Epithelial atypia in biopsies performed for microcalcifications. Practical considerations about 2,833 serially sectioned surgical biopsies with a long follow-up

This study analyzes the occurrence of epithelial atypia in 2,833 serially sectioned surgical breast biopsies (SB) performed for microcalcifications (median number of blocks per SB:26) and the occurrence of subsequent cancer after an initial diagnosis of epithelial atypia (median follow-up 160 months). Epithelial atypia (flat epithelial atypia, atypical ductal hyperplasia, and lobular neoplasia) were found in 971 SB, with and without a concomitant cancer in 301 (31%) and 670 (69%) SB, respectively. Thus, isolated epithelial atypia were found in 670 out of the 2,833 SB (23%). Concomitant cancers corresponded to ductal carcinomas in situ and micro-invasive (77%), invasive ductal carcinomas not otherwise specified (15%), invasive lobular carcinomas (4%), and tubular carcinomas (4%). Fifteen out of the 443 patients with isolated epithelial atypia developed a subsequent ipsilateral (n = 14) and contralateral (n = 1) invasive cancer. The high slide rating might explain the high percentages of epithelial atypia and concomitant cancers and the low percentage of subsequent cancer after a diagnosis of epithelial atypia as a single lesion. Epithelial atypia could be more a risk marker of concomitant than subsequent cancer.
Introduction
Breast biopsies for infraclinical lesions are more frequent with mammographic screening programs, but the distribution of the corresponding histological lesions and their associations are still imprecise. Difficulties encountered in following up patients without cancer account for the fact that the clinical significance of certain non-malignant lesions and the management of patients are still debated. Moreover, the problem of surgical biopsy sampling has never been fully investigated and has added additional confusion in appreciating the distribution and clinical significance of such lesions. In 1981, breast epithelial atypia were hardly mentioned and not clearly defined in the World Health Organization/International Union Against Cancer (WHO/UICC) histologic classification of breast tumors [73]. The histologic classification of noncancerous lesions has been mainly based on studies analyzing for each lesion the associated risk of subsequent cancer. These studies were initiated by the survival studies of Dupont and Page [15, 46] based on lesions discovered by palpation before the era of mammography. Thereafter, further studies [6, 9, 17, 24, 31, 51, 62, 67] substantiated these results, which were ratified in 2003 by the new American Joint Committee on Cancer (AJCC)/UICC classification of breast tumors [70]. Schematically, this classification differentiates benign epithelial lesions (usual ductal hyperplasia and other lesions) from atypical lesions of ductal or lobular type. Although this historical classification is challenged by a new classification [68], it remains the most widely used in practice. Interestingly, the occurrence of epithelial atypia was low in Page’s study [46] and has increased with mammographic screening programs [60, 66] and with the development of percutaneous large core needle biopsy (CNB) methods using stereotactic mammography or ultrasound guidance. At present, CNB is frequently used for the initial evaluation of clinically occult breast lesions, thus generating dilemma for the subsequent management of certain noncancerous lesions. At our institution, surgical biopsies (SB) have always been managed in the same way, and most patients with atypical and malignant lesions have been followed. The objectives of our work were to analyze the occurrence of epithelial atypia and their association with a concomitant cancer in a large series of SB performed for microcalcifications without a palpable tumor and to assess the subsequent cancer probability in the group of patients with an initial diagnosis of epithelial atypia. Finally, we provide some practical considerations for the management of patients with epithelial atypia in this era of mammographic screening and CNB.
Materials and methods
Selection of patients
At Institut Bergonié, from January 1975 to December 2002, 3,166 breast biopsies for diagnostic purposes, 2,833 SB and 333 CNB, were performed for microcalcifications without any palpable mass in 2,708 patients (mean age 51.8 years, range 19.7–81 years). Among them, 248 (9%) had several biopsies in the same or contralateral breast. Since 1998, microcalcifications have been classified according to the classifications of the American College of Radiology [2]. SB for diagnostic purposes were defined before 1998 by the absence of a preoperative diagnosis based on the clinical–mammographic–cytologic triplet and by the absence of a positive frozen section and, since 1998, by the presence of epithelial atypia on CNB. Excluded from this study were 132 cancers and 139 non-atypical benign lesions diagnosed on CNB as well as 49 re-excisions performed elsewhere than in our center. Thus, 2,833 SB in 2,375 patients were available for analysis, among which 13 corresponded to re-excision after a CNB with epithelial atypia. Since 1989, needle localization, intraoperative specimen radiography, and post-excisional biopsy mammography have been performed in most cases.
Surgical biopsies and tissue sampling: serial macroscopic sectioning
SB was removed in one fragment and measured more than 3 cm in 94% of the cases (mean size 60 mm, 5–250 mm). For SB margin assessment, either the surface of the specimen was inked or the surgeon during the same operation removed additional tissue in the remaining cavity after excision of the specimen (surgical margins). After fixation in Holland Bouin, SB and margin specimens were serially sectioned in their entirety into numbered slices every 2 mm [12]. In most cases (89%), careful macroscopic examination of the specimen failed to reveal any lesion. Each numbered slice was put in as many numbered separate cassettes as necessary and paraffin-embedded in sequence. The median number of blocks per SB was 26 (from 2 to 180) and 8 (from 1 to 44) for surgical margins. Each block was examined on one hematoxylin–eosin–safran stained slide.
Classification of lesions and review of slides
Since 1975, all patients have been prospectively included in our clinical, histologic, and biologic database by senior pathologists (IM, GMG, IS, JMC). For each SB and each lesion, we prospectively entered in our pathologic database morphological descriptive criteria by using 65 pathological items for noncancerous lesions and 181 for cancers. Definitions and terminologies given in the literature were used to report columnar cell lesions (CCL), non-atypical ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ (DCIS), and lobular carcinoma in situ (LCIS) [1, 4, 7, 10, 17, 19, 21–28, 38, 43–45, 47, 49, 50, 56, 59, 61, 69, 72, 74, 75]. The interest of our database was to collect morphological descriptive criteria of nearly all the breast lesions without labeling them. In fact, labels and definitions of breast lesions have varied throughout the past 30 years, while neither lesions nor their corresponding descriptive criteria (i.e., size, type, architecture, cellular and nuclear features, etc...) have changed. The only changes during this period were the definitions and/or the names given to these lesions. As we have listed for each lesion all the corresponding descriptive criteria among the 236 available items, we have been able to reclassify each lesion according to the “new” criteria recommended by referent authorities for a new definition, by selecting in our database the “new” correspondent descriptive criteria corresponding to this new definition. Consequently, this provided a homogeneous approach to the pathological lesions at the time of our study. For example, low-grade DCIS ≤ 2 mm have been reclassified as atypical ductal hyperplasia/ductal intraepithelial neoplasia (ADH/DIN) 1B (n = 30) according to the new AJCC/UICC classification of breast tumors [70], and lesions that we used to term before 1997 [71] as clinging carcinoma of the monomorphic type [4] have been reclassified as flat epithelial atypia (FEA)/DIN 1A or columnar cell change (CCC) with atypia (n = 84) [61]. About half of these 114 cases have been systematically reviewed by one (IM) or two senior pathologists (IM and GMG or IS), and there was a complete concordance between the second review and the initial descriptive criteria listed in the database. Similarly, lesions that we used to name LCIS before 1997 have been renamed lobular neoplasia (LN) since 1997, corresponding either to atypical lobular hyperplasia (ALH) or to LCIS. On the contrary, all the cases with micropapillary lesions were reviewed (n = 155) because there was no item corresponding to precise descriptive criteria of micropapillations (number, topography around the duct, type).
Atypical ductal hyperplasia: definition and sizing
Among the group of ADH/DIN 1B, we individualized two morphologic types of ADH. Neither had any high-grade cytological atypia or necrosis.
ADH “mimicking DCIS” (Fig. 1). In this type, architectural atypia were qualitatively insufficient to allow a diagnosis of DCIS, therefore this “mimicking” DCIS lesion was classified as ADH whatever its size. Tufts and short micropapillations formed by cells had a broad base and were cohesive. There was no polarization of cells, i.e., no true cribriform spaces. Pseudo-cribriform patterns comprised irregular or relatively round microlumina with incomplete polarization of surrounding epithelial cells. Cellular bridges were wavy without any cellular polarization. Cells corresponded either to columnar cells with uniform ovoid to elongated nuclei or to cells with a slight increase in the nuclear/cytoplasmic ratio with more or less distinct cell borders and round or ovoid nuclei. These cells were sometimes admixed in the same lesion displaying a morphological gradient, but there was no regular arrangeent. Nuclear chromatin was evenly dispersed, homogeneous, or slightly marginated, and nucleoli were inconspicuous. Apical snouts, intraluminal secretions, and psammoma-type calcifications were frequently present.
