Global coverage of prevention of mother-to-child (PMTCT) services reached 53% in 2009. However the number of pregnant women who test positive for HIV in antenatal clinics and who link into long-term HIV care is not known in many resource-poor countries. We measured the proportion of HIV-positive pregnant women in Mwanza city, Tanzania, who completed the cascade of care from antenatal HIV diagnosis to assessment and engagement in care in adult HIV clinics.Thirty antenatal and maternity ward health workers were interviewed about PMTCT activities. Nine antenatal HIV education sessions were observed. A prospective cohort of 403 HIV-positive women was enrolled by specially-trained clinicians and nurses on admission to delivery and followed for four months post-partum. Information was collected on referral and attendance at adult HIV clinics, eligibility for highly active antiretroviral therapy (HAART) and reasons for lack of attendance.Overall, 70% of PMTCT health workers referred HIV-positive pregnant women to the HIV clinic for assessment and care. Antenatal HIV education sessions did not cover on-going care for HIV-infected women. Of 310 cohort participants tested in pregnancy, 51% had received an HIV clinic referral pre-delivery. Only 32% of 244 women followed to four months post-partum had attended an HIV clinic and been assessed for HAART eligibility. Non-attendance for HIV care was independently associated with fewer antenatal visits, poor PMTCT prophylaxis compliance, non-disclosure of HIV status, and non-Sukuma ethnicity.Most women identified as HIV-positive during pregnancy were not assessed for HAART eligibility during pregnancy or in the first four months post-partum. Initiating HAART at the antenatal clinic, improved counselling and linkages to care between PMTCT and adult HIV treatment services and reducing stigma surrounding disclosure of HIV results would benefit on-going care of HIV-positive pregnant women. Mother-to-child transmission (MTCT) of HIV has been virtually eliminated in developed countries through effective prevention of mother-to-child transmission (PMTCT) programmes, but, although countries such as Botswana have demonstrated considerable success in reducing MTCT rates, significant challenges and gaps in service remain in most developing countries Data from South Africa on the operational performance of onward referral from PMTCT services of women identified as HIV-positive in pregnancy suggest that, whilst a high proportion of pregnant women may be HIV tested and receive ART prophylaxis for PMTCT at the ANC, a significant proportion are not assessed during pregnancy for eligibility to receive lifelong HAART or do not receive HAART if they are assessed as needing it The study was located in Mwanza city in Tanzania, a low-income country with an estimated adult HIV prevalence of 5.6% Data were collected between September 2008 to July 2009 from health worker interviews, from observation of PMTCT antenatal services at three antenatal clinics in Mwanza and from a cohort study of HIV-positive women recruited at the maternity wards of two large government hospitals in Mwanza city. These two hospitals also housed the main adult HIV care and treatment clinics for the city. All three antenatal clinics had an adult HIV clinic on site. In two of these sites the HIV clinics had recently opened. However, because of concern that patients might not like to attend a clinic near their place of residence, pregnant women were usually asked where they would like to attend and could be therefore be referred to HIV clinic services 5–10 km away.The study was approved by the Ethics Committees of the Tanzanian National Institute for Medical Research and the London School of Hygiene & Tropical Medicine. Informed written or finger-printed consent was obtained from all cohort participants.A structured questionnaire with open and closed questions was administered to all health workers at the three largest antenatal clinics and the maternity wards of the two largest government hospitals in Mwanza city by RB or a trained nurse or clinician interviewer. Data gathered included key work activities, PMTCT and other training received, perceived challenges in implementing PMTCT services and PMTCT-related knowledge.We enrolled 403 of 442 HIV positive women admitted for delivery between September 2008 and February 2009.HIV status was routinely recorded in the antenatal card that pregnant women brought with them on admission for delivery and these results were routinely recorded in the maternity ward admissions book. All consecutively-admitted eligible HIV-positive women were seen in private by a counsellor to inform them about the study. Women were not eligible if they were resident outside Mwanza city or were unable to provide informed consent. Of the 39 women not enrolled, 27 lived outside Mwanza, 6 refused, 4 were missed, and 2 were unable to give informed consent.3 or being diagnosed as having WHO Stage 3 or 4 Those who agreed to participate were given further verbal and written information about the study by a research assistant and were asked to sign or fingerprint a consent form. They were interviewed about socio-demographic characteristics, outcomes of previous pregnancies and past contraceptive use. Information collected about the current pregnancy included details on antenatal care, uptake of PMTCT services, referral and attendance at an adult HIV clinic during that pregnancy, CD4 cell count results if available and HAART status. In the currently-used 2007 Tanzanian National PMTCT guidelines, eligibility for HAART is based on a CD4 cell count of less than 200 cells/mmPrior to discharge from hospital, data were collected on whether the mother had been tested for HIV in the maternity suite and on any onward referral. Enrolled women were followed up at their monthly visits at the under-five reproductive and child health (RCH) clinic or four months post-partum. At each visit, participants were interviewed about referral, disclosure of HIV status to others, their progress at the HIV clinic and were asked to show the interviewer their HIV clinic card which documented attendance dates, CD4 cell count results by date, and date of initiation of HAART, if applicable. Those who had not attended an HIV clinic were asked about reasons for lack of attendance. They were then re-referred to