peters | Contribution of bone marrow-derived endothelial cells to human tumor vasculature[END_REF]. We relied on study of an adequate number of genetic polymorphic markers and found a chimeric, genetically mixed population in the patient's normal and neoplastic epithelia, suggesting that mucosal repopulation originated from host and donor stem cells, as expected.

Although genetic analysis was restricted to tissue sections with more than 75-90% epithelial breast cancer cells as judged by hematoxylin-eosin staining, host stromal cells probably contributed to extracted DNA. However, the prevailing tumour signature remained that of the sister. In fact, laser microdissection may have allowed us to test the possibility of epithelial reconstitution solely from donor cells. Even so, given the 75-90% percentage of tumour The numbers in cells represent the identified variant alleles for each locus and refer to complete four base pair repeat units for each allele. For example, the D8S1179 8 and 13 alleles contain 8 and 13 complete four base pair repeat units, respectively a The alleles after the dash (/) represent the patient's own genetic pattern b Loss of heterozygosity probable epithelial cells in tissue sections analysed, we feel confident that the bulk of DNA was extracted from breast cancer cells. Our observation, along with others, adds weight to the theory of derivation of normal and neoplastic epithelia from pluripotent stem cells and emphasizes the pivotal role of host stroma to malignant transformation. Neoplastic stem cells have been postulated to conserve self-renewal and repopulation properties in addition to possessing aberrant proliferation and genetic repair [START_REF] Pardal | Applying the principles of stem-cell biology to cancer[END_REF]. Normalappearing host stroma was shown to modulate malignant clone emergence, proliferation and regression in preclinical models [START_REF] Li | Beyond tumorigenesis: cancer stem cells in metastasis[END_REF]. In our case, the donor who contributed the stem cell graft is healthy, while a tumour originated from this same graft in the recipient. It should be emphasized that the donor stem cells, which subsequently gave rise to the malignant clone, had not been exposed to the deleterious effects of ionising radiation or cytotoxic agents, in contrast to the host stroma and epithelia. It is possible that conditioning chemotherapy and radiotherapy administered before transplantation inflicted genetic damage to host stromal cells. Further research is needed at a basic and translational level in order to illuminate the complex and poorly understood mechanisms of stem cell growth and epithelial-stromal interactions.

Table 1

 1 Genetic make-up of studied biological material STR locus Patient's blood Patient's saliva a Patient's tumor a Patient's sister's blood Patient's father's blood Patient's mother's blood

	D8S1179	8, 13	8, 13	8, 13	8, 13	13, 13	8, 14
	D21S11	30, 31.2	30, 31.2/29	30, LOH b	30, 31.2	29, 30	30, 31.2
	D7S820	8, 10	8, 10	8, 10	8, 10	10, 10	8, 10
	CSF1PO	10, 11	10, 11	10, 11	10, 11	11, 11	10, 12
	D3S1358	14, 18	14, 18/15	14, 18/15	14, 18	14, 16	15, 18
	TH01	6, 6	6, 6/9.3	6, 6/9.3	6, 6	6, 9.3	6, 9.3
	D13S317	8, 12	8, 12/11	8, 12/11	8, 12	11, 12	8, 9
	D16S539	9, 9	9, 9/11, 13	9, 9/11, LOH b	9, 9	9, 11	9, 13
	D2S1338	20, 24	20, 24/23	20/23	20, 24	23, 24	19, 20
	D19S433	13, 14	13, 14/13.2	13, 14/13.2	13, 14	13, 13.2	14, 15
	vWA	17, 17	17, 17/15	17, 17/15	17, 17	15, 17	15, 17
	TPOX	9, 11	9, 11	11	9, 11	8, 11	9, 11
	D18S51	13, 16	13, 16/15, 17	13, 16/17, LOH b 13, 16	15, 16	13, 17
	Amelogenin X, X	X, X	X, X	X, X	X, Y	X, X
	D5S818	11, 12	11, 12/13, 14	11, 12/13, 14	11, 12	11, 14	12,13
	FGA	20, 22	20, 22	20, 22	20, 22	19, 22	20, 24

Breast Cancer Res Treat (2009) 113:211-213


Lei

R J Damstra
email: r.damstra@nijsmellinghe.nl

Ae H G J Voesten

Ae W D Van Schelven

Ae B Van Der Lei

H G J Voesten

W D Van Schelven

B Van Der Lei

Lymphatic venous anastomosis (LVA) for treatment of secondary arm lymphedema. A prospective study of 11 LVA procedures in 10 patients with breast cancer related lymphedema and a critical review of the literature

Keywords: Lympho-venous anastomosis (LVA), Microsurgery, Evidence-based medicine, Lymphoscintigraphy, Inverse water volumetry, Review, Breast cancer related lymphedema

    Lymphatic venous anastomosis (LVA) for treatment of secondary arm lymphedema. A prospective study of 11 LVA procedures in 10 patients with breast cancer related lymphedema and a critical review of the literature

Introduction

Lymphedema is a chronic disease caused by impairment of the lymphatic transport capacity, resulting in edema, excess of tissue proteins, and in latter stages, inflammation and irreversible changes such as fibrosis and excess of adipose tissue [START_REF] Brorson | Adipose tissue dominates chronic arm lymphedema following breast cancer: an analysis using volume rendered CT images[END_REF]. Lymph transport impairment and clinical signs of lymphedema can be acquired (secondary) or congenital (primary). Treatment of lymphedema is challenging. Therapeutic approaches consist of both non-operative and operative methods. The overwhelming majority of patients can effectively be treated by non-operative means such as complex decongestive therapy (CDT) in combination with manual lymphatic drainage, bandaging, physical exercises, skin care and elastic stockings. In the long lasting maintenance phase, therapeutic elastic stockings are mandatory. The goal of all non-operative treatment modalities is to reduce capillary filtration, improve drainage of interstitial fluid and macromolecules, and therefore reduce swelling, inflammation, recurrence of erysipelas, and improve quality of life [START_REF] Mortimer | Therapy approaches for lymphedoedema[END_REF]. Operative treatment is only indicated in a few cases as a last resort.

