Petrova
email: gpetrova@pharmfac.net

Manoela Mrs

M.Sc Manova

M.Sc.; Manoela Manova
email: manoela_manova@yahoo.com

Ph. D.; Assena Stoimenova
email: assena_stoimenova@yahoo.com

Prof. D. Sc.; Fabrice Clerfeuille
email: fabrice.clerfeuille@univ-nantes.fr

Prof. D. Sc Guenka I Petrova

Manova M Manoela

Assistant Professor M Pharm

IMPACT OF THE GENERIC COMPETITION ON MARKET SHARE AND PRICES OF CARDIOVASCULAR MEDICINES ON THE BULGARIAN PHARMACEUTICAL MARKET

Keywords:

Impact of generic competition on market share and prices of cardiovascular medicines on the Bulgarian pharmaceutical market

Introduction

Line 9: Please correct ".such as Angiotensin-Converting Enzyme (ACE inhibitors)." to "such as inhibitors of Angiotensin-Converting Enzyme (ACE inhibitors).".-it is done Line 13: The point is missing. -it is done Line 27: Please remove the word "new" from sentence "To give an example, a new molecule, the ramipril,." because ramipril is an already known ACE inhibitor. -it is done Conceptual framework and Literature review Line 6: Please give the bibliographical reference for "... and from some of them more than a thousand generic products have originated." . It is changed to: "lots of generic products have originated" (reference added Rossy (Ed) Line 23: Please correct ".influencing the generic medicines market." to ".influencing the generic medicine market.". It is changed Main objective Line 33: Considering grammar you should write "The aim of this study was to analyse.". Agree and corrected Methods Line 14: Please delete "Appendix 1" because the statement of this sentence is not presented in this appendix. Done Please take the same spelling for the word "mg" in the whole article. Corrected Line 45: Please delete "Furthermore" because two following sentences have the same beginning. Done Line 2: These two sentences are inconsistent: "In the group of sartans generic competitors were available only for losartan." (line 2, page 10), "Within the group of sartans, valsartan and losartan have expired patents and generics available on the market"(line 17, page 8). Please check this. Checked and removed Line 2: "In the group of sartans generic competitors were available only for losartan.": Please write "In the group of sartans generic competitors were only available for losartan.". Changed Results Line 12: Please correct "ACE Inhibitors" to "ACE inhibitors" and take the same spelling in the whole article. Done Line 14: Please write ".and the generics for ramipril.". Done Line 16: These two sentences are inconsistent: "In the therapeutic class of statins we also found 4 INNs with generics: the simvastatin, pravastatin, atorvastatin and the lovastatin." (line 16, page 10), "The statins therapeutic group is composed of 6 molecules from which 3 have expired patent." (line 24, page 8). Please check this. Done Analysis of the market share of products Line 26: Considering grammar you should write ".when we begun our study." instead of ".when we begin our study.". Corrected Line 1: Please correct ".after the introduction of the generic competitor to lisinopril in 2006" to ".after the introduction of the generic competitors to lisinopril in 2006". Corrected Line 46: Please write ". in market share of the belonging INNs". Corrected Line 52: Please take the same spelling for the word "National health insurance fund". Table 4 and Appendix 1 Corrected Considering the word "BGL" you should write "BGN" because the ISO 4217 currency code for the new Bulgarian lev is BGN Corrected.

Reviewer #2: The major limitation of the study is still that the ATC/DDD system was not used. http://www.whocc.no/filearchive/publications/drug_utilization_research.pdf Dear Mr. Reviewer, As we answer in our previous correspondence the calculations using the DDD methodology are already in a process of analysis but some of the authors included in this manuscript are not participating within the other calculations and analysis, as well as some new colleagues have been included. We consider that it is not ethical to change now the teams during the evaluation process.

We know the WHO methodology and it is really the gold standard, but we also found lots of articles using the approach of measuring the utilization in units of active ingredients, and especially in milligrams. The comments for the reasoning of such an approach are added in the discussion section, as follows also and below:

Comparison of the market share and prices in milligrams has its own reasoning for international market tender purposes (Al-Abbadi 2009; [START_REF] Karam | World Health Organization -Health Action International Project on Medicine Prices[END_REF][START_REF] Zaheer | Evaluating Drug Prices, Availability, Affordability, and Price Components: Implications for Access to Drugs in Malaysia[END_REF], as well as for reference prices comparison. Such a price comparison per milligram of active substance is also regularly performed by the Management science for health and World health organisation when they publish the International Drug Price Indicator Guide and all prices are shown per milligrams of active substance [START_REF] Frye | International drug price indicator guide[END_REF]. Some countries are using the generics' price per milligrams in the process of establishment of the lowest price per product within the reimbursement system [START_REF] Mrazek | Comparative approaches to pharmaceutical price regulation in the European Union[END_REF]. Other authors consider [START_REF] Who | Introduction to drug utilization research[END_REF] that, if medicines utilization is given in terms of grams of active ingredients, drugs with low potency will account for a larger fraction of the total than drugs with high potency, and that the combined products may also change their market figure. In our study we did not observe any combination so our figures could not be affected by such an utilization data. We also did not observe significant changes in the prices per milligram for the medicines with different potency, thus this is not also the case for consideration. The international prices comparison per milligrams of active substance could be used by the regulatory authorities. [START_REF] Grootendorst | Impact of reference-based pricing of nitrates on the use and costs of anti-anginal drugs[END_REF] analysed the impact of referenced -based pricing on nitrates usage and cost reduction by using also the cost per milligram. Similar approach was found useful in case of comparison of the medicines prices for particular therapeutic groups [START_REF] Wang | The Impact of Cost Sharing on Antidepressant Use Among Older Adults in British Columbia[END_REF] , which is also the case in our study.

Introduction

During the last three decades a tremendous progress has been made in the discovery and development of a lot of new molecules in many pharmaceutical areas. The therapeutic groups of cardiovascular medicines, such as inhibitors of Angiotensin-Converting Enzyme (ACE inhibitors), sartans and statins, are good examples of the aforementioned development and creation of therapeutic families [START_REF] Fyhrquist | Renin-angiotensin system revisited[END_REF][START_REF] Raizada | ACE 2: a new target for cardiovascular disease therapeutics[END_REF].

These families of real innovative treatment comprise of "originator" molecules (first molecule released, which can be under patent or with expired patent), as well "generic" version of those originator molecules, whose patent has expired. Usually the patent expires 15 to 20 years after the creation of the originator molecule. These last products (what we call "generics") have the same chemical structure and have been proven to be essentially similar in bioavailability [START_REF] Morgan | Breakthrough "drugs and growth in expenditure on prescription drugs in Canada[END_REF]).

To give an example, a molecule, the ramipril, in the family of ACE inhibitors has been launched by the pharmaceutical laboratory SANOFI AVENTIS under the name of Triatec  .

When the patent of this product has expired, a competitor, the pharmaceutical laboratory QUALIMED launched the same product in terms of molecule and effects, so, a generic, called Ramipril Qualimed  . However, within the same therapeutic group, MSD had already launched enalapril under the name of Renitec  that is a "therapeutic competitor", possessing small variation in the chemical structure. Following the same approach of modification of the first molecule the perindopril, trandolapril, fosinopril and so on were later developed and thus the whole therapeutic family started to expand.

Conceptual framework and Literature review

The generics create competition and drive medicine prices down [START_REF] Lee | Me-too" products --friend or foe?[END_REF]). Many studies show in fact that when a new medicinal product (a new molecule giving an originator or the same molecule giving a generic) appears on the pharmaceutical market, it affects the market patterns as well as prices of the existing generic and/or therapeutic competitors [START_REF] Dirach | New safe medicines faster: A new concept for drug development[END_REF].

To what extent and for how long time it happens depends on the specific characteristics of the national market, the laws concerning the generics and the pharmaceutical companies' policy.

Blinded Manuscript Click here to view linked References

A common perception is that the original medicinal product ensures new therapeutic options for severe diseases, while the generic medicines support the sustainability of healthcare provision and contribute to the maintaining of control over the pharmaceutical expenditure (Fiona 1997). Some countries use laws to promote the prescription and the delivery of generics, which leads to a decrease in the health expenditures. Studies focusing on the market policy of the pharmaceutical manufacturers consider that every new product, within the same therapeutic class, is characterised by simultaneous entry of competitors rather than sequential (Fiona 1997). Basically, what this means is that the expiration of a product patent is accompanied by the simultaneous entry, sometimes the same day, of a different generic. Models have been created to calculate the aggregated demand and supply features, influencing the generic medicine market [START_REF] Gascon | On macroeconomic characteristics of pharmaceutical generics and the potential for manufacturing and consumption under fuzzy conditions[END_REF].

During the last two decades a reference pricing approach was developed, which began to be used in a lot of European countries [START_REF] Huttin | Experiences with reference pricing[END_REF]. The reference prices are established after an international or national comparison of prices of the generic or therapeutic alternatives [START_REF] Mestre -Ferrandiz | Reference prices and generic medicines: What can we expect?[END_REF]. The reference price could be either the lowest or the average price within the compared group of products [START_REF] Mrazek | Comparative approaches to pharmaceutical price regulation in the European Union[END_REF]. This policy stimulates research in the area of price comparison and analysis of the factors influencing price changes, as are the generics' policy and competition.

The cardiovascular medicines are of a particular interest for studying the competition between originators and generics. They are one of the leading therapeutic classes on the global medicine market today, with more or less 10% of the market sales in value [START_REF] Who | Introduction to drug utilization research[END_REF]. For this particular reason, this class presents a big interest for the pharmaceutical manufacturers and we can currently find new innovative products and new generics. This interest is also shared by the health authorities, which want to control the increasing expenditures, analysing their prescription and their usage and creating national rules for price control.

