INK4a as a possible diagnostic marker of cervical cancer. To evaluate whether the specificity of p16INK4a expression in dysplastic and neoplastic cervical epithelium is sufficient for such application we undertook a broader immunochistochemical registration of this protein with a highly p16INK4a-specific monoclonal antibody.Cervical carcinomas are second most frequent type of women cancer. Success in diagnostics of this disease is due to the use of Pap-test . However Pap-test gives significant portion of both false-positive and false-negative conclusions. Amendments of the diagnostic procedure are desirable. Aetiological role of papillomaviruses in cervical cancer is established while the role of cellular gene alterations in the course of tumor progression is less clear. Several research groups including us have recently named the protein p16Paraffin-embedded samples of diagnostic biopsies and surgical materials were used. Control group included vaginal smears of healthy women and biopsy samples from patients with cervical ectopia. We examined 197 samples in total. Monoclonal antibody E6H4  was used.INK4a-positive cells. Overexpression of p16INK4a was detected in samples of cervical dysplasia (CINs) and carcinomas. The portion of p16INK4a-positive samples increased in the row: CIN I – CIN II – CIN III – invasive carcinoma. For all stages the samples were found to be heterogeneous with respect to p16INK4a-expression. Every third of CINs III and one invasive squamous cell carcinoma  were negative.In control samples we did not find any p16INK4a is typical for dysplastic and neoplastic epithelium of cervix uteri. However p16INK4a-negative CINs and carcinomas do exist. All stages of CINs and carcinomas analyzed are heterogeneous with respect to p16INK4a expression. So p16INK4a-negativity is not a sufficient reason to exclude a patient from the high risk group. As far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages application of immunohisto-/cytochemical test for p16INK4a may be regarded as a supplementary test for early diagnostics of cervical cancer.Overexpression of the protein p16 Cervical cancer makes up about 10–12% of total women cancers [The main screening test for cervical cancer is the cytological smear staining technique developed by G. Papanicolaou  and knowHistological analysis of a biopsy sample, more laborious in preparation and study as compared with that of a cytological smear, is also not absolutely efficient owing to a substantial rate of interobserver discrepancies among expert pathologists examining the same material .Infection with human papilloma viruses (HPV) belonging to so-called high-risk group is the main risk factor of cervical cancer incidence . To deteINK4a for a possible supplementary marker of dysplastic and neoplastic cervical epithelium lesions. This protein belongs to the group of cyclin-dependent kinase Cdk4/6 inhibitors [INK 4a . Gene INK4a plays an important role in the regulatory pathway Cdk-Rb-E2F. The product of this gene p16INK4a prevents pRb phosphorylation by inactivating Cdk4/6; pRb keeps on binding E2F transcription factors and as a result cells stay in G1 phase not passing to DNA replication. In various tumor types INK4a as a bona fide tumor suppressor undergoes homozygous deletions, is inactivated by point mutations, LOH or hypermethylation; p16INK4a expression is reduced or ceases under such conditions or the protein function may be impaired [For the recent years several research groups ,6-16 inchibitors  and is eimpaired .INK4a and its protein becomes of no importance for a cell so far as they function upstream of the site of breakage. Cells with thus inactivated pRb pass cell cycle checkpoint G1/S without any obstacle.Peculiarity of cervical carcinomas is due to the ability of HPV oncoprotein E7 to interact with pRb and inactivate it . As a reINK4a commonly found in human permanent cell lines  [INK4a protein as a marker of premalignant and malignant cervical epithelium cells. Functionally active gene RB was shown to be able to negatively regulate the expression of INK4a on a transcriptional level, but details of this negative feed-back loop remain obscure [Reciprocallity between status of pRb and that of p16ultures) ,20,21 asultures) ,22,23 se obscure .INK4a as a marker of dysplastic and neoplastic alterations in cervical epithelium cells we analyzed the expression of this protein utilizing a highly p16INK4a – specific monoclonal antibody. We examined 197 samples in total. The materials studied included: 1) samples of normal epithelium of healthy women , 2) diagnostic biopsy samples from patients with cervical ectopia, 3) samples of CINs of various stages, 4) samples of invasive cervical cancer, 5) samples from different normal utery body and cervix tissues from women with gynecological diseases not associated with dysplastic lesions of epithelium  and 6) samples of cells from 3 cervical carcinoma cell lines. We also compare our data with the results presented by other groups working in similar directions [To estimate the applicability of p16rections ,12-16.INK4a is typical for some samples of dysplastic and neoplastic epithelium of cervix uteri. We have not found at least a single sample overexpressing p16INK4a among control samples. The portion of p16INK4a-positive samples increases in the following row: CIN I – CIN II – CIN III – invasive carcinoma. However p16INK4a-negative CINs and carcinomas have also been found. All stages of CINs and carcinomas analyzed turn out to be heterogeneous with respect to p16INK4a expression: side by side with the samples which express p16INK4a in 25% of cells or more we detect samples which are stained poorly or lack any staining. So p16INK4a-negativity does not seem to be a sufficient reason to exclude a patient from the high risk group when results of Pap-test, HPV detection by PCR or histological investigation warn about possibility of poor prognosis. As far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages of CINs and carcinomas application of immunohisto-/cytochemical test for p16INK4a may be regarded as a supplementary  test for early diagnostics of cervical cancer.We demonstrate in the present work that overexpression of the protein p16Immunohisto-/immunocytochemical study was performed on 197 samples in total. Those included 6 samples from normal epithelium of healthy women , as well as the following surgical and diagnostic biopsy materials: 37 samples from patients with cervical ectopia, 113 samples of CINs of different stages including cancer in situ, 26 samples of invasive cervical cancer (21 squamous cell carcinomas and 5 adenocarcinomas), 12 samples of normal tissues from uterus body (myometrium) and cervix from patients with different gynecological diseases, 3 cervical cell line samples taken for positive control (see below). Apart from 9 smears  the rest 188 materials were paraffin-embedded histological blocks. The quality of smears turned out to be satisfactory for immunochemical analysis Fig.  and 1d.Neutral formalin-fixed paraffin embedded samples of biopsies and surgery materials were received from archives of N.N.Blokhin Cancer Research Center (Moscow), P.A. Gertzen Institute of Oncology (Moscow), Central Clinical Hospital (Moscow) and Fourth Clinical Hospital .For immunohistochemical analysis 4–5 μm serial sections were transferred on Histobond slides with adhesive layer . The first section was stained with hematoxylin-eosin for traditional morphological analysis and verification of diagnosis by not less than two independent pathomorphologists.Presence of high risk HPV in cervical CIN and carcinoma cells was verified by PCR . Some ofVaginal normal epithelium smears obtained during regular examination from healthy women were transferred on HistoBond slides. Absence of abnormal cells was confirmed in Cytology Department of Cancer Research Center on a parallel slide. Smears were fixed for 5 min in 10% formaldehyde, washed with flowing water, and processed for further development as described for deparaffinized histological slices . Cells oINK4a-specific monoclonal antibodies E6H4  according to protocol by Klaes et al [Immunohistochemical staining was carried out using p16es et al .INK4a in earlier immunocytochemical studies with the same antibody [INK4a-positive one in our previous studies (on preceeding cuts). The analysis of the given slide series was carried out only if the positivity of the control sample was confirmed. Thus neither slide series in the present study was completely p16INK4a-negative.1). In the beginning of the study as a whole cells of three cervical cell lines were stained first. They were SiHa (HPV16-positive), C33a and HT-3 (both HPV-negative); all of them had been characterized as expressing the protein p16antibody . 2). OneINK4a-expressing material was included into every slide series meant for staining  which was processed in a usual way but PBS was applied instead of p16INK4a-specific antibody. The results were regarded as valid if this slide was negative. 