Fig. 1a–d. ADH “mimicking DCIS.” a Tufts and short micropapillations with a broad base. b Pseudo-cribriform spaces. c Microlumen with incomplete polarization of surrounding epithelial cells. d Cellular bridges without cellular polarization. Cells are parallel to the axes (arrows)
ADH corresponding to “mini DCIS” (Fig. 2). In this type, architectural and cytologic atypia corresponded to a low-grade DCIS but were quantitatively insufficient to allow a diagnosis of DCIS, therefore this “mini” DCIS lesion was classified as ADH when ≤2 mm. Tufts and short micropapillations had a tight base, were present on over all the periphery of the duct, and were non-cohesive with small free papillary tufts in the lumen. There were true cribriform patterns with a polar organization of cells around glandular spaces and/or variants of cribriform patterns (i.e., trabecular bars, cartwheel formations, and Roman bridges, Fig. 3) with polarized cells arranged perpendicular to the axes. Some solid areas with regular arrangement of cells were also present. Cells were often small, monomorphous, sometimes without a columnar change, with a distinct cytoplasmic membrane and a spaced regular round nucleus with uniformly dispersed chromatin without prominent nucleoli. Intraluminal secretions and calcifications (amorphous or psammoma-type) were also frequently present (Fig. 4). When one mini DCIS focus was found in one partially or completely involved duct/ductular cross-sections in one terminal ductal lobular unit (TDLU), it was classified as ADH when it measured ≤2 mm and as DCIS when it measured >2 mm. When there were several foci of “mini” DCIS in close duct/ductular cross-sections in the same TDLU or in TDLUs located in the same field at low power magnification (2.5), the lesion was classified as ADH when its size, i.e., its largest diameter, was ≤2 mm and as DCIS when >2 mm. When there were several foci of “mini” DCIS in distant duct/ductular cross-sections in the same TDLU or in close TDLUs, the size of each focus was assessed separately. FEA, rare and scattered single micropapillations, and cribriform variants were not taken into consideration for sizing, even if located in the same TDLU.
Fig. 2a–e. ADH corresponding to “mini DCIS.” a A solid mini DCIS focus measuring less than 2 mm in one TDLU. b Tufts and short micropapillations over the entire periphery of the duct with small free papillary tufts in the lumen. c Short micropapillations with a tight base. d True cribriform spaces. e Microlumen with complete polarization of surrounding epithelial cellsFig. 3a–c. Variants of cribriform patterns. Polarized cells arranged perpendicular to the axes. a Trabecular bars. b Cartwheel formations. c Roman bridgesFig. 4a and b. a Mild cytologic atypia. b Columnar cells with uniform ovoid nuclei, intraluminal calcifications
FEA were present either as a single lesion or in association with ADH in the same TDLU and since 1997 have been included in the ADH group. The distinction of FEA from columnar change without atypia was based on the criteria given by the WHO for the definition of FEA. Furthermore, columnar change without atypia was characterized by one or two layers of columnar cells without nuclear atypia, i.e., no increase in the nuclear/cytoplasmic ratio, no prominent nucleoli. Nevertheless, some cases of columnar change with progesterone impregnation, especially in the second part of the cycle, might display a lobular distension with a secretory material and large nuclei with prominent nucleoli. In such cases, myoepithelial cells displayed the same alterations with clarified cytoplasms, thus facilitating the diagnosis. The distinction of ADH mimicking DCIS from usual hyperplasia (UDH) was based on morphological criteria. Architectural pattern and cytologic criteria of usual ductal hyperplasia were easy to identify in most cases. UDH corresponded to a proliferation of epithelial cells in solid or fenestrated areas without any polarization of surrounding cells. Cells were haphazardly arranged with overlapping nuclei or were parallel with characteristic streamings. They were elongated or pseudo epithelioid, but there was no columnar metaplasia. Cytoplasms were more or less abundant with indistinct borders. Nuclei had irregular size and shape and sometimes contained a prominent eosinophilic inclusion. In some rare cases, immunohistochemical staining with cytokeratin 5/6 [41] was used and was negative in ADH mimicking DCIS and strongly positive in UDH. In some lesions, differential diagnosis between ADH and low-grade DCIS was all the more difficult because there were intermediate and intricated morphological aspects in the same TDLU. In practice, diagnosis of micropapillary lesions was often difficult. Extensive micropapillary lesions were classified as DCIS when quantitative and qualitative criteria were simultaneously present, i.e., lesion sizing more than 2 mm corresponding to micropapillations with a tight base over the entire periphery of the ducts. Additional sections could be useful for demonstrating more or less qualitative or quantitative diagnostic criteria. When malignancy remained equivocal, the case was classified as ADH. When a concomitant cancer was diagnosed, histologic size was assessed, and in DCIS, the percentage of blocks with cancer (“positive blocks”) was specified [13]. Presence and topography of microcalcifications were also assessed. Lastly, when FEA and/or “mimicking” DCIS foci were found on excision margins of a SB with DCIS, a further surgical resection was not performed.
Follow-up of patients with epithelial atypia as a single lesion
There were 443 patients with epithelial atypia in one or several SB, without any previous or synchronous carcinoma in the same or contralateral breast and treated by biopsy alone (median follow-up 160 months, 7 to 315). Only 28/443 (6%) were lost to follow-up. Among the 415 other patients, 180 were monitored at our institute and 235 outside by correspondent specialists working in close relationship with our institute. All patients received a clinical examination and mammography once a year. When a new biopsy was necessary, it was performed at our institute.
Statistical analyses
Comparison of clinical and histologic characteristics was conducted by using the chi-square test. For women with epithelial atypia, the probability of developing in situ or invasive cancer was calculated from the date of the first biopsy to the earliest event: breast cancer (ipsi- or contralateral), death, or last contact (last consultation for the group monitored at our institute and checkpoint date, i.e., 1 March 2004, for the others). Probabilities were calculated according to the Kaplan–Meier method (SPSSv11).
Results
Occurrence of epithelial atypia in the 2,833 surgical biopsies
Epithelial atypia were recorded in 971/2,833 SB (34%). They were found with and without a concomitant cancer in 301/971 (31%) and 670/971 (69%) of the cases, respectively. Thus, isolated epithelial atypia were found in 23% of the cases (670 out of the 2,833 SB). Calcifications were present at histologic examination in 98.6% of SB with cancer and were located in benign, cancerous, and both lesions in 10, 39, and 51% of the cases, respectively. In several cases, cancerous foci without any microcalcifications were located at points distant from those with calcifications detected by needle localization.
Types of epithelial atypia
Among the 971 SB with epithelial atypia, there were 101 SB with FEA as a single lesion (11%), 342 (35%) with ADH, 223 (23%) with LN, and 305 (31%) with ADH and LN. Thus, ADH was encountered in 647/971 SB (66%).
Types of cancers associated with epithelial atypia
Cancers associated with epithelial atypia corresponded to DCIS and micro-invasive carcinoma (DCIS-MI) in 233 cases (77%). Among invasive carcinomas (n = 68), there were 13 (9%) lobular and 11 (6%) tubular carcinomas (Table 1). Cancers were small (≤5 mm in 46% of invasive carcinomas, fewer than half of the blocks positive in 76% of DCIS). They were non-high grade in 78 and 67% of DCIS and invasive carcinoma, respectively. In most cases, ADH and cancer were situated close to each other. FEA alone were less frequently associated with a concomitant cancer than ADH and/or LN (p = 5 × 10−4).
Table 1Types of concomitant cancers (n = 301) in the 971 surgical biopsies with epithelial atypiaEpithelial atypiaFEA (n = 101)ADH (n = 342)LN (n = 223)ADH + LN (n = 305)No. of cases (%)No. of cases (%)No. of cases (%)No. of cases (%)Without cancer8483220641396222774With cancer17171223684387826DCIS/DCIS-MI12121033058266020IDC/NOS––164.7178113ILC1110.36352TC44213121FEA Flat epithelial atypia; ADH atypical ductal hyperplasia; LN lobular neoplasia; DCIS ductal carcinoma in situ; DCIS-MI DCIS with micro-invasion; IDC infiltrating ductal carcinoma; ILC infiltrating lobular carcinoma; TC tubular carcinoma
Cancers without epithelial atypia (malignancy alone)
There were 821 malignant SB without epithelial atypia [590 micro-invasive carcinomas, 206 infiltrating ductal carcinomas (IDC), and 25 infiltrating lobular carcinomas (ILC)].
