Open Access Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast
2005

Ethel-Michele De Villiers
Division for the Characterization of Tumorviruses
Deutsches KrebsforschungszentrumHeidelbergGermany

Robert E Sandstrom
Lower Columbia Pathologists
LongviewWashingtonUSA

Harald Zur Hausen
Division for the Characterization of Tumorviruses
Deutsches KrebsforschungszentrumHeidelbergGermany

Charles E Buck
Lower Columbia Pathologists
LongviewWashingtonUSA

Open Access Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast

Breast Cancer Res
71200510.1186/bcr940)htReceived: 10 May 2004 Revisions requested: 9 Jul 2004 Revisions received: 7 Aug 2004 Accepted: 7 Sep 2004Available online R1 Research article Corresponding author: Ethel-Michele de Villiers, firstname.lastname@example.com
Background Viruses including Epstein-Barr virus (EBV), a human equivalent of murine mammary tumour virus (MMTV) and human papillomavirus (HPV) have been implicated in the aetiology of human breast cancer. We report the presence of HPV DNA sequences in areolar tissue and tumour tissue samples from female patients with breast carcinoma. The presence of virus in the areolar-nipple complex suggests to us a potential pathogenic mechanism.Methods Polymerase chain reaction (PCR) was undertaken to amplify HPV types in areolar and tumour tissue from breast cancer cases. In situ hybridisation supported the PCR findings and localised the virus in nipple, areolar and tumour tissue.ResultsPapillomavirus DNA was present in 25 of 29 samples of breast carcinoma and in 20 of 29 samples from the corresponding mamilla. The most prevalent type in both carcinomas and nipples was HPV 11, followed by HPV 6. Other types detected were HPV 16, 23, 27 and 57 (nipples and carcinomas), HPV 20, 21, 32, 37, 38, 66 and GA3-1 (nipples only) and HPV 3, 15, 24, 87 and DL473 (carcinomas only). Multiple types were demonstrated in seven carcinomas and ten nipple samples. Conclusions The data demonstrate the occurrence of HPV in nipple and areolar tissues in patients with breast carcinoma. The authors postulate a retrograde ductular pattern of viral spread that may have pathogenic significance.

Introduction

Breast cancer is the most frequently diagnosed cancer among women in the USA, and the incidence of breast carcinoma has increased by more than 40% over 25 years [1]. The aetiology of breast cancer remains unknown. Many risk factors have been associated with the pathogenesis of this disease, including family history, hormones, cigarette smoking and alcohol consumption [2][3][4][5][6][7]. Hormones, cigarette smoking and family history have also been demonstrated to enhance infections with papillomaviruses, mainly the highrisk human papillomavirus (HPV) types involved in the aetiology of cervical carcinoma [8].

Viral infection as an aetiological factor has been addressed in other studies. The data obtained from studies investigating the presence of viral sequences in breast cancer biopsies and cell lines have been controversial. A role for herpes viruses, and specifically Epstein-Barr virus (EBV), in the aetiology of this cancer has been questioned [9,10].

A human equivalent of the murine mammary tumour virus (MMTV) has been described in breast tumours, as well as in breast cancer cell lines [11]. These results have been questioned and negated by others (R Schmidt and H zur Hausen, unpublished results) [12]. Immortalisation of normal breast epithelial cells by the HPV types HPV 16 and HPV 18 [13] has been used to study the functions of the viral early genes E6 and E7 in different cellular pathways [14][15][16][17][18]. However, the presence of HPV in malignant tumours of the breast has been controversial. Several studies reported positive results [19][20][21][22][23][24][25], whereas others reported negative results [26][27][28]. In the present study we report the presence of HPV DNA in biopsy samples from mammary carcinomas, as well as these viral sequences in the tissue of the corresponding mamilla.

R2

Materials and methods

Samples and DNA extraction

Samples from breast tumour tissue for the HPV analysis were supplied by two of the authors (RES and CEB). The 29 cases used for the retrospective study and demonstration of papillomavirus sequences were selected from routine mastectomy specimens undertaken in a course of treatment for carcinoma of the breast. Case selection was based on the availability of adequate nipple and tumour tissue from the same patient and the presence of histological features suggestive of HPV infection in nipple and areolar sections. The patients, all female, ranged in age from 30 to 88 years, with an average age of 61.3 years. Twelve tumours arose in the right breast and 17 in the left. Five tumours were subareolar and 24 were located at a distance from the nipple and areola. Tumours ranged in size from 6 mm to 15 cm. One patient received cytoxan chemotherapy before surgery. In all other cases primary treatment was surgical as represented by the specimen. One patient had undergone contralateral mastectomy ten years earlier for a localised breast cancer, which was judged clinically and pathologically to represent a separate primary tumour. Among the 29 patients there were three with remote histories of malignancy without evidence of recurrence (one cutaneous malignant melanoma, one renal cell carcinoma and one soft-tissue malignancy). One patient had undergone hysterectomy for high-grade cervical dysplasia. There were no patients with documented cervical carcinoma. No patients were immune-impaired or immunosuppressed.

Formalin-fixed paraffin-embedded samples from both the nipple (n = 29) and the carcinoma of the breast (n = 29) from the same patient were sectioned, and the total DNA was extracted from each sample. Great care was taken during sectioning to avoid any contamination between samples. Two different individuals sectioned the blocks in several small random groups at different time intervals over a period of one week. The microtome was cleaned thoroughly and exposed to ultraviolet between samples; in addition, new blades were used for each sample. Deparaffinisation was performed by rotation overnight in 1 ml of xylene, followed by centrifugation and subsequent removal of the supernatant. This step was repeated twice (each for one hour) with fresh xylene. The xylene was in turn removed by rotation in 1 ml of 100% ethanol for one hour followed by centrifugation and subsequent replacement of the supernatant with 90% ethanol for 45 min, and repeated by steps of 80% ethanol for 45 min and 70% ethanol for 45 min. Samples were freeze-dried, and total cellular DNA was extracted as described previously [29]. The DNA was digested with Proteinase K and subjected to extraction with phenol followed by extraction with chloroform/isoamyl alcohol. The extracted DNA was precipitated with ethanol and the pellet was resuspended in 10 mM Tris/HCl, pH 8.0.