2). As an intrinsic negative control served adjacent to CIN or carcinoma normal tissues including stromal elements. In neither case did we find any staining in these tissues (fig. 1). One more serial cut made from p16ues fig. .INK4a was expressed in nuclei and/or in a cytoplasm.In addition to four categories of staining defined by Klaes et al. ,- poor , sporadiINK4a-specific staining of cervix uteri normal, dysplastic and cancer cells are presented in Fig. Various types of p16INK4a  may be detected but in a cytoplasm in the majority of cells while in a number of cells both in nuclei and cytoplasm, why in some cervical cell cultures it is found solely in cytoplasm and in other cervical cell lines – in nuclei also. We did not find at least a single sample with an exceptionally nuclear staining; similar were the results by other investigators [INK4a-specific staining varies to such a degree we scored as positive every sample in which the staining was expressed either in a cytoplasm or in both nuclei and cytoplasm.We did not register any staining in either of 6 smear samples taken from healthy women Fig. . An examtigators . With keThe data of the analysis of the cervical epithelium preparations stained with monoclonal antibodies E6H4 are summarized in Table INK4a-positive cells were observed with low frequencies: 34 samples were negative and 3 ones  were stained poorly.In all 37 samples from patients with cervical ectopia p16INK4a-negative. In 7 samples (about 14%) sporadic or focal  were attributed to sporadic Fig , focal oive Fig. .INK4a-positive cells  stained poorly, 3 (21.4%) stained sporadically, 4 (28.4%)-focally and 5 (35.8%) expressed diffuse staining.As to invasive cancers, only 1 out of 26 samples (3.8%) lacked p16lls Fig. . One samlls Fig. . Among 1INK4a expression was more pronounced in cells belonging to a more advanced lesion  the results of staining with p16es Table .INK4a by monoclonal antibodies shows that the overwhelming majority of invasive cervical carcinoma samples differ from normal cervical epithelium of healthy women. We found no cases of sporadic, focal or diffuse staining with E6H4 antibodies among 6 vaginal smears from healthy women as well as among 37 samples from patients with cervical ectopia including those in which ectopically localized cervical epithelium koilocytes or condyloma had been registered. In addition, in 11 samples of normal glandular and stromal cervical tissues and one sample of uterus body normal myometrium tissue which were obtained from patients with gynecological disorders p16INK4a was not expressed at all.Immunochemical detection of p16INK4a-expressing. However in 2 samples not expressing any p16INK4a or expressing it poorly high-risk HPV DNA sequences were detected by both PCR and Southern blot hybridization. These data are in a good agreement with those by Klaes et al., who described two samples of p16INK4a-negative but HPV-positive cervical cancer [INK4a in less than 1% of cells . DCS-50.1/H4-NA29 , 2). 375P , 3). ZJ11 and JC8 , 4) 05–418  and 5). J175–405 . The results of the comparative experiments by Klaes at al [Monoclonal antibodies E6H4 originally described by Klaes et al  had beenINK4a-positivity  in a comparatively small number of CIN I and CIN II samples (13–14 %). Among CINs III the portion of such samples increased up to 54,2%, with diffuse staining in every sixth case.We observed strong p16The data we have obtained for CINs of all stages rather differ from the results by Klaes et al. . All of INK4a expression in dysplastic and neoplastic cervical epithelium in the course of progression.Nevertheless the coincidence between our results and the data by Klaes et al  concerniINK4a-negative in control group. Similar was the situation in the reference group: 12/12 negative. Among CINs I, CINs III and invasive carcinomas these indices made up 32/51 (63 %), 8/24 (33%) and 1/26 (4 %) respectively.This trend is especially evident in our study when negative cases only are taken into consideration. All 6 samples were p16INK4a when detected immunohistochemically may be regarded as a marker of dysplastic and neoplastic lesions in cervical epithelium. Positive correlation between p16INK4a expression and morphological stage of the disease on the same slide (when combined cases with both dysplastic and neoplastic lesions were found) also favors this inference.The data presented herein allow us to conclude that hyperexpression of p16INK4a was suggested to be performed on cytological smears [INK4a to become rather widely used not only as an addition to surgery but also for regular examinations in outpatient clinics. In this connection it seems important to estimate frequencies of a feasible false-negativity of the test.In a number of recent communications immunochemical staining for p16l smears ,13-15. CINK4a-negative invasive carcinoma samples , to utilization of different types of monoclonal antibodies, to different criteria used by different research groups for the results interpretation, etc.Keeping in mind that dysplastic lesions of all stages frequently regress and do not convert into invasive cervical carcinomas , we summINK4a expression among previously developed tests for early detection of cervical carcinomas. In this connection the following points deserve mentioning. A pathologist in the course of common histological investigation usually registers some morphologic features such as zones of active mitotic divisions, multipolar mitoses, vicinity of condilomas and other formations. However when performing an immunocytochemical analysis on a smear a cytologist cannot bring in a correlation with morphological structure results of staining with p16INK4a-specific antibodies in separate cells. According to the results of the present study about 33% of CINs III as well as about 5% of invasive squamous cell carcinomas of cervix uteri do not differ from normal cervical epithelium with respect to p16INK4a expression. Thus immunochemical staining for p16INK4a  does not seem to be the approach that can help to fully overcome absolutely all existing ambiguities of cervical cancer early diagnostics.It seems important to realize the proper place of immunocyto-/ immunohistochemical analysis of p16INK4a the study by Agoff et al [INK4a – specific antibody E6H4. So far as endometrial cells may occur on vaginal smears the test does not seem to enable one to fully avoid false positivity.As to possible sources of false positivity of immunochemical detection of the protein p16INK4a encoded by tumor suppressor gene INK4a is a characteristic of displastic and neoplastic alterations of cervical epithelium. The portion of p16INK4a-positive samples increases in the following row: CIN I – CIN II – CIN III – invasive carcinoma. All stages of CINs and carcinomas analysed are heterogeneous with respect to p16INK4a expression: side by side with the samples which expressed p16INK4a in 25% of cells or more we detected samples which were stained poorly or lacked any staining. According to the data we present herein p16INK4a-negative cervical neoplasms and carcinomas do exist. Thus the lack of p16INK4a-positive cells in samples from different types of cervical lesions should not be regarded as a sufficient reason for excluding a patient from the high-risk group. Despite this as far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages of CINs and carcinomas application of immunohisto-/cytochemical test for p16INK4a may be regarded as an additional  test for early detection of precancerous lesions in cervical epithelium.Overexpression of the protein p16none declaredF.K., D.S., G.V. and G.F. contributed to study conception and design; E.K. and A.B. delt with sample obtaining and preliminary diagnosis; Yu.A., G.F. and V.E. were independent pathologists who confirmed the diagnosis; G.V., L.Z. and D.G. performed immunohistochemical staining and analysis; A.B. carried out HPV typing; G.V. drafted the manuscript. All authors read and approved the final version.The pre-publication history for this paper can be accessed here:Data on high risk HPV genome detection by PCR in CIN I and CIN II samples. the data are presented on high-risk HPV genetic material detection in 9 CIN I and 9 CIN II samples.Click here for file


Swedish patient associations for breast cancer patients (PABCPs) offer patients with breast cancer unlimited meetings with a breast cancer survivor, a contact person (CP). We applied the voluntary action perspective in this interview study with members of Swedish PABCPs in order to explore how women with breast cancer experienced their contact with a CP from a PABCP.Audio-taped narratives from 8 women were analysed using Reissman's monitoring and Gee's analysis structure.Three themes appeared: 1. Shared experiences give new perspectives on having cancer, 2. Feelings of isolation are a part of the identity of the illness and 3. Relations with others enable self-help. However, the relationship with the CP is sensitive to timing, correct information and understanding.CPs act as sounding boards and should optimally have capacity for listening, gives support and act as partner in this conversation. On the other hand, CPs should be aware that their presence and limited general medical knowledge could at times disturb the patient's psychological recovery and strengthen feelings of isolation. Thus, PABCPs must be careful in selecting CPs and offer relevant educational activities related to the themes identified herein. Reach to Recovery Program (RtoRP), a world-wide rehabilitation program initiated in the U.S. in 1952 and was later established also in Europe. RtoRP is based on the opportunity to meet with individuals with similar experiences and thereby learn different ways of dealing with disease-related feelings and problems  understandswhat I mean, like she understandsthat I can float above itand thought like it wasn't so serious (informant 8)yes, I think thatshe took me seriouslywhen I felt like it was hardnot to be able to identify myself in the group [the PACP group] (informant 8)One woman described being one among others like oneself by talking about the special feeling that came over her when she was welcomed into the association. It was not necessary to say that she had had a breast cancer operation since everyone there knew; this was obvious in the association. Another woman described the importance of meeting people in the same age and situation in life, which took place at regular theme meetings for younger women. A younger woman exemplified the importance of being the same age by saying that women of the same age had common questions to discuss – questions that could not be talked about with health professionals. The following two stanzas illustrate how the women used the CP meetings to talk with women with similar problems:it could for example behow I could make up my eyes nowwhen I don't haveany eyelashes (informant 6)if there was anyonewho had childrenbecause the ones I'd met were only older peoplewho didn't have children living at home (informant 6)At each contact meeting, the women felt that they could gain knowledge through the different experiences that were described about different forms of treatment and their effects on well-being. One woman describes how she could balance her worry in this environment by talking about what she thought about, which is illustrated in the stanzas below:how will I go through this psychologicallyin the futurewill I trust this answeror how much will I worry (informant 2)alot of questions like thatthat I've thought aboutand that I couldtalk about with other people (informant 2)Another woman's reactions to some of the meetings were feelings of sadness, while other meetings could give her hope for the future. However, while the meetings made her react differently, she felt it was important to come to the CP meetings regularly to see with her own eyes that the other women were alive and led good lives. It came out in another narrative how it is gradually possible to talk