Many reconstructive techniques have been described, such as lymphovenous anastomosis (LVA) [START_REF] O'brien | Microlymphaticovenous anastomoses for obstructive lymphedema[END_REF], lymphovenous-lymphatic (LVL) transplant (especially in the presence of venous hypertension [START_REF] Campisi | the use of vein grafts in the treatment of peripheral lymphedemas: long term results[END_REF]), and forms of lymph vessel transplantation [START_REF] Baumeister | Die autogen gefasstransplantation zur microchirurgischen Rekonstruktion des Lymphgefassysteems[END_REF]. LVA has been the most frequently used type of operation.

So far, most studies on LVA have both demonstrated a lack of significant volume reduction and considerable heterogeneity in the study populations. Therefore, we decided to evaluate the effectiveness of LVA in the treatment of one-sided breast cancer related lymphedema in a limited prospective study using objective, validated measurement methods with very strict inclusion criteria. These results will be discussed along with a review of the literature.

Patients, materials and methods

Study design and population

The study was conducted in the Lymphedema Department of Nij Smellinghe Hospital in Drachten, The Netherlands, from 1999 to 2002. A systematic review of English and German language literature retrieved from MEDLINE, COCHRANE and Cinahl databases up to April 2007 was performed. The articles were classified in levels of scientific evidence according the criteria for evidence-based medicine.

The study population consisted of 10 BCRL patients with severe lymphedema and persistent complaints, not responsive to maximal non-operative lymphedema treatment and who met the inclusion criteria for operative treatment. The patients were hospitalized in the lymphedema clinic to undergo a LVA according to our protocol.

Inclusion and exclusion criteria

In order to be included in this prospective study, patients with unilateral breast cancer related lymphedema had to meet the following criteria:

• No volume reduction after 3 months of complex decongestive treatment including manual lymph drainage, compression therapy, and physiotherapy, with persistent complaints such as heaviness, pain, shoulder function impairment, and recurrent attacks of erysipelas • Persistent volume excess of more than 800cc measured by inverse water volumetry • Proven scintigraphic signs of obstruction by absence of liver uptake and highly pathological transport index • No recurrent malignancy • Good patient compliance and willingness to wear therapeutic elastic stockings • Operability.

Data collection and lymphoscintigraphy

All patients underwent objective measurements with validated methods. The quality of life was measured preoperatively and 6 months postoperatively using the validated SF-36 quality of life assessment questionnaire [START_REF] Aaronson | Translation, validation, and norming of the Dutch language version of the SF-36 health survey in community and chronic disease populations[END_REF]. Volume measurements were performed prior to LVA and at 3, 6, 12, and 24 months after LVA. Volumetry was performed with the inverse water volumetry method (gold standard) [START_REF] Damstra | The validation of the inverse water volumetry: New gold standard for arm volume measurement[END_REF]. Furthermore, indirect circumferential measurements were taken according the Herpertz method [START_REF] Herpertz | Messung und Dokumentation von O ¨demen[END_REF] for unilateral swelling, correlating the difference between the lymphedemic limb and the normal one.

The following clinical classification [START_REF] Vignes | Quantitative evaluation and qualitative results of surgical lymphovenous anastomosis in lower limb lymphedema[END_REF]

Scintigraphic protocol

Unilateral lymphoscintigraphy was performed with focus on the liver uptake. On the anterior and posterior images, counts were determined in the liver and in the total field of view respectively. Subsequently, geometric means were calculated for the liver and the total field of view. The liver fraction was calculated. In cases of very early detection of liver uptake, the possibility exists for technical failure resulting from unintentional intravenous injection of radioactive tracer: the fast venous transport causes the tracer to show up in the liver very early. In the case of correct subcutaneous administration of the radioactive tracer, the liver can be visualized after some time through uptake via blood vessels. The radioactive tracer reaches the blood stream either via the thoracic duct or, in the case of a functioning lymphatic venous anastomosis, via this shunt. We defined any uptake at 120 and 180 minutes as sign of lymphatic drainage from the arm, which was preoperatively absent. Results were analyzed preoperatively and one year postoperatively.

In unilateral lymphoscintigraphy, 99m Tc-Nanocolloid was administered subcutaneously in the second interdigital space of the edematous arm. A dose of 80 MBq and a volume of 0.1 ml were used. Mobilization was standardized by asking the patient to make a fist repetitively during a fixed period of time. During the first 40 min following the injection, dynamic images were taken, including the entire arm, the axilla and the liver. Then images were made 60, 120, and 180 min after the injection.

The images were interpreted in three ways: visual (qualitative) interpretation, semi-quantitative interpretation using the Transport Index, and quantitative interpretation through calculation of the liver fraction after 60, 120, and 180 min.

In clinical practice, visual interpretation is the method most often used, as it is easy to perform; however, small differences between pre and postoperative studies may be difficult to recognize.

Kleinhans et al. have described the Transport Index [START_REF] Kleinhans | Evaluation of transport kinetics in lymphoscintigraphy: follow-up study in patients with transplanted lymphatic vessels[END_REF]. In this index, five parameters describe the lymph flow: lymphatic transport kinetics (K), distribution pattern (D), time lapse to appearance of lymph nodes (T in minutes, multiplied by 0.04), assessment of lymph nodes (N), and assessment of lymph vessels (V). Each parameter can be given a score from 0 to 9, where 0 means that there are no abnormalities and 9 indicates that the condition for a given parameter could not be worse. The five scores are then added, resulting in the Transport Index, which can vary from 0 (normal) to 45 (most abnormal).