PCR analysis and cloning

DNA (50-100 µg) of each sample was amplified by polymerase chain reaction (PCR). The quality of the DNA obtained from the fixed samples was controlled by amplification with primers to detect the β-actin gene [30]. The primers RS42 and KM29 were initially used to amplify the 536-base-pair fragment of the β-actin gene. If this size of fragment could not be amplified, the primers PC03 and PC04 were used to amplify the 110-base-pair (bp) fragment of the β-actin gene. Papillomavirus sequences were amplified by three different methods, each targeting highly conserved regions within the L1 open reading frame of papillomaviruses. These included the GP5 + /GP6 + primers [31], the CP primers using modified conditions as previously described [32] and the FAP primers [33]. All three primer combinations are routinely used on individual samples tested in our laboratory. We initially modified the PCR conditions for each described method to include the amplification of the largest number of individual HPV types possible. Cloned DNA of each known HPV type was used to optimise these conditions. Amplification with the GP primers was performed in 2 mM MgCl 2 using 40 cycles with an annealing temperature of 40°C. The expected size of the amplicon was 140-150 bp. The expected size with the FAP primers is 480 bp after 45 cycles in 3.5 mM MgCl 2 and an annealing temperature of 50°C. The nested amplification approach with the CP primers results in an amplicon size of 370-400 bp. The initial amplification was performed in 2 mM MgCl 2 and 40 cycles at 50°C annealing temperature, after which an aliquot was re-amplified with nested primers but with the same PCR conditions. All amplicons were eluted after gel electrophoresis, purified and cloned. At least ten fragments of each amplicon were sequenced. Sequencing was performed with either the Sequenase 2.0 DNA Sequencing Kit (USB, Cleveland, OH) or an ABI Model Sequencer with Big Dye Terminator chemistry (Perkin Elmer Applied Biosystems Division, Dreieich, Germany). All precautions were taken to avoid contamination before or during extraction of the DNA from the embedded formalinfixed samples. After initial results had been obtained, a second sectioning was performed on the same embedded samples and DNA was again extracted to ensure that no contamination had taken place during the first round of experiments. This second round of analyses was performed in different rooms and by different individuals.

Sequence analysis

Sequences were compared with all available data banks with the aid of the Husar software package (Deutsches Krebsforschungszentrum).

In situ hybridisation

In situ hybridisation was performed as described by Kawase and colleagues [34]. Papillomavirus probes were digoxigenin-labelled by PCR in accordance with the manufacturer's instructions (PCR DIG Probe Synthesis Kit, catalogue no. 1636090; Roche, Mannheim, Germany). The FAP primers were used to label HPV 6 and HPV 11 DNA and the CP primers to label HPV 16 DNA. Denaturation was performed in a Biozym cycler for 5 min at 95°C followed by hybridisation overnight at 37°C under stringent conditions as described previously [35]. This was followed by washing with solutions in accordance with instructions in the Genpoint Kit (catalogue no. K0620; Dako, Hamburg, Germany). Anti-digoxigenin-AP antibodies (anti-digoxigenin-AP, Fab fragments, catalogue no. 1093274; Roche), blocking reagent (catalogue no. 1096176; Roche) and Nitro Blue Tetrazolium/5-bromo-4-chloroindol-3-yl phosphate (catalogue no. 1681451; Roche) were applied for visualisation of the signals. Sections from each sample were used for in situ hybridisation. Reading of the samples was blinded and performed by one of us (HzH).

Results

Nipple specimens were reviewed and selected by two of us (CEB, RES) for histological features consistent with human papillomavirus infections. Features identified included epithelial hyperplasia, hypergranulosis, parakeratosis, and distorted nuclear shape associated with nuclear clearing and pyknosis suggestive of koilocytosis. In addition, squamous metaplasia of lactiferous ducts and shedding of metaplastic cellular elements into lactiferous ducts and sinuses were noted ( Fig. 1). In addition to the 29 invasive carcinomas, ductal carcinoma in situ was identified in lactiferous ducts in four samples and small benign papillomas were present in three samples. Most invasive carcinoma samples (18 of 29) displayed a duct cell carcinoma pattern (Table 1). Other types included medullary carcinoma (five cases), tubular carcinoma (three cases), lobular carcinoma (two cases) and mucinous carcinoma (one case). DNA extracted from the samples taken from the nipple and the corresponding carcinoma of each breast (29 pairs) was analysed by PCR for the presence of papillomavirus sequences. The quality of the DNA was controlled by amplification of the β-actin gene. The 536-base-pair fragment of this gene was amplified in 47 of 58 samples. The 110-base-pair fragment was amplified in the remaining 11 samples (Table 2). Intact papillomavirus particles are resistant to degeneration. Extraction of DNA from fixed tissue can harbour intact viral genomes even under conditions in which the cellular DNA has been degraded. The three different methods used for amplification of the papillomavirus DNA were chosen to ensure amplification of all known papillomavirus types, as well as putative new types. All three primer combinations (GP, CP and FAP) are routinely used on individual samples tested in our laboratory. We initially modified the PCR conditions for each described method to include the amplification of the largest number of individual HPV types possible. Cloned genomic DNA of each known HPV type was used to optimise these conditions. The GP primers have been designed to amplify the majority of papillomavirus types associated with mucosal lesions. The FAP primers, in contrast, were initially designed to amplify the majority of papillomavirus types associated with cutaneous lesions. The amplicon size described is 480 bp, but in our hands smaller fragments identified as papillomavirus sequences (after cloning and sequencing) were also generated, depending on the HPV type. The CP primers were originally designed to amplify all the epidermodysplasia verruciformis-associated HPV types, but under modified conditions a large spectrum of mucosal types are amplified as well. Combining the three methods with the modified PCR conditions allows us to amplify all known HPV types with more or less equal efficiency. The amplicons generated, using any of the described methods, often constitute cellular sequences depending on the viral load of the sample.

Normal breast and areolar tissue was not available for use as control samples. The described methods of testing have been used routinely in our laboratory over several years in the analyses of tissues from a large variety of organs. We have analysed large numbers of samples from benign and malignant tumours of the oesophagus, head and neck and skin, as well as normal skin tissue for the presence of papillomavirus DNA with identical methods. HPV positivity varied depending on the type of tumour and normal tissue [29,[36][37][38] (E-MdV, unpublished data). The present study describes the first series of tissue of the breast analysed in our laboratory for the presence of papillomavirus DNA. Sequence analyses showed that 25 (86%) of the breast carcinoma samples and 20 (69%) of the samples from the mamilla harboured papillomavirus sequences. The results for each sample using each of the primer combinations are presented in Table 2. Only one pair of samples were both negative for HPV DNA. HPV DNA was present in 17 (59%) pairs of samples, namely nipple as well as carcinoma, whereas papillomavirus DNA was detected only in the carcinomatous tissue in eight cases (28%) and only in the nipple in three cases (10%) ( Table 3). HPV 11 DNA was present in both carcinoma and nipple samples in seven cases, HPV 6 in one case and HPV 57 in two cases. The most prevalent HPV types were HPV 11 (19 carcinomas and 8 nipples) followed by HPV 6 (7 carcinomas and 6 nipples) ( Table 4)  Histological features of the carcinomatous breast (magnification × 100) Histological features of the carcinomatous breast (magnification × 100). Detailed histology of (a) is seen in (b-d). AS, anucleate squames; asterisks, interstitial lymphocytic infiltrates; G, galactophore; K, keratin; LS, lactiferous sinus; LD, lactiferous duct; SG, sebaceous gland (tangenial cut); SGD, sweat gland duct; SH, simple epithelial hyperplasia; PH, papillary epithelial hyperplasia.