with any of the members of the association that have experience even though it is the CP that is the person one initially talks with. Thus in the long run the experiences are felt as being most important, where the CP acts to open the door to them.Time, in the sense of a particular phase in the process, is important when the women want to make contact with others with experience of having breast cancer. One woman felt that she was not ready to contact the association and meet others with similar experience despite having many questions. At first, she said, she wanted most just to crawl into herself and manage on her own without involving other people. Nevertheless, this woman was content having received a card from the CP early in the process, telling her about the possibility of establishing a contact. The possibility of getting a contact is thus given higher priority than hearing others' experiences at an early stage.One woman evaluated the meeting with the CP positively in part because the CP was a trained CP and in part because the CP knew what she needed to hear. This is illustrated in the following stanza:it was most of all goodto hear [name of CP]maybe because she had trainingknew what you needed to hear (informant 8)Another woman described how she had used her CP as a "sounding board" in her choice of surgical method. It comes out in this narrative that the sounding board function and not the counselling function made the woman finally decide in favour of a surgical method that the CP had not recommended on the basis of her own experience. The fact that the CP had given an opinion helped the woman to form an opinion herself. Thus it is not necessary to share the exact same experience; at times it is enough to share the experience of cancer.One of the women said that she would have appreciated there being someone with experience to contact when she was forced to wait for a long period before having her operation. Access to a CP for one's own personal problems is illustrated in the following stanza:I know she's the contact personwhich makes me thinkthat I can turn to herand talk about my special problems (informant 8)Women describe not only the benefit of the CP meetings but also say that there can be a feeling of more or less indifference in the contact with the CP. In one example the CP acted as a guide to the association's premises but had otherwise not spoken of anything in particular. Another woman had come into contact with the association's CP at an educational class but had never contacted a CP herself. For these women it was sufficient to contact with other women in educational classes organised by professionals.Since the CP activity is central within the PABCPs, we aimed to investigate how women with breast cancer described their experiences from having contact with a CP. The narratives allowed the women to reflect and formulate themselves through this opportunity to tell their own stories with the aim to analyse their experiences . The strThe narratives in our study illustrate the importance of meeting women of the same age and in a similar life situation and of being in an environment (CP meeting) that is free of need to explain. Meeting others and experiencing their' reactions help these women feel normal . "CatchiEven though all patients do not wish to be confronted with the experiences of others via personal contact with a CP, they may be ready to be confronted with the disease in other ways. A confrontation with women who have undergone partial or complete mastectomies can take place in water therapy sessions and in the sauna. The results show that this meeting also gave a feeling of fellowship. This may mean that the person who chooses only this kind of activity is ready only for visual impressions. Breasts mean different things to different women, and only the individual woman can define how great a handicap breast surgery is. Langellier and Sullivan  studied It is also important that a CP is able to manage timing, in the sense of the phase in the process at which contact is made with others who have experience or when the person is ready to confront others' experiences. Great sensitivity towards individual needs is required and there are individual variations as to the optimal time for a meeting, which are related to when the individual is ready to encounter other people's experiences. In terms of being ready, thoughts arise as to the many visual and auditory impressions that cancer patients receive during a treatment session, e.g. in a waiting room and when given treatment, regardless of whether they are ready for these or not.It is conceivable that some individuals do not have any need to meet with other people and share their experiences since recovery can also occur without involving others. However, it is not only the initial need for contact that varies. Women also have different needs in terms of the duration of the contact. For one person, contact with a CP may simply mean guidance in the association's activities while for another a CP can act as a support person for a period of several years and the information-seeking behaviour can also change over time .For the woman who felt great isolation in the illness the meeting with the CP strengthened the feelings of isolation, which demonstrates how the meeting with the CP led to a non-intended consequence. This demonstrates the need for the PABCPs to be meticulous in the choice of CPs; the requirements must not only be that they have been rehabilitated but also that they have substantial knowledge about cancer, treatment possibilities, and psychological reactions to disease. Although self-help may come through a relation with other individuals with a shared experience  the assoThe results also demonstrate that some questions could not be answered by health-care professionals, but rather by individuals with personal experience from cancer. Hence, new knowledge developed in the meeting with the CP  declare that they have no competing interests.CC: study design, data collection, data analysis, and writing the manuscript.MN: supervising the study and participation in writing the manuscript.KN: study design, data analysis, supervising the study, and participation in writing the manuscript.The pre-publication history for this paper can be accessed here:


Human papilloma viruses (HPV) are the necessary cause of invasive cervical cancer (ICC). Of the many different types identified so far, only a few of them account for the great majority of cases worldwide, with geographical differences in their distribution. Data on the local distribution are now of interest in view of the soon-to-come introduction of HPV type-specific prophylactic vaccines.We have investigated HPV type distribution in samples of 48 ICC cases occurred in women living in North-East Italy in the years 1997–1999. Cases were extracted from the Venetian Tumour Registry files, as incident cases whose specimens had been processed in two Pathology Departments. Search and typing were performed by polymerase chain reaction (PCR) using GP5+/GP6+ primers, followed by direct sequencing or reverse dot blot. Three cases were PCR negative using the housekeeping primers and hence excluded. One case was negative by all HPV tests used. HPV 16 was present in 32 (72.7%) cases, as single infection in 28, in mixed infection in 4. Of the 44 positive cases, HPV 16 and HPV 18 accounted for 33 (75%), as single or mixed infections. The other high risk HPV types accounted for 11 (25%) of the remaining infections. Of the 32 HPV 16 positive cases, sequencing of the E6 gene could be performed in 25; the prototype isolate was identified in 7, and the variant T350G in 18; in 4 cases one or more additional mutations were present.Our results suggest that HPV 16 has a very high prevalence among women with invasive cervical cancer in Italy; therefore, the use of a prophylactic vaccine for HPV types 16 and 18 could prevent up to 75% of invasive cervical cancers in Italy. Persistent infection with high risk HPV is a necessary cause for invasive cervical cancer; studies on HPV type distribution among ICC cases worldwide have shown some geographical variation . LimitedA total of 48 ICC cases were selected; 43 squamous cell cancers (SCC) and 5 adenocarcinomas. Median age at diagnosis was 55 yrs (range 28–89). DNA was extracted from formalin-fixed paraffin-embedded samples by using the QIAamp DNA minikit , according to the manufacturer's instructions. To verify DNA quality, amplification of a 268 bp fragment of the Beta-globin gene was performed using PC04/GH20 primers. HPV detection was conducted using GP5+/GP6+ general consensus primers (which amplify a fragment of approximately 140–150 bp), as previously described [Of the 48 cases analyzed, 3 SCC resulted negative for the housekeeping gene and were excluded, and 1 SCC was negative for HPV DNA with all the tests used. The remaining 44 cases (97.8%) were positive for HPV sequences; 39 single infections and 5 (11.4%) double infections, as described in Table Overall, 44 out of the 45 valuable ICC Italian cases occurred during 1997–1999 were HPV positive. HPV 16 was detected in 32 cases (72.7%), a result indicating a very high prevalence in Italy, in keeping with other studies . Indeed,Sequence analysis of the E6 gene in 25 HPV 16 positive ICC cases showed the presence of the T350G mutation in the great majority , in 4 cases also accompanied by other mutations; among the mutations identified, A257G, C335T, T350G, A442C lead to amino acid changes , while C256T, T286A, A289G are silent not leading to amino acid changes . InteresHPV 33 was detected in 4 cases, resulting the second most frequent type (together with type 18), a result observed also in The Netherlands .In conclusion, our results suggest that HPV 16 has a very high prevalence among women with invasive cervical cancer living in Italy, implying that a prophylactic vaccine for types 16 and 18 could prevent up to 75% of cancer cases in Italy.HPV (Human papilloma viruses); ICC ; SCC (squamous cell carcinoma); CIN3 ; CIS (carcinoma in situ); PCR (polymerase chain reaction).ADM planned and coordinated the study, analyzed the data and drafted the manuscript. HFS and RT performed the molecular analyses. RB participated in the sequence alignment and helped to draft the manuscript. AP, EB, PZ and DM participated in the design and coordination of the study, and in the collection, assembly and review of the patients' data. All authors read and approved the final manuscript.