Operating technique

All LVA procedures were carried out under general anesthesia. A skin incision was made in the upper arm, about 7-10 cm above the elbow. No pneumatic tourniquet was used for exsanguination. Intradermal and subcutaneous injections of Methylene Blue were used 1 to 2 cm distal to the skin incision lines to outline the lymphatic system. All operative procedures were carried out by an experienced micro-vascular surgeon (BvdL) using an operating microscope. After skin incision, careful dissection was performed to identify small-sized veins (1-3 mm in diameter). After identifying a suitable vein, and collecting lymphatics (including some lymphatic vessels), lymphatic capillaries were dissected in the surrounding area. The diameters of these lymphatic vessels were 0.3 mm or less. With micro instruments, end-to-side anastomoses were made according to the Degni-Cordeiro procedure [START_REF] Degni | New techniques of lymphatic-venous anastomosis for the treatment of lymphedema[END_REF] using 11-0 micro sutures; at least 3-4 anastomoses were made. Average completion time of the LVA procedure was about 60 min. Antibiotics were used perioperatively; the extremity was bandaged and elevated at night. Elastic stockings were continued permanently during the follow-up.

Results

A total of 11 LVA procedures in 10 female patients were included in our study in the period from 1998-2002. The mean age of the patients was 58.7 years (D 46-68). All patients had stage 3 breast cancer related lymphedema according to Campisi, and were treated with mastectomy, axillary lymph node dissection, radiotherapy, and chemotherapy. Lymphedema was present for a mean period of 5.3 years (D 3-14 years) before LVA. None of the patients had responded sufficiently to our standardized conservative treatment program over 3 months. After removal of any pitting component, no further volume reduction could be achieved.

Quality of life

After 6 months, 5 of 10 patients had subjective relief of their complaints according to the SF-36 questionnaire.

Volumetry (See also Table 1) After 3 months, there was a slight but insignificant reduction of volume. After one year this reduction diminished to almost zero (Table 1). The preoperative volume difference between both arms was 988cc.

After one year, the mean volume difference was 1075cc (D500-1856).

The Herpertz circumferential measurement demonstrated improvement of 4.8% after one year. The initial volume reduction with water displacement of 16% at three months was lost after one year, when no more than 2% volume difference was observed.

Scintigraphy (see also Table 1)

According to the Kleinhans transport index, there was no significant difference in transport before and one year after the operation: 43.0 (D 28-45) versus 42.2 (D 30-43).

Qualitative scintigraphic observation demonstrated an absence of lymph transport, dermal backflow and few signs of any organized transport, comparable with longstanding lymph-obstruction. In one patient, a lymph node in the elbow was visualized. After one year, a quantitative scintigraphical evaluation demonstrated no liver uptake after LVA, as compared with an initial lymphoscintigraphy.

Long term follow-up

Beyond the initial study design, at long term follow up in 2007 (mean follow-up 8 years), 2 patients had stable lymphedema using elastic stockings, 3 patients had died of metastasis, 4 patients had complete volume reduction after circumferential suction-assisted lipectomy (Bro ¨rson method [START_REF] Brorson | Liposuction in arm lymphedema treatment[END_REF]) and 1 patient was lost to follow up. The main characteristics and results of 10 patients are presented in Table 1.

Discussion

This prospective study clearly demonstrates that, although there was an initial period of relief of the subjective complaints in 5 of 10 patients, there were no significant improvements after LVA in our series of patients with chronic lymphedema; neither volume measurements nor scintigraphic measurements showed any effect.

When reviewing the literature of the last decades (see Table 2), there is a striking lack of prospective and comparative studies concerning the effect of LVA on chronic lymphedema. All studies reviewed initially used nonoperative treatment by CDT for a period ranging from 3 days up to 6 months. Although this might create a bias when selecting patients for LVA, the alternative is that many patients would have unnecessary operative treatment with perhaps detrimental effects. Most patients with early stages 1-3 of lymphedema can be treated successfully with CDT [START_REF] Fo ¨ldi | The lymphedema chaos: a lancet[END_REF][START_REF] Szuba | Decongestive lymphatic therapy for patients with cancer related or primary lymphedema[END_REF]. Moreover, in many studies information is missing about the follow-up protocol and many do not report whether or not elastic stockings are mandatory after an LVA procedure. In order to compare treatment options, an adequate classification is necessary. According to stages 1 to 3, conservative treatment of lymphedema is sufficient in most patients [START_REF] Fo ¨ldi | The lymphedema chaos: a lancet[END_REF][START_REF] Szuba | Decongestive lymphatic therapy for patients with cancer related or primary lymphedema[END_REF]. In stage 3 lymphedema, the pitting component can often be treated successfully; however the fibrosis and access of adipose tissue will remain. Matsubara [START_REF] Matsubara | Long term results of microscopic lymphatic vessel isolated vein anastomosis for secondary lymphedema of the lower extremities[END_REF] studied LVA in 9 women with secondary lymphedema of the legs refractionary to non-operative management. He obtained the best results when operative procedures were combined with continued postoperative physiotherapy.

In a recent personal communication, Campisi [START_REF] Campisi | Microsurgery for treatment of peripheral lymphedema: long term outcome and future perspectives[END_REF] suggested that from his point of view, LVA is most suitable in the early stages 1B, II and early III. No other postoperative therapeutic modalities such as elastic stockings, decongestive treatment or anti platelet clotting medication were mentioned. Campisi advised initial physical treatment for peripheral lymphedema. Additionally, in later stages (3, 4 and 5), in the absence of scintigraphical visualization of lymphatics, it is standard procedure to reduce the lymphedema by non-operative methods first, before considering microsurgery. According to Campisi LVA is further contra-indicated for lymphaticlymphnodal aplasia and stage 5 lymphedema.

For these stages Brorson [START_REF] Brorson | Liposuction in arm lymphedema treatment[END_REF] described a promising method (circumferential suction-assisted lipectomy) that introduces reductive surgical therapy with good long-term results, demonstrating more than 100% volume reduction. Elastic stockings must be worn to maintain these excellent results. From this perspective, we used this method successfully in 4 of our 10 patients refractionary to LVA.