In situ hybridisation was performed on all samples that had previously been positive by PCR amplification for HPV 6, HPV 11 or HPV 16. Positive signals were clearly distinguishable from the surrounding negative tissue (Fig. 2). All carcinoma samples that had been negative for one of these three HPV types by PCR were also subjected to in situ hybridisation with each of the respective labelled HPV probes. No positive hybridisation signal was observed in any of these samples. In situ hybridisation supported the presence of HPV 6, 11 and 16 in nipple and areolar samples and in all tumour tissue in which these type-specific HPV sequences were detected by PCR analysis.

Discussion

The ubiquitous distribution of papillomavirus infections of the skin and genital and oral mucosa has been documented [39][40][41]. Infection with specific papillomavirus types has been shown to be a necessary but not sufficient cause in the pathogenesis of malignant genital tumours. The malignant tumours generally develop after long latency periods during which additional cellular modifications occur within the infected cell [8]. The E6 and E7 genes of the most prevalent high-risk HPV types, HPV 16 and HPV 18, modulate cellular pathways, thereby regulating proliferation and cell survival [8,42]. In contrast, the E6 and E7 proteins of the low-risk types, HPV 6 and HPV 11, do not influence these cellular pathways in the same manner, although they have occasionally been demonstrated in premalignant and malignant tumours. Additional cofactors are probably needed to modulate cellular proteins so as to immortalise and transform the infected cells [8]. The mechanism through which other HPV types, mainly associated with cutaneous lesions, might be involved in the pathogenesis of tumours has received little attention. However, preliminary data indicate that several molecular pathways are probably followed, depending on the HPV type involved [43].

The detection of papillomavirus infections in tissues largely depends on the method used. Described methods vary greatly in their sensitivity and specificity. The HPV types HPV 6, 11, 16, 18, 31, 33, 35, 39, 52 and 58 are frequently associated with genital lesions and are therefore most often targeted for HPV detection. Tests to demonstrate any of the 96 characterised HPV types in tissues require extensive analyses, including the sequencing of cloned amplim-  ers. Most published studies therefore used methods restricted to the detection of specific, single, or combinations or groups of HPV types. The use of type-specific primers may increase the number of positive samples but is biased with regard to the HPV types involved, because other HPV types present cannot be detected.

The present study investigated breast samples by using DNA PCR amplification of two conserved regions within the L1 open reading frame of the papillomavirus genome. Two of the primer combinations used (CP and GP primers) amplify an overlapping region within the L1 open reading frame. By using all three primer combinations to amplify the DNA of a single sample we are able to detect all known HPV types and also putative new HPV types. In addition, the cloning of the amplified product, in combination with sequencing of the cloned inserts, allows us to distinguish individual HPV types present in one sample, including multiple infections in one sample. Studies applying the techniques described here have demonstrated a larger spectrum of papillomavirus types associated with different types of benign and malignant tumours and argue that the historical grouping into 'mucosal' and 'cutaneous' HPV types can no longer be upheld [44]. We are not aware of any study outside our group that has analysed tissues for the presence of infection by any HPV types as extensively as the study described here.

Other investigators have analysed nipple and areolar specimens for the presence of HPV DNA. HPV 6/11 was detected in one of 20 papillomas of the nipple, whereas ten nipple duct adenomas were negative [45] and seven lowrisk and high-risk HPV types were not present in 20 cases of Paget's disease of the nipple [46]. Single cases of papillomas of the nipple resembling condylomata acuminata harboured HPV 6 DNA [47] and HPV 41 DNA [48]. Reports on the detection of HPV 16, HPV 18 and/or HPV 33 DNA in breast carcinoma samples range between 11% and 68% positivity [19][20][21][22][23][24][25]. The present study detected HPV 16 DNA in 7% of the carcinoma and in 10% of the nipple samples. The only other high-risk HPV type, HPV 66, was present in two samples from the nipple. The reasons for the differences in published reports are unclear, but may be attributed to numbers of samples tested, methodological differences or the demographics of the samples tested. In contrast, several other studies failed to demonstrate HPV sequences in tumours of the breast: all of 15 papillomas and 28 breast carcinoma samples were negative for HPV 6, 16 and 18 DNA [26], no HPV 16 and HPV 18 DNA could be detected in 26 fine needle aspirates and four breast carcinoma biopsies [28], and no HPV DNA was found in 80 samples of breast carcinomas [27]. HPV 16 and HPV 18 oncogenes immortalise normal human mammary cells under in vitro conditions [14][15][16][17][18]. It is unclear whether these data truly represent the in vivo situation.

The present study detected papillomavirus sequences in 86% (25/29) of the breast carcinoma samples and 69% (20 of 29) of the nipple samples. HPV 6 and HPV 11 infections were most prevalent in our collection of samples. Either of the two HPV types was present in 20 of 29 (69%) of the breast carcinoma biopsies and in 12 of 29 (41%) of the samples of the mamilla. We detected the simultaneous presence of more than one HPV type in about one-third of the samples. HPV types associated with mucosal lesions, and also several associated with skin lesions, were detected. HPV 27 and HPV 57 are closely related HPV types. HPV 27 is very frequently found in cutaneous warts, whereas HPV 57 has been demonstrated in mucosal and cutaneous lesions [49]. Several other HPV types found in the breast samples have historically been associated with the rare hereditary disease epidermodysplasia verruciformis but have more recently been shown in nonmelanoma skin cancers and their precursors in both immunosuppressed and immunocompetent patients [44].

The confirmation of PCR-derived evidence of HPV sequences in nipple, areolar and tumour tissue by in situ hybridisation in our hands lends substantive support to the conjecture that viral sequences are present and not an artefact in the tissues we have examined. The absence of HPV DNA in several samples, as well as the detection of different HPV types in the carcinoma versus the nipple of the same patient, might be attributed to the viral load at the time of sampling in combination with the sensitivity of the tests performed. The tissue sections also harboured unaffected tissue, leading to the dilution of virus-containing cells. In addition, the amplification of viral DNA by PCR with degenerate or consensus primers will detect replicating viral sequences and is not sensitive enough to detect all individual HPV types with equal sensitivity in a mixed population of cells. The in situ hybridisation technique is also less sensitive and will not detect a single viral genome copy per cell.

HPV 6 and HPV 11 are regarded as low-risk HPV types because they have only rarely been demonstrated in premalignant or malignant tissue, and the early genes E6 and E7 do not immortalise primary keratinocytes in vitro [50]. HPV 6 DNA has been found in carcinomas [51,52] and giant condylomas (Buschke-Löwenstein tumours) [53,54]. The molecular mechanism by which these low-risk HPV types induce or participate in the transformation of cells has not been resolved. Duplications of the long control region [54][55][56][57], minimal sequence dissimilarities [58,59] and integration [60] of HPV 6 and/or HPV 11 genomes have been shown in malignant tissue.