Accurate and standardized testing methods are of prime importance to ensure the proper classification of the patient’s her2/neu status. A meeting of pathologists from across Canada was convened to update the Canadian her2/neu testing guidelines. This National her2/neu Testing Committee reviewed the recently published American Society of Clinical Oncology/ College of American Pathologists (asco/cap) guidelines for her2/neu testing in breast cancer. The updated Canadian her2/neu testing guidelines are based primarily on the asco/cap guidelines, with some modifications. It is anticipated that widespread adoption of these guidelines will further improve the accuracy of her2/neu testing in Canada.Testing for  This asco/cap guideline was reviewed by the panel of Canadian pathologists. The data from that review and the experience of the panel members were used to create the current update of the Canadian her2 testing guidelines and algorithm  and Frances P. O’Malley md Wedad Hanna md , Richard Berendt md , Louis Gaboury md , Anthony Magliocco md , Norman Pettigrew md , Susan Robertson md , Sandip Sengupta md , Bernard Têtu md , and Thomas Thomson md Penelope Barnes asco/cap Guideline Recommendations for her2/neu Testing in Breast Cancer.The information that follows is based both on the experience of the consensus participants and on the recently published her2/neu gene has proved to be a significant prognostic and predictive biologic marker in breast cancer her2/neu at the time of diagnosis The ihc) or in situ hybridization (ish) as a predictor of benefit from Herceptin therapy. The testing algorithm proposed for all invasive breast cancers is based on the very high concordance between her2/neu gene amplification and protein expression as determined using accurate and reproducible assay methods. Thus, the Canadian consensus agrees with the asco/cap guidelines and is based on testing first with ihc and then retesting equivocal cases using ish [either fluorescent (fish) or another validated brightfield ish method such (as chromogenic cish) or silver-enhanced (sish)] fish methodology is not exclusive; labs can use other validated ish methods to assess equivocal cases both for clinical case and quality assurance (qa) activities.The panel agreed that standardized and validated tests should be performed. Available data do not show superiority for either immunohistochemistry  documentation must be in place.Stringent compliance to validated standard operating procedures developed in assay validation must be adhered to and quality control (The controls should include positive (overexpressed/ amplified) and negative (not overexpressed/not amplified) cases, plus a low amplified/low protein over-expression case if possible. The control tissue should be fixed and processed in the same manner as the patient samples.The use of correctly operated automated staining protocols and equipment are acceptable, but validated methods must be used. Maintenance and service records should be regularly updated and filed in the laboratory.Use of image analysis systems can be useful to enhance reproducibility of scoring; pathologists must supervise all image analyses.ihc The panel agreed that mandatory reporting elements testing include these items for Patient identification informationPhysician identificationDate of serviceSpecimen identification (case and block number)Specimen site and typeSpecimen fixative type Time to fixation (if available)Duration of fixation (if available)Antibody clone and vendorMethod used (test and vendor)Image analysis method (if used)Adequate controlsAdequacy of sample for evaluationResults:Percentage of invasive tumour cells exhibiting complete membrane stainingUniformity of staining: present or absentHomogenous, dark circumferential pattern: present or absentInterpretation :her2 protein expression)Positive (for fish will be done and reported)Equivocal (her2 protein expression)Negative (for Not interpretablefish testing include these itemsThe panel agreed that mandatory reporting elements for Patient identification informationPhysician identificationDate of serviceSpecimen identification (case and block number)Specimen site and typeSpecimen fixative type Time to fixation (if available)Duration of fixation (if available)Identification of probe (or probes)Method used (specifics of test and vendor)Image analysis methodAdequate controlsAdequacy of sample for evaluation (adequate number of invasive tumour cells present)Results:Number of invasive tumour cells countedNumber of observers her2 signals per nucleus or tileaAverage number of cep 17) signals per nucleus or tileaAverage number of chromosome enumeration probe 17 EquivocalNegative (not amplified)Not interpretableihc is being done because of problems with assay or results, that fact should also be includedIf ihc fish, then at least 100 fish tests should be performed annually. However, given the complexity of the procedure and the experience needed, it is advisable and preferable to perform at least 200 fish tests annually.Per the United Kingdom guidelines, each lab should tests perform at least 250 qc and qa practices. Technologists should undergo appropriate training, including ongoing education in fish technology and interpretation.Appropriate training for pathologists should take place, and the number of tests performed by each pathologist should be considered to ensure competency. Test volume should be assessed in conjunction with the lab’s adherence to strict qc and equipment maintenance. Initial and ongoing education of laboratory personnel, training, and competency assessment should also be implemented. The use of standardized operating procedures, including routine use of control materials, should be enforced, and modified procedures should be revalidated. Finally, ongoing competency assessment and education of pathologists should take place.The panel recommends that initial test validation should take place together with ongoing The panel agreed that participation in an external proficiency testing program with at least two testing events (mailings) annually is mandatory. Also, satisfactory performance requires at least 90% correct responses in graded challenges for either test. Unsatisfactory performance will require a laboratory to respond according to accreditation agency program requirements.qa results and processes, results, and reports should be put into place. Unsatisfactory performance results in suspension of laboratory testing for her2 for that method.Onsite inspection should take place every other year with an annual requirement for self-inspection. Review of laboratory validation, procedures, “Sensitivity” is defined as the percentage of positive test results obtained when evaluating only specimens that are truly positive. “Specificity” is defined as the percentage of negative test results reported when only truly negative specimens are evaluated. “Overall accuracy” (concordance) combines sensitivity and specificity into a single measure of the percentage of cases (positive and negative) for which the assay result agrees with the true status. Note that overall accuracy (concordance rate) can be strongly influenced by the positive–negative mix of the test case set if the sensitivity and specificity rates are not similar.qa programs include these:Examples of external cap (www.cap.org)neqas: www.ukneqas.org.uk)U.K. National External Quality Assessment Service (www.nordiqc.org)Nordic Immunohistochemical Quality Control (NordiQC: www.qmpls.org) and Cancer Care Ontario (www.cancercare.on.ca)]Ontario [overseen by Quality Management Program–Laboratory Services (ihc and then, in equivocal cases, moving to fish or to another validated brightfield in situ hybridization method such as cish or sish. In cases in which the available sample is small or the sample is a core biopsy in the neoadjuvant setting, it is preferable to perform ish as the initial test (The panel agreed that pathologists should test at diagnosis, starting with ial test .her2/neu status in invasive breast cancer, her2/neu testing performed at the time of initial diagnosis is now the standard of care. In line with the updated asco/cap guidelines, the Canadian consensus guidelines provide recommendations on how to evaluate and report her2/neu over-expression and gene amplification. In summary, the guidelines outline specific criteria that laboratories must meet before engaging in her2/neu testing.Considering the prognostic and predictive significance of determining her2/neu testing algorithm integrates both the ihc and the fish algorithms as published in the asco/cap guidelines. It is important to note that the cut-offs for positivity for both ihc and fish have been updated; a positive ihc result is now defined as >30% strong complete membrane staining; a positive fish result is defined as a ratio > 2.2 or a her2 gene copy > 6.0.The 2007 her2/neu testing accuracy, the testing guidelines and algorithm will be regularly updated to convey current practices.With continual efforts being made to optimize


West Virginia is the only state that lies entirely within Appalachia. West Virginians tend to be poorer and more likely to lack health insurance than the general U.S. population. The purpose of this qualitative study was to 1) obtain an understanding of attitudes about breast and cervical cancer screening among women aged 25 to 64 years; 2) determine factors that motivate women to be screened for breast and cervical cancer; and 3) evaluate educational materials about breast and cervical cancer screening for use in this population.The West Virginia Breast and Cervical Cancer Screening Program (WVBCCSP) is a comprehensive public health program, funded by the Centers for Disease Control and Prevention, dedicated to removing barriers to breast and cervical cancer screening and providing screenings to underserved women aged 25 to 64 years. The program partnered with RMS Strategies, Inc, to conduct six focus groups in three communities in West Virginia. Women were recruited by telephone based on program eligibility guidelines.Results indicated that women were concerned about health care costs and lack of health insurance. Cost, fear, and embarrassment were identified as the top barriers to breast and cervical cancer screening. Participants believed that community-based educational campaigns would increase screening and promote use of the WVBCCSP.Understanding why low-income Appalachian women do not get screened for breast and cervical cancer and determining motivational factors that encourage screening are important to increase screening rates among this population. Breast and cervical cancer efforts that use the words, knowledge, and suggestions of the women they serve are more likely to be effective and have a larger impact. West Virginia is the only state in which all of the counties lie entirely within the Appalachian region. The region, as well as West Virginia, is largely rural and demographically homogeneous. West Virginia's population is 95% white, compared with 92% for Appalachia ,2. ApproIn 2000, the median age of West Virginians was 38.9 years, 3.6 years older than the national median . AccordiWest Virginia ranks 48th among all U.S. states and the District of Columbia for high school graduates and 51st for college graduates  women aged 40 years and older in West Virginia had a mammogram within the past year. Women aged 65 and older are slightly less likely to have had a mammogram in the past year 57.9%) than women aged 40 to 64 (61.9%). Nearly two in five (38.7%) women aged 40 and older in West Virginia reported not having any kind of health insurance  is part of a nationwide comprehensive public health program funded through the Centers for Disease Control and Prevention's National Breast and Cervical Cancer Early Detection Program (NBCCEDP). The goal of the program is to help uninsured and underserved women gain access to screening services for the early detection of breast and cervical cancer . ResearcThe WVBCCSP partnered with RMS Strategies, Inc , an independent research firm, to conduct focus groups of women in three communities in West Virginia. Qualitative research methodology, such as use of focus groups, allows for interpretation of the human experience ,12 and pSix focus groups were conducted among women aged 25 to 64 years in three communities of West Virginia — Beckley (south), Charleston , and Morgantown (north). These communities were selected based on the high percentage of low-income households, high percentage of uninsured or underinsured individuals, and low rates of breast and cervical cancer screening.Sixty-nine women participated. Participants included three groups of women aged 25 to 49 years (n = 33) and three groups of women aged 50 to 64 years (n = 36). Participants were recruited randomly by telephone. The project used a random-digit–dialed sample from GENESYS . It included the location  and telephone numbers of households in the three study communities. The random sampling was managed by the computer-assisted telephone interviewing (CATI) system.Eligibility requirements for participation were age, lack of health insurance, and low income. Low income was defined as earning less than 200% of the federal poverty level based on size of household .Focus groups were led by a professionally trained facilitator with expertise in conducting cancer research groups among Appalachian women. Each session lasted 2 hours and included 10 to 12 participants. Participants received $50 for participating and signed a consent form before the focus groups began. Each session was video and audio recorded, and the facilitator took additional notes. WVBCCSP staff members observed the sessions from an adjacent room.The facilitator used a discussion and interview guide that was developed by the research team to lead the participants through a discussion of breast and cervical cancer attitudes and behaviors and best methods for educating low-income women about the WVBCCSP . At the Participants reviewed four brochures. One brochure was from New Mexico Breast & Cervical Cancer Early Detection Program , one wasParticipants were asked to complete a questionnaire about the educational materials after the group discussion . The queQualitative methods were used to analyze data . The facCost of health care and lack of health insurance were the dominant concerns among these women. These two issues kept women from routinely seeing a health care provider. A majority of the participants lacked the financial means to purchase health insurance for themselves or their families if their employer did not provide it: "My husband is going to retire in 2 years, and I have to think of how that is going to affect us with our medication and health care."Older women tended to believe that they were "falling through the cracks" when it came to health insurance. Women who retired early (in their 50s), lost their jobs, or who were divorced or widowed had a difficult time paying for health care and health insurance; they were too young for Social Security and Medicare or did not qualify for Medicaid:For me it is the affordability of the insurance. When my husband died, I had to drop our insurance because by the time I paid my health insurance, it left me with $250 per month to live on. If you are older than 55 and younger than 65, you are falling through the cracks because there is nothing out there to help you.Cancer was a personal and emotional topic among participants. Nearly every participant had a personal story or experience of how cancer affected her life: "I almost didn't come tonight because my mother told me a half hour before I left that she has cancer in her lymph nodes and lungs." Religion was identified as a comforting factor that helped individuals and families get through trying times. Several participants were cancer survivors themselves: "Cancer concerns me because I have had it."Overall, participants were generally more knowledgeable about breast cancer than cervical cancer. However, they were able to identify the screening tests associated with both types of cancer. Women unanimously agreed that having a Pap test and a mammogram were extremely important and that the examinations could detect cancer in the early stages. Several participants mentioned that it was even more important for women who have a family history of cancer to be screened more frequently and at an earlier age: "If you are having problems or anyone in your family has had problems, then do it more often."Women cited friends and family as their sources of information about breast and cervical cancer. As a result, several women shared misconceptions relating to breast and cervical cancer. For example, when asked about breast cancer, one woman said that it was more prevalent and harder to detect in women with large breasts and that women with breast implants were unable to have mammograms. Cervical cancer misconceptions included tampons causing cervical cancer and women who have had a hysterectomy, regardless of reason, no longer needing Pap tests.Knowledge about the recommended frequency of Pap tests and mammograms varied by region and age. Women in Charleston and Morgantown were more knowledgeable about the recommended frequency of Pap tests and mammograms than were women in Beckley. Younger women expressed more uncertainty as to the frequency and purpose of Pap testing than older women.Participants reported that they did breast self-examinations; however, some reported doing it only when they remembered rather than monthly. Older women were more likely to perform breast self-examinations than younger women. Several participants mentioned that they rely on breast self-examinations and rarely have mammograms because of cost and lack of insurance: "I lost our insurance and relied on the self-exam. I went 4 years without a mammogram."Women discussed the role that health insurance played in relation to breast and cervical cancer screening recommendations. Some women perceived that because of their lack of insurance, doctors did not recommend Pap tests or mammograms: "As much as we want to think that they just want to make us better, they know about the insurance underneath it all. I think they know they are not making a profit."The major barriers that prevented women from getting a Pap test or a mammogram were cost ("We just don't go because of the cost") followed by fear of getting a mammogram  and embarrassment during a Pap test ("How much more vulnerable can you get when you are lying on your back with your legs in stirrups?"). Other barriers mentioned include discomfort, lack of transportation, denial, and lack of time .Health care providers have the power to help women overcome some of these barriers. For example, to overcome fear and embarrassment, participants expressed a desire for health care providers to talk to them during the procedure, letting them know what they were doing and why they were doing it.According to focus group participants, the best motivators for screening were women sharing their cancer stories and testimonials, the buddy system, health care providers, and educational campaigns focused on increasing knowledge. Several women mentioned an educational campaign similar to "Janet's story" as being an effective campaign model for the WVBCCSP. Janet's story, a smoking cessation educational campaign, is about a woman from southern West Virginia who died in her early 40s from lung cancer caused by smoking.Nearly all women believed they would be motivated to schedule a Pap test or mammogram if a family member or friend would go with them to the appointment. They stated that this type of buddy system would help them overcome their fears and encourage them to follow through with their appointments.Women acknowledged the important role health care providers play in motivating women to get a Pap test and a mammogram. They noted that health care providers educating them on the examination and its purpose, along with recommending the test, would motivate them to make appointments. However, several women admitted to canceling their appointments out of fear, despite their doctors' recommendations: "It depends on how serious the doctor thinks the problem may be." However, they said that if the health care provider talked to them during the procedure, it would help eliminate their fears and reduce their embarrassment. It was also discovered that women preferred a female physician over a male physician:I had a male gynecologist for years when I was young, and there is no comparing them to a female. They are gentler and understand where you are coming from, the emotional ups and downs. They are also sensitive.A majority of women were unfamiliar with the WVBCCSP; however, they were interested in knowing more about the eligibility guidelines and available providers in their areas. Participants who knew about the program stated that they first learned about it through their health care provider or local Department of Health and Human Resources. Those aware of the WVBCCSP went on to state that they educated their friends and family about the program.Women who lived in Charleston and Morgantown were more likely to have heard about the WVBCCSP than women living in Beckley. Familiarity with the program tended to increase with the availability of health care providers in the focus group area. Several participants expressed concern about the limited number of health care providers in their area who participated in the program and perceived doctors' biases against patients who participated in federal and state health insurance programs.A majority of women believed an educational campaign would increase breast and cervical cancer awareness and knowledge and use of the WVBCCSP. By creating an educational campaign to educate women about breast and cervical cancer and the WVBCCSP, participants believed that barriers could be overcome: "I think it would definitely get more women to get the exams." Participants also felt that it was important to begin educating women at a younger age: "Start in the schools. Educate young girls about the importance of Pap smears and mammograms."The campaign's message should be consistent, strong, and serious in nature: "We'll get through to the humor after we get through the crisis." Suggested campaign messages included: "A few moments of embarrassment are not worth risking your life over" ; "We want you for LIFE"; "Stop cancer before it stops you"; "Your life may depend on reading this"; and "Don't be a cancer statistic." Women felt that knowledge was empowering: "Maybe educate girls about it when the teachers are talking to them about getting their period. Let them know that part of becoming a woman is making sure your body is taken care of." The media were viewed as a great resource for spreading the message: "I think advertising done right is the most powerful tool we have."The messengers of the educational campaign need to accurately portray the program's target audience, West Virginia women. The message should be delivered by multiple women from across West Virginia from diverse age groups and racial and ethnic backgrounds:I don't think the messenger should be one person. It should be many women — older, younger, and backwoods women. In this area [Beckley], you have a lot of people who are not going to respond to advertising from strangers.The messenger would encourage women to talk to their doctors and get screened for breast and cervical cancer.The educational campaign should also include direct mail, television, and radio in addition to a grassroots campaign. Radio and television educational campaigns did not necessarily mean advertisements, but rather a combination of news stories and informational pieces to educate women about breast and cervical cancer and the WVBCCSP. Television and radio news stories or informational pieces should be adequately promoted so women know to tune in. Direct mail campaigns should be colorful, bold or "something of interest." The likelihood of reading direct mail varied by age and location. Younger women were more likely to read all of their mail than older women. At the same time, women in the Beckley area were more likely to read their mail than women in Morgantown and Charleston.Information women would like included on a brochure or in an advertisement included: WVBCCSP eligibility guidelines, the importance of screening, cost of screening, and breast and cervical cancer statistics. Additionally, participants indicated that it would be beneficial to have a Web site or telephone number available to contact program staff about participating providers in their area and gain more information about the free or low-cost procedures offered.Educating health care providers about the WVBCCSP should be an area of focus for the campaign. Women tended to rely on their doctor or clinician to recommend routine Pap tests and mammograms. Providers should also be educated about the important role they play as motivators.The focus groups enabled us to meet the project's objectives. Findings from the focus groups suggest that more needs to be done in West Virginia to adequately educate women about breast and cervical cancer and programs that provide free or low-cost breast and cervical cancer screening services for uninsured, low-income women.As expected, this population of Appalachian women was most concerned about the cost of health care and lack of health insurance. Major barriers identified through this project  were also not surprising, given the cultural identity of the area. The lack of health insurance is a major obstacle for women and should be addressed to increase their use of screening services, such as those provided by the WVBCCSP. Our findings seem to coincide with the findings of several national surveys which suggest that poor, less educated, and uninsured populations are more likely to underuse breast and cervical cancer screening services .Despite the fact that programs exist to assist low-income, uninsured, or underinsured women in receiving breast and cervical cancer screening services, many women lack knowledge and awareness of them. A primary goal of the WVBCCSP should be to increase program recognition throughout the state. To accomplish this goal, a community-based, culturally sensitive educational campaign must be developed to help women learn about the program and thereby increase breast and cervical cancer screening rates among this population. These types of campaigns have been proven to be effective in increasing screening rates among other populations of rural women, such as African Americans and Hispanics ,17.Public health programs and health care providers need to openly communicate with each other about the population that they serve. The WVBCCSP communicated the findings of this project to its health care providers, particularly the information about women viewing health care providers as motivators and suggested techniques that could be used during tests to help reduce the patient's fear and ease her embarrassment. This type of information exchange could help promote a more comfortable atmosphere for the patient.The WVBCCSP has reviewed program brochures and begun updating them based on focus group findings. The general program brochure has been redesigned to include the information that the focus groups indicated they would like to see in a brochure. The program will continue to review its educational materials to evaluate whether the information accurately reflects the needs of its targeted population. Breast and cervical cancer efforts that use the words, knowledge, and suggestions of the women they serve are more likely to be effective and have a larger impact.There were limitations to this project. Because women were recruited for this study by telephone, the opinions expressed in the focus groups may not reflect the opinions of some of the poorest residents of the state who are most likely to not have telephones. As is possible with all focus group discussions, dominant personalities may have intimidated shyer participants from fully engaging in the discussion. It is also probable that some discussion may have been inhibited because of the sensitive nature of the subject.