From a pathophysiological perspective, the goal for LVA is to reduce interstitial volume by improving lymphatic drainage. To exemplify the results of LVA, we chose objective outcome parameters for improvement of lymphtransport capacity, volume changes and quality of life. Kleinhans et al. demonstrated the usefulness of the Transport Index to evaluate the results of lymphatic grafting [START_REF] Baumeister | Treatment of lymphedemas by microsurgical lymphatic grafting: what is proved?[END_REF][START_REF] Weiss | Post-therapeutic lymphedema: scintigraphy before and after autologous lymph vessel transplantation; 8 years of long-term follow-up[END_REF][START_REF] Vaqueiro | Lymphoscintigraphy in lymphedema: an aid to microsurgery[END_REF].

In general, lymphoscintigraphy is performed bilaterally, offering objective evidence to distinguish (late stage) lymphatic pathology from non-lymphatic causes of extremity edema. Criteria for lymphatic dysfunction include delayed ([30 min), asymmetric, or absent visualization of regional lymph nodes, and the presence of ''dermal backflow''. Additional findings include asymmetric visualization of lymphatic channels and collateral lymphatic channels. All of these parameters are correlated with a clinical diagnosis of lymphedema [START_REF] Yuan | The role of radionuclide lymphoscintigraphy in extremity lymphedema[END_REF][START_REF] Scarsbrook | Pearls and pitfalls of radionuclide imaging of the lymphatic system: part 2: evaluation of extremity lymphedema[END_REF]. Liver fraction scanning [START_REF] Ketterings | Use of the C-scan in evaluation of peripheral lymphedema[END_REF] is only of merit when the radionuclide is administered in the edematous extremity. Late uptake in the liver (after 3 h) is an indication of lymphatic transport out of the injected arm. The theoretical advantage of using the liver fraction as an objective means to judge lymphovenous shunt efficacy is clear.

In the current study, we considered any uptake as a positive result. Therefore, we postulated, with an open and well functioning shunt, in due time the radionuclide would be transported from the arm via the shunt into the veins of the arm, followed by further transport to the reticuloendothelial system, which is mainly located in the liver. A perceptible liver fraction on the postoperative images, when compared to the preoperative situation, thus should indicate an open, well functioning shunt. In this study we could not find any significant improvement in the objective parameters for volume change or liver uptake of 99m Tc-Nanocolloid. In the literature, convincing, validated, and objective methods for visualization of the effects of a LVA are missing.

Conclusions

In this first prospective study on the LVA treatment of unilateral breast cancer related lymphedema, despite the limited number of patients, no effect could be measured using objective validated measurement methods. The effects of LVA in literature are also not proven; there is a lack of standardization of the patient population, and the measurement methods are not validated. Most studies are retrospective and often involve a mix of primary, secondary arm and leg lymphedema in the same study. On top of this, classification of lymphedema varies, and the indications and criteria for LVA are unclear. Therefore we conclude that, considering our results and the literature review, LVA is not effective for the treatment of unilateral breast cancer related lymphedema. Although the LVA has been performed and studied for more than 3 decades, this method still has not had a breakthrough and will never become a treatment of choice in daily practice.

Non-operative, decongestive treatment combined with the regular use of elastic stockings is the corner stone of treatment for most patients with lymphedema, especially in its early stages when it involves few irreversible changes; however, in later stages with more irreversible changes, LVA still does not effectively reduce volume.

Table 1

 1 Summary of results

	Mean age (n = 10)	58.7 Years (D 46-68)
	Duration of lymphedema	5.4 years (D 3-14)
	Clinical classification by Campisi	Stage 3: n = 10
	Inverse water volumetry
	Total volume pre OP	4253 cc (D 2817-6456)
	Volume difference normal side pre OP	988 (D 532-1400)
	Volume difference normal side at 3 months	841 (D 232-1256)
	Volume difference normal side at 6 months	994 (D 500-1789)
	Volume difference normal side at 12 months	1075 (D 500-1856)
	Herpertz method at 0-12 months: (at four points)
	Mean Volume difference pre OP	35.2% (D 20-50%)
	Mean volume difference 12 months	33.5% (D 18-49%)
	Visual signs lymphoscintigraphy
	Dermal back flow	10
	Lymph vessels	0
	Lymphnode in the elbow	1
	Kleinhans transport index at 0-12 months
	TI pre OP	43.0 (D 28-45)
	TI post OP	42.2 (D 30-43)
	Liver uptake at 0-12 months
	LC pre OP (after 120 and 180 min)	0
	LC post OP (after 120 and 180 min)	0
	SF 36 questionnaire 0-6 months	Slight subjective improvement in 5 patients who felt less disabled

Table 2

 2

		Results		Reduction = 126	St 1: 0	St 2: &15% +	St 3: *32% +	St 3: *35%	St 4/5: *30%	[75% (n = 616)	50-75% (n = 202)	\25% (n = 25)	No scintigraphic change	TI: 2% *	Circumference: 4.8% + 1	year	Volume: 16% + 3 month/	9% + after 1 year
		Addional Follow up	treatment (month)	NM N = 126	([15 years)	Total group:	N = 843								Elastic stockings 8 years
		N		St 1: n = 25	St 2: n = 346	St 3: n = 413	= 59 St 4/5: n								10
		Lymphoscintigraphy Intervention		Yes LVA arms and legs	(arms: n = 231)	194 SL	37 PL	(legs: n = 612) 323	SL	289 PL			Yes + index (TI) LVA arms in	stadium 3
		Method of volume measurement		Volumetry	Clinical signs	Conservative treatment first	Staging according to Campisi								Inverse volumetry	Circumference according to herpertz	SF-36	Staging according to Campisi
	continued	Study		Retro	PL/SL	A/L	CTF(6)								Pro	SLA	CTF(3)
		First	author	Campisi	[21]										Damstra

NM = Not mentioned; PL = Primary lymphedema; SL = Secondary lymphedema; Pro = Prospective study; Retro = Retrospective study; A = Arm lymphedema; L = Leg lymphedema; CTF = Conservative treatment first considered unsuccessful (months)

Breast Cancer ResTreat (2009) 113:199-206

Acknowledgements Contributions R.J. Damstra was the lead author in the overall conceptualization and design of the study and the manuscript. He collected the data and prepared the databases. H.G.J Voesten participated in revising and editing the text. W.D. van Schelven participated in the design and the execution of the (semi) quantitative scintigraphic studies, as well as the analysis and the interpretation of the scans. He had the primary responsibility for the scintigraphic paragraphs. B. van de Lei performed all microsurgical procedures, participated in drafting the text and provided overall supervision of the manuscript.