the counsellor at the RCH clinics to reiterate why assessment of their own health was important and to encourage them to attend the HIV clinic for this. They were followed up at home by a field worker if they did not attend the clinic visit and had given consent for home visits. If the field worker believed that the interview could not be conducted in private at the home then the interview was postponed and the participant was asked to come to the health facility.The study aimed to enroll 330 HIV-positive women. Allowing for a 20% loss to follow-up over four months, it was estimated that 264 women attending the four month visit would allow us to measure the proportion of HIV-positive pregnant women referred to and attending an HIV clinic with adequate precision if the proportion attending HIV clinic services was between 40–70%.Data were double-entered in EpiData 3.1  and cleaned and analysed using Stata 11.0 . Data from the cohort study were used to determine the proportion of women who successfully completed the PMTCT/maternal health uptake intervention cascade. A Piot-Fransen model was built up to show the fall-off at each step of the referral and assessment cascade. This used the denominator of women who successfully completed the previous step based on data from women who tested HIV-positive for the first time during the current pregnancy and who were seen at the fourth monthly post-partum follow-up visit. This was to allow inclusion of women who had complete information up to the end of the study. This model was adapted from an evaluation model of case finding and treatment for tuberculosis control programmes and has been used to evaluate other programmes such as those for sexually transmitted diseases To identify potential client and provider factors in the cohort study that were associated with successful completion of the referral and treatment cascade, crude odds ratios (OR) with 95% CI were estimated using logistic regression analysis. Factors whose univariable association with the outcome reached statistical significance at p<0.1 were included in an initial multivariate model and retained in the final model if independently associated with the outcome (p<0.1). Associations with p-values below 0.01 are interpreted as showing strong evidence, p-values around 0.05 as moderate evidence, and p-values around 0.1 as weak evidence.In total, 403 HIV-positive women admitted for delivery were enrolled. The mean age of cohort participants was 28.3 years (range 16 to 48 years). Seven women did not know their age. Overall, 164 (40.7%) were Catholic, 140 (34.7%) were other Christians, 97 (24.1%) were Muslims and 2 (0.5%) had no religion. Most  had primary school or higher education; 192 (47.6%) were housewives, 196 (48.6%) reported having unskilled work and 15 (3.7%) reported being in skilled work. Two hundred and eighty five (70.7%) were married, 47 (11.7%) were single, 53 (13.2%) were divorced or separated and 18 (4.5%) were widowed. Two hundred and twenty seven (56.3%) were Sukuma, the largest ethnic group in Mwanza Region.Overall 93 of 403 enrolled women reported that they had been tested and diagnosed with HIV before the current pregnancy and were excluded from this analysis. Of the remaining 310 women who tested HIV-positive through PMTCT services, 240 (77.4%) were tested during pregnancy, 23 (7.4%) were tested at the maternity ward before delivery, and 47 (15.2%) were tested at the maternity ward after delivery.3, three of whom (60.0%) had been started on HAART.At enrollment, of the 240 women who were tested at antenatal PMTCT services, 123 (51.3%) had received a referral to an HIV clinic. Of those referred, 71 (57.7%) had attended prior to delivery of whom only 36 (50.7%) could confirm their attendance by providing a clinic card. Only 30 (24.3%) of those referred (83.3% of those with a clinic card) had evidence of a CD4 test result . Five . No referrals to the HIV clinic were given by maternity ward staff to HIV-positive women who had not yet attended HIV care and treatment services, including those who were identified as HIV-positive for the first time in the maternity ward.A total of 244 (79.0%) women were seen at the four month follow-up visit, and by this time 199 (81.6%) had received referral to an HIV clinic. Approximately half received this referral pre-delivery and half received the referral post-delivery, when they attended the under-five clinic or when they visited the maternity wards for infant diagnosis .3 and 13 (65.0%) were started on treatment sometime between diagnosis and four months post-partum. Because not all women attended all follow-up visits, the proportion reporting referral to and attendance at an HIV clinic and assessment with a CD4 cell count was examined at each visit  of 310 women were referred to and attended an HIV clinic within four months post-delivery and 95 (71.4%) of these had a CD4 cell count. Twenty (21.1%) had a CD4 cell count below 200 cells/mmch visit . The proUsing data on the attrition along the patient cascade for HIV-positive pregnant women who attended the four month follow-up visit, the Piot-Fransen model  showed t3). Assuming 56 (23.1%) of 244 HIV-positive women were eligible to start HAART  of 123 women tested through PMTCT services who had received a referral to an HIV clinic prior to delivery had not attended prior to admission. Thirty-eight women were asked about their reasons for not attending an HIV clinic (others were not asked because they had not brought their HIV clinic cards to the maternity ward). Reasons included not understanding why it was important to attend an HIV clinic soon after diagnosis (n = 17), too ill to attend (n = 5), afraid to disclose their HIV status (n = 4), wanting to wait until after delivery (n = 4), distance to the HIV clinic (n = 2), being informed services were not available when they attended the HIV clinic (n = 2), lack of time (n = 1), did not believe the HIV results (n = 1), and not feeling it was necessary to attend HIV clinic services because she felt well (n = 1).By four months post-partum, 199 women had been referred to an HIV clinic but 76 had not attended. On univariate analysis, there was strong evidence of an association of non-attendance was associated with being of non-Sukuma ethnicity, not having disclosed HIV status and poor compliance with PMTCT ART prophylaxis, moderate