Imagine you are late for your train. As you approach within sight of the station, the last car pulls off to the left. So you start running in a diagonal to catch up with it. The difficulty is deciding how much to bear left. A different angle will be needed depending on both your speed and the train's speed. For a given set of speeds, mathematics dictates that there is only one bearing angle that will put you on that train.Determining that optimal bearing and staying the course are the essence of constant bearing (CB), a strategy that sailors, ballplayers, and animals on the prowl use to intercept (or avoid) moving targets. CB is the optimal strategy as long as the target follows a predictable course. Using mental projections of the target's and their own trajectories, a fish pursuing sinking bait and an outfielder tracking a fly ball compute their optimal bearing and move in a straight line toward the point of intercept, adjusting their course along the way if necessary. With targets that change speed and direction unpredictably, a pursuer may still use CB successfully over the stable segments of the trajectory, which is how dogs catch swerving Frisbees. But erratic targets, which change speed and direction quickly and randomly, may not leave the pursuer enough time for CB computations.Zeroing in on erratic targets is a matter of survival for the big brown bat, which must snag fitfully flying insects before they return to the safety of foliage. Still, a second or two is all a bat needs to locate and catch a straying beetle on the wing, suggesting bats have an efficient way of tracking their prey's erratic motions. Kaushik Ghose, Cynthia Moss, and colleagues observed big brown bats capturing flying insects in a laboratory setting. They report in a new study that, rather than CB, the bats rely on constant absolute target direction (CATD), a strategy that can be shown mathematically to be most time efficient for tracking erratic motions and is similar to that used by search missiles to latch onto moving targets.Big brown bats use sound rather than light to sense their surroundings. They emit short ultrasonic shrieks into the air and listen to the returning echoes to detect objects around them, quickly adjusting their flight to approach an insect or avoid an obstacle. The researchers released a big brown bat and a flying insect in a large dark room. Using high-speed infrared cameras and a battery of microphones, they recorded the flight path of bats and insects, as well as the bats' shrieks and echoes. Computer analyses of the recordings allowed them to measure the speed and direction of the flights, and calculate a theoretical optimal bearing for each point on the bat's path. From the direction of the shrieks and echoes, they could deduce the orientation of the bat's head relative to the insect, a situation equivalent to detecting the gaze of visual animals.They found that after spending some time locating the insect, the bats quickly adopted the same flight path as would be predicted if they continuously recalculated bearing angles to match their prey's erratic trajectory. Rather than concluding that bats are geometry wizards, the researchers propose that they use a simple trick to cheat on the math.One of the geometrical properties of the bat's flight path is that theoretical lines drawn from the bat to the insect would appear as a series of parallels to an external observer (hence the term CATD). Previous research has shown that the big brown bat locks its head direction to the target position—continuously looking at the target during pursuit, like a baseball player keeping his eye on the ball. The researchers suggest that when adopting the CATD strategy with its head locked to the target, all the bat has to do to simplify the math is to ensure its head does not rotate in space as its body swoops and glides to follow its prey.In fact, the bat may possess a reflex similar to the one that links sensations of imbalance in our inner ear to a posture adjustment. This reflex would allow the bat to quickly correct any deviation of its body's alignment with the prey, thus insuring its unerring aim.


First, far from “deliberate selection of data,” we followed standard snowball methodology  for searNothing in Jenkins’ letter contradicts our conclusion that he and his colleagues presented the data from the 16 Cities Study in a way that conformed to the stated objective of the Tobacco Institute’s “OSHA Projects” to “encourage adoption of a ventilation standard and to discourage adoption of a smoking ban or of a standard that requires separate ventilation for areas where smoking is allowed” . Indeed,


This strategy preferentially removed older individuals who died before reaching the sixth exam, thus the intersection datasets are smaller (n = 1114) and significantly younger than the full datasets. We performed variance components linkage analysis on these intersection datasets and on their sex-specific subsets.Previously, we reported significant linkage of body mass index (BMI) to chromosomes 6 and 11 across six examinations, covering 28 years, of the Framingham Heart Study. These results were on all individuals available at each exam, thus the sample size varied from exam to exam. To remove any effect of sample size variation we have now constructed six subsets; for each exam individuals were only included if they were measured Results from the sex-specific genome scans revealed 11 regions in which a sex-specific maximum lodscore was at least 2.0 for at least one dataset. Randomization tests indicated that all 11 regions had significant (p < 0.05) differences in sex-specific maximum lodscores for at least three datasets. The strongest sex-specific linkage was for men on chromosome 16 with maximum lodscores 2.70, 3.00, 3.42, 3.61, 2.56 and 1.93 for datasets 1–6 respectively.Results from the full genome scans revealed that linked regions on chromosomes 6 and 11 remained significantly and consistently linked in the intersection datasets. Surprisingly, the maximum lodscore on chromosome 10 for dataset 1 increased from 0.97 in the older original dataset to 4.23 in the younger smaller intersection dataset. This difference in maximum lodscores was highly significant (p < 0.0001), implying that the effect of this chromosome may vary with age. Age effects may also exist for the linked regions on chromosomes 6 and 11.Sex specific effects of chromosomal regions on BMI are common in the Framingham study. Some evidence also exists for age-specific effects of chromosomal regions. Body Mass Index (BMI) is one of the most heavily studied measures of obesity MIM #601665). In a previous article [665. In aThe scientific literature on the genetics of obesity is extensive and has been reviewed in depth by Perusse et al.  in the lAn examination of the tables presented by Perusse et al. indicate that while association studies often report sex-specific effects on obesity phenotypes, linkage results are rarely sex-specific. One of the few sex-specific linkages showed that severe obesity in women, but not men, was strongly linked to chromosome 4 in the Utah Tree Study . A searcThe relative effect of a gene could change over the lifetime of an individual. The Framingham study, with its longitudinal data, is one of the few studies capable of age-specific tests. In our initial analyses , there wTable All multipoint maximum lodscores greater than 1.0 for the intersection sets are shown in Table D6S1009, GATA184A08, D6S2436 and D6S305. These chromosome 6 results are consistent with our results on the original datasets [The highest lodscore over all intersection datasets was 4.53 on chromosome 6q23-25 in dataset 1. The five other datasets all had lodscores greater than 1.0 in this region. This region on chromosome 6 contains the markers D10S1430, D10S1423, D10S1426, and D10S1208. This is in contrast to the results on the original datasets [smaller in size than the original dataset 1 due to the exclusion of older individuals  supported linkage in this region with maximum lodscores of 1.60, 2.43, 1.71, 2.29, and 2.70, respectively. This region of chromosome 11 contains the markers The randomization tests for differences between the original and intersection datasets indicated that all three chromosomes with significant linkage 6, 10, and 11) had some evidence for age-specific effects. For chromosome 6 the maximum lodscore of 4.53 at 149.6 cM was higher (p = 0.025) than expected given such a large decrease in sample size between the original set 1 and intersection set 1 ) for the randomization tests of sex-specific linkage on the linked chromosomal region. Each red line corresponds to the test of the difference in lodscores between men and women (shown in the graph directly above) for one of the datasets. For example, a value of 3 on the graph corresponds to a p-value of 0.001. The light blue horizontal line represents a nominal p-value of 0.05.The graph directly underneath the lodscore graph displays p-value curves . We believe that this range of peak lodscores from 52 cM to 63 cM is within the bounds of error for localization of a QTL across multiple independent studies.We found strong evidence (maximum lodscore 4.23) for linkage of BMI to families  found sifamilies  also conThe evidence for widespread sex-specific linkage effects on BMI of varying chromosomal regions in the Framingham Heart Study seems overwhelming. Regions on 11 different chromosomes showed significant differences between maximum lodscores in men and women. In nine of those regions the sex-specific maximum lodscore was higher than the maximum lodscore in the full dataset, in spite of the fact that the full datasets had larger sample sizes. In these cases it seems clear that the presence of the unlinked sex in the full dataset was actually attenuating the linkage signal and lowering the lodscore. The ubiquitous nature of the sex-specific results has implications for other family studies of BMI, especially those with sufficient sample size to have viable sex-specific subsets. We believe that linkage analysis of sex-specific subsets should become standard practice for BMI.It is puzzling that, of the eleven sex-specific regions, eight were linked to men and only three to women. The two significant lodscores in women  there were six randomization tests. Each test was a comparison between the original dataset and the intersection dataset. The strongest evidence for differences between the original and intersection datasets tended to occur in datasets 1–3, which had the largest age differences between original and intersection. This was especially true for chromosome 10 where a maximum lodscore of 0.97 in the older original dataset 1 actually increased to 4.23 in the younger intersection dataset 1. As Table 0, and 11The tendency for stronger linkage in younger individuals extends to the sex-specific subsets. Table detectable single locus effects are more likely to be in younger samples simply because fewer genes affect BMI at younger ages. If this conjecture is correct, then it is unfortunate that most studies of common disease are designed to maximize heritability and therefore have ascertained older subjects.Table The statistical test we use to infer significant sex-specific and age-specific is a randomization test, which is the emerging standard in complex genetic analysis. However, the difference in the way we used the test here should be noted. The sex-specific test structured the randomization directly, based on sex. The age-specific test was indirect; the randomization was structured to achieve a uniform sample across all six datasets. The inferred age-specific effect was secondary to the