The mere presence of the low-risk HPV 6 and 11 in most of the mamillae and in the concomitant breast carcinoma samples does not prove its role in the aetiology of the disease, although it merits further investigation. Unfortunately,   because of the previous fixation of our samples, RNA from these tumours was not available for the detection of viral transcripts as additional support of our results. Relatively few studies have been performed in an attempt to unravel the intracellular mechanisms through which HPV 6 or 11 may immortalise cells, and information is still fractional in comparison with data on the high-risk HPV types. Introduction of the HPV 6 E6 into normal mammary cells leads to immortalisation and reduced levels of the p53 protein [61]. Binding of the HPV 6 E6 protein modulates the function of the cellular proteins Bak [62], Gps2 [63] and Zyxin [64]. Additional in vitro studies investigating the influence of hormones, smoke adducts and genetic factors on the interaction of the HPV 6 and 11 proteins with cellular proteins should provide valuable information on a possible role for HPV 6 and 11 in the pathogenesis of breast carcinoma. The data presented in this study also indicate a strong need for epidemiological studies correlating HPV, cigarette smoking, hormone use and family history to substantiate in vitro findings.

This study suggests that human papillomavirus may infect the epithelium of the nipple and areola. Human papillomavirus infection may be identified with recognisable histological features comparable to human papillomavirus infection at other sites. Furthermore, this study is consistent with a pathogenic mechanism involving transfer in a retrograde fashion via the nipple, areola, lactiferous ducts and sinuses of the human papillomavirus. If confirmed, the pathogenic mechanism proposed may have significant implications for the prevention and treatment of breast carcinoma and the identification of individuals at risk for carcinoma development. Examination of cellular samples of nipple and areola inclusive of cytological examination and methods for identifying the presence of human papillomavirus might aid early diagnosis and perhaps therapy.

Conclusions

The data demonstrate the occurrence of HPV in nipple and areolar tissues in patients with breast carcinoma. The presence of papillomavirus DNA in most mamillae and concomitant breast carcinoma samples merits further investigation.

Competing interests

The author(s) declare that they have no competing interests.

EBV = Epstein-Barr virus; HPV = human papillomavirus; MMTV = murine mammary tumour virus; PCR = polymerase chain reaction. Breast Cancer Research Vol 7 No 1 de Villiers et al.

. Other HPV types detected were HPV 16 (2 carcinomas and 3 nipples), HPV 57 (2 carcinomas and 3 nipples), HPV 27 (2 carcinomas and 4 nipples), HPV 66 (2 nipples), candHPV 87 (4 carcinomas), HPV 37 (4 nipples) and the putative new HPV type DL250 (HPV 9-related) in two carcinomas. HPV types detected in single samples of the nipple were HPV 20, 21, 23, 32, 38 and GA3-1 (HPV 8-related) and HPV 3, 15, 23, 24 and DL473 (HPV 15related) in carcinoma samples. Multiple HPV types were demonstrated in eight carcinomas (24%) and ten nipple (34.5%) samples.

Figure 1
1Figure 1

Figure 2
2Papillomavirus sequences detected by in situ hybridisation Papillomavirus sequences detected by in situ hybridisation. (a) Case 10012, in the nipple epithelium (magnification × 40). (b) Case 10041, in the nipple epithelium (magnification × 40). (c) Case 10012, in tumour tissue (magnification × 100). (d) Case 10014, in tumour tissue (magnification × 100).

Table 1
1Samples analysed for HPV sequencesCarcinoma type
Location
Number tested
HPV DNA positive

Invasive ductal
Carcinoma
18
16

Nipple
11

Medullary
Carcinoma
5
4

Nipple
3

Tubular
Carcinoma
3
3

Nipple
3

Lobular
Carcinoma
2
1

Nipple
2

Mucinous
Carcinoma
1
1

Nipple
1

Total carcinoma
Carcinoma
29
25 (86%)

Nipple
20 (69%)

HPV, human papillomavirus.

Table 2
2Presence of HPV DNA: results obtained after amplification with respective primersbp, base pairs; HPV, human papillomavirus.Carcinoma type Patient number Location
β-actin
HPV

KM29/RS42
(536 bp)

PC03/PC04
(110 bp)

GP
CP
FAP

Invasive duct
cell

1
Carcinoma
+
HPV11
-
HPV11

Nipple
+
HPV16
HPV27
HPV11, HPV57

2
Carcinoma
+
HPV11
-
HPV11

Nipple
-
+
HPV11
-
HPV6, HPV11

3
Carcinoma
+
-
-
HPV11

Carcinoma
+
HPV16
HPV27
-

4
Carcinoma
+
-
-
HPV11

Nipple
-
+
-
-
-

5
Carcinoma
+
-
-
HPV 11

Nipple
+
-
-
HPV3, HPV11,
HPV20, HPV21

6
Carcinoma
+
HPV11, HPV16 HPV6, HPV27,
HPV57

HPV3, HPV6,
HPV57

Nipple
+
-
-
HPV6, HPV11,
HPV57

7
Carcinoma
+
-
-
-

Nipple
+
-
-
-

8
Carcinoma
+
-
DL250 (HPV9-
related)

-

Nipple
+
-
-
-

9
Carcinoma
+
-
DL473 (HPV15-
related)

-

Nipple
+
-
-
-

10
Carcinoma
+
-
HPV6
-

Nipple
+
-
-
-

11
Carcinoma
+
-
HPV23
-

Nipple
+
-
GA3-1 (HPV8-
related)

-

12
Carcinoma
+
HPV11
-
-

Nipple
+
-
HPV37
-

13
Carcinoma
-
+
HPV11
-
HPV11

Nipple
-
+
-
-
HPV6

14
Carcinoma
-
+
-
HPV 24
-

Nipple
+
-
-
-

15
Carcinoma
+
HPV11
HPV6, DL250
HPV11, HPV87

Nipple
+
HPV66
-
-

16
Carcinoma
+
-
-
HPV11

Nipple
+
-
-
-

17
Carcinoma
+
-
-
-

Table 3
3Papillomavirus sequences demonstrated Infection with multiple human papillomavirus (HPV) types was demonstrated in eight carcinoma samples (27.6%) and ten nipple samples (34.5%). b One pair harboured HPV 6, HPV 11 and HPV 57 DNA.Tumor pairs
Number
HPV types a

Carcinoma + nipple +
17
Same type in both samples: 11 (7×) b , 6 (1×) b , 57 (2×)
Different types: 3, 15, 16, 20, 21, 23, 27, 37, 38, 66, 87, DL250, DL473, GA3-1

Carcinoma -nipple +
3
6, 11, 32, 37, 66

Carcinoma + nipple -
8
6, 11, 24, 87, DL250, DL473

Carcinoma -nipple -
1

a

Table 2 (Continued)
2Presence of HPV DNA: results obtained after amplification with respective primers