In this cardiovascular category of treatment, three groups of therapeutics present a bigger interest due to the presence of innovative molecules and the arrival of generics: the families of inhibitors of Angiotensin-Converting Enzyme (ACE inhibitors), sartans and statins (drugs.com 2008;[START_REF] Luno | The reno-protective effect of the dual blockade of the renin angiotensin system (RAS)[END_REF]Rossi et al 2006;[START_REF] Levy | How to explain the differences between renin angiotensin system modulators[END_REF].

The ACE inhibitors appeared with the discovery of the captopril molecule in 1975 that was considered as a breakthrough, due to its novel mechanism of action and the revolutionary development process. Nowadays this therapeutic group includes 16 different molecules with a similar structure, and from some of them lots of generic products have originated (Rossi S (Ed.) 2009). Currently, only 2 molecules are still under patent protection.

The sartans (also named "Angiotensin Receptor Blockers" or "Angiotensin II Receptor Antagonists") modulate the rennin-angiotensin-aldosterone system and thus lead to a decrease in blood pressure. The tetrazole group is a main part of the chemical structure of losartan, irbesartan, olmesartan, candesartan and valsartan. In addition, losartan, irbesartan, olmesartan, candesartan and telmisartan include one or two imidazole groups. Within the group of sartans, valsartan and losartan have expired patents and generics available on the market.

The statins are lipid lowering agents with first marketed INN lovastatin and soon after, the simvastatin was placed on the market [START_REF] Stossel | The Discovery of Statins[END_REF]). The statins therapeutic group is composed of 6 molecules from which 4 have expired patent.

Main objective

The aim of this study was to analyse the changes in the drug usage and prices in these three pharmaco-therapeutic groups, acting on cardiovascular system, on the Bulgarian market between the years 2005-2007.

Hypothesis tested

Due to the fact that there is no law regulation on the generic medicines in Bulgaria, we want to test two hypotheses: To test the second hypothesis, we first performed a two-way ANOVA analysis to evaluate the statistically significant differences between the prices of active substances, per milligram, for the products within each therapeutic group and during the observed years. If there was a difference established we next perform a one way ANOVA analysis to evaluate them per INNs and among the therapeutic alternatives.

Results

Out of all collected medicinal products in the therapeutic class of ACE inhibitors, we found 3 INNs with generics: the enalapril, lisinopril (during 2005-2007) and the generics for ramipril appears during the third year of observation ( 2007). In the therapeutic class of statins we also found 4 INNs with generics: the simvastatin, pravastatin, atorvastatin and the lovastatin. In the last therapeutic class studied, the sartans, we found 1 INN with a generic, the losartan. The other INNs within these three therapeutic groups are considered as therapeutic competitors.

All these results are in Appendix 1. Some of the fields in Appendix 1 are marked with "0" due to the lack of sales data in the National Health Insurance Fund's database for the particular year observed.

Analysis of the market share of products

In the product class of ACE inhibitors, the enalapril in milligrams is the leader of the class with 93.1 % of the market share in 2005 -when we begun our study. It's not possible to see it in this appendix but the enalapril permanently increased its' market share in the years before We can say that the fast growth rate of the therapeutic group and introduction of 2 different new molecules within the class lead to a decrease in the market share of enalapril during 2006 with 4,5 % and during 2007 with 2,24% (Figure 1). On the other hand, the market share of the therapeutic competitors slowly and permanently increases every year thus taking part of the enalapril market share.

It is also necessary to point out that after the introduction of the generic competitors to lisinopril in 2006, its market share increase with a bigger percentage (from 2.34 % in 2005 to 3.65 % in 2006) than the share of the other therapeutic competitors within the group and this increase continued still, even after the exclusion of one of the products during the next year. The product class of statins is the most dynamic in terms of changes of generic and therapeutic competitors. In this group, we observed that after the increase of the number of generic competitors under the same INN, the relative market share of the INN increased -as is the case for simvastatin. We can consider that the introduction of new generics did not deprive the sales data of the originator immediately, but, in general, contributed to the accumulation in market share of the belonging INNs. In this group we also observed that the introduction of a new product affects the sales data of competitors, as is the case with the inclusion of rosuvastatin.

Placed

Placed here -Figure 3 1). The test confirmed that the sales data of the observed therapeutic groups changed statistically significantly during the analysed years.

Placed here -Table 1: Results of the Kolmogorov Smirnov test

To test the differences among the INNs' sales data, the z-test analysis was applied. The results of the z-test analysis confirm that for almost all the products, except for the perindopril and trandolapril in the class of ACE inhibitors, and irbesartan from class of sartans, the changes in the market share are statistically significant (Table 2). For all of these three products there is no generic competitor. So we can conclude that the introduction of generics changes the market share of the INNs and contributes to the changes in the share of the therapeutic groups. The changes in the market share products without generics could be explained with the marketing policy of the companies -a fact that was not explored in this study.

Thus our first hypothesis is confirmed.

Placed here -Table 2. Results of the z-test analysis

Analysis of the price changes

Concerning the prices, all of them decreased during the observed period from 2005 to 2007 but it was mainly due to the changes in the regulation (Appendix 1) (Ministry of health, Drug price registry 2005,2006,2007). The regulatory changes affect mainly the decrease of the distributors' margins (wholesale and retail), almost half for the medicines paid by the National Health Insurance Fund.

For the group of ACE inhibitors, the two way ANOVA analysis confirms that the prices per milligram differed among the observed products (p<0.05), but did not differ statistically significant during the years for the whole group (p>0.05) (Table 3). Multiple comparison among the prices of different INNs established statistically significant differences (p < 0.05) between the prices per milligram of trandolapril and all other INNs within the group (benazepril; cilazapril; enalapril; fosinopril; lisinopril; perindopril; quinapril and ramipril).

The one way ANOVA analysis revealed significant differences (p < 0.05) in the prices during the years only for lisinopril (Table 4). It is necessary to point out that the mean price decreased in the year 2007, when one of the generics was excluded from the database, which was the most expensive one.

The statines group included 6 INNs with 20 to 29 dosage forms per year. In this group, we also did not find significant price differences among the years (p>0.05), but prices of the INNs within the group differs (p<0.05) (Table 3). By applying multiple comparisons, the difference among lovastatin and rosuvastatin was evaluated as statistically significant. No differences among the prices for particular INNs were observed (Table 4).

For the group of sartans, we also found, through the two way ANOVA analysis, that the prices per milligram differ among the observed products (p<0.05), but did not differ significantly during the observed years for the whole group (p>0.05) (Table 3). Multiple comparisons among the INNs within the therapeutic groups revealed significant price differences between telmisartan and eprosartan. No differences between the prices for particular INNs were evaluated separately (Table 4).

The results from two way and one way ANOVA analysis revealed that there are no statistically significant changes in the prices of the observed INNs, except for lisinopril, but there are significant price differences between the therapeutic competitors within the groups.

Thus our second hypothesis is not fully confirmed.

Discussion, limitations and further research

The cardiovascular medicine market characteristics are of importance for the national markets for a lot of reasons, for example: their permanently growing usage, progress in new molecules discovery, changes in life expectancy of the population, creation of the national rules for price control, and cost containment measures.

We do observe a dynamic national market of cardiovascular medicines that correspond to the world tendencies, but the competition is mostly evident at the therapeutic groups level than at the generic ones ( drugs.com. 2008).

Our results confirm those of similar studies -that the generic competition, in general, changes the market in the observed country. These changes benefit the generic products and decrease the medicines' prices.

The generic competition is not regulatory supported in the country and this fact could influence negatively our study because it does not correspond to the world tendencies (Law on Medicinal products for human medicine 2007; Ministry of health 2005, 2006, 2007).

Furthermore, our results confirm some other publications -that the creation of a sustainable generic pharmaceutical market requires active regulatory and marketing measures at all levels, including incentives for manufactures, physicians, and dispensers [START_REF] Simoens | Sustainable generic medicines market in Europe[END_REF][START_REF] Adriaen | Pricing strategies of originator and generic medicines following patent expiry in Belgium[END_REF]. It is not only sufficient to shorten the marketing authorisation process for generic medicines at a European level, but every country should perform its own measures as well.

Our study possesses some limitations in terms of the fact that we compare the mean prices per milligram and not per Defined Daily Dose (DDD), which is more therapeutically compatible.

Despite the fact that DDD is established in milligrams for most of the medicines, we consider that in the future such analysis should be done for the changes in prices per DDD.

Comparison of the market share and prices in milligrams has its own reasoning for international market tender purposes [START_REF] Al-Abbadi | One-year assessment of joint procurement of pharmaceuticals in the Public health sector in Jordan[END_REF][START_REF] Karam | World Health Organization -Health Action International Project on Medicine Prices[END_REF][START_REF] Zaheer | Evaluating Drug Prices, Availability, Affordability, and Price Components: Implications for Access to Drugs in Malaysia[END_REF], as well as for reference prices comparison. Such a price comparison per milligram of active substance is also regularly performed by the Management science for health and World health organisation when they publish the International Drug Price Indicator Guide and all prices are shown per milligrams of active substance [START_REF] Frye | International drug price indicator guide[END_REF]. Some countries are using the generics' price per milligrams in the process of establishment of the lowest price per product within the reimbursement system [START_REF] Mrazek | Comparative approaches to pharmaceutical price regulation in the European Union[END_REF].

Other authors consider [START_REF] Who | Introduction to drug utilization research[END_REF] that, if medicines utilization is given in terms of grams of active ingredients, drugs with low potency will account for a larger fraction of the total than drugs with high potency, and that the combined products may also change their market figure. In our study we did not observe any combination so our figures could not be affected by such an utilization data. We also did not observe significant changes in the prices per milligram for the medicines with different potency, thus this is not also the case for consideration.