Subsequent cancer in patients with an initial diagnosis of epithelial atypia as a single lesion
At 5 and 10 years, the probabilities of developing invasive breast cancer in the group of 443 patients with epithelial atypia were 2.8% [95%CI = 1.4 to 5.5] and 5.5% [95%CI = 3.3 to 9.9], respectively (Fig. 5). Among the 18 subsequent carcinomas, 15 were invasive (11 IDC and 4 ILC), and 3 corresponded to DCIS. Most subsequent carcinomas were encountered in the homolateral breast (n = 14) and before 10 years (n = 16). Seven carcinomas occurred in the group of patients with an initial diagnosis of LN, in the same (n = 5) or contralateral (n = 2) breast. They corresponded to infiltrating ductal (n = 6) or lobular (n = 1) carcinomas. The interval of development was 4, 5, 6 (n = 2), 9, and 12 (n = 2) years. Seven carcinomas occurred in the group of patients with an initial diagnosis of ADH, in the same (n = 3) or contralateral (n = 4) breast. They corresponded to DCIS (n = 2) and to infiltrating ductal (n = 4) or lobular (n = 1) carcinomas. The interval of development was 1, 2, 3 (n = 3), 9, and 12 years. Four carcinomas occurred in the group of patients with an initial diagnosis of ADH associated with LN, in the same (n = 3) or contralateral (n = 1) breast. They corresponded to DCIS (n = 1) and to infiltrating ductal (n = 2) or lobular (n = 1) carcinomas. In the four cases, the interval of development was 4 years. There was no subsequent carcinoma in the group of patients with FEA.
Fig. 5Probability of developing subsequent invasive breast cancer in the group of 415 patients with epithelial atypia (dotted line: confidence interval 95%)
Discussion
Application of the WHO classification: practical considerations
For a long time, DCIS was diagnosed even if the characteristic features were found in only one ductal space [6]. Thereafter, some authors introduced quantitative criteria for distinguishing between ADH and DCIS [46, 67], while others [23] rejected them. More recently, Rosen [57–59] and Schnitt and Vincent-Salomon [61] described CCL comprising CCC and columnar cell hyperplasia (CCH) with and without atypia. Nasser [40] challenged this classification based on columnar shape and limited the group of lesions to proliferations characterized by a low-grade atypicality, “atypical columnar cell lesions,” (ACCL) rather by a columnar cell configuration. In the WHO classification, ADH includes various not clearly defined types of lesions (Table 2). On one hand, there are lesions with arcades, moundings, and micropapillary formations, but without any true cribriform/complex architectural patterns [34]. This type of ADH corresponds to the CCH with atypia of Schnitt and Vincent-Salomon [61] termed category 3 in Simpson’s study [51], to the definition of ADH by Koerner [34] and to ADH “mimicking” DCIS in our study. On the other hand, there are lesions displaying architectural and cytologic atypia. This type of ADH corresponds to the complex architectural pattern with cytologic and architectural atypia of Schnitt and Vincent-Salomon [61] termed category 5 in Simpson’s study [64], to the definition of “microfocus of DCIS” by Koerner [34] and to “mini DCIS” in our study. In Simpson’s study, the ADH/category 5 contained chromosomal changes and the same total mean number of changes to that observed in DCIS/DIN IC, unlike the other CCL. Lastly, because there is still no consensus for measuring ADH, there is no clear-cut distinction between ADH and DCIS, and the cut-off at 2 or 3 [52] mm or at two completely involved spaces [70] seems arbitrary. While awaiting a definitive molecular classification, the simplest attitude could be recommended in routine practice. Only mini foci of ADH obviously similar to low-grade DCIS foci could be measured and classified as ADH when equal to or less than 2–3 mm [52] and as DCIS when more than 3 mm. Although the mode of measurement in our study is not under consensus, it is simple and can been routinely applied.
Table 2Terminologies used for intraductal proliferative lesions with low-grade cytologic atypia, so-called atypical columnar cell lesionsSpectrum of lesions1, 3–5 LayersNo polarization*With polarization Occasional mounding Mounding, arcades Cribriform spaces and their variants No or rare arcades and micropapillary formations Cohesive micropapillary tufts with a broad base Non-cohesive micropapillary tufts with a tight base[70]Flat epithelial atypia/DIN 1AADH/DIN 1B ≤ 2 mm; or in two spacesDCIS/DIN 1C[59]Columnar cell hyperplasia with atypiaADH if not extensiveDCIS if extensive[61]Columnar cell change (CCC) with cytologic atypiaComplex structures with architectural and cytologic atypia[34]Columnar cell lesions + ADHMicroscopic focus of DCISDCISInstitut BergoniéEx-clinging carcinoma of monomorphic typeADH “mimicking” DCISADH corresponding to “mini” DCIS ≤ 2 mmDCIS[64]CCC with cytologic atypiaCCH with architectural atypiaCCH with architectural atypiaDCISOrAndCCH with cytologic atypiaCytologic atypiaRegrouping?DIN 1AADH/DIN 1BADH/DIN 1CDCIS/DIN 1CNot measuredMeasured: ≤3 mm>3 mm*Or incomplete polarization
Occurrence of epithelial atypia and their association with a concomitant cancer: practical considerations
In our study, the proportion of epithelial atypia is high (23%), a result difficult to compare to others in the literature, as the methodologies used by teams are different. In the Page and Dupont case-control studies [15, 46], ADH and ALH were found in 2.1 and 1.6% of the cases, respectively. In mammographic screening programs, epithelial atypia and cancers increase as the number of biopsies performed for microcalcifications increases, especially as ACR4/ACR5 lesions are more often excised than ACR3. However, as underlined by Page [46], “the most direct relationship of epithelial atypia incidence is to slide rating.” The number of slides per SB in our study (median 26) was higher than in the other studies on benign breast lesions: 1–5 in 93% of the cases in the study of Page et al. [46] (n = 283), 3 (range 1–25, n = 674) in the study of Shaaban et al. [62], and a mean of 1.6 slides per cm of tissue (n = 199) in the study of Tavassoli and Norris [67]. In a recent study conducted in the south west of France in women aged between 50 and 75 with mammographically detected non-palpable breast lesions, a similar proportion of atypical lesions were found when biopsies were serially sectioned [39]. Furthermore, this high slide rating allowed the detection of small concomitant cancers in the vicinity of epithelial atypia in 31% of our cases, with a skew towards low-grade lesions (high proportions of DCIS and low-grade invasive carcinomas, especially tubular carcinomas). Our results strengthen the hypothesis that FEA and ADH are risk markers of low-grade cancers. This has been confirmed by the study of Simpson et al. [64] on molecular genetic profiles of CCL. In some of them, there are both a morphological and a molecular continuum in the degree of proliferation and atypia, supporting the hypothesis that “CCL are a non-obligate, intermediary step in the development of some forms of low grade in situ and invasive carcinoma.” The association of epithelial atypia with a concomitant cancer in nearly one third of the cases in our study parallels previous findings concerning the frequency of cancers found in SB performed for atypia in CNB. Thus, approximately 30% [20, 29] and 15 to 21% [5, 8, 14, 18, 35, 45, 53, 63, 76] of excisions after CNB with ADH and LN, respectively, were proven to have cancer. Consequently, excision is recommended [45] for all patients in whom ADH is identified on CNB and may be justified in patients with FEA, as they are included in the spectrum of ACCL. Excision remains a controversial issue in patients with LN. Some authors have advocated it [3, 18, 33, 63], while others have rebutted it [55], especially when LN is an incidental non-extensive finding [48] with no radiologic–pathologic discordance [18] and without any synchronous mass lesion [37]. SB corresponding to re-excision should be processed in its entirety by serial macroscopic sectioning [32, 65]. When pathologic examination is exclusively focused on mammographic calcifications, the risk is to underestimate the DCIS size/extension because cancerous foci without any calcification (10% in our study vs 6% in Owing’study) [42] may be located at points distant from those with benign breast tissue containing calcifications.
Subsequent cancer after an initial diagnostic of epithelial atypia as a single lesion: practical considerations
In the literature, 4 to 22% (average interval 8.3 years follow-up) [6, 42, 67] and 15 to 20% [16, 30, 56] of patients developed invasive carcinomas after a diagnosis of ADH and LN, respectively. The risk of developing cancer increases with extended follow-up, but many cancers after a diagnosis of LN have a good prognosis and a low mortality [36]. These results are difficult to compare to ours because the methodologies are different. The low probabilities of subsequent invasive cancer in our study could be due to the high slide rating, allowing the detection of small concomitant cancer that might have been missed with a low slide rating and inadequate patient management [11, 54].