evidence of an association with having fewer antenatal visits, and weak evidence of an association with being Muslim and a primigravida . On multChallenges to implementation that were mentioned included a lack of tracking systems to record whether women were referred and attended an HIV clinic. Health workers reported that, because adult HIV care and treatment services were busy, pregnant women often were not enrolled and assessed for eligibility for HAART during pregnancy. One health worker reported that a particular HIV clinic also sometimes refused to enroll women who had been sent from the maternity ward to the HIV clinic in the same hospital during their admission for delivery. The HIV clinic staff mistakenly assumed that HIV-positive pregnant women had received all the care they required at the antenatal PMTCT services or in the maternity ward. There were few suggestions on how the system might be improved, but those mentioned included asking PMTCT health workers to accompany pregnant women to an HIV clinic, and designated HIV care and treatment services for pregnant women.In total, 134 pregnant women attended the nine observed antenatal HIV education sessions, facilitated by a nurse in Swahili. The average length and average number of participants for the sessions was 26 minutes (range 15 to 35 minutes) and 15 (range 7 to 22), respectively. No participants asked any questions in four of these sessions. Most questions in other sessions focused on PMTCT, and women were interested to learn about the effectiveness of ART for PMTCT. However, there were no questions about interventions that focused on the woman’s own health if she tested positive despite this not being mentioned in any of the sessions.Current WHO guidelines recommend that HIV-infected pregnant women should be initiated on HAART for their own health if their CD4 cell count is less than 350 cells/mm3 or if they are in WHO clinical stage 3 or 4 Concerns that the health of mothers has been neglected in PMTCT programmes have been raised previously, since programme emphasis had largely focused on preventing transmission to infants Women who did receive referrals to an HIV clinic described a number of barriers to attending during pregnancy, such as lack of understanding why they should attend and fear of disclosure of their HIV status. The latter can be associated with stigma which has been shown to influence willingness to access HIV treatment services in other studies Delays in accessing care after diagnosis amongst adult HIV patients are common in sub-Saharan Africa Lack of understanding among staff about when women should be referred after being given their HIV results is likely to have contributed to the low referral rate. Inadequate health worker knowledge was found to impact on a number of steps in the PMTCT care continuum in South Africa There is evidence that HAART in pregnancy provides a substantially greater reduction in MTCT of HIV compared with simpler regimens. In South Africa, for example, MTCT of HIV was 11–13% with single-dose NVP compared to 0.3% in women who received more than 7 weeks of HAART mothers2mothers program which works with HIV-positive “mentor mothers” may also be effective in offering HIV-infected pregnant women additional support as they transition between ANC and adult HIV care and treatment services Even with WHO Option B, improvement in linkages between services will still be required to ensure that women are assessed to determine whether they need to stay on HAART after breastfeeding and to ensure they are linked into HIV care and support services to monitor their own health. Whilst our study demonstrated the importance of continuing to deliver information post-partum about enrollment into HIV treatment services, steps to link women into HIV care will have to happen more promptly and efficiently in order to avoid treatment interruptions. HAART may therefore need to be available at both ANC and RCH post-delivery services if these are not integrated. Strategies such as the Strengths of our study were the prospective cohort design and multiple data collection methods. Limitations include the fact that cohort participants were enrolled at delivery, and therefore the study did not include pregnant women testing HIV-positive in antenatal services but who did not deliver in hospitals. In addition, women from remote rural areas in Tanzania were not included and we were not able to determine if women lost in the treatment cascade sought care elsewhere. We have assumed that the proportion of HAART-eligible women who not attend an HIV clinic is the same as those who do attend a clinic but this assumption may be incorrect. The proportion of women who reported receiving a referral to an HIV clinic increased post-partum, and it is possible that the research team contributed to this improvement.In conclusion, we observed multiple missed opportunities to link HIV-positive pregnant women into HIV treatment programmes. Changes in the training and supervision of health workers implementing PMTCT and health system modifications are needed for staff to successfully enable women who test HIV-positive during antenatal or delivery care to access HIV treatment and care services for their own health, and to identify women who have not been assessed. Initiating HAART at the ANC for all pregnant women should reduce MTCT and maximise the health benefits for women who require HAART for their own care. Further research should be conducted to explore the effectiveness of these interventions and to explore the barriers to attending HIV care and treatment that pregnant women or mothers face when they leave PMTCT services, since linkage between the services will be essential at some point during pregnancy or the post-partum period.