The international prices comparison per milligrams of active substance could be used by the regulatory authorities. [START_REF] Grootendorst | Impact of reference-based pricing of nitrates on the use and costs of anti-anginal drugs[END_REF] analysed the impact of referenced -based pricing on nitrates usage and cost reduction by using also the cost per milligram. Similar approach was found useful in case of comparison of the medicines prices for particular therapeutic groups [START_REF] Wang | The Impact of Cost Sharing on Antidepressant Use Among Older Adults in British Columbia[END_REF], which is also the case in our study.

The fact that we could not establish strong evidences supporting our second hypothesis could be explained with the short period of observation and slow entrance of generic medicines in the positive drug list of the National Health Insurance Fund. Generic competitors were found for only 8 out of 20 compared INNs and for 4 of them (ramipril, pravastatin, atorvastatin, losartan) only one competitor that limits the evaluation of the impact of generic competition on prices existed. We didn't analyse the way the pharmaceutical laboratories communicate with the prescribers, which can also have an effect on the market.

Further studies should be organised to evaluate the changes in prices during a longer period, when more generic competitors will be available in the country. The national generic medicines policy is also of importance as it stimulates the medicines' prices competition.

Conclusion

There are two main conclusions from our study. The first one is that for the therapeutic groups, which include a lot of similar molecules, it is important to consider the competition between the molecules and not only the generic entrance. Originating pharmaceutical companies seem to compete among themselves at a therapeutic level and with generic companies at a price level.

The second conclusion is that the introduction of generic medicines did not affect the mean market prices immediately, which means the market needs more than 3 years of stronger competition to benefit.

From the managerial point of view, it is necessary to point out that if the government wants to benefit from the generic drug policy, and thus to reduce the medicines prices in the country, it should create a stronger supporting environment affecting all the participants in the pharmaceutical sector in the country.

Hypothesis 1 :

 1 The introduction of new medicinal products (originators or generics) affects the sales data and/or market share within the group.Hypothesis 2: The introduction of new medicinal products (originators or generics) decreases the prices of the competitors within the group.MethodsUsing the officialNational Health Insurance Fund's database, we collected information for the prescription of 20 different INN in the cardiovascular classes of medicines: 9 ACE inhibitors, 5 sartans and 6 statins, authorized for sale on the Bulgarian market in the years 2005-2007. A database was created, which included the prescribed packages of different trade names and dosage forms of medicinal products, sold under the observed 20 International Nonproprietary Names (INN). To summarise the differences between the packages, the usage was recalculated in milligrams for every INN during the particular year observed. The information for the drug prices was gathered from the official register of the Ministry of Health for all authorised trade names of observed medicines (Ministry of health 2005, 2006, 2007). Then, the prices were recalculated, to establish the price per milligram for every dosage form and within the group of products under the same INN. To calculate the mean price per INN and per milligram a descriptive statistic was applied. To test the first hypothesis we first applied the Kolmogorov Smirnov (K-S) test towards the sales data during the observed years. The K-S test seeks differences between the distribution function of sales data in 2006 in comparison with 2005, and 2007 in comparison with 2006.The test provides information for the differences between the observed years by rejecting the null hypothesis if no differences exist.To analyse the possible differences among the sales data of particular INNs during the observed years, the market share of every INN, out of all sold milligrams of INNs within every therapeutic group, was recalculated. Furthermore, in a second step, we calculated the proportions, using the z-test analysis for every INN assuming 95% CI for the changes in proportions of INNs, comparing the data for the year 2005 versus the year 2006 and the year 2006 versus the year 2007. The z-test provides information for the statistically significant differences among the proportions of INNs sold during the years 2005, 2006 and 2007.

  2005. The market share of enalapril decreased in 2006 (88.6 %), and 2007 (86.34 %) despite the introduction of new generics in 2006. All other products within the group, except the quinapril (which decreased in 2007 versus 2006) perform a steady increase in their usage, measured in kg, of active substance sold (Appendix 1).

  Here -Figure 1: Relative Market Share of ACE inhibitors sales during 2005, 2006 and 2007. In the product class of sartans, the biggest market share in 2005 was that of telmisartan, probably because of the promotional price of the originator. In the second year the only one product with a generic version (losartan) increased its market share almost 6 times, thus taking a leading position in the group. The market dynamic in proportions of INNs sold within the group of sartans is mostly evident in comparison with the previous group, but we can suppose that these changes are contributed mainly to the active market development of the products within the group. Placed here -Figure 2: Relative Market Share of sartans sales during 2005, 2006 and 2007.

  : Relative Market Share of statins sales during 2005, 2006 and 2007. After applying the Kolmogorov Smirnov test, we observed that there is a statistically significant difference among the sales data during 2006 and 2005, as well as during 2007 and 2006 (Table

Table 1 :

 1 Results of the Kolmogorov Smirnov test

	Table
		Sales data	Sales data	Sales data
		(mg)	(mg)	(mg)	K-S
	INN	2005	2006	2007	2006/2005 K-S 2007/2006
	enalapril	1906795530 2287185280 2362110040
	lisinopril	47860510	94173850	154909420
	perindopril	14378880	19729440	23778840
	ramipril	5880420	29455406	55117400
	quinapril	47253900	96471300	68185500
	fosinopril	23618840	47761840	56553000
	trandolapril	3322444	6061288	7445446	Null	Null
	cilasapril		553630	3979220	hypothesis	hypothesis
	benazepril		692160	3899840	rejected	rejected
		Sales data	Sales data	Sales data
		(mg)	(mg)	(mg)	K-S
	INN	2005	2006	2007	2006/2005 K-S 2007/2006
	simvastatin	13140540	26303860	125098060
	lovastatin	199510800	310969800	300126000
	pravastatin	5301900	9335100	12384300
	fluvastatin	81453120	84938560	68862080	Null	Null
	atorvastatin	16486500	31483800	36445200	hypothesis	hypothesis
	rosuvastatin		157360	4523400	rejected	rejected
		Sales data	Sales data	Sales data
		(mg)	(mg)	(mg)	K-S
	INN	2005	2006	2007	2006/2005 K-S 2007/2006
	losartan	15715000	196378000	485893800
	valsartan	58464000	70936320	68051200
	telmisartan	169261120	225245440	207121600	Null	Null
	irbesartan	231000	252000	0	hypothesis	hypothesis
	eprosartan	28089600	143152800	131695200	rejected	rejected

Table 3 .

 3 Differences among the prices among the therapeutic competitors and among the years (Results of the two-way ANOVA analysis)

	Therapeutic	Prices differences	Statistical	Differences	Statistical
	group	among the	significance	among the prices	significance
		therapeutic		during the years
		competitors
	ACE inhibitors	p< 0.05	Yes	p > 0.05	No
	Statins	p< 0.05	Yes	p > 0.05	No
	Sartans	p< 0.05	Yes	p > 0.05	No

Sales and prices values for the observed therapeutic groups and INNs of cardiovascular medicines in 2005, 2006, and 2007

  Figure 1: Relative market share of ACE inhibitors sales during 2005, 2006 and 2007. Relative market share of sartans sales during 2005, 2006 and 2007. Relative market share of statins sales during 2005, 2006 and 2007.

	Figure
	93.05 Figure 2: 5.78 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 % s a l e s 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 % s a l e s 63.16 10.00 40.00 50.00 60.00 70.00 80.00 % s a l Figure 3: 4.16 30.00 e s 20.00	21.51 1.68	62.28 25.79	88.58 5.68 30.88 New generic 11.15 67.14 2.02 generic New generic 0.09 10.34 5.22 New New generic	35.42 18.34	86.34 Generic products New 0.04 22.51 0.03 6.80 excluded 22.85 54.43 Generic excluded 7.62 23.20 54.82 2.26 12.58 Generic 0.00 Excluded 14.75 product 0.83 6.66 excluded New product
	0.00				2005 year				2006 year			2007 year
	10.00 0.00	2.34	0.70	0.29 2005 year 2.31 1.15	0.16	losartan	3.65 valsartan 0.76	1.14 2006 year 3.74 INN 1.85 telmisartan INN	0.23 irbesartan 0.02 0.03	5.66 eprosartan	0.87	2.01 2007 year 2.49 2.07	0.27	0.15	0.14
					2005 year		simvastatin	lovastatin	2006 year pravastatin fluvastatin	atorvastatin	rosuvastatin	2007 year
											INN
						enalapril	lisinopril	perindopril	ramipril	quinapril	fosinopril	trandolapril	cilasapril	benazepril

Conflict of interest

The authors confirm that there are no relevant associations that might pose a conflict of interest.


Brunella Pilato

Marianna Martinucci

Katia Danza

Rosamaria Pinto

Daniela Petriella

Rosanna Lacalamita

Michele Bruno

Rossana Lambo

Angelo Cosimo D'amico

D ' Cosimo

Angelo Amico

Stefania Paradiso

Tommasi

PhD Stefania Tommasi
email: s.tommasi@oncologico.bari.it

Mutations and polymorphic BRCA variants transmission in breast cancer familial members

Keywords: BRCA1, BRCA2, Polymorphism, Breast cancer risk, Familiarity

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

SNPs are sequence's variants present in more than 1% of human population.

SNPs seem to be related to person's genetic predisposition or resistance to peculiar disease determining the severity or progression of disease [START_REF] Risch | The future of genetic studies of complex human diseases[END_REF]. A large number of SNPs has been demonstrated to influence protein expression or gene function either through aminoacid change and modification in protein function or through indirect epigenetic changes in synonymous or non-coding SNPs. In familial breast cancer, this has been shown to be peculiar of genes involved in double-stranded break repair [START_REF] Sehl | Associations between single nucleotide polymorphisms in double-stranded DNA repair pathway genes and familial breast cancer[END_REF].