Ana Osorio
email: aosorio@cnio.es

Alicia Barroso

Maria J García

Beatriz Martínez-Delgado

Miguel Urioste

Javier Benítez

Ana Osorio Ae

Alicia Barroso Ae

Maria J Garcı ´a Ae

Beatriz Martı ´nez-Delgado

A E Miguel

Urioste Ae

Javier Benı ´tez

Á A Barroso

Á M J Garcı

Á B Martı ´nez-Delgado

Á M Urioste

Á J Benı ´tez

Evaluation of the interacting genes and in familial breast cancer susceptibility

Keywords: Hereditary breast cancer, RAP80, CCDC98, BRCA1

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

Introduction

Only around 25% of the inherited susceptibility to breast cancer is explained by germline mutations in the known high penetrance susceptibility genes BRCA1 and BRCA2 [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] Osorio | Molecular analysis of the BRCA1 and BRCA2 genes in 32 breast and/or ovarian cancer Spanish families[END_REF][START_REF] Antoniou | Evidence for further breast cancer susceptibility genes in addition to BRCA1 and BRCA2 in a population-based study[END_REF]. Mutations in genes such as TP53 and PTEN have been linked with high risk for breast cancer within specific cancer syndromes [START_REF] Borresen | Screening for germ line TP53 mutations in breast cancer patients[END_REF][START_REF] Lynch | Inherited mutations in PTEN that are associated with breast cancer, cowden disease, and juvenile polyposis[END_REF] and germline variants in other genes such as CHEK2 [START_REF] Meijers-Heijboer | Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations[END_REF] and ATM [START_REF] Renwick | ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles[END_REF] and more recently the Fanconi Anemia genes BRIP1/FANCJ and PALB2/ FANCN [START_REF] Seal | Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles[END_REF][START_REF] Rahman | PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene[END_REF] have been found to confer modest risk to breast cancer. However alterations in these additional genes account for less than 5% of the hereditary cases and are sometimes restricted to specific populations. Despite most of the familial aggregation remains unexplained, it is worth to note that all the genes linked so far with breast cancer predisposition are functionally related and converge in a common pathway involved in the preservation of genomic integrity [START_REF] Walsh | Ten genes for inherited breast cancer[END_REF]. Therefore, analysis of other members of this pathway seems a good rationale to identify new susceptibility genes. Particularly good candidates are those known to have a direct interaction with the BRCA genes, as it has been the case of the last two susceptibility genes identified BRIP1/FANCJ and PALB2/FANCN that interact with BRCA1 and BRCA2 respectively.

The RAP80 (also known as UIMC Ubiquitin-Interacting Motif-Containing) and CCDC98 (or Abraxas) genes encode for proteins that have been very recently identified as BRCA1-interactors and seem to be necessary for its role in DNA damage response [START_REF] Sobhian | RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites[END_REF][START_REF] Wang | Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response[END_REF][START_REF] Yan | The Ubiquitin-Interacting Motif-Containing Protein RAP80 Interacts with BRCA1 and Functions in DNA Damage Repair Response[END_REF][START_REF] Kim | CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response[END_REF][START_REF] Liu | CCDC98 targets BRCA1 to DNA damage sites[END_REF]. Upon DNA damage, BRCA1 undergoes hyperphosphorylation by the sensors ATM, ATR and Chk2 [START_REF] Chen | Ataxia telangiectasia-related protein is involved in the phosphorylation of BRCA1 following deoxyribonucleic acid damage[END_REF][START_REF] Cortez | Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks[END_REF][START_REF] Tibbetts | Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress[END_REF][START_REF] Zhang | Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair[END_REF]. BRCA1 forms nuclear foci that co-localise with H2AX, a marker of DNA damage sites, and RAD51, implicating BRCA1 in homologous recombination (HR) during DNA double strand break repair and meiosis [START_REF] Paull | A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage[END_REF][START_REF] Scully | Association of BRCA1 with Rad51 in mitotic and meiotic cells[END_REF]. RAP80 and CCDC98 act upstream of BRCA1, being crucial for the formation of BRCA1 foci in response to ionizing radiation [START_REF] Sobhian | RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites[END_REF][START_REF] Wang | Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response[END_REF][START_REF] Yan | The Ubiquitin-Interacting Motif-Containing Protein RAP80 Interacts with BRCA1 and Functions in DNA Damage Repair Response[END_REF][START_REF] Kim | CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response[END_REF][START_REF] Liu | CCDC98 targets BRCA1 to DNA damage sites[END_REF][START_REF] Kim | Ubiquitin-binding protein RAP80 mediates BRCA1-dependent DNA damage response[END_REF]. A model has been proposed in which RAP80 binds to the N terminus of CCDC98, forming a stable complex that localises to damage sites depending on the UIM domain (Ubiquitin Interacting Motif) of RAP80; BRCA1 is then recruited by CCDC98 through the interaction of its C-terminal phosphorylation motif with the BRCA1 BRCT domain [START_REF] Kim | CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response[END_REF]. Abolishing the expression of each of these three genes with short interfering RNAs (siRNAs), have demonstrated that RAP80 is necessary for the formation of both CCDC98 and BRCA1 foci, while CCDC98 is required for formation of only BRCA1 foci [START_REF] Kim | CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response[END_REF].