Table 4
4Types of papillomavirus detectedCarcinoma type Location
HPV types

6
1 1
1 6
2 7
3 7
5 7
6 6
8 7
D L 2 5 0
O t h e r s

Intraductal
Carcinoma
3
11
1
1
-
1
-
1
2
3, 23, 24, DL473

Nipple
5
4
2
2
3
2
2
-
-
20, 21, GA3-1

Lobular
Carcinoma
-
1
1
1
-
-
-
-
-
-

Nipple
-
1
-
-
-
-
-
-
-
32

Tubular
Carcinoma
-
2
-
-
-
1
-
1
-
15

Nipple
1
1
1
2
1
1
-
-
-
-

Medullary
Carcinoma
3
4
-
-
-
-
-
2
-
-

Nipple
-
2
-
-
-
-
-
-
-
23

Mucinous
Carcinoma
1
1
-
-
-
-
-
-
-
-

Nipple
-
-
-
-
-
-
-
-
-
38

Total carcinoma
(%)

Carcinoma 7 (24.1) 19 (65.5) 2 (6.9)
2
-
2
-
4 (13.8)
2

Nipple
6 (21)
8 (27.6) 3 (10.3)
4
4
3
2
-
-

AcknowledgementsWe thank Corinne Whitley, Elsbeth Schneider, Romana Cop, Karin Gunst, Helene Rahn and Imke Grewe at the Deutsches Krebsforschungszentrum and Kari Bradshaw, Joyce Kangas and Connie Gibbons at Lower Columbia Pathologists for excellent technical support. This study was supported in part by The Healthcare Foundation, Cowlitz County, Washington. The study was reviewed by the IRB committee at St John Medical Center, Longview, Washington (acting chairperson Dane Moseson MD) and accepted as conforming to the institution's guidelines with regard to retroactive studies employing human tissue samples.
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Power and Fairness in a Generalized Ultimatum Game
2014

G L Ciampaglia
S Lozano
D Helbing
Power and Fairness in a Generalized Ultimatum Game

PLoS ONE
96990392014Received March 12, 2014; Accepted May 6, 2014;Dirk Helbing Chair of Sociology, in particular of Modeling and Simulation, ETH Zü rich, Zü rich, Switzerland 0099039 Editor: Matjaz Perc, University of Maribor, Slovenia Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data may be accessed from Figshare under the The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
Power is the ability to influence others towards the attainment of specific goals, and it is a fundamental force that shapes behavior at all levels of human existence. Several theories on the nature of power in social life exist, especially in the context of social influence. Yet, in bargaining situations, surprisingly little is known about its role in shaping social preferences. Such preferences are considered to be the main explanation for observed behavior in a wide range of experimental settings. In this work, we set out to understand the role of bargaining power in the stylized environment of a Generalized Ultimatum Game (GUG). We modify the payoff structure of the standard Ultimatum Game (UG) to investigate three situations: two in which the power balance is either against the proposer or against the responder, and a balanced situation. We find that other-regarding preferences, as measured by the amount of money donated by participants, do not change with the amount of power, but power changes the offers and acceptance rates systematically. Notably, unusually high acceptance rates for lower offers were observed. This finding suggests that social preferences may be invariant to the balance of power and confirms that the role of power on human behavior deserves more attention.

Introduction

At virtually all levels of human societies, socio-economic transactions are determined by the balance of power between parties. In fact, it is difficult to underestimate the importance of power in human social dynamics [1]. In the past, bargaining situations, and the Ultimatum Game (UG) in particular, have received large attention in the scientific literature, mostly because they highlight the importance of social preference in explaining human motivations beyond the standard rationality assumption [2]. In fact, under selfish rationality assumptions, as they have classicaly been made, Game Theory predicts that in the UG people would share as little money as possible and accept any share of money from others. However, since early pioneering work by Güth and colleagues [3], it has been observed experimentally that people have other-regarding preferences that favor fairnessoriented, pro-social outcomes. That is, small offers are rejected and accepted offers tend to come close to equity [2,4]. This holds, albeit with certain caveats, across a large variety of cultural and social contexts [5,6] (for a recent overview of the literature on ultimatum bargains, see the review by Güth and Kocher [7]).

Therefore, social preferences have been further incorporated into a fairness-oriented rational framework, which assumes inequality aversion [8]. Subsequently, various authors have developed evolutionary game theoretical models to explain human fairness preferences [9,10]. Experimental results, however, indicate that some people have selfish preferences, while most people have fairness preferences, the degree of which varies from one person to another [11]. Such a broad spectrum of selfish and fairness preferences has been recently explained with a model that explains the evolution of both, a selfish ''homo economicus'' and an other-regarding ''homo socials'' [12]. In contrast to this model, which implies a level of cooperativeness and fairness that depends on the context, specifically on the behaviors in the social neighborhood, most models assume that preferences are stable with respect to the specific situation in which the bargain is performed.

Experiments show that situational and context-specific factors do affect preferences [13], and that what is perceived as selfish in one context can be construed as fair in another [14,15]. Even unconscious cues may have a profound impact on the willingness of people to act fairly [16].

The effect of power on basic human cognition is clear: power is known to increase the amount of top-down stereotyping [17], to enhance goal-oriented behavior, and to increase the chances of goal-consistent action [18]. In general, the powerful tend to respond more to rewards, whereas the powerless more to threats [19,20]. Power may act as an inhibitory factor of specific psychological systems [21] and has been associated with a decreased neural capability for processing social inputs [22].

The effects of power on social outcomes, however, are more nuanced: according to some authors, high social class, and hence higher power, is merely associated with more anti-social behavior [23]. However, others point out that exchange-oriented individ-uals (in the sense of the work of Clark and Mills [24]) when given power tend to act in a self-interested fashion, while in similar situations communally oriented individuals act more altruistically [25]. Power may also activate norms of social restraint in counterintuitive ways [26], but so far previous scientific literature has devoted little attention to the role of power on individual preferences in bargaining situations, and it remains largely unclear.

In this work we set out to understand the role of bargaining power on social preferences, and we use the Ultimatum Game (UG) as a testbed for doing so. The UG is a stylized example of a bargaining situation in which the two players have different kinds of power. The proposer sets the stage and the responder has the power of the last word. We modify the payoff structure of the standard UG to investigate three situations: two cases in which the power balance is either against the proposer or the responder, and a balanced situation.

Our generalization of the classical UG is akin to manipulating power by means of a role-play with a well-defined balance of power. In the literature on the social psychology of power, it has been pointed out that having participants role-play a powerful position may trigger informal norms of social restraint [18,26,27]. To avoid possible interpretations of our results in terms of informal norms, we decided to design a completely double-blind setting in which confidentiality and anonymity were guaranteed among participants and between the participants and the experimenter.

We found that, while offers and acceptance rates vary almost predictably with the balance of power, other-regarding preferences, as measured by the amount of money donated by participants to the International Committee of the Red Cross (ICRC), do not change with the amount of power. We also find that, even when controlling for both fairness-oriented and self-oriented players, a significant fraction of bargains cannot be explained in purely rational terms.

This result shows that social preferences may be invariant to the balance of power. This is somewhat surprising, given the common belief that 'power corrupts' and in view of recent results about the increased chances of observing unethical behavior in higher strata of society [223]. It also adds to the growing literature on the role of power in affecting basic cognition [17][18][19][20][21]25] and emphasizes that the role of power on human behavior deserves further scientific attention.