In conclusion, when epithelial atypia are present, they are associated in nearly one third of the cases with a concomitant close cancer and are found as isolated lesions in nearly 23% of SB performed for microcalcifications. In practice, ADH should be more clearly defined with simple guidelines for measuring lesions. When malignancy remains equivocal and/or when sizing is difficult, it is better to classify the lesion as ADH. Epithelial atypia could be more a “risk factor” of a concomitant geographically small close cancer than a risk marker for a subsequent cancer, as they form part of a spectrum of lesions [64].

epithelial atypia
breast
cancer
atypical ductal hyperplasia
lobular neoplasia


HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C

Background Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at −20°C for prolonged periods (0.5–4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4°C for 1 year.
Introduction
The availability of clinical specimens that can be easily collected, stored and transported is advantageous in areas that lack appropriate infrastructure for blood processing. Whole blood samples collected by finger stick and dried onto a filter paper [dried blood spots (DBSs)] represent an attractive alternative to the conventional collection of blood in tubes. DBSs have been extensively used for HIV-1 antibody testing,1 molecular diagnostics2 and virus load (VL) quantification3–5 and are now considered a convenient alternative to plasma for HIV-1 drug resistance testing. We recently found that drug resistance genotypes generated from DBSs were similar to those derived from plasma in antiretroviral-naive and -experienced patients.6,7 We also found that resistance testing from DBSs can be as sensitive as with plasma when using a genotypic assay that amplifies a large (1.8 kb) pol fragment.6 The high success of amplification was noted using DBSs stored at −20°C in the presence of desiccant, suggesting that −20°C may represent a feasible storage temperature for DBSs. Although these findings were encouraging, storage at −20°C is not always possible in resource-limited settings where 4°C or room temperature may represent a more realistic alternative. Studies on HIV-1 VL determinations using DBSs have reported the efficient amplification of short HIV-1 RNA sequences from filter papers stored at 4°C or room temperature.3,8,9 However, genotypic assays usually rely on the amplification of large pol fragments and are thought to be particularly sensitive to HIV-1 RNA degradation that may occur under suboptimal storage conditions. Here, we report on the efficiency of amplification and drug resistance genotyping from DBSs stored at 4°C for 1 year.
Methods
Preparation and storage of DBSs
We prepared a total of 40 DBSs from residual diagnostic specimens collected from highly antiretroviral-experienced HIV-1 subtype B-infected persons. A more detailed description of the study population including antiretroviral drug treatment has been reported elsewhere.6 Drug resistance genotypes were available for the matched plasma specimens.6 The median plasma VL in these patients was 13 680 RNA copies/mL (range 518–676 694, Versant HIV-1 RNA 3.0 Assay, Bayer HealthCare Diagnostic Division, Tarrytown, NY, USA) (Table 1). DBSs were prepared by pipetting 50 µL of whole blood onto pre-marked circles on 903 filter paper cards (Schleicher & Schuell, Keene, NH, USA). Cards were dried overnight at room temperature (25°C), placed in a gas-impermeable, sealable plastic bag (Fisher Scientific Company, Pittsburgh, PA, USA) containing a silica gel desiccant (Mini Pax Sorbent, Multisorb technologies, Buffalo, NY, USA) and stored at 4°C until they were shipped to CDC in dry ice. Upon arrival, individual plastic bags were put in a larger bag containing three extra desiccants and stored at 4°C. All desiccants were evaluated for humidity at 6 months; 21 of the 40 bags had evidence of humidity as indicated by the colour indicator in the desiccant. Therefore, a new desiccant was added to each individual bag after allowing the spots to equilibrate at room temperature for 30 min. None of the specimens had mould after 12 months of storage at 4°C.
Table 1
Frequency of amplification of HIV-1 pol from plasma or DBSs stored at −20°C for 6 months or at 4°C for 12 months
Specimen ID
Plasma VL (log10 copies/mL)
CD4 cells/mm3
Plasmaa
DBS (−20°C, 6 months, ViroSeq assay)b
DBS (4°C, 12 months, ViroSeq assay)
DBS (4°C, 12 months, in-house assay)
1
676 694
32
+
+
+
+
2
266 612
195
+
+
+
+
3
214 330
195
+
+
+
+
4
191 674
48
+
+
+
+
5
170 289
663
+
+
+
+
6
90 758
324
+
+
+
+
7
63 261
169
+
+
+
+
8
51 598
324
+
+
+
+
9
52 342
457
+
+
+
+
10
49 805
450
+
+
+
+
11
37 010
152
+
+
+
+
13
30 267
525
+
+
+
+
14
28 742
378
+
+
+
+
16
23 352
221
+
+
+
+
17
23 321
168
+
+
+
+
18
21 896
391
+
+
+
+
19
17 563
289
+
+
+
+
20
16 359
609
+
+
n.s
+
21
14 622
483
+
+
+
+
22
13 952
396
+
+
+
+
23
13 407
224
+
+
+
+
24
12 452
374
+
+
−
+
25
12 307
465
+
+
+
+
26
12 264
1320
+
+
+
+
27
11 999
81
+
+
−
+
28
11 663
336
+
+
n.s
+
29
10 998
442
+
+
n.s.
−
30
9955
342
+
+
−
+
31
8316
288
+
+
−
+
32
4717
+
+
−
+
33
4349
990
+
+
−
+
34
4213
1080
+
+
−
+
36
3880
434
+
+
n.s.
+
37
3455
468
+
+
−
+
38
2240
154
+
+
+
+
39
1929
416
+
+
−
+
42
1254
858
+
+
−
n.s.
44
1045
360
+
+
−
+
45
929
63
+
+
n.s.
+
46
518
240
+
+
−
+
n.s., HIV-1 RT and protease were amplified but failed to generate full-length sequences.
aPlasma genotypes were generated using the Viroseq assay except for 1, 3, 18, 32, 33, 39 and 42 (TrueGene assay).
bAmplification from these DBSs stored at −20°C has been reported elsewhere.6
Nucleic acid extraction
Total nucleic acids were extracted from one spot after 1 year of storage at 4°C using a modification of the Nuclisens assay described previously.7 Briefly, a whole spot containing 50 µL of blood was cut with scissors and added into 9 mL of Nuclisens lysis buffer (bioMérieux, Inc., Durham, NC, USA). Special care was taken to avoid contact of the scissors with the blood spots; scissors and forceps were sprayed with 70% ethanol after each use and wiped dry. After 2 h of incubation with lysis buffer at room temperature under gentle rotation, the supernatant was clarified by centrifugation at 250 g for 5 min and then transferred to a clean 15 mL conical tube. Nucleic acids were then extracted following the manufacturer’s instructions, resuspended in 45 µL of elution buffer and stored at −80°C until use.
Drug resistance testing
Resistance testing from DBSs was performed using the ViroSeq HIV-1 Genotyping System (Abbott Molecular, Des Plains, IL, USA) and an in-house reverse transcriptase (RT)-nested PCR method described previously.7 The ViroSeq assay amplifies a 1.8 kb pol fragment and has a sensitivity of detection of 2000 RNA copies/mL plasma. The in-house assay amplifies a 1023 bp fragment of HIV-1 pol comprising amino acids 15–99 of the protease and 1–256 of the RT. This assay has been validated only for HIV-1 subtype B viruses and has a sensitivity of detection of 1000 RNA copies/mL of plasma.7 Genotypes were interpreted using the Stanford Genotypic Resistance Interpretation Algorithm (version 4.2.6) available at http://hivdb.stanford.edu/pages/algs/HIVdb.html.
Results
When the ViroSeq assay was used, only 23 of the 40 (57.5%) DBS specimens stored at 4°C were successfully genotyped; 22 of the successful amplifications were from specimens that had plasma viraemia >10 000 RNA copies/mL (Table 1). An additional five specimens produced amplicons that did not generate full-length RT and protease sequences. Visual inspection of the desiccant bags after 6 months of storage at 4°C showed evidence of humidity in 21 of the 40 specimens. Of these 21 specimens, only those with plasma VL higher than 14 000 RNA copies/mL (12 specimens) were successfully genotyped by the ViroSeq assay at 12 months (data not shown).
As we previously noted using parallel DBSs stored at −20°C, resistance genotypes generated from DBSs and matched plasma specimens were highly concordant. Of the 163 drug resistance mutations identified in these 23 plasma sequences, 158 were also found in sequences generated from DBSs. Of the five mutations absent in DBSs, three were protease mutations, two were major (V82A and I54M) and one minor (L10IT), and two were polymorphisms at codon 333 of the RT (G333E and G333D) (data not shown). We also compared the efficiency of amplification seen in these DBSs with that previously seen in DBSs prepared in parallel from the same patients and stored at −20°C for 6 months.6 Under storage at −20°C, all of the 40 DBS specimens were successfully amplified and genotyped (Table 1).