BRCA1 mutation carriers with metastatic breast cancer.The purpose of this investigation was to evaluate the efficacy of cisplatin chemotherapy in BRCA1 were treated with cisplatin 75 mg/m2 intravenously every 3 weeks as part of a 21-day cycle for 6 cycles. Restaging studies to assess response were performed after cycles 2 and 6, and every three months thereafter.In a phase II, open-label study, 20 patients with metastatic breast cancer who carried a mutation in Between July 2007 and January 2009, 20 patients were enrolled. Baseline characteristics were as follows: 65% had prior adjuvant chemotherapy, 55% had prior chemotherapy for metastatic breast cancer; mean age was 48 years (ranges 32 to 70); 30% estrogen receptor (ER) or progesterone receptor (PR)+, 70% ER/PR/Human Epidermal Growth Factor Receptor 2 (HER2)- and 0% HER2+. Overall response rate was 80%; nine patients experienced a complete clinical response (45%) and seven experienced a partial response (35%). Overall survival was 80% at one year, 60% at two years and 25% at three years. Four of the 20 patients are alive four years after initiating treatment. The median time to progression was 12 months. The median survival from the start of cisplatinum treatment was 30 months. Cisplatin-related adverse events, including nausea (50%), anemia (5%) and neutropenia (35%) were mostly mild to moderate in severity.BRCA1 mutation and metastatic breast cancer and is generally well tolerated.This phase II study demonstrates that cisplatin chemotherapy has high activity in women with a NCT01611727.This trial is registered retrospectively on the clinical trials website ClinicalTrials.gov. The identifier is  BRCA1 mutation and breast cancer, choice of chemotherapy is a critical issue. There are emerging data which suggest that mutation carriers may respond differently than non-carriers to particular agents [BRCA1-associated cancers differ from non-hereditary cancers for a range of pathologic and molecular factors, including tumor grade and histologic appearance [BRCA1-associated breast cancer reflects the underlying tumor biology, in particular, the impairment of the DNA damage response and repair pathways, and that it is possible to exploit the sensitivity of BRCA1-associated cancers to DNA damage [Among women with a r agents . BRCA1-apearance -7. SeverA damage ,9.BRCA1 mutations. There are three BRCA1 founder mutations in Poland , which account for the great majority of all BRCA1 mutations in Polish families [This study was performed as a component of a large, multi-center research program conducted in Poland at the Pomeranian Medical University, which is designed to characterize the hereditary burden of breast cancer in the country and to identify and evaluate means of prevention, screening and treatment for women with families -12.BRCA1-positive women with early breast cancer, that a high rate of complete pathologic response was achieved using cisplatin chemotherapy as a single agent in the neo-adjuvant setting [BRCA1 carriers with metastatic breast cancer. The primary objective was to determine the objective response rate of cisplatin in BRCA1 carriers with metastatic breast cancer. The secondary objectives were to determine three-year survival and to evaluate the toxicities of cisplatin in BRCA1 carriers with metastatic breast cancer.We have previously reported, in a small study of  setting . It is eBRCA1 mutation, were eligible. In addition, the following were required: adequate hematologic, renal and hepatic function; adequate recovery from recent surgery and/or radiation therapy; recovery from all prior treatment-related toxicities ; life expectancy of at least 12 weeks; and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients could have received up to four prior chemotherapies for metastatic disease. Patients with known brain metastases were not eligible. Patients previously treated with a platinum-based chemotherapy were not eligible.Female patients age ≥18 years, with measurable  criteria ) metastaThe protocol was approved by the Ethics Committee of the Pomeranian Medical University with jurisdiction over the specific sites that registered patients on the study. The study was approved by Ethics Committee of the Pomeranian Medical University BN-001/83/07Patients were recruited from three academic hospitals in Poland. All patients provided written informed consent before enrollment.2 intravenous (IV) infusion every three weeks, for six cycles. Dexamethasone (8 mg)  was administered once daily for three days after chemotherapy. Ondansetron (Zofran™)  chemotherapy was administered as a 75 mg/mA total of 18 of the 20 patients received additional lines of chemotherapy subsequent to platinum therapy at the time of progression. These included AT  (eight patients) ACT  (two patients), FAC  (two patients) and AC   and complete metabolic profile. Hormone (estrogen receptor (ER)/progesterone receptor (PR)) and Human Epidermal Growth Factor Receptor 2 (HER2) receptor status were performed locally. During treatment, these assessments were performed in the same manner at the start (Day 1) of each cycle, except radiological examination of the tumor which was performed after Cycle 2 and Cycle 6, and every three months thereafter.Objective tumor response rate, defined as the percentage of patients who achieved a complete response or partial response by RECIST criteria , was theBRCA1 mutation . Secondary objectives for this study included estimating the one-, two- and three-year rates of overall survival and the evaluation of toxicity. The intent-to-treat (ITT) population was defined as all eligible patients enrolled in the study that had no major violations of protocol inclusion and/or exclusion criteria.The primary objective of this study was to determine the overall response rate of cisplatin in metastatic breast cancer patients with a known The response rate was calculated as the number of responders  divided by the number of patients enrolled. Survival was calculated using the Kaplan-Meier methods. Patients were followed from the date of first receiving cis-platinum until the date of first evidence of progression, or the date of death, depending on the analysis.BRCA1 founder mutations and had been found to be positive. Eighteen patients were treated in Szczecin and two were treated in Krakow.Between July 2007 and January 2009, 20 women were enrolled in the study. No prospective patient was found to be ineligible or declined to participate. All study patients had been tested previously for the presence of three BRCA1 mutations, and predominance of triple-negative cancers (70%). More than one-half of the patients had been treated previously with chemotherapy for metastatic disease. The median disease-free interval from the first diagnosis of breast cancer until the time of metastatic disease was 2.2 years. No woman had a prior history of ovarian cancer or of another form of cancer.Patient