Given that these two proteins are required for the correct function of BRCA1, the absence of any of them could lead to a similar phenotype as the caused by the inactivation of BRCA1 itself and places RAP80 and CCDC98 as excellent candidate familial breast cancer susceptibility genes. In order to investigate their role in breast cancer, in the present study we have performed for the first time a molecular analysis of the RAP80 and CCDC98 genes in a series of 168 non-BRCA1/2 breast cancer families.

Material and methods

Non-BRCA1/2 families

Index cases from 168 breast cancer high-risk families recruited through the CNIO were included in the study, most of them previously reported [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] Osorio | Molecular analysis of the BRCA1 and BRCA2 genes in 32 breast and/or ovarian cancer Spanish families[END_REF]. Briefly, families contained at least three females affected with breast cancer or at least two first-degree females affected with breast cancer (at least one of them diagnosed before 50) or at least one case of female breast cancer and at least one case of either ovarian, female bilateral breast or male breast cancer. DNA extracted from peripheral blood of the 168 cases, had been fully screened for mutations in BRCA1 and BRCA2 by a combination of DHPLC and direct sequencing and found to be negative.

Controls

DNA samples from 855 women aged between 25 and 65 years and without personal or familial antecedents of any cancer were analyzed to establish the frequency in the general population of some of the variants found in RAP80 and CCDC98 genes. Molecular analysis of RAP80 and CCDC98 genes DNA from the 168 index cases was subjected to Whole Genome Amplification by using the GenomiPhiV2 Amplification Kit (GE Healthcare UK Limited, Buckinghamshire, UK) according to manufacturer's instructions. Primers were designed to amplify the whole coding sequence, exon-intron boundaries and part of the 5 0 and 3 0 untranslated regions (UTR) of RAP80 and CCDC98, using the program Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). 29 primer pairs were used to cover the 14 coding exons of RAP80 (Gen-bank NM_016290) and 9 coding exons of CCDC98 (Gen-bank NM_139076) (Supplementary Table 1). PCR-products were analysed by DHPLC on the WAVE HT system (Transgenomic, Omaha, NE) using an acetonitrile gradient and scrutinised for aberrant profiles with the Navigator TM software (Transgenomic, Omaha, NE). Samples representative of each DHPLC pattern were bidirectionally sequenced using the Big Dye Terminator Cycle sequencing kit and a 3730 automated sequencer (ABI Perkin Elmer). All non-described variants were confirmed by sequencing a fresh aliquot of the non-WGA stock DNA. Those missense variants found in either gene that were not reported in the http://www.ncbi.nlm.nih.gov/SNP/, were analysed by DHPLC in the 855 control samples to investigate whether they were common polymorphisms and establish their frequency in our population.

Immunohistochemistry for DNA repair proteins detection.

Five millilitres of peripheral blood was collected from three females (two carriers and one non-carrier) of the family harbouring the variant p.Met299Ile in CCDC98. Lymphocytes were extracted using Histopaque 1077 (Sigma Aldrich). After centrifugation at 1,500 rpm for 30 min, lymphocytes were collected and grown for 24-48 h in RPMI 1640 medium and stimulated with 5 lg/ml phytohaemagglutinin PHA (Invitrogen). Cells were then non-irradiated or irradiated with 10 Gy incubated for 2 h after irradiation and spun down onto microscope slides. Cells were fixed with 2% paraformaldehyde in PBS 0.5% Triton X-100 for 15 min. To visualize the DNA-damage induced focus formation, staining was done incubating 10 min with primary antibodies anti phospho-H2AX (Upstate, clone JBW301) and CCDC98 (kindly provided by Dr J Chen). After incubation with the corresponding secondary antibody, lymphocyte nucleuses were stained with 0.1 lg/ml DAPI (Invitrogen) for 2 min at room temperature.

Results

Screening of the RAP80 gene

The whole coding sequence and exon-intron boundaries of the RAP80 gene were analysed in the index cases of the 168 non-BRCA1/2 families. During the screening we identified 10 different variants (Table 1), half of which had been already described in the NCBI SNP database (http:// www.ncbi.nlm.nih.gov/SNP/) and were considered as common polymorphisms. We found five not previously described variants, four of which, 5 0 UTR 160A [ G, c.1677-39C [ T, c.1913-86A [ G and p. Lys562Lys were considered as non-pathogenic, as the first one was located in the 5 0 UTR, two were located in introns not affecting to the consensus acceptor splice site and the last one in exon 12 represented a synonymous change. The fifth not previously described variant, p.Tyr564His located in exon 12, was the only one that could be suspected to have a pathogenic effect, given that represented a non-synonymous change, located in the second zinc finger-like motif of the protein, in a position that is highly conserved across different species (http:// www.ncbi.nlm.nih.gov/blast/). To further investigate its potential role in breast cancer susceptibility, we estimated the frequency of Tyr564His in the general Spanish population, performing a screening by DHPLC in 855 controls. None of the control samples harboured the variant, confirming that it was not a common polymorphism. The variant was found in a woman affected with ovarian cancer at 39 years of age (Fig 1a, III-1), who had one maternal aunt affected with colon and breast cancer at the ages of 51 and 55 respectively and another maternal aunt diagnosed with bilateral breast cancer at the ages of 45 and 55 (Fig. 1a). We could analyse the DNA sample from the woman affected with colon and breast cancer (Fig 1a, II-1) but she did not carry the Tyr564His variant.