The ViroSeq assay amplifies a large (1.8 kb) pol fragment in a single round of PCR and may be particularly sensitive to the degradation of HIV-1 nucleic acids that may occur during long-term storage. We, therefore, assessed if the rate of amplification from DBSs stored at 4°C could be improved by using an in-house assay that amplifies a smaller (1023 bp) fragment and uses a nested PCR step.7 Using this method, viruses from 38 of the 40 DBSs (95%) were successfully genotyped (Table 1). Despite the use of a different assay to genotype viruses from these 38 DBS specimens, resistance genotypes were highly concordant with those generated from plasma using the ViroSeq assay; 275 of the 291 mutations found in plasma viruses were also found in viruses from DBSs (data not shown).
To investigate if the decreased amplification success rate observed with the ViroSeq assay was due to insufficient or degraded HIV nucleic acids, we performed serial dilutions of RNA extracts from high VL specimens and tested them by both the in-house and the ViroSeq assays (Figure 1). Although both assays have comparable sensitivity, amplification signals were only maintained when the shorter pol fragment was amplified by the in-house method. Figure 1 shows that such signals were consistently lost when the larger pol fragment was amplified using the ViroSeq assay, suggesting the possibility of nucleic acid degradation during storage at 4°C. Such a possibility was further supported by parallel testing of serial dilutions of RNA extracts prepared from specimens stored at −20°C. Figure 1(c) shows that amplification signals generated from these specimens using the in-house assay were generally stronger than those obtained from the same DBS specimens stored at 4°C (Figure 1b).
Figure 1
Agarose gel showing the HIV-1 pol amplification signals obtained from DBS specimens stored at 4 or −20°C. Two serial 10-fold dilutions (1/10 and 1/100) of specimens 2 (266 612 RNA copies/mL), 3 (214 330 RNA copies/mL), 4 (191 674 RNA copies/mL) and 10 (49 805 RNA copies/mL) were prepared in Nuclisens elution buffer and were tested in parallel using the ViroSeq assay (1.8 kb) or the in-house RT-nested PCR method (1023 bp). (a) DBS specimens stored at 4°C and amplified using the ViroSeq assay. (b) DBS specimens stored at 4°C and amplified using the in-house method. (c) DBS specimens stored at −20°C and amplified using the in-house method.
Discussion
Our study on a small number of specimens suggests that storage at 4°C may represent a feasible alternative to −20°C for long-term storage of DBSs. However, we noted that the efficient genotyping from these DBS specimens required the use of an in-house nested PCR assay and that the ViroSeq assay failed to genotype a substantial proportion of specimens. The results with the ViroSeq assay differed from our previous findings with this assay showing a high success of genotyping from DBSs stored at −20°C and suggest that some degradation of RNA may have occurred during long-term storage at 4°C possibly due to suboptimal storage temperature, humidity or both. Such a possibility was suggested by the weaker amplification signals observed in RNA extracts from specimens stored at 4°C compared with those stored at −20°C upon serial dilutions. Although different efficiencies of RNA extraction could partially explain these differences, we found that a high proportion of the specimens stored at 4°C had evidence of humidity at 6 months, whereas none of the specimens stored at −20°C did. High humidity conditions are thought to be detrimental to resistance testing from DBSs given the extreme sensitivity of HIV nucleic acids to degradation in the presence of humidity.10 Nonetheless, we showed that it is possible to overcome potential losses in HIV-1 RNA integrity and efficiently genotype from DBSs stored at 4°C by using an in-house nested PCR protocol that amplifies a smaller fragment using stringent, quality-controlled reagents. These results are encouraging and expand on our earlier findings showing the efficient genotyping of HIV-1 pol from DBSs stored at −20°C for up to 4 years.6,7 A high success of genotyping has also been noted from DBSs stored for 3 months at 37°C and 85% humidity in the presence of desiccant.10 Under these extreme conditions, viruses from ∼85% of the DBS specimens could be genotyped by using in-house nested PCR assays that amplified small (700 bp), overlapping pol sequences.10 The use of nested PCR protocols and/or the amplification of small fragments may likely result in higher amplification success rates from DBSs stored under less optimal conditions. Such a possibility was supported by our findings, showing a better maintenance of amplification efficiencies using the in-house method upon serial dilution of RNA extracts. Despite having comparable sensitivity, amplifications were rapidly lost when a larger fragment was amplified using the ViroSeq assay.
The main objective of our study was to evaluate the amplification success rate from DBSs stored at 4°C for a prolonged period of time. We used an in-house assay validated for HIV-1 subtype B since our study population included patients infected with subtype B viruses. It is important to note, however, that information on the performance of our assay with non-B subtypes is limited to a small number of specimens.7 Therefore, we caution that resistance testing from DBSs collected in areas with prevalent non-B subtypes will require in-house assays appropriately validated for other HIV subtypes.
The ability to collect blood samples on filter paper represents an advantage for HIV drug resistance surveillance and monitoring, particularly in areas that lack the appropriate infrastructure for plasma processing and transport. Much effort is currently underway to define the best conditions that will facilitate resistance testing from DBSs including the most appropriate storage temperature and time. The findings reported here suggest that 4°C may represent a feasible storage temperature for long-term storage of DBSs and add to the promise of DBSs as a convenient specimen type for HIV-1 drug resistance testing.
Funding
Work at the CDC was done with intramural funding. Work at Hospital Carlos III was supported by grants from the Fondo de Investigación Sanitaria (FIS, CP06/284 and PI06/1826).
Transparency declarations
None to declare.
Disclaimers
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.

resistance testing
903 filter paper
viroseq assay


Effects of the noradrenergic agonist clonidine on temporal and spatial attention

Rationale Recent theories posit an important role for the noradrenergic system in attentional selection in the temporal domain. In contrast, the spatially diffuse topographical projections of the noradrenergic system are inconsistent with a direct role in spatial selection.
Introduction
The locus coeruleus–norepinephrine (LC–NE) system is one of several brainstem neuromodulatory nuclei with widely distributed, ascending projections to the neocortex (Berridge and Waterhouse 2003). The conventional view of NE is that it has broad, nonspecific functions such as regulating arousal (Jouvet 1969). However, recent studies suggest that NE may have more specific cognitive functions, which are nevertheless closely related to the concept of arousal (Robbins 1997; Aston-Jones et al. 2000; Yu and Dayan 2005; Chamberlain et al. 2006). In particular, it has been proposed that NE has a key role in facilitating the responses to decision-making processes and in regulating the balance between exploitation and exploration (Aston-Jones and Cohen 2005).
Recent views on the role of the LC–NE system in decision making have been based on neuronal recordings of the primate LC during the performance of simple decision-making tasks. These recordings have indicated that under alert conditions (i.e., conditions typical of human subjects in our experiments), LC phasic responses are selectively observed after task-relevant and otherwise motivationally significant (e.g., highly salient or infrequent) stimuli (Aston-Jones et al. 2000). Furthermore, stimulus-elicited phasic LC bursts reliably precede (by ∼200 ms) and are closely coupled in time with the behavioral response (Bouret and Sara 2004; Clayton et al. 2004). The noradrenergic innervation associated with such LC responses results in a system-wide, transient increase in the responsivity of efferent target neurons, which is thought to facilitate processing in response to the eliciting stimulus (Berridge and Waterhouse 2003). These observations have led to the suggestion that the LC phasic response can be thought of as a temporal attentional filter (1) that selects for the occurrence (i.e., timing) of task-relevant (Aston-Jones and Cohen 2005) or unexpected (Dayan and Yu 2006) events, much like cortical attentional systems filter the content (e.g., spatial characteristics or color) of stimuli and (2) that facilitates the processing of and responding to these stimuli to help optimize task performance (Aston-Jones and Cohen 2005; Nieuwenhuis et al. 2005a; see also Coull 1994).
In the current study, we evaluated the following two hypotheses: (1) According to the temporal filtering hypothesis outlined above, the LC–NE system should be involved in temporal attention modulations operating at the phasic time scale (e.g., in the order of tens of seconds); (2) In contrast, because of the spatially nonspecific pattern of LC projections to the forebrain, the LC–NE system cannot be directly involved in spatial attention shifts. That is, although the LC–NE system projects to multiple brain areas, including many known to be involved in spatial attention, the diffuse character of these projections is inconsistent with the direct biasing of attention in any specific direction (i.e., the LC does not “know” the relevant spatial location). To test these two hypotheses, we contrasted, in human subjects, the effects of a noradrenergic drug on measures of temporal and spatial attention. Most previous studies examining the functional significance of the LC–NE system have employed drugs acting on α2 receptors. In this study, we used the α2 adrenoceptor agonist clonidine, which at low doses decreases LC firing and attenuates the release of NE from axon terminals (Svensson et al. 1975).