characteristics are summarized in Table Of the 20 patients enrolled in the study, 17 completed six cycles of cisplatin chemotherapy. Two patients did not complete treatment due to disease progression. One patient discontinued therapy after four cycles due to grade 4 neutropenia. Five patients delayed treatment due to neutropenia or anemia. One patient required blood transfusion. One patient continued to exhibit tumor shrinkage after six cycles and thus, at the treating physician's discretion, received nine cycles of chemotherapy.P = 0.6; Fishers exact test). Of the nine women who had a complete response, all were determined on the basis of the scan four months after stopping treatment. An example of CT scans, pre- and post-treatment for a woman who experienced a complete response is shown in Figure Responses of the patients are summarized in Table All 20 patients have now experienced progression. The median time to progression was 12 months (range 1 to 36 months). The proportion of patients experiencing progression was 55% at one year, 80% at two years and 95% at three years Figure . Among tThe actuarial survival rate for the cohort was 80% at one year, 60% at two years and 25% at three years Figure . The medTreatment-related toxicities are summarized in Table BRCA1 mutation and metastatic breast cancer who received cis-platinum chemotherapy as a single agent. The median survival time from the start of cisplatinum treatment was 30 months.There has been a recent resurgence in interest in evaluating platinum-based chemotherapies in patients who are known to have an inherited deficiency in DNA repair . An earlBRCA1 patients. In a recent study from the Netherlands, Kriege et al. reported on 93 women with a BRCA1 mutation and metastatic breast cancer who were treated with conventional chemotherapy  [BRCA1 carriers were included [BRCA1-positive subgroup are not given). Thus, the patients in the current study who have been treated with cisplatin appear to have superior outcomes to these historical controls. However, it is difficult to compare the survival experience of different patient cohorts who may have different disease patterns and different baseline characteristics. The numbers of BRCA1 carriers in these studies are not large and some of the variation may be due to random fluctuation.There are few studies which report on the survival experiences of cohorts of e-based) . The objincluded . The medIt is of interest to try to identify predictors of response in these patients to determine which women are more likely to benefit from cisplatin chemotherapy. Our study was too small to be definitive in this regard, but it is of interest that the past use of chemotherapy for metastases was associated with a lower response rate, as was the presence of disease at multiple sites. Also, only 1 of 5 patients with ER-positive breast cancer had a complete response, compared to 8 of 15 ER-negative breast cancers. However, the numbers of patients in these subgroups were small and it is too early to make definite conclusions in this regard.BRCA1 carriers who received neo-adjuvant chemotherapy, 10 of 12 women with a BRCA1 mutation who were given cisplatin experienced a complete pathologic response (83%), compared to 14 of 90 patients (16%) treated with other regimens  [et al. found that patients with ovarian cancer and a BRCA1 mutation often responded to cisplatin on multiple occasions during the course of their treatment [et al., who reported that women with ovarian cancer and a BRCA1 mutation were more likely than non-carriers to respond well to cisplatin [at al. documented revertant mutations in four of the six recurrent platinum-resistant ovarian cancers [BRCA1 protein. We do not have data on the mechanisms of eventual resistance to cis-platinum and it will be of interest to establish whether revertant mutations are also a source of platinum resistance in breast cancer.The results of this study are consistent with our earlier results of the use of cisplatin as a neo-adjuvant treatment of breast cancer. In a study of 102 ct test) . To datereatment . This isisplatin . Swisher cancers . These sBRCA1 mutations in mammary tissue, Drost et al. found that tumors associated with the C61G mutation had, on average, a poorer response to platinum-based drugs than did mice with tumors that were homozygous null for BRCA1 [In a mouse model which expressed or BRCA1 . There in = 20) and they originate from three institutions. Nevertheless, this is among the largest series of BRCA1 carriers with metastatic breast cancer to be studied to date, and the underlying Polish population is relatively homogeneous. We conducted computed tomography (CT) scans after two and six cycles due to the constraints of the institutions and, therefore, it is challenging to accurately assess stable disease, duration of response and time to progression. Ideally, we would have had more frequent scans and we would have assessed disease at 16 weeks as well. However, the data regarding overall survival are perhaps the most striking and this endpoint is straightforward to evaluate. A total of 18 of the 20 patients were treated with subsequent lines of chemotherapy after platinum therapy and, therefore, it is not possible to dissociate the effect of the cis-platinum therapy from that of other drugs.There are several limitations to this study. This is a relatively small sample of patients  where BRCA2 founder mutations are prevalent  [Future studies should evaluate the potential of using this therapy for the treatment of hereditary breast cancer due to other genes, including  Quebec) . Other aACT: adriamiycin/cyclophosphamide taxol; AT: adriamycin/taxol; CBC: complete blood profile; CTC: Common Toxicity Criteria; ECOG: Eastern Cooperative Oncology Group; FAC 5: flurouracil, adriamycin, cyclophosphamide; HER2: Human Epidermal Growth Factor Receptor 2; ITT: intent-to-treat; OR: odds ratio; pCR: pathologic complete response; RECIST: Response Evaluation Criteria In Solid TumorsThe authors declare that they have no competing interests.TB supervised clinical activities and data collection, and drafted and edited the manuscript. RD helped with study design and edited the manuscript. PB, MF-K and EM recruited patients and helped with clinical monitoring. JG, TH and CC performed genetic testing and recruited patients. RC acted as a clinical advisor on medical imaging, and interpreted and provided images. AE reviewed the manuscript and acted as the clinical advisor for medical oncology. JL was the principal investigator of all activities in Poland. SN was the lead investigator that designed the study, performed the analysis and edited the manuscript. All authors read and approved the final manuscript for publication.