Hereditary breast cancer syndrome was firstly associated with BRCA1 and BRCA2 genes whose mutations confer high risk to develop breast and/or ovarian cancer. However, a remarkably high number of sequence variants other than mutations have been identified, some of them are unidentified variants and some polymorphic variants. Because of the breast cancer risk in BRCA1 and BRCA2, mutation carriers have also been found to vary depending on different factors (i.e. age at diagnosis, unilateralism, other pathologies in the family, environmental factors, etc.), segregation-analysis models evidenced a variability in terms of a polygenic-modifying variance [START_REF] Antoniou | The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions[END_REF]. A recent genome-wide association study in breast cancer identified five common susceptibility alleles that are associated with an increased risk of breast cancer in the general population [START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF].

Segregation analysis of breast cancer in families can provide the logical basis and the specific genetic models for mapping and identifying genes responsible for human breast cancer. This is also true for polymorphisms mapping in genes usually mutated. Previous reported literature on this issue was based on case-control studies, while vertical transmission of SNPs, such as those related to the apoptotic trait (i.e. TP53 R72P), has been only rarely demonstrated [START_REF] Imyanitov | Gene polymorphisms, apoptotic capacity and cancer risk[END_REF].

The present study aims to clarify a possible role of BRCA1 and BRCA2 SNPs as susceptibility markers of risk besides the so defined pathological mutations present in the 2 genes. The design of the study differs from what previously reported based on case-control analyses [START_REF] Freedman | A haplotype-based case-control study of BRCA1 and sporadic breast cancer risk[END_REF][START_REF] Hughes | Use of association studies to define genetic modifiers of breast cancer risk in BRCA1 and BRCA2 mutation carriers[END_REF]; in fact, the vertical transmission of pathological mutations and polymorphisms both in carrier and non carrier families have been studied by analyzing all available family members. In particular, the polymorphisms that we previously proved to have a harmful role by in silico analyses have been considered [START_REF] Tommasi | Molecular and in silico analysis of BRCA1 and BRCA2 variants[END_REF].

Materials and Methods

Patients selection

Thirty families comprising 97 cases healthy and affected (30 probands and 67 family members) people with high risk to carry BRCA mutations have been enrolled from Counseling Program of National Cancer Institute of Bari.

All people undersigned an informed consent, previously approved by the ethical committee, to perform molecular analyses and use data for research purposes [START_REF] Tommasi | BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from Apulia, Italy[END_REF]. All patients were also characterized according to pathological features (Table 1) and family history and have been classified as having an "increased risk" to carry BRCA mutations (>10%) by BRCAPRO software [START_REF] Berry | BRCAPRO validation, sensitivity of genetic testing o f BRCA1/BRCA2, and prevalence of other breast cancer susceptibility genes[END_REF].

Among the 30 probands (median age 48 yrs), 10 carried BRCA1 mutations, 6 BRCA2 mutations and 14 were non-carriers.

The design of the study forecasts to analyze the vertical transmission of pathological mutations and polymorphisms in the considered series.

A survey asking for their detailed genealogy and medical history was administered to each proband's firstand second-degree relatives (Table 1).

Particular attention has been done to other tumor types eventually present.

On the basis of survey information a pedigree was developed for each proband (Figure 1)

Molecular analysis in nonrelated patients and family members

Genomic DNA was extracted using QIamp DNA blood midi kit (Qiagen, Valencia, CA, USA) following manufacturer's instruction. DNA was quantified and mutational screening was performed as previously described [START_REF] Tommasi | BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from Apulia, Italy[END_REF]. If a variant was identified in the proband, was requested him the consent to inform other family members. All enrolled members, healthy or affected, undersigned an informed consent and their blood analyzed for the proband's identified variant. Family's information for all members permitted to design a pedigree with the Progeny Program v6.

Statistical analyses

The statistic association between mutation frequency and the other parameters was assessed using a X2 test and the Fisher test. Differences were considered to be significant when p value was less than 0.05.

Statistical analyses were carried out with the SPSS statistical software (SPSS, inc, Chicago, IL).

Results

BRCA1/2 mutations transmission

In the present series the most frequent BRCA1 mutation was 5382insC, carried by families #2, #3, #7, #8, #10, #12, #13 and #28 and BRCA1 R1495M mutation was found in family #20. All probands were only affected by breast cancer with the exception of family #29 whose proband had a co-occurrence of breast and ovarian cancer.

As regards BRCA2 mutations: 2150insTA was carried by a male of family #4, 3034delAAC by a female of family #23, 6024delTA by a female of family #21, 6696delTC by family #30, 2024delCTTAT by family #1 and mutation 6710delAAC by one family #9's female member.

Only the probands of family #1 and #4 were males and affected by breast cancer of grading III. In family #1 we found that the mutation has been transmitted in five daughters, two affected and three unaffected. In family #4 the mutation has been transmitted to two daughters and to one son unaffected. Table 2 reported mentioned results.

BRCA1/2 polymorphisms transmission

In the current study have been investigated for polymorphisms families with and without mutation in BRCA1 and BRCA2 genes. Most of the analyzed polymorphisms were heterozygous with the exception of 871Leu in family #2, #5, #21, #23 and #24 and N372H in family #14 that were homozygous.

Among all the detected polymorphisms, we considered those more frequent in our series which resulted to have a harmful role by previous in silico analyses, too [START_REF] Tommasi | Molecular and in silico analysis of BRCA1 and BRCA2 variants[END_REF].

The SNPs in aminoacidic position 871, 1038 and 1613 often segregated together and patients with 871Leu-1038Gly-1613Gly cluster seemed to have peculiar pathologic features presenting association with higher differentiated tumors (83%, p=0.000) and inverse relation with higher proliferative activity (81% lacking cluster presented Mib-1 ≥ 10%, p=0.04).

As regards the analysis of the association of different polymorphisms in families with pathological mutation transmission, the group 871Leu-1038Gly-1613Gly cluster has been found in family #2 with the 5382insC BRCA1 mutation and in families #9 and #23 with BRCA2 mutations. In family #23, these polymorphisms were also associated to the presence of BRCA2 intronic variant IVS16-14 T>C. In the families with R1495M mutation the association of 1183Arg and 1038Gly BRCA1 polymorphisms was present. In the mutated family #21, these polymorphisms were also associated to 871Leu and BRCA2 372His. The analysis of polymorphisms with low frequency showed that 289His (exon 10) and 991Asp (exon 11) in BRCA2 gene were only present in non mutated families and resulted always associated with the same gene intron variant: IVS14+53 C>T (p=0.01), which was previously verified to be a possible site for alternative splicing [START_REF] Tommasi | Molecular and in silico analysis of BRCA1 and BRCA2 variants[END_REF][START_REF] Dunning | Common BRCA1 variants and susceptibility to breast and ovarian cancer in the general population[END_REF]. These 3 polymorphisms also resulted always related to BRCA1 exon 11 (871Leu, 1038Gly and 1183Arg) and exon 16 (1613Gly) SNPs (p=0.003). Table 3 and Table 4 showed the transmitted haplotype blocks in mutated and BRCAX families in family available members.

Only 3 probands (families #7, #12 and #25) resulted triple negative and did not present any polymorphic sites. Families #7 and #12 were BRCA1 mutated, whereas family #25 was BRCAX with an age of onset of 29 years.

Mutated and non mutated families were considered for the occurrence of the polymorphisms. The SNP transmission frequency in the relatives of mutated probands was similar to that in relatives of non mutated probands with exception for 1183Arg BRCA1 which resulted significantly more frequent in mutated families. (Table 5)

Follow up study over a period of 24 months showed the occurrence of breast cancer in relatives of mutated and non mutated families. Family members of mutated and BRCAx families that developed a breast cancer had at least one polymorphism. There was a comparable percentage of each SNP between the two groups except 1183Arg and 372His polymorphisms.

(Table 5)

Discussion

The aim of the present paper was to investigate the transmission of BRCA1 and BRCA2 variants in families enrolled and to evaluate the risk of breast and/or ovarian cancer associated with BRCA1/2 polymorphisms. Specific variants association have been studied in relation to proband pathological features, BRCA pathological mutations, other tumors present in each family, characteristics of transmission among relatives. Our hypothesis was that, also in hereditary breast cancer, multiple SNPs, belonging to the same haplotype, could be associated to an increased risk, even if slight. This happens for example in NAT2 polymorphisms which have been shown to be associated with a lower bladder cancer risk [START_REF] Olden | Commentary: From phenotype, to genotype, to gene-environment interaction and risk for complex diseases[END_REF].

In our previous study [START_REF] Tommasi | Molecular and in silico analysis of BRCA1 and BRCA2 variants[END_REF] we found that 80% of patients presented at least one polymorphism of which 1038Gly (52%), 871Leu (36%), 1613Gly (34%), 1183Arg (17%) and 372His (48%) were the most representative; and, by in silico analysis, we evidenced their higher impact on the two proteins function.

The present study arose from these observations and introduced a different design of the study. Currently, studies on polymorphic haplotypes have been conducted in a few case-control series [START_REF] Lange | Family-based samples can play an important role in genetic association studies[END_REF], while the type of vertical transmission in the family has been investigated only for known pathological mutations. In the present series, vertical transmission of both pathological mutations and polymorphisms has been followed up in 30 families in which all alterations resulted mendelian transmitted, as supposed, in maternal and paternal lineage.

In particular, haplotype 871Leu-1038Gly which in the previous series identified high differentiated tumors when associated to pathological mutations, confirmed their role also in the present series but in association with 1613Gly. Their function on BRCA1 exonic splicing silencer binding site and on transcription factors binding sites seemed to be stressed by the fact that the two SNPs were always transmitted together among all relatives.

The peculiar association of 289His-991Asp-IVS14+53 C>T in BRCA2, peculiarly present in BRCAX families, evidenced the pathological role of this cluster being each of these alterations able to affect alternative splicing [START_REF] Tommasi | Molecular and in silico analysis of BRCA1 and BRCA2 variants[END_REF].

Analyzing separately the polymorphism occurrence inside the group of mutated and non mutated families, all the most frequent SNPs, with the exception of K1183R, were transmitted among relatives with the same rate.