randomization and based on observed age differences between the original sets and the intersection subsets. Thus, while we believe the age difference is the most parsimonious explanation, it is certainly possible that some other unobserved factor is responsible for the significant difference in the two datasets. Thus, the direct evidence for the sex-specific effects should be regarded as stronger than the indirect evidence for age-specific effects.This study offers insight into the replication problem. The inability to replicate significant linkage results has bedevilled this field for years and has been a serious hindrance to progress. Some of the reasons cited for lack of replication have been different study designs, different ascertainment schemes, ethnic differences, genetic heterogeneity, and random error. This study of BMI adds two factors to that list. The first factor is sex. A study that is exclusively or predominantly of one sex may show quite different patterns of genetic effect than a study that is of the other sex or both sexes. The second factor is age. If it is generally true that some single locus effects are more easily detectable in a certain age range, then age differences between studies could hinder replication.The unique nature of the Framingham cohorts needs to be emphasized. Unlike many family studies Framingham is population-based and not ascertained on a particular disease trait for which obesity may be a risk factor. The intersection datasets, which are crucial to this study, required, by definition, that each individual be present at all six examinations. The natural consequence of this design is that the subjects of this study are the youngest and healthiest individuals in what was already a study of normal health. A final caveat about this study is that the first dataset, where so much of the significant linkage was detected, is from examinations that took place in 1971–1975. It is unlikely that any current study could replicate the dietary and physical activity patterns that were prevalent at that time.Sex and age specific effects of chromosomal regions on BMI are common in the Framingham study. When we accounted for these effects we were able to detect two new regions that showed significant linkage to BMI on chromosomes 10 and 16.The Framingham Heart Study (FHS) has been described in detail elsewhere . BrieflyAt each exam, each subject undergoes an extensive data gathering protocol. For this study we use only five variables: sex, age, height, weight, and cohort status. BMI was derived as weight (in kg) divided by the square of height (in meters).we constructed six new datasets that comprise only those individuals who had measurements on all six exams. In each dataset, individuals who did not have phenotype measurements at all six exams were set to missing. Family structures were not changed. Logically, the individuals with data in these six new sets are the intersection of the individuals with phenotype data in the six original datasets. Therefore, we will refer to these six new datasets as the 'intersection datasets' and the six datasets that were used in our original linkage analysis [Initially, we constructed six datasets corresponding to the six offspring exams matched to the cohort exam that corresponded in time . These danalysis  as the '2, and cohort status in the model.Linkage analysis was performed using a variance components approach ,15 as imSex-specific linkage analysis for both sexes was performed by setting all phenotype values for one sex (or the other) to missing and performing the usual linkage analysis with the corresponding sex-specific mean.All variance components are estimated by maximum likelihood. Linkage is tested by a likelihood ratio test in which a null hypothesis of the QTL variance component being equal to zero is compared to it being greater than zero. The resulting chi-square statistic is converted to a traditional lodscore by dividing by 2*ln(10). The proportion of the total phenotypic variation due to the QTL can be estimated, however, it has been shown ,19 that There are significant differences in age between the original sets and the intersection sets  phenotype information on e individuals randomly selected (without regard to age) from the original set. Then we repeated the linkage analysis on this new set and retained the lodscores. We repeated this drop-then-link procedure for 10,000 replications, choosing a new set of size e, without replacement, randomly at each replication. The 10,000 linkage scans of this region form the null distribution of no effect of excluding e individuals from the original dataset. The p-value at each point in the region is the proportion of the 10,000 that are greater than or equal to the lodscore at that point in the intersection set.Let w be the number of women in the full intersection set. First, we formed two subsets; in the first subset all phenotype information on w randomly selected individuals  was set to missing, while the second subset retained those same w individuals and set the other n-w individuals to missing. Then we repeated the linkage analysis on both of these new sets and retained the difference in lodscores at each point. We repeated this drop-then-link procedure on the two subsets for 10,000 replications, choosing a new set of size w randomly at each replication. The resulting 10,000 differences in lodscores at each point form the null distribution of no sex-specific effects of this region. The p-value at each point on the chromosome is the proportion of the 10,000 that are greater than or equal to the difference in the true sex-specific lodscores. These p-values will be plotted as -log10(p). While this p-value is for a one-tailed test, the chosen tail depends on which sex has the greater lodscore. The graph of this p-value will be labeled 'men' or 'women' to indicate which sex had the larger lodscore in the full intersection datasets. When both sexes had larger lodscore at different points in the region, we will plot the p-value both ways (see e.g. chromosome 7 in figure We also performed randomization tests to determine if there were sex-specific effects of linked regions in the intersection sets. In each of the six intersection sets we constructed two subsets, one all men and one all women, and then performed the linkage on both subsets noting the difference in lodscores at each point. Under the null hypothesis of no sex-specific effect the difference in lodscores should be zero. If the difference in lodscores significantly deviates from the null hypothesis then we have evidence for a sex-specific effect at that point. Let LDA is the director of the Framingham Obesity Genetics project. He directed the analysis and wrote the manuscript. NLHC contributed to the design and performed most of the analysis. CSF is the physiologist on the Framingham Obesity Genetics project and provides expertise on the mechanism of obesity. CEJ contributed to the design, assisted in the analysis, and edited the manuscript. LAC is a senior statistical geneticist on the Framingham Heart Study and contributed to the design and edited the manuscript.


There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes.Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV.There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples.Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none. Infection with human papillomaviruses (HPV) is causal in the development of genital and oral cancers ,2. HPV iThe goal of this study was to determine if HPV could be detected in amniotic fluid from women with intact membranes.Human subject approval from the University of Michigan was obtained to use residual amniocentesis samples that were to be discarded. To be eligible for the study, pregnant women had to be undergoing clinically indicated amniocentesis and have intact membranes. The only information on the women available to the investigators was age, race/ethnicity, and number of fetuses present at time of sampling. All clinical assays related to chromosomal assessment of the fetus had been completed, and the residual amniotic fluid and amniotic cell culture pellets were retrieved from the cytogenetics laboratory.Amniotic fluid samples (8–12 ml) were spun at 4500 rpm for 30 minutes. After decanting the supernatant fluid, the pellet was suspended in 150 μL of TrisEDTA buffer. The cultured cell pellets were resuspended in 200 μL PBS. A 150 μL volume of each sample was extracted using the Roche MagNA Pure LC instrument and the DNA isolation kit I resulting in a final volume of 150 μL.We used a direct PCR method followed by a nested PCR method to detect HPV. The Roche line blot assay, based on L1 consensus PCR with biotinylated PGMY09/11 primer sets and β-globin as an internal control for sample amplification ,18 was uFor the nested reaction, five microliters of each L1 amplicon was added to a PCR reaction mix containing GP5+/GP6+  primers The HPV assays were repeated using separate extractions, increased volumes of extracts in the PCR assays and re-amplification of L1 consensus PCR products with PGMY primers.The data was analyzed using SPSS 11 with frequency distributions of study participants by age, race, and ethnicity by HPV status.The residual amniotic fluid and cell pellets were collected from 142 women. There were 146 paired samples representing 138 singleton pregnancies, 2 twin pregnancies, 1 triplet pregnancy, and 1 singleton pregnancy with an egg donor. The women's average age was 35.2 years with a range from 23–55 years. The race and ethnicity was 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. No other data were available on the women. No HPV was detected in any of the amniotic fluid or pellet cell samples. All samples were β-globin positive. There was no evidence of technical barriers to detecting HPV in samples if it was present.In this study, no HPV was detected in amniotic fluid or culture cell pellets from 142 pregnant women with intact amniotic membranes. Other investigators have reported the upper 95% confidence interval for detection of perinatal transmission from women with any evidence of genital HPV is only 2.8% . Worda aThis study was not able to actually determine the HPV status of the study participants or their risk for having HPV. It is possible that none of the women had ever been infected or were not currently infected with HPV. Other studies addressing the issue of vertical transmission of HPV have reported that 12–36% of pregnant women asymptomatic for HPV were positive for HPV ,10,16,20Perinatal transmission of HPV clearly does occur; juvenile onset recurrent respiratory papillomatosis is a documented rare outcome of infection with HPV. This occurs in 7 of every 1000 infants born to a mother with genital warts . Some inThe author(s) declare that they have no competing interests.MR and JB participated in concept development, study design, implementation, and coordination; data analysis, and drafting the manuscript. DR collected the data and coordinated the sample numbers with demographic data. DRL, RAT, DCS, and ERU participated in the HPV assays and drafting the manuscript.The pre-publication history for this paper can be accessed here:


The expected ratio of male to female births is generally believed to be 1.05, also described as the male proportion of 0.515.We describe trends in sex ratio at birth and in fetal deaths in the United States, in African Americans and in whites, and in Japan, two industrial countries with well-characterized health data infrastructures, and we speculate about possible explanations.Public health records from national statistical agencies were assembled to create information on sex ratio at birth and in fetal deaths in the United States (1970–2002) and Japan (1970–1999), using SPSS.Sex ratio at birth has declined significantly in Japan and in U.S. whites, but not for African Americans, for whom sex ratio remains significantly lower than that of whites. The male proportion of fetal death has increased overall in Japan and in the United States.Sex ratio declines are equivalent to a shift from male to female births of 135,000 white males in the United States and 127,000 males in Japan. Known and hypothesized risk factors for reduced sex ratio at birth and in fetal deaths cannot account fully for recent trends or racial or national differences. Whether avoidable environmental or other factors—such as widespread exposure to metalloestrogens or other known or suspected endocrine-disrupting materials, changes in parental age, obesity, assisted reproduction, or nutrition—may account for some of these patterns is a matter that merits serious concern. In modern nations today, the proportion of couples reporting difficulties with reproduction ranges from one of every five to one in four. The survival of any population depends on the capacity of individuals to reproduce successfully if and when they chose to do so. It is increasingly clear that exposures to foreign substances in either parent before conception can have a broad range of teratogenic effects, including reproductive failure, structural or functional defects or altered expression of sex at birth , 1998b. SRY (sex-determining region Y) gene located on the Y chromosome determines the sex of a fertilized egg. But events occurring after conception may differentially affect the viability of a male conceptus. There is a normalized rate of the proportion of male and female births: Most often, for every 100 female births, 105 male births typically occur  from the consumption of contaminated Great Lakes fish resulted in decreased sex ratio of offspring . The oddWe conducted separate analyses for the United States and Japan. For Japan, we examined sex ratio at births and of fetal deaths from 1949 to 1999. We relied on national statistics provided by the Japanese Vital Statistics Bureau , which aFor the United States, we examined trends in sex ratio overall, for African Americans, and for whites, from vital statistics data  from 197Data for the United States were taken from vital statistics published by the National Center for Health Statistics . Data toOur data show distinct and unexplained trends in sex ratio in Japan and in the United States. Since 1970, sex ratio (SR) has declined significantly in U.S. whites and Japanese. But in African Americans, SR has increased modestly over time, while remaining lower than that of whites.−5 with an R2 of 0.70. Over the study period of 1970–1999, SR for Japan declined significantly (p < 0.01) from 0.5172 to 0.5135. This is equivalent to a decline of 37 males per 10,000 births. If the 1970 male proportion had remained constant, this would correspond to a shift from male to female births of approximately 127,000 over these three decades.−3, nearly seven times greater than that of the proceeding 72 years. The proportion of fetal deaths that are male has gone from just over half to nearly two-thirds, a figure that held throughout the 1990s. Male fetuses in Japan, then, are clearly at greater risk of dying than are female fetuses. Although fetal death rates have generally fallen, from around 100 per 1,000 in 1960 to 32 per 1,000 in 1999, the proportion of fetal deaths that are male has continued to increase, from 56% of fetal deaths in 1960 to 67.7% in 1999.In Japan, fetal death rates are typically more than twice those of the United States, and range to as much as five times as large. For example, in 1999 Japan reported 31.6 deaths per 1,000, whereas the 1999 U.S. fetal mortality rate for all races was 6.7 per 1,000. p < 0.001) . When U.S. non-Hispanic whites and U.S. African Americans are examined separately, distinct differences are apparent. For U.S. whites, the sex ratio decreased from 105.9 to 105.0 between 1970 and 2002 (and was as low as 104.7 in 2001). This can be expressed as a decline from 0.5143 to 0.5122, equivalent to a drop of 21 males per 10,000 births. This trend for whites has a regression coefficient of −0.029 (p < 0.001); that is, the trend for whites was for the sex ratio to drop −0.029 annually since 1970. Assuming that the 1970 sex ratio for all births in the United States had remained constant over these three decades, this is equivalent to a shift from male to female births of approximately 135,000.Next consider data for the United States, shown in p < 0.01). In sum, the data show a reduction in the sex ratio for non-Hispanic whites, but no comparable decline for African Americans, whose rates are significantly lower.For U.S. African Americans, however, the sex ratio increased from 103.1 to 103.2 , and remained well below that of U.S. whites. This trend has a regression coefficient of 0.013 , and hypospadias (displacement of the urinary opening toward the scrotum) have been increasing in industrial countries . Each ofWhatever their causes, trends in the sex ratio of fetal death can have important impacts on trends in SR. With respect to the different sex ratio trends in U.S. African Americans and whites, it is important to consider that fetal loss is more common with male fetuses, as indicated by the male dominance in sex ratio of fetal deaths in all populations studied. Improvements in obstetric care for the general population may account for increases in SR that occurred before 1970 in many countries. If fetal deaths in Japan are recorded at 12 weeks and in the United States at 20 weeks, this could account for why the death rates differ so much. But the increasing proportion of males in fetal deaths in the United States and Japan is noteworthy.In the United States, quality of and access to prenatal obstetric care for African Americans historically has lagged behind those of whites. To the extent that improvements in obstetric care lead to reductions in fetal deaths that occur disproportionately in African-American males, then these advances in care could affect sex ratio at birth. It is possible that improvements in prenatal care may reduce male fetal death rates, with a time lag in the African-American population, as advances have reached this population at a later point in time. The effect of any exposure causing a decline in SR would be more clearly reflected in the white population than the African-American population, because for African Americans recent changes in SR are subject to the confounding effect of drops in the male fetal death rate, as a result of improved prenatal care. As male fetal loss rates decline because of better obstetric care for U.S. African Americans, more male fetuses are expected to survive to birth, leading to an increase in SR. For all years, U.S. African Americans have a lower SR than U.S. whites. Both worldwide and within the United States, sex ratios vary by a few males per 100 females when one examines different racial and ethnic groups. In the United States, this difference could be partly the result of differences in fetal death rates combined with sex ratio of fetal deaths. African Americans have a higher fetal death rate and a higher male proportion of fetal deaths. Combined, these factors could account for reductions in SR for U.S. African Americans compared with U.S. whites by eliminating a larger proportion of male fetuses between conception and birth. Thus, the slight increase in SR for U.S. African Americans since 1970 may be the result of relatively recent advances in prenatal care.Racial differences in sex ratio in the United States may seem puzzling, though it is possible that what is “normative” varies slightly among groups of different racial heritage. Among the reasons why sex ratio in African Americans could be consistently lower than that of whites are factors that contribute to parental hormonal differences. Some studies have found that greater rates of early onset puberty and premature menarche in young African-American women e.g.,  whereas One other possibility deserves mention. Among African-American young women, obesity is nearly 50% higher than among their white counterparts. Among non-Hispanic white adults 20–74 years of age, 46.8% of women are overweight or obese (body mass index ≥ 25.0). Among non-Hispanic African Americans in the same age group the percentage is 68.3 . In the Many developed countries where sex ratio has declined have also seen substantial increases in average body weight and obesity . MoreoveHinoeuma, females born in certain years are believed to be stronger than males and will ultimately kill their husbands. Based on this belief, every 60 years when Hinoeuma occurs, female babies have been killed by midwives or sometimes even by their family immediately after birth. This is believed to have resulted in a rise in sex ratio in 1906 and again in 1966  differentially affects the timing of meiosis in the ovary and testes and could influence sex. Stra8 effects occur during embryogenesis in the female, but occur only after birth in the male , an active derivative of vitamin A, meiotic initiation will not occur. RA is sufficient to induce Stra8 expression in embryonic testes and in vitamin A–deficient adult testes in vivo. Cytochrome P450 (CYP)-mediated RA metabolism prevents premature Stra8 expression in embryonic testes. Treatment with an inhibitor of RA-metabolizing enzymes shows that a CYP from the 26 family (CYP26) delays Stra8 expression in embryonic testes. Sex-specific regulation of RA signaling thus plays an essential role in meiotic initiation in embryonic ovaries and precludes its occurrence in embryonic testes.In mammals, meiosis occurs at different time points in males and females, although the mechanism accounting for this is unknown. Although female germ cells begin meiosis during embryogenesis, male embryonic germ cells do not; males undergo Gp = 0.01)  . Similar = 0.01) . Others Although the percentage declines in SR reported here are small, because they have occurred in populations of > 400 million, they represent large numbers with considerable public health implications. Declines in the proportion of males born in Japan from 1970 through 1999 and for whites in the United States from 1970 through 2002 are equivalent to a shift from male to female births of 127,000 and 135,000 births, respectively. Fetal death rates are five times higher in Japan than in whites in the United States. Disturbances in SR may provide a sentinel indicator of larger ecologic patterns that can only be corroborated by detailed studies of smaller, more defined cohorts, with specified exposures and well-characterized information on socioeconomic, cultural, and racial factors.While noting that sex ratio has declined in many regions, one of the foremost authorities on the topic questions whether environmentally prevalent endocrine disrupters could ever be demonstrated to account for such broad patterns . He addsAlthough population-wide trends in sex ratio cannot be attributed to any single set of risk factors, given the importance of reproduction for the health of any species, the continuing trends in reduced births of males in U.S. whites and Japan reported here are matters that merit concern. Efforts should proceed to examine sex ratio in smaller groups with defined exposures as a potential indicator of environmental contamination, and to develop geographic and temporal analyses of patterns in sex ratio as a means of generating hypotheses about avoidable or controllable factors that may account for the trends reported here.