Screening of the CCDC98 gene

As for RAP80, the whole coding sequence and exon-intron boundaries of the CCDC98 gene were analysed in the index cases of the 168 non-BRCA1/2 families. During the screening we identified 6 different variants (Table 2), those described in the NCBI SNP database (http://www. ncbi.nlm.nih.gov/SNP/) being considered as polymorphisms. We found four not previously described variants, three of which, p.Thr141Ile, c.542-30G [ A and 3 0 UTR 1696T [ C were considered as non pathogenic, the first one because despite representing a non-synonymous change was found with a frequency of heterocigotes of 0.02 in a control sample of 855 individuals and the other two because they were located in intron 5 (not affecting to the consensus acceptor splice site) and 3 0 UTR respectively. The forth not previously described variant, p.Met299Ile located in exon 9, was the only one that could be suspected to have a pathogenic effect, given that represented a nonsynonymous change and was found only in one of the 168 index cases and in none of the 855 controls. The affected amino-acid was not within the N-terminal or C-terminal regions known to be necessary for the interaction of CCDC98 with RAP80 and BRCA1 respectively, however the position is highly conserved across different species (http://www.ncbi.nlm.nih.gov/blast/). The variant was found in a woman affected with invasive breast cancer at 36 years of age (Fig 1b, III-1), who had two sisters affected with ductal carcinoma in situ (DCIS) and invasive breast cancer respectively both at the age of 50 (Fig. 1b, III-5 and All the variants already reported in the NCBI database were considered as not pathogenic, as well as those located in introns and not affecting the consensus acceptor or donnor splice sites or those that resulted in a synonymous change III-6). Blood samples from the two affected sisters could be obtained and their DNA analysed for the presence of the Met299Ile alteration, finding that only the one affected with invasive breast cancer carried the variant while the one affected with DCIS did not (Fig 1b, III-5 and III-6). In this case, given the availability of material from the three affected sisters, inmunohistochemistry experiments were performed in order to investigate if there were any difference regarding the presence of CCDC98 in the DNAdamage induced focus upon DNA damage between the women carrying the variant and the one that did not (see Material and Methods). CCDC98 localized normally to nuclear foci lymphocytes in the three patients analyzed.

Discussion

Genes encoding for proteins that interact directly with BRCA1 and/or BRCA2 or participate in the DNA damage response are good candidates to represent additional breast cancer susceptibility genes. Several of these genes have already been investigated and a few of them such as CHEK2, ATM, FANCJ/BRIP1 and FANCN/PALB2 (the last two directly interacting with BRCA1 and BRCA2 respectively) have been found to confer modest risk to the disease [START_REF] Meijers-Heijboer | Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations[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] Rahman | PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene[END_REF]. The Ubiquitin-Interacting Motif-Containing Protein RAP80 and the coiled-coil domain-containing protein CCDC98 are two very recently identified BRCA1 associated proteins and seem to be essential for the formation of BRCA1 foci upon DNA damage [START_REF] Sobhian | RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites[END_REF][START_REF] Wang | Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response[END_REF][START_REF] Yan | The Ubiquitin-Interacting Motif-Containing Protein RAP80 Interacts with BRCA1 and Functions in DNA Damage Repair Response[END_REF][START_REF] Kim | CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response[END_REF][START_REF] Liu | CCDC98 targets BRCA1 to DNA damage sites[END_REF]. The fact that these two genes act upstream BRCA1 and are necessary for its recruitment to DNA damage sites [START_REF] Liu | CCDC98 targets BRCA1 to DNA damage sites[END_REF], make them specially good candidates as breast cancer susceptibility genes, given that the absence of any of them could lead to a phenotype similar to that caused by the absence of BRCA1.

In the present study we have investigated for the first time, the role of RAP80 and CCDC98 genes in breast cancer susceptibility by analysing their entire coding sequence in a series of 168 non-BRCA1/2 breast/ovarian cancer families. In total we have identified 10 and 6 different variants in RAP80 and CCDC98 respectively, none of them giving rise to stop codons. Around half of the variants (5 in RAP80 and 2 in CCDC98) had been already reported in the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/) and were considered as common polymorphisms (Tables 1 and2). From the not previously reported variants, those located in introns (not involving consensus splice sites), UTRs or representing silent changes were considered as not likely to be pathogenic (Tables 1 and2). Three missense not previously reported alterations were found, Tyr564His in RAP80, and Thr141Ile and Met299Ile in CCDC98. From these, Thr141Ile in CCDC98 was found with a frequency of 0.02 of heterocigotes in a control sample of 855 individuals and therefore considered as a common polymorphism.