Moreover, considering the rate of SNPs transmission among relatives affected by breast cancer, we also did not find differences between mutated and non mutated families except for K1183R and N372H. These two SNPs resulted more frequent in BRCAX tumors suggesting, in our opinion, their harmful role in the place of known pathological mutations.

Heterogeneity in relatives made the design of this study very complex, but the possible role of all variants in breast cancer risk could be obtained increasing the number of sample cases. Despite of the difficulties in collecting data from a sufficient number of relatives per family, our results

were the first reported with regard to polymorphisms transmission and strongly suggested the importance of studying also polymorphic variants in defining hereditary risk.

Conclusion

In conclusion, this study shows that the genetic screening of patients with familial breast cancer should be conducted also for polymorphisms and intronic variants analysis in all families because of their possible role as susceptibility markers.

Figure 1 .

 1 Figure 1. Example of family pedigree. : Cancer diagnosis=Breast; : Cancer diagnosis= Prostate. B1-E: BRCA1 E1038G, B1-S: BRCA1 S1613G, B1-P: BRCA1 P871L, B2-N: N372H

Table 1 : Familial characteristics of 30 probands. Patients #1 and #4 were males. Relati ves presenting other kind of neopl asia other than breast or ovary are here listed. Oc: ovari an cancer, Mel: melanoma, Blc: bladder cancer, Crc: colon rectal cancer, Lc: lung cancer, Pc: prostate cancer, Os: osteosarcoma, Tyr: tyroi d cancer, Brc: brai n cancer, Utc: utereus cancer, Gc: g astric cancer, Lr: laryngeal cancer, Leu: leukemi a, Kin: kidney cancer, Lnh: non Hodgkin lymphoma, Psc: pancreas cancer

 1

	D	Histology	Age of onset	Grading	ER	Mib1	PgR	Her2	TNM classification	Metachronous bilateral breast cancer	No. of Breast Cancer in family	Other cancer i
		IDC	72	III	70	10	90		pT4N0M x	/	3	M el-O
		IDC	41	II	25	22	15	0	pT1cN1bIIIM 0	/	1	Crc-L
		IDC	41	III	0	80	0	+--	pT1cN0M x	yes	3	Oc
		IDC	59	III	70	60	60	+++	pT2N1aM x	/	2	P
		IDC	54	III	0	90	0		pT2N1aM 0	/	1	Tyr-L
		IDC	38	III	0	8	60	+--	pT1cN0(sn)M x	/	1
		IDC	53	III	0	40	0	0	pT2N1aM x	yes	3	Pc-
		IDC	51	III	55	30	10	+++	pT1cN0M 0	yes	3	U
		IDC	33	II	0	22	0		pT2N0M 0	/	1	Brc-O
		IDC	45	II	10	10	15		pT1N0M x	/	3	Gc-O
		IDC	36	III	35	3	40	0	pT1cN0M x	/	1	Lc-
		IDC	34	III	0	70	0	0	pT1N1M 0	/	1	Oc-B
		IDC	34	III	0	70	0	++-	pT1N0M x	yes	4	G
		Inflammatory carcinoma	77		0	20	0	++-	pT1N0M x	/	4	P
		IDC	46	III	20	25	8	++-	pT3N1b1M 0	/	3
		IDC	56	II	45	0	35	+--	pT1N1M x		1	Lr-Le
		IDC	63	II	5	10	1		pT1bN0M x		2	Lc-O
		ILC	58		99	10	30	0	pT1N0M o	yes	4	L
		IDC	51	II	45	10	70		pT1N1M x	/	2
		IDC	34	III	0	75	0	++-	pT2N0M x	/	2
		IDC	41	III	10	30	10	+++	pT2NxM x	/	2
		Tubular carcinoma	48	II-III	30	3	60	++-	pT1aN0M x	/	3	Lnh-O
		IDC	30	II	40	20	30	+--	pT2N0M x	yes	2
		IDC bifocal	48	II	12	24	0	+--	pT1N0M x	/	1	O
		IDC	29	III	0	70	0	0	pT1cN0M 0	/	1	O
		ILC	56		70	8	40	+++	pTxN+M x	/	2	L
		IDC	26	III	25	35	30	0	pT3N3aM x	/	3
		IDC	52	III	0	25	0	+--	pT1bN0M 0	/	4	Brc-P
		IDC	46	III	0	60	0	+--	pT2N2aM x	yes	5
		IDC	39	III	30	5	8	+++	pT3N1aM x	/

Acknowledgme nts: Partially supported by Regione Puglia ( DIEF 2007)


Elrasheid A H Kheirelseid

Hanzali Jumustafa

Nicola Miller

Catherine Curran

Karl Sweeney

Carmel Malone

Ray Mclaughlin

John Newell

Michael J Kerin

Hanzali Jumastapha

Bilateral breast cancer: analysis of incidence, outcome, survival and disease characteristics

Introduction:

Breast cancer is the most common malignancy and second only to lung cancer as the major cause of cancer-related deaths among women in the western world [START_REF] Jemal | Cancer statistics[END_REF].

Increasing breast cancer incidence rates, improved diagnosis and management modalities and growing life expectancy have result in increasing numbers of women at risk of developing contralateral primary breast cancer. The incidence of clinically observed bilateral breast cancer is reported to range from 1.4% to 11.8% [2 -5]. There is uncertainty in the literature whether developing a contralateral tumour influences the outcome as some studies suggest poor survival while others report similar survival compared to unilateral disease. Women diagnosed with breast cancer are at increased risk of developing contralateral breast tumour. This represents two to six times greater relative risk than developing a first breast cancer in general population [START_REF] Chaudary | Bilateral primary breast cancer: a prospective study of disease incidence[END_REF]. Other factors including family history of breast cancer, initial tumour diagnosed at an early age, lobular histology of the first tumour, treatment received for the first tumour and nulliparity all contribute to the risk [START_REF] Chen | Epidemiology of Contralateral Breast Cancer[END_REF][START_REF] Heron | Bilateral breast carcinoma: risk factors and outcomes for patients with synchronous and metachronous disease[END_REF][START_REF] Newman | A casecontrol study of unilateral and bilateral breast carcinoma patients[END_REF][START_REF] Mertens | Contralateral Breast Cancer: Factors Associated with Stage and Size at Presentation[END_REF][START_REF] Hartman | Genetic implications of bilateral breast cancer: a population based cohort study[END_REF] There has been conflicting evidence on the impact of bilateral breast cancer on management of patients with regard to surgical treatment options, bilateral prophylactic mastectomy and role of magnetic resonance imaging (MRI) in patients screening. The best management of patients with bilateral breast cancer is still uncertain. Patients are often treated with bilateral mastectomy rather than breast conserving treatment although some reports confirm its efficiency in management of bilateral breast cancer as for unilateral tumours [START_REF] Lee | Efficacy of Breast Conservation Therapy in Early Stage Bilateral Breast Cancer[END_REF][START_REF] Jobsen | Synchronous, bilateral breast cancer: prognostic value and incidence[END_REF]. Secondly, there has been a dramatic increase in rates of contralateral prophylactic mastectomy after technical advances in surgical procedures and improved satisfaction with breast reconstruction, especially in the USA [START_REF] Tuttle | Increasing rates of contralateral prophylactic mastectomy among patients with ductal carcinoma in situ[END_REF]. This is because of perception of increased risk of bilateral breast cancer and related mortality, allowing patients to avoid the physical, emotional and financial costs associated with second tumour detection and treatment. However, the potential benefit of prophylactic mastectomy in reducing the rates of bilateral breast cancer diagnoses and improve survival rate is controversial [START_REF] Hartmann | Efficacy of bilateral prophylactic mastectomy in women with a family history of breast cancer[END_REF][START_REF] Quan | Incidence and outcomes of contralateral breast cancers[END_REF]. Finally, multiple reports demonstrate increased sensitivity of MRI for detection of breast cancer compared to conventional methods particularly in patients suspected or known to have cancer [START_REF] Lee | MR Imaging Screening of the Contralateral Breast in Patients with Newly Diagnosed Breast Cancer: Preliminary Results[END_REF][START_REF] Boetes | Screening women at increased risk with MRI[END_REF]. Nevertheless, its role in screening settings has not yet been outlined. This might be, in part, related to the moderate specifity of MRI and the associated deficiencies of cost, anxiety and benign biopsies and perhaps over-diagnosis.

The objectives of the study were to examine a defined group of breast cancer patients with a view to addressing the incidence, clinicopathological characteristics and outcome of bilateral breast cancer amongst these patients to study its outcome and survival compared to unilateral tumours and to analyzed the disease clincopathological characteristics in order to identify the at risk group. Moreover, we aimed to give context to our management of patients with bilateral breast cancer based on a prospectively maintained database which captures all such patients referred to our institution.

Materials and Methods:

Patient cohort:

The prospectively updated Galway university hospital breast cancer database was queried for all cases of primary breast cancer from 1988 through 2008. The resulting data was further queried for cases of bilateral breast cancer. The distinction between bilateral cancers and metastatic cancer in the contralateral breast was established by Chaudary's criteria [START_REF] Chaudary | Bilateral primary breast cancer: a prospective study of disease incidence[END_REF] which are the demonstration of in situ disease, different histological types and grades of cancer between the two breasts and no evidence of local, regional or distant metastasis. Patients with disseminated disease elsewhere in the body would be at high risk of having metastatic disease in the contralateral breast rather than a primary cancer and are not included in the group of bilateral breast cancer unless the two breasts have different histopathological features. In our series, 2 patients had metastatic disease when breast cancer was originally diagnosed without documented differences in histopathological features hence they were not considered to have bilateral breast cancer. The bilateral cancers were categorized as synchronous when a contralateral breast cancer was diagnosed within 12 months and metachronous when contralateral breast cancer was diagnosed after 12 months of the initial tumour diagnosis. The term contralateral tumour is used in this study to describe the second primary tumour to arise on the other breast following an initial breast cancer diagnosis.