The purpose of this study was to evaluate the efficacy of a guided imagery (GI) intervention for stress reduction in pregnant African American women beginning early in the second trimester. This prospective longitudinal study of 72 women used a randomized controlled experimental design with two groups conducted over 12 weeks. The intervention was a CD with 4 professionally recorded tracts designed and sequenced to influence study variables. Participants in both GI and usual care (UC) completed measures and donated 5 cc of blood at baseline, 8 weeks and 12 weeks. Participants also completed a daily stress scale. A mixed-effects linear model tested for differences between groups for self-reported measures of stress, anxiety, and fatigue as well as corticotrophin releasing hormone (CRH), a biologic marker of stress. Significant differences in perceived stress daily scores and at week 8 but not week 12 were found in the GI group compared to UC group. The GI group reported significantly less fatigue and anxiety than the UC group at week 8 but not week 12. There were no significant differences in CRH levels between groups. Results suggest that GI intervention may be effective in reducing perceived stress, anxiety, and fatigue measures among pregnant African American women. Maternal stress has been associated with pregnancy complications such as hypertension and preeclampsia as well as negative perinatal outcomes such as intrauterine growth restricted (IUGR), low birth weight (LBW) infants, preterm birth (PTB), and neuropsychological developmental delays of affected offspring –3. PerceOf equal importance is the major disparity in rates of stress and pregnancy complications between African American women and Caucasian or Hispanic women . ResearcThe pathway from maternal stress to negative health outcomes is a multifaceted and complex process which represents the connection between the mind and the body . The perGiven the mind-body connection between maternal stress and adverse health events, it is possible that a complementary and alternative medicine (CAM) intervention may be effective in reducing self-reported and biologic measures of stress during pregnancy . HoweverGI is a psychophysiological dynamic modality in which a person imagines and experiences an internal reality in the absence of external stimuli . The mecThe use of GI has been reported to reduce self-reported measures of stress, anxiety, and fatigue as well as neuroendocrine measures of stress such as cortisol among nonpregnant participants –30. HoweHospitalized pregnant women with preterm labor were found to benefit from a GI intervention. In Taiwan, this population was found to have lower state anxiety scores but not perceived stress scores compared to control group immediately after 3 sessions and over time until delivery . Women hNot all studies have reported a reduction in anxiety. For example, the use of imagery during prenatal classes reported no significant differences in anxiety state . LikewisBecause imagery links perceptions, emotions, and physiological responses, the selection of appropriate images is of utmost importance . PreviouWhile the results of these studies are promising, variations in research design, dosage of intervention, and selected study variables limit the applicability of these findings. Additional research addressing these limitations is warranted. Therefore, the purpose of this study was to investigate the effects of a 12-week professionally developed GI intervention on self-reported and biologic measures of perceived stress and the associated symptoms of anxiety and fatigue in pregnant African American women.This study used a repeated measures, two-group randomized experimental design to examine the effect of GI on perceived stress, and the associated symptoms of anxiety and fatigue, as well as CRH, a neuroendocrine stress measure.Participants were recruited from 2 academic obstetric clinics affiliated with the Virginia Commonwealth University Health System and the Riverside Regional Health System. Inclusion criteria included being 1) pregnant African American women between 14 and 17 weeks gestation, (2) ≥18 years of age or older, (3) able to read, write, and understand English, and (4) able to verbalize a source of social support. Exclusion criteria included (1) multiple pregnancy, (2) cervical cerclage, (3) current use of oral corticosteroids, (4) uterine or cervical abnormality, (5) dissociative disorders, borderline personalities, and psychotic pathology, (6) medical and/or pregnancy complications, and (7) current use of GI techniques. Criteria were selected to reduce sources of variability between and within subjects to include (1) variables that could potentially influence CRH levels such as ethnicity, development and growth of the placenta, gestational age of the pregnancy, use of glucocorticoids, and medical and/or pregnancy complications potentially associated with infection/inflammation  pregnant. CriteriA total of 148 potential participants were assessed for eligibility. Of these 148 women, 74 did not meet inclusion criteria, 2 were eligible but declined to participate, and 72 pregnant African American women between 14 and 17 weeks gestation were enrolled after completing the informed consent process .The 12-week intervention consisted of a CD with 4 GI tracts, each 20 minutes in length. The script for each CD was developed and professionally recorded by the author certified in GI. Key components included relaxation, focused breathing, and a variety of multisensory images to promote reduction of stress and anxiety as well as to restore levels of energy. The intervention contained multiple styles of imagery to include (1) feeling state imagery designed to shift the participant's mood to one of peace and calm, (2) end state imagery suggesting that participants see themselves the way they wish to be, and (3) energetic imagery focusing on restoring normal levels of energy .