Previous studies have examined the effects of clonidine on temporal and spatial attention in a visuospatial cueing task. In this task, subjects are required to respond as quickly as possible to a series of stimuli presented to the left or right of fixation. Each stimulus is preceded by a cue indicating at which location the imperative stimulus is most likely to appear, and subjects are instructed to make use of this information by switching their attention to the location indicated by the cue. The comparison of reaction times on validly versus invalidly cued trials (the “validity effect”) provides an index of the efficiency of cue-induced spatial attention shifts. Fernandez-Duque and Posner (1997) extended this paradigm by including and contrasting reaction times on neutral-cue trials and no-cue trials (the “alerting effect”), which provides an index of the transient increase in responsivity brought about by the mere presentation of the cue. The alerting effect thus serves as a measure of temporal (cue-evoked) changes in attention to the imperative stimulus.
Clonidine has been found to reduce the size of the alerting effect in humans (Coull et al. 2001) and monkeys (Witte and Marrocco 1997), suggesting that the temporal attentional effects of cues are mediated in part by the noradrenergic system. In contrast, studies have reported mixed results regarding the influence of clonidine on the validity effect: Two studies have reported a reduced validity effect (Clark et al. 1989; Coull et al. 2001), and one study has reported no effect of clonidine (Witte and Marrocco 1997). However, it is hard to interpret clonidine-related reductions in the validity effect, because they may reflect either a direct involvement of the noradrenergic system in spatial attention processes or an interaction between the alerting and attention-directing (i.e., spatial) effects of cues. Specifically, by increasing the responsivity of cortical representations after cue presentation, LC-induced phasic NE release can enhance the effects of target selection by location (e.g., the top-down modulation by prefrontal cortex), resulting in an increased validity effect. The effects of this amplification mechanism would be diminished by clonidine.
In contrast to previous studies, we used separate tasks for measuring the effects of clonidine on temporal and spatial attention: an attentional blink task, which requires the selection of targets in time and has no spatial component, and a visual search task, which requires the selection of targets in space but without the help of alerting cues.
In the attentional blink task, each trial starts with a rapid serial visual presentation (RSVP) stream containing two target stimuli (T1 and T2) and multiple distractors, each presented for about 100 ms. At the end of the RSVP stream, subjects are required to report the identity of T1 and T2.
The critical finding in this task is that subjects are heavily impaired at the identification of T2 for a few hundred milliseconds after correct identification of T1. This deficit, known as the “attentional blink,” is usually most severe around 200–400 ms (or two to four items) after T1, after which, performance gradually recovers (Raymond et al. 1992; Chun and Potter 1995). In contrast, if T2 follows T1 without intervening distractors (at “lag 1”), performance on T2 is often (partially) spared (Raymond et al. 1992; Hommel and Akyürek 2005); this phenomenon has been labeled “lag-1 sparing.” These and other findings suggest that the attentional blink task reveals important clues about the time course of fine-scale fluctuations in attention (Olivers 2007).
Recently, a theory has been proposed that explains the attentional blink in terms of the temporal dynamics of the LC–NE system (Nieuwenhuis et al. 2005b). The theory was inspired by the observation that the timing of the attentional blink coincides with the refractory period in LC activity that follows an LC phasic response to target stimuli. Specifically, although NE potentiates processing in cortical areas, local NE release within the LC is autoinhibitory because of the noradrenergic action at presynaptic and dendritic α2 autoreceptors (Aghajanian et al. 1977). This autoinhibition results in a period after the LC phasic response (∼200–450 ms after the eliciting stimulus), during which subsequent LC phasic discharge is rarely observed. According to the theory, the attentional blink may be mediated by this momentary unavailability of the LC phasic response (and attendant noradrenergic potentiation of information processing) after the LC response to T1. In other words, if T2 is presented during the LC refractory period associated with T1, it will not receive the benefit of NE-mediated facilitation and is therefore more likely to remain unidentified. A computational model implementing this theory was shown to accurately simulate the time course of the attentional blink, including lag-1 sparing (Nieuwenhuis et al. 2005b). In addition, the theory offers an account of the close relationship of the attentional blink to the P3 component of the event-related potential, an electrophysiological correlate of the phasic NE release (Nieuwenhuis et al. 2005a).
To assess the effect of clonidine on spatial attention, we used a visual search task. On each trial, subjects searched for a target among multiple distractors in a visual search array and indicated as quickly as possible whether the target was present or absent. The number of distractors (i.e., set size) was systematically varied across trials. In one condition (single-feature search), the target was defined by a salient basic feature, such that search was efficient and the number of distractors had little influence on reaction times. In another condition (conjunction search), the target was defined by a conjunction of two features, such that search was inefficient, and reaction times increased linearly with the number of distractors—as if each item needed to be examined in turn. In this task condition, the efficiency of spatial attention processes is expressed in the function relating search reaction times to set size: The more visual attention is impaired, the steeper the slope of this function (as expressed in an increased effect of set size; Tales et al. 2002).
Our predictions with regard to the effects of clonidine were the following: First, we expected that clonidine would impair performance on the attentional blink task, in particular through its stimulation of inhibitory α2 autoreceptors in the LC. In accordance with the temporal filtering hypothesis, we expected that the clonidine-induced reduction in noradrenergic activity would lead to an impaired identification of T1 but especially of T2, because it should presumably exacerbate the lack of NE associated with T2 processing under normal conditions (Nieuwenhuis et al. 2005b). Second, aside from the well-documented main effect of clonidine on response speed (i.e., general slowing; e.g., Tiplady et al. 2005), we expected no drug effects on visual search efficiency, as indexed by the effects of distractor set size. This latter prediction was based on the notion that the effects of NE are topographically diffused and hence should not affect the efficiency of selection in the spatial domain.
Materials and methods
Subjects
Thirty-two healthy young adults, aged 18–25 years, took part in a single experimental session in return for 60. Only subjects with a systolic blood pressure above 100 mmHg and a diastolic blood pressure above 70 mmHg were included in the study. All subjects underwent a medical screening and were considered to be in satisfactory health. The use of medication that could interfere with clonidine was stopped the day before. All subjects with color blindness were excluded from the study.
Subjects received an oral dose of 150 μg of clonidine or placebo in a double-blind, between-subjects design. Clonidine has well-established antihypertensive properties; therefore, blood pressure and heart rate were monitored for subject safety. Measurements were taken every 15 min starting from t = −15 until t = 120 and every 30 min from t = 120 until t = 240.
The clonidine group (N = 16, eight women, M age 21.3) and the control group (N = 16, eight women, M age 21.3) had similar mean ages. We also verified that the groups were similar in terms of intellectual functioning as assessed with the three-subtest version of the Groninger Intelligence Test (GIT), a reliable indicator of the full-scale IQ (Luteijn 1966). Estimated IQ scores were 117.1 for the clonidine group and 116.8 for the control group. The visual-search task data from one subject from the clonidine group were discarded because of an accidental failure to comply with the task instructions. The study was approved by the medical ethics committee of the VU University Medical Center and was conducted according to the Declaration of Helsinki. Informed consent was obtained from all subjects before their inclusion in the study.
Procedure
Each subject was tested at approximately the same time of the day (afternoon). Subjects were instructed to abstain from caffeine, nicotine, and other psycho-active substances from 15 h before the start of the session and to abstain from alcohol from 15 h before the start of the session until the day after the session. After the medical screening, subjects received a lactose-filled capsule with either clonidine or placebo. Immediately after that, the subtests of the GIT were administered. The two attentional tasks, described in detail below, were performed between t = 60 and t = 120 min posttreatment. Half of the subjects in each group started with the attentional blink task. The other half started with the visual search task. After completion of the tasks, the subjects were debriefed and paid. At t = 240, subjects were reevaluated and returned home by taxi if blood pressure was (near) normal.