First breast is defined as the breast in which the initial tumour diagnosed while second breast is the breast in which the contralateral tumour developed.

All patients were treated according to local protocols and followed every three month for one year, 6 monthly for two years and then annually. They also have an annual mammographic review.

Data collected included age and year of presentation for both initial and contralateral tumour, tumour size, grade, stage, histological type, extent of tumour invasion and lymph node involvement, present or absence of local or distant metastasis and time between initial tumour diagnosis and presentation of contralateral breast tumour. The estrogen, progesterone and HER-2/neu receptors status was also noted when data were available as was local therapy and the use of hormonal therapy, chemotherapy and radiotherapy.

Statistical analysis:

The risk factors for developing contralateral primary breast cancer were investigated comparing histopathological parameters of unilateral breast cancer and the first tumour of the bilateral cancers while the bilateral disease characteristics were determined comparing the unilateral cancer to the second tumour of the bilateral disease and further analyzing the synchronous and metachronous tumours. Survival studies were performed from the date of diagnosis of the first cancer and disease-free survival was regarded as zero if the patient presented with metastatic disease.

The SPSS ® 16.0 software package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Mann-Whitney U test and t-test were used, as appropriate, for comparison of continuous variables and Chi-square test for categorical variables. All tests were two sided and a result was considered significant if the calculated P value was <0.05. Survival distributions were analyzed by the method of Kaplan-Meier. The statistical significance of differences in survival between groups was determined by log rank which compares differences along all points of the curve. Multivariate analysis was done using Cox regression while for categorical data, logistic regression was used.

Results:

There were 2524 cases of breast cancer identified in the 20 year study period. Of those, 112 women had bilateral breast cancer (4.4%). Among the patients with bilateral breast cancer 52 (46.4%) had synchronous cancers and 60 (53.6%) had metachronous breast cancers. Although the annual risk of breast cancer was increasing in our population during the study period especially from 2005 -2008, the risk of BBC remained constant for both synchronous and metachronous tumours (figure 1). The mean interval between the development of metachronous cancers was 5.9 years while the median was 3.9 years. Women with BBC were younger than the unilateral group at the time of the diagnosis of their initial tumours (p=0.021). Contralateral tumours of BBC patients measured significantly smaller than contemporary unilateral tumours (p=0.001) and their associated initial tumour of BBC (p=0.006). This difference was also present for synchronous tumours as the second primary was significantly smaller than the initial tumour (p=0.003).

No significant differences were found between the study groups for histological patterns and grades of tumours. The distribution of histopathological patterns was similar for all groups of patients with more than 70% of unilateral and bilateral patients having ductal carcinoma. Nevertheless, contralateral tumours of BBC patients were diagnosed at earlier stage than the initial tumours (p<0.001), a difference that is also noted for both synchronous (p=0.001) and metachronous (p=0.004) tumours.

Contralateral primaries of BBC, synchronous and metachronous tumours were found to be stage 0 or I more frequently than initial tumours and stage II and III less often. A similar significant difference was also seen when comparing the contralateral tumours to unilateral breast cancer (p<0.001).

Moreover, contralateral tumours were significantly more likely to be associated with negative axillary lymph nodes than initial tumours of BBC (p=0.005) and unilateral breast carcinoma (p<0.001). Again, this difference was noted to occur in synchronous (p=0.028) but not the metachronous cancers. Seventy-nine percent of BBC patients who underwent axillary lymph node dissection were lymph node negative, 11.1% had 1 -3 positive lymph nodes and 9.7% had more than 3 positive lymph nodes.

Regarding receptors status, there were significant differences in the rates of progesterone receptors (PR) positivity (p=0.002) and HER2/neu expression (p=0.018) but not the rate of estrogen receptor (ER) positivity (p=0.712) between the unilateral and contralateral tumours of BBC (table 1). Approximately 72% of unilateral tumours were PR positive and 20% were HER2/neu positive compared to 56% and 33%, respectively. In case of BBC no significant differences were noted in any of the receptors status when comparing initial and contralateral tumours.

Risk factors:

Many of the previously described risk factors for BBC were identified in our series of patients. More than 43% of patients who developed BBC have family history of breast cancer, 33% were using oral contraceptive and 14.3% were on postmenopausal hormones. The characteristics of the first tumour of BBC were analyzed in order to identify the risk factors for developing synchronous or metachronous tumours. Patients who presented with stage IV progesterone receptor positive initial tumour were more likely to develope synchronous tumours (Table 3).

Detection methods:

Details of methods of diagnosis for both tumours of BBC were available for 81 cases.

The initial tumours were detected during screening in 16% of cases compared to 64% of contralateral tumours. The sensitivity of mammography was 86% and 87.3% for initial and contralateral tumours, respectively. 57% of initial tumours were detected by mammography compared with 74.3% of contralateral tumours. Only one case (1.4%) of contralateral tumours required MRI scan to diagnose cancer (table 4).

Management of BBC in our institution:

Thirty-three (30.6%) patients with contralateral tumours and 777 (42.7%) of the unilateral cancer patients received adjuvant chemotherapy. Hormonal therapy was prescribed to 92% of unilateral cancer group, 86% of bilateral cancer patients when got the initial tumour and 74% of them when developed the contralateral tumour. Patients with BBC were more likely to have bilateral mastectomy (64.7%). Breast conserving surgery of both breasts was performed in 11.3% and in combination with mastectomy of the other breast in 24.1% of BBC patients. Of those who had mastectomy of contralateral breast, 19.4% had breast reconstruction performed (13.3% immediate, 6.1% delayed). No significant differences were noted in both surgical treatment (p=0.280) and radiation treatment (p=0.093) when comparing initial and contralateral tumours of BBC. On the other hand, adjuvant hormonal and chemotherapy were differentially prescribed with p values of 0.036 and 0.006 respectively. When comparing contralateral tumours of BBC with unilateral carcinoma, there were no differences in all management modalities received by patients .

Disease-free survival (DFS):

DFS was calculated as the number of months from the diagnosis of the initial tumour to recurrence (local and distant), death or last follow up. Recurrence occurred in 42% of patients with synchronous tumours, 55% of patients with metachronous and 21% of patients with unilateral breast carcinoma. The difference in DFS was significant between the bilateral cancer patients (p=0.013) as was the difference between the bilateral and unilateral cancer patients (p<0.001) (figure 2). Median time for DFS was 52 month for synchronous tumours, 148 month for metachronous and 169 month for unilateral tumours. On Cox regression analysis, initial tumours grade (p=0.021), stage (p=0.020) and nodal status (p=0.001), contralateral tumours grade (p=0.035) and PR status (p=0.018); and adjuvant chemotherapy for both tumours (p=0.007) were independent predictors of DFS in BBC patients. After adjustment for these variables both groups did not differ significantly with p value of 0.157 for bilateral and unilateral tumours and 0.284 for synchronous and metachronous tumours.

Overall survival:

There was no significant difference in overall survival of patients with bilateral and unilateral breast carcinoma (p=0.073), however, comparison of patients with synchronous disease to those with metachronous disease yielded a significant difference with p value of 0.010 (figure 3). Women with synchronous tumours had a high mortality from breast cancer with median survival of 62 months compared to 148 months in metachronous and 154 months in unilateral cancer patients. A multivariate Cox regression analysis was used to determine the prognostic factors for overall survival for all groups. ER (p<0.001) and PR (p=0.018) positivity, lymph nodes negativity (p=0.001) and radiotherapy (p=0.002) for the second primary tumour were found to be by far the most important factors. After simultaneous adjustment of all these variables there continue to be a significant difference in survival between synchronous and metachronous tumours (p=0.026).

Discussion:

The reported incidence of BBC is variable and may reach up to 20% in patients in whom breast cancer diagnosis has been made by contralateral biopsy and mastectomy in clinically negative breasts [START_REF] Carmichael | The longterm outcome of synchronous bilateral breast cancer is worse than metachronous or unilateral tumours[END_REF]. The Variations in reported incidence could be explained by the use of different analysis methods, variations in intensity of screening for contralateral tumours and differences in duration of follow up. In keeping with previous reports, the incidence of BBC in the present study was 4.4% of which 2.1% were synchronous and 2.3% were metachronous. However, as no minimum length of follow up was defined in this study to classify patients into unilateral breast cancer group, some unilateral cases would become bilateral cases with longer follow up. The annual risk of breast cancer was noticed to increase markedly in 2005 and after in our population. This observation not necessarily indicates an increase incidence of breast cancer but may be due to the fact that the screening programme was started in that year.