Methods

Manipulating power in the UG

In the experimental literature on the psychology of power, power is manipulated essentially in two ways: priming or roleplaying [18][19][20]. In the first case, participants in an experiment are primed by asking them to recall a past experience in which they were either in a powerful or powerless situation. Despite the fact that priming only acts on the perception of power, several studies have shown that it is a powerful technique, eliciting measurable effects even at a neural level [22]. Moreover, depending on how the recall procedure is actually implemented (for example by having people write an essay) it may also let the experimenter assess its degree of activation in individual subjects -by measuring the valence of the essays in the case above. In our study priming was not a viable option because of the anonymity requirement; in writing about a past experience people might feel they are disclosing personal information and thus lose the perception of confidentiality with regards to the experimenter.

On the other hand power also can be manipulated by asking people to enact a situation in which some participants are actually assigned power over other participants. For example, in a manager/worker situation, Galinsky et al. [18] divided participants into teams and appointed a manager to each. The managers' task was to coordinate the work of the other team members, evaluate their results, and decide how much money they would be paid at the end of the experiment.

Because we were interested in studying the role of power in bargaining situations and how it may possibly affect otherregarding preferences, we chose the latter option, developing a version of the UG (described in greater detail in the following section) in which the payoff structure can assign more or less power to one of the two players.

Our experiment took place on the Web. Compared with a traditional, lab-based setting, the Internet offers several obvious advantages for implementing double-blind designs, since participants do not need to show up at the laboratory and are free to participate from home. People nowadays are more used to online collective interaction [28], and it also avoided the logistical problem of setting up multiple separate areas within our laboratory, which would have limited the number of participants that could simultaneously attend each session. Online experiments on the UG are becoming more common [29] thanks especially to online marketplaces such as the Mechanical Turk [30]. To our knowledge, what we report here is one of the earliest attempts to perform a completely double-blind design of the UG.

It is also worth noting that the confidentiality offered by the online setting provides a solution to another problem as well, raised by Galinksy et al. in the context of endowing participants with power instead of simply priming them with it [18,26]: the activation of informal norms. In enacting situations such as the manager/worker one described above, informal norms of restraint could activate during the bargain and thereby influence part if not all of the observed behaviors. In other words, if exposed to a social context, the powerful might find it socially reproachable to exploit their bargaining power to make a selfish offer or to reject an equitable offer in favor of a more advantageous one. But because we could not interact with the participants, nor could they communicate with each other, this possibility can essentially be ruled out. Thus, any pro-social behavior we see in the experiment can be exclusively accounted to other-regarding preferences.

Our online setting also required another departure from the traditional way UG experiments are designed. In the classical UG, two people have to share a certain amount of money provided by the experimenter, which in the literature is known as ''the cake''. It has been argued that this could lead participants to agree on an equal split just because the cake is a free gift from the experimenter [31]. To avoid this form of 'windfall gains', Berger et al. [31] required participants to wait for a certain amount of time before they could collect a constant payoff (the show-up pay) and asked them to decide how to split the waiting time.

Our experiment develops the setup of Berger et al. further [31]. There, sharing money was replaced by waiting time to avoid windfall gains, but despite this and also various efforts to increase the anonymity of the social interaction, the experiment delivered experimental results that are consistent with the original UG. Despite a number of further modifications in the Web experiments reported here, we find again similar sharing and acceptance behavior as in the classical UG, if the power between proposer and responder is balanced. However, varying the power of proposer and responder produces significantly different decision making results.

In our case, considering the online setup of the game, people could potentially spend their waiting time surfing on the web or taking a break from the computer, thereby defeating the whole concept of a bargain over time. We thus asked players to share the work of solving 300 additions of two single-digit numbers in case of acceptance and 600 in case of rejection. If the calculations were equally split, this amount corresponded to roughly 15-30 minutes of work for each participant. We chose simple arithmetic additions on the basis that the task should be simple enough for anyone and tedious enough to be perceived as an opportunity cost. Also, as the results of the sums were validated and stored on our server, this allowed us track effectively which participants completed the experiment without having to communicate with them.

Ethics statement

The experiment was designed in compliance with the research ethics policies of the Decision Sciences Laboratory at ETH Zürich, and was approved by the ETH Ethics Committe (EK 2012-N-63). In particular, these policies prohibit any form of deception.

Participants were drawn from the student body of the Swiss Federal Institute of Technology (ETH) in Zürich. They were contacted via email (see File S1). For each experimental session 1,000 participants were randomly selected and contacted. Each participant was contacted only once. Participants were required to review a copy of the experimental policies before taking part in the experiment and agree to them, as a form of informed consent. They were not required to disclose any personal information in order to access the experiment website, and they were not assigned any data that could potentially allow us to track their actions individually during or after the experiment, such as cookies or personalized URLs. To access the website they were given a session password that was the same for all participants of any given session. Table 1 summarizes the payoff structure of our generalized UG.

Ultimatum Game with different power structures

x p is the workload that the proposer offers to do, and x r~3 00{x p is the suggested share of the responder, if accepted (top row).

Similarly, x' p and x' r are the assignments in case of rejection. In our game offers can range from a minimum value of x p~3 0 to a maximum of x p~2 70. In the standard UG, if the second player rejects the offer of the first player, both players do not receive any money. In the variant of Berger et al., where players bargain over time, each of them was required to wait for the specified time [31]. In our case we decided to double the total workload (i.e. from 300 to 600 additions) and split it according to three different payoff functions reflecting the relative balance of power between players, corresponding to three different experimental treatments.

We will call the three payoff functions taken into account ''weak proposer'', ''weak responder'', and ''balanced situation'', as shown in Table 1 (bottom row). Their payoff structures are motivated by simple strategic considerations.

We consider two types of players: fairness-oriented and self-oriented. The payoffs were chosen such that the different treatments would be able to differentiate between self-and fairness-oriented preferences. Figure 1 graphically represents responders' expected preferences depending on their respective orientation (i.e. self-or fairness-oriented). From a self-oriented perspective, responders should always reject if this would reduce the number of calculations to be done. Thus, in a bargain involving self-oriented rational responders, they should reject all offers in the weak proposer and accept all offers in the weak responder treatment, while in the balanced treatment the response would depend on the concrete offer. Fairness-oriented responders would instead try to maximize fairness, i.e. minimize the difference in the number of calculations to be performed by both players.

Similarly, a self-oriented proposer should make the offer that minimizes the number of calculations to be done, and a fairnessoriented one should try to minimize differences in the number of calculations. However, in order to reach their goal, proposers have to make an assumption about the orientation of the responder. For example, take a self-oriented proposer in a ''weak'' responder situation. If the responder was also self-oriented, the optimal split of the workload would be 1-9, since it is the option assigning him the least workload among the ones potentially accepted by the responder (see red outlined cells in Fig. 1). However, if the responder was fairness-oriented, she should propose the split 4 -6.