Attentional blink task
Stimulus generation and response recording in both tasks were controlled by E-Prime software (Psychology Software Tools, Pittsburgh, PA). Stimuli were presented in black against a light grey background. Each trial started with a fixation cross measuring 0.5 × 0.5°, presented for 1,000 ms in the center of the display. Subsequently, the fixation cross was replaced by an RSVP stream of 15–22 uppercase letters, each measuring approximately 0.9 × 0.9°. Each letter was randomly drawn (without replacement) from the alphabet and presented for 50 ms, followed by a 30-ms blank interval. This relatively rapid pace of stimulus presentation (i.e., 80 ms between two consecutive stimulus onsets) was chosen to avoid ceiling levels of performance and consequently to increase the sensitivity of the task to individual and group differences. “I,” “O,” “Q,” and “S” were left out as they resemble digits too much. On each trial, two of the letters were replaced with digits, randomly drawn without replacement from the set 2 to 9. T2 was presented three to six temporal positions from the end of the stream. The temporal distance between T1 and T2 was systematically varied from one to five items, corresponding to lags of 80, 160, 240, 320, and 400 ms. The subject’s task was to identify both T1 and T2 by typing the digits in order on a standard keyboard after the end of the RSVP stream. Subjects were instructed to guess whenever they failed to identify a digit. The two keyboard entries were followed by the presentation of a feedback stimulus for 150 ms (e.g., ‘+, −’ to indicate that T1 was correct and T2 was incorrect). After a 1,000-ms blank screen, the next trial started. Each subject started with 15 practice trials, three with each condition, randomly intermixed. This was followed by four blocks of 50 trials each with each block containing ten repetitions of each lag. All task instructions were automated and presented on screen.
Visual search task
On each trial, subjects searched for a target among multiple distractors in a visual search array. On half of the trials, the target, a vertical red bar, was present in the array. On the other half of the trials, the target was absent. In one condition (single-feature search), the distractors in the search display were vertical green bars of equal size as the target. Because in this case the target is defined by a unique feature (color), it “pops out” from the display, resulting in a fast and efficient search process. In another condition (conjunction search), the distractors were vertical green bars and horizontal red bars. In this case, the target does not consist of a single identifying feature but is defined by a specific conjunction of features (color and orientation), resulting in a more time-consuming and error-prone search process.
Each trial started with a white fixation cross measuring 0.9 × 0.9° against a dark background, presented for 500 ms in the center of the display. Subsequently, the fixation cross was replaced by the search display, which consisted of four, eight, or 16 items that were randomly plotted in the cells of an imaginary 6 × 6 matrix (8.7° horizontally × 9.6° vertically) with some random jitter within the cells. The subject’s task was to report whether or not the target (0.7 × 1.3°) was present by giving a response with their left or right index finger using the ‘z’ and ‘m’ keys on the computer keyboard. The keyboard entry was immediately followed a 1,000-ms blank screen after which the next trial started.
Subjects performed four blocks of 96 trials each, with each block containing 16 repetitions of the factorial combination of set size (4, 8, or 16) and trial type (target present or absent) presented in random order. Search condition (single-feature search or conjunction search) was varied across blocks in an ABBA-order. Half of the subjects started with single-feature search, and the other half started with conjunction search. Subjects received written instructions and 12 practice trials before entering the experimental phase. The task instructions encouraged subjects to respond as quickly as possible while minimizing the number of errors. Performance feedback was provided at the end of each block. All task instructions were automated and presented on screen.
Results
Physiological measures
Figure 1 presents for both groups the absolute values of heart rate, systolic, and diastolic blood pressure as observed throughout the experimental session. All three measures showed a significant effect of time (all P < 0.001). Systolic [F(1, 30) = 11.8, P = 0.002] and diastolic blood pressure [F(1, 30) = 6.3, P = 0.018] both showed significant main effects of group. The main effects of time and group were qualified by significant time X group interactions for systolic [F(13, 390) = 9.8, P < 0.001] and diastolic blood pressure [F(13, 390) = 6.1, P < 0.001].
Fig. 1Systolic and diasystolic blood pressure and heart rate following administration of placebo or 150 μg of clonidine (time = 0). The grey shaded area marks the time period during which the two cognitive tasks were administered
Attentional blink task
Figure 2 shows the average T1 accuracy (left panel) and T2 accuracy (right panel; contingent on correct T1 identification) as a function of lag and group. A similar pattern of results was found if T2 accuracy was averaged across correct and incorrect T1 trials. Trials on which T1 and T2 were accurately identified but in the wrong order were treated as correct. As an analysis of variance (ANOVA) showed no significant effects of the order in which the two tasks were performed, this factor was omitted from the reported analyses.
Fig. 2Average identification accuracy for the first (T1; left panel) and second (T2; right panel) of the two targets in the attentional blink task as a function of group (clonidine vs placebo) and the lag between T1 and T2. As is usual, T2 accuracy is reported contingent on accurate identification of T1
T1 and T2 accuracy were entered in separate two-way mixed ANOVAs with lag and time-on-task (blocks 1,2 vs 3,4) as within-subject factors and group as a between-subject factor. The variable time-on-task was included to assess the possibility that a group effect emerged over time. T1 accuracy showed an increasing trend with lag [F(4, 120) = 12.3, P < 0.001]. The clonidine group (79.5% correct) performed slightly worse on T1 than the placebo group (84.6%), but the main effect of group did not reach significance [F(1, 30) = 2.0, P = 0.17]. The lag X group interaction was not significant [F(4, 120) = 1.0, P = 0.39].
The two T2 accuracy curves show a pattern that is characteristic of attentional blink research: lag-1 sparing, followed by a drop in performance for lags 2, 3, and 4 (i.e., the attentional blink), and followed by the initial recovery of performance at lag 5, at least in the clonidine group. This pattern was expressed in a significant effect of lag [F(4, 120) = 43.2, P < 0.001]. The most important for the present purposes is the finding that T2 accuracy did not reliably differ between the two groups [F(1, 30) < 1]. Furthermore, although at lag 4 there was a sizeable numerical effect of group in the expected direction, the lag X group interaction was not significant [F(4, 120) = 1.2, P = 0.30]. Time-on-task did not reliably affect T1 or T2 performance or interact with other variables.
In a separate analysis, we calculated the average T2 performance across lags 3–5 as a summary measure of attentional-blink magnitude and examined group differences in this measure. The two groups did not differ in attentional-blink magnitude, and performance at lags 3–5 did not improve with time-on-task [both F(1, 30) < 1]. Interestingly, there was a reliable interaction between group and time-on-task [F(1, 30) = 4.9, P = 0.034]: Whereas performance of the placebo group deteriorated over time (66.6 vs 62.2% correct; P = 0.65), performance of the clonidine group improved slightly (60.3 vs 62.6%; P = 0.28).
In a final analysis, we examined the percentage of order reversals at lag 1, the phenomenon that the two targets, when immediately succeeding each other, are often identified correctly but reported in the wrong order (Hommel and Akyürek 2005). The percentage of such order reversals was roughly the same in the placebo group (34.4%) and in the clonidine group [35.4%; t(30) < 1].
Visual search task: single-feature search
The total number of items in the search display (set size) was varied from trial to trial, allowing us to derive the function relating reaction time (RT) to set size. The slope of this function measures the cost for adding additional items to the display and is often interpreted as “search efficiency,” with steeper slopes indicating slower, less efficient search. As expected and illustrated in Fig. 3, slopes in the single-feature search condition were close to zero [average 1.6 ms/item; t(30) = 2.6, P = 0.014].
Fig. 3Average correct reaction times for the clonidine and placebo groups in the visual search task. Top panels show data from the single-feature search condition, and bottom panels show data from the conjunction search condition. Data for target-present trials are plotted in the left-hand panels, data for target-absent trials are plotted in the right-hand panels
Correct RTs were entered in a three-way mixed ANOVA with target presence (target present vs target absent) and set size (4, 8, or 16) as within-subject factors and group as between-subject factor. Despite the relatively shallow RT slopes, the main effect of set size was significant [F(2, 58) = 6.3, P = 0.006]. Furthermore, the clonidine group was overall slower than the placebo group [F(1, 29) = 5.6, P = 0.025]. Group did not interact with target presence [F(1, 29) = 1.2, P = 0.29] or set size [F(2, 58) = 2.5, P = 0.10]. The three-way interaction was also not significant [F(2, 58) < 1]. Error rates were generally low (average 2.3%). An ANOVA yielded only a significant main effect of target presence [F(1, 29) = 8.9, P = 0.006], with errors occurring more often on target-present trials (2.9%) than on target-absent trials (1.8%).
Visual search task: conjunction search
As expected, slopes in the conjunction-search condition were sizeable [average 25.0 ms/item; t(30) = 13.8, P < 0.001], indicating that visual search was time consuming and inefficient. A three-way mixed ANOVA yielded significant main effects of target presence [F(1, 29) = 31.5, P < 0.001] and set size [F(2, 58) = 152.9, P < 0.001] and a significant interaction of these two variables [F(2, 58) = 26.7, P< 0.001], indicating that set-size effects were larger for target-absent trials. Importantly, the main effect of group was not significant [F(1, 29) = 2.1, P = 0.16], and group did not reliably interact with any of the task variables (all F < 1). Error rates were again low (average 3.3%). An ANOVA yielded the same pattern of effects as the RT analysis, indicating that there was no speed–accuracy trade-off, and that there were no significant group differences.