The amino-acid changes Tyr564His in RAP80 and Met299Ile in CCDC98 were the only variants further investigated as putatively involved in breast cancer susceptibility, as they were found each in one of the 168 index cases and in none of the 855 controls and could potentially affect the functionallity of the respective proteins. Tyr564His in RAP80 is located outside the recently identified Abraxasinteracting domain or the UIM motifs, neccesary for the localization of RAP80-CCDC98 complex in the DNA damage induced nuclear foci [START_REF] Kim | CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response[END_REF][START_REF] Wang | Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage[END_REF]. Met299Ile in CCDC98 is outside the N-terminal or C-terminal regions known to be necessary for its interaction with RAP80 and BRCA1 respectively [START_REF] Wang | Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response[END_REF][START_REF] Kim | CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response[END_REF][START_REF] Wang | Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage[END_REF]; however, just by their position, we could not rule out that these changes could have any effect on the functionality of the proteins, especially taking in account that it is thought that multiple regions in both proteins are required for the interaction between RAP80 and CCDC98 [START_REF] Liu | CCDC98 targets BRCA1 to DNA damage sites[END_REF]. Actually, both missense variants were located in highly conserved positions and in the case of Tyr564His in RAP80, the change was located in the second zinc finger-like motif of the protein, whose exact function is not known, but is a type of motif usually involved in DNA recognition and protein-protein interactions. Analysis of the families in which the two variants were identified was more revealing, as none of them were found to show a complete segregation with the disease. In the case of Tyr564His in RAP80, only one of the two affected relatives of the index case, could be analyzed for the presence of the variant and was found not to carry it (Fig. 1a), which suggested that at least it could be ruled out as a high penetrance allele. For the p.Met299Ile in CCDC98, the analysis was more complete, given that blood samples from the two affected sisters of the index case could be obtained and their DNA analysed for the presence of the Met299Ile alteration. Only one of the sisters was found to carry the variant while the other did not (Fig. 1b). Although again the segregation was not total, from the three affected sisters (considering the index case), the only non carrier was the one affected with DCIS. In the case of BRCA1/2 families, there is controversy about the consideration of DCIS as a criterion for BRCA risk assessments, given that a diagnosis of DCIS seem not be as suggestive for the presence of a underlying BRCA mutation than a diagnosis of IBC [START_REF] Hwang | Ductal carcinoma in situ in BRCA mutation carriers[END_REF][START_REF] Smith | BRCA mutations in women with ductal carcinoma in situ[END_REF]. The same could be applicable to the case of a potential new susceptibility gene, which made in our case, the segregation analysis of the variant in CCDC98 difficult to interpret. Further investigation of the variant was performed by analysing the nuclear CCDC98 foci formation in lymphocytes from the three affected sisters. Inmunohistochemistry analysis revealed that, upon DNA damage prompted by ionizing radiation, both wild-type and Met299Ile harbouring CCDC98 localized normally in the nuclear foci, suggesting that this variant is not affecting to the correct localization of CCDC98 in the DNA breaks sites. Therefore segregation and inmunohistochemistry analyses both suggested that the Met299Ile variant in CCDC98 is not a high susceptibility allele either. In summary, this first screening of the RAP80 and CCDC98 genes in 168 index cases from non-BRCA1/2 families, suggests that they can be ruled out at least as high penetrance breast cancer genes. However, we can not exclude the possibility that allelic variants in these two genes could be associated with a low or modest risk to the disease similar to the found for other genes [START_REF] Meijers-Heijboer | Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations[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] Rahman | PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene[END_REF].

Fig. 1

 1 Fig. 1 (a) Summarized family history of the index case (indicated by an arrow) harbouring the Tyr564His variant in RAP80. Coloured-in circles represent individuals with breast or ovarian cancer, individuals with other cancers are indicated by circles with diagonal lines and type of cancer indicated. The age at diagnosis is indicated under each individual. (b) Family harbouring the variant Met299Ile in CCDC98

Table 1

 1 Genetic variants identified in the RAP80 gene Mutation nomenclature listed uses GenBank reference sequences NM_016290.3 and NP_057374 .3 with numbering starting at the A of the first ATG, following the journal guidelines (www.hgvs.org/mutnomen) except for the variants located in the UTR for which the numbering starts at the first nucleotide of the mRNA sequence b The rs number is indicated for those SNPs previously reported in the NCBI database http://www.ncbi.nlm.nih.gov/SNP/. These variants were considered as common polymorphisms or at least not pathogenic variants c Frequency based on the number of heterocigotes found within the 168 cases analysed. Frequencies in bold are those established by analyzing the corresponding variant in the 855 control individuals d

	Nucleotide change a	Protein change	rs number b	Frequency of heterocigotes c	Pathogenicity d
	5 0 UTR 160A [ G	-	-	0.02	No
	c.43 C [ T	p.Trp15Arg	rs13167812	0.01	No
	c.228T [ C	p.Ile76Ile	rs1700490	0.472	No
	c.1677-39C [ T	-	-	-	N o
	c.1344A [ C	p.Thr448Thr	rs365132	0.463	No
	c.1686G [ A	p. Lys562Lys	-	-	No
	c.1690T [ C	p. Tyr564His	-	\0.001	Unknown
	c.1849-17A [ G	-	rs 2292255	0.442	No
	c.1913-86A [ G	-	-	0.05	No
	c.1913-78A [ G	-	rs2292256	0.442	No

a

Table 2

 2 Genetic variants identified in the CCDC98 gene Mutation nomenclature listed uses GenBank reference sequences NM_139076 and NP_620775 with numbering starting at the A of the first ATG, following the journal guidelines (www.hgvs.org/mutnomen) except for the variants located in the UTR for which the numbering starts at the first nucleotide of the mRNA sequence b The rs number is indicated for those SNPs previously reported in the NCBI database http://www.ncbi.nlm.nih.gov/SNP/. These variants were considered as common polymorphisms or at least not pathogenic variants c Frequency based on the number of heterocigotes found within the 168 cases analysed. Frequencies in bold are those established by analyzing the corresponding variant in the 855 control individuals d All the variants already reported in the NCBI database were considered as not pathogenic, as well as those located in UTRs or introns (not affecting the consensus acceptor or donnor splice sites), those that resulted in a synonymous change or those non-synonymous changes found to have a frequency of heterocigotes [1% in the control population

	Nucleotide change a	Protein change	rs number b	Frequency of heterocigotes c	Pathogenicity d
	c.422C [ T	p.Thr141Ile	-	0.02	No
	c.542-30G [ A	-	-	0.01	No
	c.897G [ A	p.Met299Ile	-	\0.001	Unknown
	c.1042G [ A	p.Ala348Thr	rs12642536	0.431	No
	c.1117G [ A	p.Asp373Asn	rs13125836	0.115	No
	3 0 UTR 1696T [ C	-	-	0.01	No

a

Acknowledgements We thank V. Ferna ´ndez, R. Alonso and F. Ferna ´ndez (Sapnish National Cancer Centre) for excellent technical assistance and L. Robles (Doce de Octubre Hospital, Madrid), P. Zamora (La Paz Hospital, Madrid) and C. San Roma ´n (Ramo ´n y Cajal Hospital, Madrid) for their contributions in the ascertainment of part of the families included in the study. This study was partially supported by the Fancogene project (Fundacio ´n Genoma Espan ˜a) and the Spanish Health Ministry FIS 04/2240, and FIS PI 070378.