Numerous studies have found that patients with BBC were significantly younger at the time of diagnosis of their initial cancer and considered age as the most important predictor for contralateral breast cancer [START_REF] Chen | Epidemiology of Contralateral Breast Cancer[END_REF][START_REF] Carmichael | The longterm outcome of synchronous bilateral breast cancer is worse than metachronous or unilateral tumours[END_REF][START_REF] Adami | Age at first primary as a determinant of the incidence of bilateral breast cancer: Cumulative and relative risks in a population-based case-control study[END_REF][START_REF] Brenner | Clinical epidemiology of bilateral breast cancer[END_REF]. The earlier a woman develops a first breast cancer, the higher the risk of developing a contralateral tumour [START_REF] Rauschecker | Eigh-year results of a prospective non-randomized study on therapy of small breast cancer. The German Breast Cancer Study Group ( GBSG)[END_REF]. This finding was also observed in our series of patients. Increased risk in younger group might be due to increased life expectancy and the fact that patients with family history of breast cancer develop their cancer at an early age. Furthermore, we demonstrated smaller contralateral tumour size and earlier stage when compared with unilateral tumours and the initial tumours of BBC. These differences were also observed for both synchronous and metachronous diseases. Some studies described similar differences between initial and contralateral tumours of BBC [START_REF] Heron | Bilateral breast carcinoma: risk factors and outcomes for patients with synchronous and metachronous disease[END_REF][START_REF] Mertens | Contralateral Breast Cancer: Factors Associated with Stage and Size at Presentation[END_REF][START_REF] Abdalla | The impact of contralaterl breast cancer on the outcome of breast cancer patients treated by mastectomy[END_REF][START_REF] Samant | Diagnosis of metachronous contralateral breast cancer[END_REF] while others reported conflicting results [START_REF] Newman | A casecontrol study of unilateral and bilateral breast carcinoma patients[END_REF][START_REF] Brenner | Clinical epidemiology of bilateral breast cancer[END_REF][START_REF] Gajalakshmi | Survival from contralateral breast cancer[END_REF][START_REF] Irvine | Prognosis of synchronous bilateral breast cancer[END_REF]. These findings might be related to increased compliance with screening after development of the initial tumour. ER positivity was found to be predictive of bilateral disease previously [START_REF] Lesser | Multicentricity and bilaterality in invasive breast carcinoma[END_REF][START_REF] De La Rochefordiere | Simultaneous bilateral breast carcinoma: a retrospective review of 149 cases[END_REF], however, Coradini et al [START_REF] Coradini | Is steroid receptor profile in contralateral breast cancer a marker of independence of the corresponding primary tumour?[END_REF] described no differences in ER and PR positivity of the initial and contralateral tumours. In the current study, there was low rate of PR positivity in patients with bilateral tumours compared to those with unilateral disease.

Examining the HER2 status, we found that initial tumours of bilateral cancers significantly overexpress HER2/neu rather than unilateral tumours (p=0.018). Based on this we can postulate that HER2 over-expressers are more likely to develop contralateral tumour.

With regard to risk factors for developing BBC in our study, there was significant association of early stage (stage 0 and I) and progesterone receptor negative initial tumours and the development of metachronous tumours. The breast conservative surgery (BCS) for the initial tumour is a confounding factor as it usually performed in early stage disease.

Multiple reports in the literature demonstrated the increased sensitivity of MRI for detection of breast cancer compared with mammography and ultrasound, with MRI sensitivity approaching 100% [START_REF] Lee | MR Imaging Screening of the Contralateral Breast in Patients with Newly Diagnosed Breast Cancer: Preliminary Results[END_REF][START_REF] Lehman | MRI Evaluation of the Contralateral Breast in Women with Recently Diagnosed Breast Cancer[END_REF][START_REF] Kriege | Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition[END_REF][START_REF] Viehweg | MR imaging of the contralateral breast in patients after breast-conserving therapy[END_REF][START_REF] Lehman | Added cancer yield of MRI in screening the contralateral breast of women recently diagnosed with breast cancer: results from the International Breast Magnetic Resonance Consortium (IBMC) trial[END_REF]. However, most of these reports evaluated MRI in diagnostic settings. Lehman et al [START_REF] Lehman | MRI Evaluation of the Contralateral Breast in Women with Recently Diagnosed Breast Cancer[END_REF] investigated the role of MRI in detecting cancer in the contralateral breast that is missed by mammography and clinical examination at time of the initial tumour diagnosis. They were able to detect cancer in only 3.1% of the women who were enrolled in the study with biopsy performed depending on MRI results on 12.5%. Furthermore, Moore et al [START_REF] Moore | Costeffectiveness of MRI compared to mammography for breast cancer screening in a high risk population[END_REF] concluded that the use of breast MRI compared to mammography for screening in young women at high risk doesn't appear to be cost effective. In this study we showed that mammography is still a valid and convenient method of screening for breast cancer in women with initial breast cancer and MRI was able to detect only one mammography and clinically occult contralateral tumour. Although MRI can improve the detection of contralateral tumours, its use should be preserved for high risk patients and in conjunction with thorough clinical examinations and mammographic evaluation.

Adjuvant therapy generally is given to improve survival and to reduce incidence of contralateral breast cancer. Both chemotherapy and tamoxifen were found to be associated with reduced risk of contralateral breast cancer. Both Bertelsen et al [START_REF] Bertelsen | Effect of systemic adjuvant treatment on risk for contralateral breast cancer in the Women's Environment, Cancer and Radiation Epidemiology Study[END_REF] and Vogel et al [START_REF] Vogel | Effects of tamoxifen vs raloxifene on the risk of developing invasive breast cancer and other disease outcomes: the NSABP Study of Tamoxifen and Raloxifene (STAR) P-2 trial[END_REF] observed a significant reduction of incidence of contralateral primary breast cancer among women receiving adjuvant hormonal therapy. Adjuvant hormonal therapy also reported to reduce the incidence of BBC by 40% in both ER positive and ER negative patients [START_REF]Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists' Collaborative Group[END_REF]. Consistent with what was previously stated, in this study, adjuvant therapy was significantly associated with improved disease-free and over-all survival. Furthermore, adjuvant hormonal therapy was significantly more prescribed in unilateral cancer group compared to initial tumours of BBC, which might indicate its role in reducing the risk of developing a contralateral tumour The surgical management of BBC patients is an area for debate. The controversy is due to the conflicting data regarding the role of contralateral prophylactic mastectomy in preventing BBC, the belief of superiority of mastectomy over breast conserving surgery for management of contralateral tumours and the increasing risk of developing contralateral tumour after conserving breast surgery and radiotherapy for the initial cancer. Bedrosian et al. in their study using SEER database, showed that contralateral prophylactic mastectomy is associated with small improvement in 5-years survival, mainly in young women with stages I-II disease ER-negative breast cancer [START_REF] Bedrosian | Population-Based Study of Contralateral Prophylactic Mastectomy and Survival Outcomes of Breast Cancer Patients[END_REF].

However; most of previous reports confirmed that contralateral prophylactic mastectomy is unlikely to be beneficial in improving disease specific survival if performed outside of the existing recommendations [START_REF] Quan | Incidence and outcomes of contralateral breast cancers[END_REF][START_REF] Peralta | Contralateral prophylactic mastectomy improves the outcome of selected patients undergoing mastectomy for breast cancer[END_REF]. Moreover, measurable morbidity rates and negative psychological impacts were found to be associated with prophylactic mastectomy [START_REF] Zion | Reoperations after prophylactic mastectomy with or without implant reconstruction[END_REF][START_REF] Frost | Satisfaction after contralateral prophylactic mastectomy: the significance of mastectomy type, reconstructive complications, and body appearance[END_REF]. Secondly, BCT was described as a safe and efficacious treatment option for treating early stage cancer [START_REF] Lee | Efficacy of Breast Conservation Therapy in Early Stage Bilateral Breast Cancer[END_REF][START_REF] De La Rochefordiere | Simultaneous bilateral breast carcinoma: a retrospective review of 149 cases[END_REF][START_REF] Gollamudi | Breast-conserving therapy for stage I-II synchronous bilateral breast carcinoma[END_REF][START_REF] Yamauchi | Bilateral Breast Cancer: Results and Consideration of Radiation Technique[END_REF]. Yamauch et al [START_REF] Yamauchi | Bilateral Breast Cancer: Results and Consideration of Radiation Technique[END_REF] demonstrated no local recurrence or distant metastasis in patients who underwent bilateral BCT after a median follow up of 95 months. Lee et al [START_REF] Lee | Efficacy of Breast Conservation Therapy in Early Stage Bilateral Breast Cancer[END_REF] also noticed no differences in survival in bilateral and unilateral caner patients treated with BCT. In addition, Rochefordiere et al [START_REF] De La Rochefordiere | Simultaneous bilateral breast carcinoma: a retrospective review of 149 cases[END_REF] found no significant difference in survival comparing 51 patients with synchronous BBC treated with bilateral BCT to a group of patients treated with either bilateral mastectomy or unilateral BCT. Finally, most studies have demonstrated no significant increase risk of contralateral breast cancer after radiation treatment for initial tumour [START_REF] Bernstein | Risk factors predicting the incidence of second primary breast cancer among women diagnosed with a first primary breast cancer[END_REF][START_REF] Gajalakshmi | Risk factors for contralateral breast cancer in Chennai (Madras), India[END_REF][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], while others documented the opposite [START_REF] Boice | Cancer in the contralateral breast after radiotherapy for breast cancer[END_REF][START_REF] Storm | Adjuvant radiotherapy and risk of contralateral breast cancer[END_REF][START_REF] Gao | Risk of second primary cancer in the contralateral breast in women treated for early-stage breast cancer: a population-based study[END_REF]. We observed no significant association between initial tumour radiation and the development of contralateral cancer. Also we noticed that the second tumours were less likely to get radiation therapy compared to the unilateral tumours due to the fact that most of the contralateral cancer patients end up with mastectomy rather than BCT.

Previous studies comparing survival between women with bilateral versus unilateral breast cancer have yielded inconsistent results. Some studies found that BBC had a poorer prognosis than unilateral cancer [START_REF] Jobsen | Synchronous, bilateral breast cancer: prognostic value and incidence[END_REF][START_REF] Abdalla | The impact of contralaterl breast cancer on the outcome of breast cancer patients treated by mastectomy[END_REF][START_REF] Gajalakshmi | Survival from contralateral breast cancer[END_REF], whereas others showed similar prognosis for both types [START_REF] Heron | Bilateral breast carcinoma: risk factors and outcomes for patients with synchronous and metachronous disease[END_REF][START_REF] Newman | A casecontrol study of unilateral and bilateral breast carcinoma patients[END_REF][START_REF] Irvine | Prognosis of synchronous bilateral breast cancer[END_REF][START_REF] Takahashi | The impact of bilateral breast cancer on the prognosis of breast cancer: a comparative study with unilateral breast cancer[END_REF][START_REF] Verkooijen | Survival after bilateral breast cancer: results from a population-based study[END_REF]. These variations might be related to the small number of patients, variable definitions of synchronous and metachronous cancers and the use of different methods as calculation of survival from the time of the initial or the second tumour development. Carrying out multivariate analysis we were unable to identify significant differences in both disease-free and over-all survival between bilateral and unilateral breast cancer patients, while in BBC patients synchronous tumours were significantly associated with poor over-all survival. One of the reasons metachronous tumours seem to do better is because they are smaller and detected earlier by surveillance. We also identified factors like adjuvant chemotherapy, PR and ER positivity, degree of invasion and grade of the contralateral tumour as important prognostic indicators in BBC patients. In contrast, factors like histological types have no effect on BBC survival. This finding is in keeping with Mhuircheartaigh et al [START_REF] Mhuircheartaigh | Prospective matched-pair comparison of outcome after treatment for lobular and ductal breast carcinoma[END_REF] who found no difference in outcome of invasive ductal and lobular breast cancer in our unit

Conclusion:

Significant differences in some characteristics of bilateral compared to unilateral breast cancer were identified in this study. However; BBC does not appear to have a major impact on outcome beyond the initial primary tumour. The increasing practice of bilateral mastectomy is not supported by this large single institutional experience which allows confident prediction of outcome based on accurate follow-up.