Notice that a wrong assumption about the responder's orientation might lead to a completely unsatisfactory outcome for the proposer [15]. For instance, in the example discussed above, if the proposer mistakenly assumes that the responder is self-oriented, the 1-9 split would be rejected in favor of a more fair 9-11 split.

Taking all this into account, we derived the expected behaviors of both players based on their respective preferences, i.e. assuming that a player is either self-oriented or fairness-oriented and the beliefs of the proposer concerning the preferences of the other player. Figure 2 shows the predictions of the model in form of a decision tree. Each level of branching represents the choice of one of the above variables. We start at the root node with the preference of the proposer; the intermediate nodes stand for the belief of the proposer regarding the responder's orientation ('ASSUMPTION'), which determines the rational proposal. Finally, the branching leading to the leaves ('ACTUAL') represents the two possible beliefs of the responder and determines the expected outcome of the bargain.

Since the assumption of the proposer regarding the orientation of the responder might be wrong, our model captures a possible source of variability that is often observed in experimental settings [15].

Results

Turnout rate and game completion

We performed four experimental sessions, sending out 4,000 invitation emails in total. Weather conditions and time of the day were similar in all sessions. Altogether, 246 participants visited the website of the experiment. This amounts to a 6.15% show-up rate. Typical response rates in web surveys are slightly higher [32], but web experiments are usually also more demanding in terms of the involvement requested to participants, and ours was no exception.

Not all participants who logged into the website completed the experiment: 32 left before being matched to another player: 13 during the proposal phase (weak proposer: 7; weak responder: 3; and balanced: 3), and 5 after the game started (weak proposer: 2; balanced: 3). In summary, a total of 98 games in which both players stayed until the end of the experiment were completed. Broken down by treatment these are: 38 in weak proposer; 30 in weak responder; and 30 in the balanced treatment. Given the size of the workload they were required to bargain over (300 or 600 calculations), the number of participants who stayed until the end was surprisingly large.

Effect of power on bargains

Introduction of power led to strong divergence from previously reported observations in the UG. Figure 3 shows the proportion of accepted offersp p as a function of the workload x p proposers offered to do. Notably, unusually high acceptance rates for lower offers (20%ƒx p v30%) were observed (lower bound of the 95% Agresti-Coull approximate confidence intervalẑ z 1{ 1 2 a~0 :36). Here, all treatments are taken together. Moreover, there was a considerable amount of 'hyperfair' offers (x p w50%), e.g. n~17 for 60%ƒxv70%. There were even n~3 offers for x~90% -the maximum allowed offer -and, looking at the responses to those offers, not all of them were accepted, which is very surprising.

Compared with the traditional UG, varying the balance of power has thus dramatic consequences on the observed bargaining behavior. In particular, the somewhat surprising rejections for hyperfair offers (x~90%) mentioned above have occurred in the ''Balanced'' situation, which could be explained as a form of inequality aversion (see Fig. 1). In line with our predictions, we found that different balances of power of the three treatments led to different sharing behaviors (x 2~5 .28, p~0:07). Figure 4 shows the breakdown by experimental treatment. In the balanced treatment (left panel), for example, the highest lower bound on the probability of acceptance does not correspond to the fair split but is attained for 60%ƒx p v70%. Moreover, almost no offer below 40% was made. In the other two treatments fair splits still correspond, in terms of lower bound, to the modal rate of acceptance, but, besides this detail, the distributions look very different from each other. In the weak responder case, the most advantageous offers for the proposers (x p v20%) were made more often (n~5 vs n~2), compared to the weak proposer case, and almost no offer (n~1 vs n~6) was observed in the range of moderately advantageous splits (30%ƒx p v50%)) which is usually observed experimentally in the standard UG with no power.

These results are further substantiated by regression analysis of the proposed workload, using beta-distributed errors [33] and controlling for various confounding factors induced by the experimental design (see Section S3 of File S1); these include the donation, whether the show-up pay was eventually collected, the time spent by the proposer making the offer, the average outside temperature, which we use to measure opportunity costs, and others. The overall effect of power was significant (x 2~9 :8,p~0:0074). In particular, relative to a balanced scenario, proposers made smaller (i.e. more selfish), proposals in both the

Donations and other-regarding preferences

Using a web form at the end of the game we asked participants whether they wanted to donate part of their show-up pay of 17 CHF to the International Committee of the Red Cross (ICRC). This method was used in order to measure other-regarding preferences without breaking the perceived level of confidentiality among participants. While the majority of subjects did not make any donations, 31.7% of them donated part of their money, with an average non-zero donation of 8:40+6:79 Swiss Francs. If non-donors are also taken into account, the average donation was instead 2:63+5:44 Swiss Francs.

To understand whether power affected other-regarding preferences, we matched each game according to the predictions of our model, see Figure 2. In particular we first determined the proposer's preference and their assumption about the responder's type by matching the observed offer x p ,x r À Á with any of the expected proposals. In doing so we used equally sized bins of 30 calculations. Then, based on the observed response, we determined the actual other-regarding preference of the responder.

The possible outcome of this exercise could result in matching a game to single, multiple, or no leaves of the decision tree at all, depending on the concrete case. This corresponds to assigning any of the three variables to either one, multiple, or no classes, respectively. For example, a weak responder game in which the proposal (divided by 30) is 5 -5 and the response is acceptance is compatible with two different leaves (the fifth and the sixth from the top of Fig. 2), both related to a fairness-oriented proposer (lower branch, top level), who assumes she is dealing with a selforiented responder (upper branch, 'ASSUMPTION'). Regarding the preference of the responder, both branches ('ACTUAL'), corresponding to either self-or fairness-oriented preferences would be compatible with the observed outcome, thus resulting in a 'Multiple' assignment.

Applying this procedure on the experimental data resulted in 85 participants matching fairness-oriented preferences, 13 matching selfish-oriented preferences, 30 matching multiple classes, and 58 matching none of the classes. In particular, we found that 31.2% of the games were consistent with both players being fairnessoriented and a correct assumption of the proposer. On the other hand, 28% of the bargains did not match any of the predictions of the rational model (i.e., neither self-nor fairness-oriented). It is interesting to compare the donation data for both types of inferred other-regarding preferences. Figure 5 shows such a comparison, further broken down by the role (proposer or responder) of participants.

We then regressed the donated amount over the inferred preferences and the amount of power endowed to participants. We used the inferred preference instead of the actual proposed workload because what is a fair split depends on the treatment. Specifically, we considered a linear model including second-order interactions between the participants' role and their preference, the experimental treatment, the proposal, and the response. Moreover, as the data showed strong overdispersion (h~0:15), we employed a negative binomial regression model in lieu of a standard Poisson linear model. We tested several other models, including logistic regression, and only the negative binomial one could fit the data in a satisfactory manner.