Discussion
The main findings of the current study can be summarized as follows. First, in contrast to our predictions, clonidine did not have a deleterious effect on attentional blink performance. Although the numerical group differences in T1 and T2 accuracy were in the expected direction, these differences were small compared to the sizeable performance differences between the individuals within each group (for a discussion of these individual differences, see Martens et al. 2006). The results from the visual search task were as expected: Although clonidine slowed the overall response speed, it did not affect the efficiency of the visual search for a target in a two-dimensional array of stimuli. Below, we will discuss the implications of these principal findings.
The absence of a significant drug effect on attentional blink performance appears to be at odds with the temporal filtering hypothesis, which suggests that phasic activity of the LC–NE system is important for the selection of important or infrequent stimuli in the temporal domain (Aston-Jones and Cohen 2005; Dayan and Yu 2006). The attentional blink results also seem inconsistent with the predictions of a recent theory that explains the attentional blink in terms of the temporal dynamics of the LC–NE system (Nieuwenhuis et al. 2005b). One possible explanation for these discrepancies is that the predicted group differences do in fact exist, but that our task and experimental design were not sufficiently sensitive to reveal them. Some critical factors in this regard may be the choice of a between-subject design and the sample size of 16 subjects per group. However, previous studies using between-subject designs, similar group sizes, the same subject population, and the same task have had no difficulty detecting group-levels effect on attentional blink performance of various manipulations aimed at distracting subjects from the RSVP stream (Olivers and Nieuwenhuis 2005, 2006). Indeed, our choice of design was based on these previous studies and on the assumption that the effect of clonidine would be of at least the same size as the effect of these behavioral manipulations. In this context, it is worth noting that the interindividual variance in attentional blink performance was comparable to that in the Olivers and Nieuwenhuis studies, discounting increased variance as a reason for our failure to detect group differences.
A more principled reason for the use of a between-subject design was that subjects can learn to adopt a ‘mental state’ that greatly benefits attentional blink performance (Olivers and Nieuwenhuis  2006), and we wanted to avoid the transfer of such learning between drug conditions to exclude a potential contaminating source of variance. In addition, Coull et al. (1995a, b) have found that in within-subject designs, the order of treatments (i.e., clonidine before or after placebo) can show complicated interactions with task performance, another source of variance that we aimed to exclude.
Another design feature that may have led to a failure to detect an existing effect is the timing of test administration relative to the treatment (t = 60–120 min). In particular, the systolic and diastolic blood-pressure time series reveal a maximal drug effect after the test period, suggesting that cognitive effects may have peaked later too. However, there are at least three arguments against this possibility. First, Tiplady et al. (2005) have investigated the time course of effects of 150 μg of orally administered clonidine on cognitive test variables and found that such effects were already present at t = 45 min but were decreased at t = 135 min. Second, our task-block comparisons show that, if anything, the group difference in attentional blink performance became smaller over time, a trend opposite from that expected if the effects of clonidine needed more time to develop. A third and related argument is that we found no significant interaction between group and task order, corroborating the notion that group effects did not become larger over time. Alternatively, we cannot rule out the possibility that group effects peaked earlier than our test period. However, our choice of test period seems justified by the finding of significant cognitive effects of clonidine in various previous studies using similar test periods (Halliday et al. 1989, 1994; Coull et al. 2001). Thus, our methods and results do not seem to contain any clear indication that the observed null effects reflect a lack of sensitivity. However, it is of course possible that a higher drug dose would yield more robust group differences. This is a possibility that can be addressed in future research (note that such research should take into account that the primary mode of action of clonidine changes with increasing dose; (Arnsten and Cai 1993). At low doses, presynaptic receptors and LC receptors are stimulated, leading to a reduced NE activity. In contrast, at higher doses, postsynaptic receptors are activated, resulting in a boost of NE activity. This has important implications for the expected effects of clonidine on cognitive function).
Another possible explanation for the absence of significant drug effects on attentional blink performance is that the attentional blink may not be mediated by the LC–NE system. This possibility would be particularly bothersome for the theory of Nieuwenhuis et al. (2005b), which directly relates the attentional blink to a refractory period in activity of the LC–NE system. Although there is substantial indirect evidence in support of this theory (e.g., the relationship between the attentional blink and the P3), the basic tenets of the theory are based on cell recordings in animals and are hard to validate directly in humans. Indeed, the current study is perhaps the most direct test to date of a relationship between the attentional blink and the LC–NE system. In this context, it is important to note that another research group has reported preliminary evidence consistent with such a relationship (De Martino et al. 2005). This study, using a between-subject design, found that attentional blink performance was significantly impaired after intake of the ß-adrenergic receptor antagonist propranolol (40-mg oral dose compared to placebo). Although this finding is not predicted by the theory of Nieuwenhuis et al. (2005a, b), which relates the attentional blink to the dynamics of inhibitory α2 autoreceptors in the LC, it will be informative to determine to what extent the current findings generalize to other task designs and other noradrenergic drugs. A further avenue for future research will be to test and evaluate the effects of noradrenergic drugs on temporal attention using alternative experimental paradigms, such as the temporal analogue of the spatial cueing paradigm (Posner et al. 1980; Coull and Nobre 1998; see Coull et al. 2001). Eventually, accumulating evidence obtained in such human psychopharmacological studies can be used to accommodate theories about the role of NE in temporal attention.
The results from the visual search task were consistent with our predictions: Although clonidine led to a general slowing of response speed, we found no evidence for drug effects on visual search efficiency as indexed by the effects of the distractor set size. This pattern of results was expected based on the notion that the effects of NE are topographically diffuse (i.e., not selective with regard to any specific spatial representations) and hence should not affect the efficiency of selection in the spatial domain. Previous studies using visuospatial cueing tasks have found that clonidine reduces the cue validity effect (Clark et al. 1989; Coull et al. 2001), which has led to the suggestion that the LC–NE system is directly involved in visuospatial orienting. For example, it has been proposed that the LC–NE system plays an important role in the disengagement of visuospatial attention from invalidly cued spaces (Clark et al. 1989; Posner and Petersen 1990). This proposal is consistent with the little available evidence, which suggests that the reduced validity effect after clonidine administration is mainly due to attenuated performance costs on invalidly cued trials. However, we propose an alternative interpretation for these results to reconcile them with our theoretical framework. According to our interpretation, the presentation of the cue causes a spatially nonspecific, transient LC–NE response that boosts the processing of any stimuli presented for a brief period thereafter (i.e., the alerting effect). Although not supported by the current attentional blink findings, this assumption is based on various sources of evidence in the animal literature (Aston-Jones and Cohen 2005; Witte and Marrocco 1997). The cue-induced LC–NE response speeds up cortical spatial selection processes involved in localizing the cue and/or subsequent imperative stimulus. This improves performance on validly cued trials and impairs performance on invalidly cued trials thus increasing the validity effect. Administration of clonidine counteracts this amplifying effect of phasic LC–NE activity thereby reducing the validity effect (note that this hypothesis predicts both improved performance on invalid trials and impaired performance on valid trials). This type of interaction between the alerting effect and spatial selection processes is absent in the visual search task, which has no clear alerting component. Therefore, the current results are consistent with the notion that the LC–NE system is not directly involved in visuospatial orienting but can only indirectly modulate the effects of spatial attention mechanisms implemented elsewhere in the brain.
One drawback of the current study is that we did not include a task that has already been shown to be sensitive to clonidine in previous studies. Therefore, a possible interpretation of the reported null findings is that the administered dose of clonidine failed to affect general cognitive function in our subjects, and that the main effect of drug on visual search RT reflects drug-induced motor slowing rather than a central cognitive deficit. Although we deem this possibility unlikely, given that previous studies using similar doses of clonidine have found significant effects on cognitive function (Coull et al. 2001; Tiplady et al. 2005), a more extensive study with a larger task battery is needed to adequately address this issue.
There is rapidly growing interest in the specific role of the LC–NE system in human cognition (Cohen and Aston-Jones 2005). Psychopharmacological data from humans will be of critical importance in testing and further developing the increasingly sophisticated hypotheses that are based on neurophysiological observations in animals (e.g., Yu and Dayan 2005). The current research provides a valuable contribution toward a better understanding of the role of the LC–NE system in human attention, both in the spatial and the temporal domain.

clonidine
attention
attentional blink
visual search
norepinephrine
locus coeruleus