Figure 1 :Figure 2 :

 12 Figure 1: Annual risk of bilateral breast cancer compared to unilateral tumours diagnosed in the period from 2005 -2008

Figure 3 :

 3 Figure 3: Over-all survival analysis. (A) In bilateral compared to unilateral breast cancer patients. No significant difference in over-all survival between both groups as determined by Kaplan-Meier survival analysis (X 2 =3.2, p=0.073) and Cox-regression (p=0.639). (B) In synchronous compared to metachronous bilateral breast cancer. The significantly reduced over-all survival in synchronous tumours was confirmed by both Kaplan-Meier analysis (X 2 =6.6, p=0.010) and Cox-regression (p=0.026).

Table 1 :

 1 Characteristics of bilateral breast cancer compared to unilateral disease First breast of bilateral tumours vs. unilateral, b Second breast of bilateral tumours vs. unilateral, c First breast vs. second breast of bilateral cancer, Φ t-test, # Mann-Whitney u test, *statistically significant.

	Variable	Unilateral	Bilateral (n =112)	P value a P value b P value c
		(n=2524)	1st breast	2nd breast
	Age in yrs (SD) Φ				0.021*	0.725	0.045*
	Mean	57.98 (13.71)	55.1 (12.9)	58.45 (13.3)
	Median (range)	57 (20-96)	54 (29-92)	58 (33-93)
	Tumour size (mm) #				0.461	0.001*	0.006*
	Mean	23.1	27.24	18.23
	Median (range)	20 (1-160)	20 (1-130)	15(1-140)
	Nodal status				0.327	<0.001* 0.005*
	0	736 (48.7%)	47 (54.7%)	57 (79.2%)
	1-3	388 (25.7%)	23 (26.7%)	8 (11.1%)
	>3	388 (25.7%)	16 (18.6%)	7 (9.7%)
	UICC stage				0.256	<.0.001* <0.001*
	0	167 (8.6%)	3 (3.1%)	15 (15.2%)
	I	456 (23.3%)	26 (26.8%)	29 (29.3%)
	II	744 (38.1%)	33 (34%)	22 (22.2%)
	III	436 (22.3%)	26 (26.8%)	9 (9.1%)
	IV	150 (7.7%)	9(9.3%)	24 (24.2%)
	PR status				0.226	0.002*	0.271
	Positive	979 (72.5%)	44 (65.7%)	46 (56.8%)
	Negative	372 (27.5%)	23 (34.3%)	35 (43.2%)
	Her-2 status				0.938	0.018*	0.168
	Positive	203 (20.4%)	9 (20.9%)	20 (33.3%)
	Negative	790 (79.6%)	34 (79.1%)	40 (66.7%)
	Surgical treatment				<0.001* <0.001* 0.280
	Mastectomy	1442 (68.3%)	85 (78.7%)	78 (72.9%)
	BCS	668 (31.7%)	18 (16.7%)	18 (16.8%)
	No surgery	0	5 (4.6%)	11 (10.3%)
	Hormonal therapy				0.026*	<0.001* 0.036*
	Yes	1652 (92.5%)	90 (86.5%)	68 (74.7%)
	No	133 (7.5%)	14 (13.5%)	23 (25.3%)
	Chemotherapy				0.217	0.013*	0.006*
	Yes	777 (42.7%)	54 (48.6%)	33 (30.6%)
	No	1044 (57.3%)	57 (51.4%)	75 (69.4%)
	Radiotherapy				0.153	<0.001* 0.093
	Yes	1058 (59.2%)	57 (52.3%)	42 (40.8%)
	No	728 (40.8%)	52 (47.7%)	61 (59.2%)

a

Table 2 :

 2 Bilateral breast cancer characteristics (synchronous vs. metachronous) Synchronous first breast vs. synchronous second breast, b Metachronous first breast vs. Metachronous second breast, c Synchronous first breast vs. Metachronous first breast, d Synchronous second breast vs. Metachronous second breast, Φ t-test, # Mann-Whitney u test, *statistically significant.

		Synchronous Bilateral Metachronous Bilateral P value a P value b P value c P value d
	Variable	1st breast 2nd breast 1st breast 2nd breast
	Age in yrs Φ				0.890	0.006* 0.056	0.676
	Mean (SD)	57.5 (13.9)	57.9 (14.4)	52.8 (11.6)	58.9(12.3)
	Median (range)	57 (33-92)	57 (33-93)	52 (29-82)	58(33-84)
	Tumour size (mm) #				0.003* 0373	0.063	0.394
	Mean	32.5	18.77	22.27	17.85
	Median (range)	24 (1-130)	12 (1-140)	20 (1-100)	15 (1-82)
	Nodal status				0.028* 0.083	0.567	0.586
	0	22 (55%)	27 (84.4%)	24 (53.3%)	30 75%)
	1-3	9 (22.5%)	3 (9.4%)	14 (31.1%)	5 (12.5%)
	>3	9 (22.5%)	2 (6.3%)	7 (15.6%)	5 (12.5%)
	UICC stage				0.001* 0.004* 0.048* 0.374
	0	0	10 (22.2%)	3 (6.3%)	5 (9.3%)
	I	10 (20.4%)	12 (26.7%)	15 (31.9%)	17 (31.5%)
	II	17 (34.7%)	11 (24.4%)	16 (34%)	11 (20.4%)
	III	14 (28.6%)	3 (6.7%)	12 (25.5%)	6 (11.1%)
	IV	8 (16.3%)	9 (20%)	1 (2.1%)	15 (27.8%)
	PR status				0.133	0.584	0.003* 0.249
	Positive	31 (79.5%)	23 (63.9%)	12 (44.4%)	23 (51.1%)
	Negative	8 (20.5%)	13 (36.1%)	15 (55.6%)	22 (48.9%)
	Her-2 status				0.152	0.800	0.043* 0.464
	Positive	4 (13.3%)	8 (28.6%)	5 (41.7%)	12 (37.5%)
	Negative	26 (86.7%)	20 (71.4%)	7 (58.3%)	20 (62.5%)
	Surgical treatment				0.292	0.194	0.011* 0.709
	Mastectomy	42 (85.7%)	36 (73.5%)	42 (72.4%)	42 (72.4%)
	BCS	3 (6.1%)	7 (14.3%)	15 (25.9%)	11 (19%)
	No surgery	4 (8.2%)	6 (12.2%)	1 (1.7%)	5 (8.6%)
	Reconstruction				0.816	0.012* 0.003* 0.495
	Immediate	9 (19.6%)	7 (15.2%)	0	6 (11.5%)
	Delayed	3 (6.5%)	4 (8.7%)	7 (13.5%)	2 (3.8%)
	No	34 (73.9%)	35(76.1%)	45 (86.5%)	44 (84.6%)
	Hormonal therapy				0.572	0.028* 0.444	0.034*
	Yes	42 (89.4%)	35 (85.4%)	48 (84.2%)	33 (66%)
	No	5 (10.6%)	6 (14.6%)	9 (15.8%)	17 (34%)
	Chemotherapy				0.036* 0.060	0.130	0.254
	Yes	29 (56.9%)	18 (36%)	25 (42.4%)	15 (25.9%)
	No	22 (43.1%)	32 (64%)	34 (57.6%)	43 (74.1%)

a

Table 3 :

 3 Characteristics of the first tumour of BBC and the risk of developing synchronous vs. metachronous contralateral tumour.

		First	Second tumour of BBC
	Variable	tumour of BBC	Synchronous Metachronous	P value
	UICC stage				0.048*
	0	3	0%	100%
	I	25	40%	60%
	II	33	51.5%	48.5%
	III	26	53.8%	46.2%
	IV	9	88.9%	11.1%
	PR status				0.004*
	Positive	43	72.1%	27.9%
	Negative	23	34.8%	56.2%
	Surgical treatment				0.011*
	Mastectomy	84	50%	50%
	BCS	18	16.7%	83%
	No surgery	5	80%	20%

Φ t-test, # Mann-Whitney u test, *statistically significant.

Table 4 :

 4 Diagnosis of bilateral breast cancerfirst breast vs. second breast

	Variable	1st breast	2nd breast
	Presentation
	Symptomatic	65 (83.3%)	29 (35.8%)
	Screening	13 (16.7%)	52 (64.2%)
	Mode of detection
	Clinically	27 (41.6%)	16 (21.6%)
	Ultrasound	1 (1.5%)	2 (2.7%)
	Mammogram	37 (56.9%)	55 (74.3%)
	MRI	0	1 (1.4%)
	Mammogram efficiency
	Detected tumours	37 (86%)	55 (87.3%)
	Undetected tumours	6 (14%)	8 (12.7%)

Acknowledgements

The authors would like to acknowledge the National Breast Cancer Research Institute (NBCRI) for their financial support of the study