“I am calm, I am relaxed, I am energized, I am invigorated with a powerful sense of well-being”). It is these images that potentially mediate the communication between perception, emotion, and physiologic change and may affect a physiologic process such as reducing the stress reaction and the related stress symptoms [letting the feelings of calmness and relaxation carry over with you into a fully alert state” to promote sustained effects of the GI. CD tract number 4 (week 4) included a shortened version of focused breathing and relaxation along with feeling, end-state, and energetic images associated with decrease in stress, anxiety, and fatigue. Because pregnancy-related worries have been identified as major stressors and sources of anxiety in African American women [Imagine your baby tucked away safe inside your body—safe and cozy—cushioned and protected” combined with positive affirmations such as “More and more I can let go of worrying about things and focus on my own inner peace and stillness” were also included to target potential pregnancy-specific stressors.The content and order of the CDs were sequenced to focus on the desired outcomes and were based on examples in the professional literature and the Academy for Guided Imagery , 38–41. symptoms . CD tracsymptoms . This tran women , feelingThe R-GI group continued usual care with the addition of the GI intervention. Participants were instructed to listen to the CD once a day for 12 weeks in a recommended order for weeks 1–4 and used in any order for weeks 5–12. This plan was based on a study reporting that some women got tired of hearing the same script after three weeks  as well Participants in the UC group continued their usual obstetric care. To reduce the possible effect of researcher attention, a member of the researcher team called participants every week to ask if they have completed the NRSS measures for that week.A self-reported demographic and health history questionnaire was developed for this study to assess participants' basic demographic information as well as medical, mental, and/or obstetric risk factors and current health behaviors. Estimated gestational age (EGA) was determined by the first day of the last menstrual period and, if possible, confirmed by ultrasound .This is a 10-item, self-reported measure of global perceived stress that measures the degree to which a respondent appraises one's life as being stressful during the past month . The scono stress and 10 representing the worse stress imaginable. A numeric rating scale is considered to be a simple yet sensitive measure of subjective phenomenon and has been used in anxiety, relaxation, and GI research in the general and pregnant population [This is a unidimensional scale developed for the purpose of quantifying the intensity of stress , 51. Thepulation , 52, 53.not at all (1) to very much so (4), whereas the trait anxiety responses range from 1  to 4 . Scores for each subscale range from 20 to 80 with a higher score indicating a higher level of state or trait anxiety. The STAI is considered a reliable measure with Cronbach's alpha reliability reported between 0.86 and 0.93 [This is a self-report measure consisting of two 20-item subscales measuring state anxiety and trait anxiety . The staand 0.93  and has and 0.93 , 55. CroThis is a 9-item self-report measure that assesses the severity of fatigue and interference on daily functioning over the past 24 hours . Three iFrequency of GI CD tract use, barriers to use, and perceived benefits of the intervention were assessed by participants' documentation on a daily practice log developed for this study.Venous blood samples were drawn from a subset of participants using standard venipuncture procedures in the clinic during a prenatal care visit. Blood samples were collected into tubes containing the anticoagulant ethylenediaminetetra-acetic acid (EDTA) and the protease inhibitor aprotinin (0.6 TIU/mL of blood) to prevent degradation of the sample . The samEligible women who consented and agreed to participate were randomized using a computer generated masked assignment list using envelopes to the GI or UC group. At time 1, participants completed the demographic and health history questionnaire and the PSS, STAI, and BFI. Blood was drawn for plasma CRH. Folders containing the daily logs, instructions for completion of the NRSS, and contact information were given to all participants. Participants in the GI group received the CD, CD player, and extra batteries and were individually introduced to GI by a member of the research team.Approximately 8 weeks (EGA 22–25 weeks) from the initial visit, a member of the research team met with the participants at the prenatal visit. Participants completed the PSS, STAI, and BFI and had blood drawn for plasma CRH. The logs from the previous 8 weeks were collected.Approximately 4 weeks from time 2 (EGA 26–29 weeks), participants completed the PSS, STAI, and BFI and had blood drawn for plasma CRH. The logs from the previous 4 weeks were collected.The NRSS was completed daily. The UC group was instructed to complete it in the evening and the GI group before and after listening to the CD. In addition, the GI participants were asked to record any perceived benefits or barriers to using the CD on the daily practice log. The research team telephoned all participants weekly to inquire about their wellbeing and any difficulties in completing the NRSS forms.t-tests for continuous variables and chi-square tests for categorical variables.Descriptive statistics were used to summarize demographic characteristics. At baseline (Time 1), groups were compared using two-sample A mixed-effects linear model was used to test for differences between the GI intervention group and the UC group across time for the behavioral variables  and biologic variable (CRH). Following the intent to treat principle, all participants with postbaseline data were used in the mixed effects linear model. The fixed effects included visit number (baseline (visit 1), visit 2, and visit 3), intervention group (GI and UC), and visit by intervention group interaction. Participant was modeled as a random effect. CRH was positively skewed; thus, a log-transformation served the dual role of normalizing the data and stabilizing the variance.NRSS scores were obtained daily from each participant. In the GI group, two NRSS sores were obtained (prior