The residual deviance of the model was D~117:68 for 166 degrees of freedom, indicating a good fit (p~0:99). Table 2 reports the results of the regression analysis, including the coefficients and standard goodness-of-fit measures. Predictably, and consistent with the presence of other-regarding orientation, higher proposals tend to be associated with higher donations among proposers and, interestingly, with lower donations of responders. Proposers whose offers were rejected had a tendency to donate less, perhaps  because the higher workloads resulting from rejection may have increased their sense of entitlement towards the earned pay. Crucially, however, in no treatment was power significantly related with increased or decreased donations. This is true for both roles, and supports the idea that other-regarding preferences may be invariant with respect to power. We also tested a simpler model with no interactions, but it could not capture any of the above results and was inferior to the model presented in Table 2. It is also worth noting that non-rational behavior, i.e. behavior consistent with neither self-nor fairness-oriented preferences (preference = 'None'), was associated with significantly lower donations.

Summary and Discussion

Decades of experimental research have shown that in bargaining situations people do not follow the tenets of Homo Economicus and that other-regarding preferences, the hallmark of Homo Socialis [12], are the norm rather than the exception [6]. However, much is still to be understood about how pro-social behavior unfolds, and power is unarguably one of the most important factors affecting human interactions.

Here we studied how the balance of power in a bargaining situation affects other-regarding preferences of its actors by designing a double-blind variant of the classical one-shot UG to understand this. Our first finding is that, compared to a traditional setting, introducing power leads to strikingly different bargaining behaviors: whereas offers below 30% are seldom accepted in the classical UG, the Agresti-Coull 95% lower confidence bound on the estimated rate of acceptance for offers between 20% and 30% of the cake (all treatments) wasẑ z 1{ 1 2 a~0 :36, and manipulating the balance of power between players explained overall 19% of the variance in observed proposals. The fact that power shifted proposals towards smaller amounts of work thus provides further evidence that changing the process by which a bargain is performed changes also the perception of what is considered to be fair. Thus power may be affecting process-regarding preferences [14,34].

While power changed the strategic component of the bargain dramatically, we also found that it did not affect the predisposition of people to donate to a humanitarian organization, i.e., otherregarding preferences do not seem to be influenced by assigning experimental subjects to a position of greater or smaller power. This is somewhat surprising, given recent results indicating that greater social status, and thus to a large extent greater power, is associated with greater chances of performing more selfish behavior [23].

Regarding the bargaining behavior of proposers, our anonymous setup allows us to rule out any explanation in terms of norms of social restraint. Fairness-oriented preferences show up in two independent ways -altruism and reciprocity. Regarding the latter, since we contacted our participants using their ETH student email address, we cannot entirely exclude the possibility that some form of group identification might have occurred. However, we have measured other-regarding behavior separately by asking people to donate to the ICRC -a generic universal institution. Therefore, we believe that explanations in terms of reciprocating behavior, at least for the donation behavior, can essentially be ruled out.

How can we make sense of this finding? We propose that the way participants framed the situation conditioned their propensity to engage in pro-social behavior or not. Power has been reported to enhance the likelihood of acting regardless of whether a social dilemma is framed as a public-goods or commons type of contribution [18,25]. Thus, considering that donating to the ICRC corresponds to making a contribution (i.e. a public-good type of situation), this would predict more donations from the powerful. However, in order to avoid participants from inadvertently skipping the donation phase altogether, the donation screen was designed such that participants had to enter the amount of '0 CHF' in case they did not want to donate any money. Consequently, they were prompted to act regardless of their intentions.

Given that they had to work in order to earn their reward, donating to the ICRC must have been framed as a loss of property that subjects were entitled to own. Further experimental work is thus needed to tease apart the effect of action, power, and loss aversion on other-regarding preferences. More work is also needed to incorporate these findings into a coherent theoretical framework. Recent work proposes an evolutionary framework for the emergence of pro-social and self-regarding preferences in a society [12,29,35]. It would, therefore, be interesting to assess whether such an approach can explain the invariance of preferences with respect to power or whether novel theoretical explanations need to be developed. Figure S1 Test screen. Before taking part in the ultimatum bargain, participants were required to perform three calculations to get an idea of the subsequent workload.

Supporting Information

Figure 1 .
1Rational choice predictions for self-and fairness-oriented preferences. For each panel, the proposal and the alternative workload in case of rejection are shown. The values displayed multiplied by 30 correspond to the actual number of arithmetic calculations to be done according to Table 1. Colors represent the orientation of responders in terms of their rational preferences (i.e. self-or fairness-oriented). doi:10.1371/journal.pone.0099039.g001 weak proposer (pv0:05) and weak responder (pv0:005) treatment. The log-likelihood of the model is 45:81 on 11 degrees of freedom, with a pseudo-R 2~0 :19. This quantity can be interpreted as the amount of variance explained by different treatments after controlling for relevant confounding factors.

Figure 2 .
2Rational choice predictions based on the proposer preference (root), proposer belief (intermediate nodes), and responder preference (leaves). In the intermediate nodes, the triplets indicate the expected proposal in each treatment (divided by 30). In the leaves, the expected outcomes (divided by 30) after the responder decision are shown. doi:10.1371/journal.pone.0099039.g002

Figure 3 .
3Acceptance rate as a function of proposed workload. Error bars are 95% Agresti-Coull approximate confidence intervals. We find monotonic acceptance rate, except for few hyperfair rejections. doi:10.1371/journal.pone.0099039.g003

Figure 4 .
4Probability of acceptance as a function of proposed workload and power treatment. Error bars are 95% Agresti-Coull approximate confidence intervals. We find monotonic acceptance rate, except for few hyperfair offers in the ''balanced'' treatment. Different treatment elicit different bargaining behaviors. doi:10.1371/journal.pone.0099039.g004

Figure 5 .
5Donations by inferred orientation and role. doi:10.1371/journal.pone.0099039.g005

Figure
S2 Proposal screen. This screen was showed to the proposer during the proposal phase. (TIFF) Figure S3 Response screen. This screen was showed to the responder during the response phase. (TIFF) Figure S4 Structure of the experiment performed. The online part of the experiment, during which subjects played the ultimatum game, is indicated by a solid line. (TIFF) File S1 Supporting material for Power and Fairness in a Generalized Ultimatum Game. (PDF)

Table 1 .
1Payoff functions for proposers and responders in our three experimental treatments.Top row: workloads x p and x r (number of two-digits additions) assigned, respectively, to the proposer and the responder in case of acceptance. Bottom row: workloads x' p and x' r assigned in case of rejection in each of the experimental treatment. doi:10.1371/journal.pone.0099039.t001Offer
x p [½30,270 x r~3 00{x p

Rejection
Weak proposer
Weak responder
Balanced

x' p~xp z330
x' p~xp z240
x' p~6 30{x p

x' r~2 70{x p
x' r~3 60{x p
x' r~xp {30

Table S1
S1Control variables included in the analysis of the proposed workloads and responses. (PDF)
PLOS ONE | www.plosone.org
June 2014 | Volume 9 | Issue 6 | e99039
AcknowledgmentsThe authors would like to thank Silvana Jud, James Kitts, Stefan Wehrli, and Heiko Rauhut for helpful discussion and support in the initial setup of the experiment, and John McCurley for his help in editing the manuscript.Author Contributions
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