to and after listening to the GI intervention) and one NRSS score in UC group was obtained. These daily data were averaged to obtain weekly means (weeks 1–12). Thus, each subject had as many as 12 weekly averages. Two different questions are posed regarding these data. The first is “do the weekly means differ from before to after intervention for the subjects within the GI group?” The mixed-effects linear model used to test this question contained fixed effects for week (1–12), time (pre- or post-CD), and a week by time interaction and a random effect for participant. In order to model the doubly repeated measures (pre/post and across 12 weeks), an unstructured variance structure was used to model the repeated time (pre/post) and a compound symmetric variance structure was used to model the repeated week (1–12). The second question asked of the daily data was “do the post-GI group means differ from the UC group means across the weeks?” The data used to answer this question was the post-GI data and the UC group data. The mixed-effect linear model used to test this question contained fixed effects for week (1–12), intervention group (GI versus UC), and a week by intervention group interaction and a random effect for participant.Because this was a pilot study, no adjustment for multiplicity was used; the results from this trial should be regarded as exploratory and should be confirmed in a larger trial. JMP and SAS were used for all data analyses.The study protocol was approved by the Institutional Review Boards (IRB) of Virginia Commonwealth University and Riverside Regional Medical Center.Participants ages ranged from 18 to 39 years with a mean age of 24.26 years (1.06) . The meaThe mean PSS scores for the GI and UC groups for each time point are presented in P < 0.001). The week (1–12) (P = 0.081) and week by time interaction (P = 0.457) were not significant. P < 0.019). The week (1–12) (P = 0.255) and week by time interaction (P = 0.376) were not significant. The first question of the NRSS variable was “do the weekly means differ from before to after intervention for the subjects within the GI group?” From the mixed model analysis, time (pre- versus post-CD) was statistically significant  were used for the CRH analysis. The median (pg/mL) and range for CRH levels are presented in The frequencies of CD tract usage from week 5–12 are presented in One hundred percent of GI group participants wrote in their daily logs at least 1 benefit they had received by listening to the intervention and most participants documented multiple benefits. Comments such as “definitely helped me calm down and not think of anything negative,” “I felt like a weight was lifted,” “it helped me deal with stress better,” “I let the energy come so I could be stress-free,” “ it brings me closer to my baby,” “ease feelings I was having about the baby,” “tiredness and headache went away,” and “learned how to control myself when I am in a tight spot” are just several of many comments describing the perceived benefit of the intervention.The majority of participants reported no perceived barriers. Of those documented, all barriers pertained to the delivery system  or environmental disruptions that made it difficult to concentrate.I know my baby is tucked away inside my body—safe and cozy—cushioned and protected and images of how secure and comfortable and safe the baby is in your body every cell in your body supporting this wonderful baby” promoted a sense of maternal wellbeing and a reduction in perceived stress. While there was a significant difference in PSS scores at 8 weeks (time 2), this was not the case at 12 weeks (time 3). This did not appear to be due to an increase in PSS mean scores in the GI group but rather a decrease in the UC group mean PSS scores, while the GI group mean remained constant. However, even though the GI participants were able to maintain their initial changes at 12 weeks, they did not demonstrate further reduction in PSS scores.The purpose of this study was to investigate the effects of GI on self-reported and biologic measures of stress and the associated symptoms anxiety and fatigue in pregnant African American women. There was a statistically significant reduction in perceived stress scores at 8 weeks (time 2) but not at 12 weeks (time 3) in the GI compared to UC group. The significant decrease in PSS scores for the GI group at 8 weeks has not been reported in previous studies with pregnant women , 32. A pThe NRSS weekly mean stress scores were significantly lower in the GI compared to UC group and, in the GI group, the NRSS scores were significantly lower after intervention compared to before intervention. These findings are consistent with prior reported research  and add While the GI group's state anxiety scores trended downward and the UC group's state anxiety scores remained fairly constant at 8 weeks, there were no significant differences in the state anxiety scores at time 2 or 3. While previous studies , 32 repoThere was a significant difference in trait anxiety scores with the GI group reporting decrease levels at time 8 but not time 12 compared to UC group. Prior studies , 34, 35 Fatigue at 8 weeks (time 2) was decreased in the GI group compared to UC group. We believe this is the first published study to document the effects of GI on fatigue in pregnant African American women. This finding is consistent with reports of researchers examining the effects of GI on fatigue in other populations , 65. TheAs expected, the CRH increased at each measurement. In a normal pregnancy, levels of CRH in maternal blood increase exponentially from weeks 15 to 36 gestation with a significant rise at weeks 26 to 30 . It is tThe intervention was well received by the GI group. The GI participants had never used GI prior to this study and were open to participating and listening to the intervention. As evidenced by the written comments in the daily practice log, the participants found the intervention helpful in feeling less stressed and calmer, relieving symptoms, and soothed pregnancy-related concerns. The positive comments coupled with the fact that only one participant withdrew because the intervention was “too much bother” suggest that CAM interventions are potentially acceptable to African American pregnant women. This is an important finding given the current health disparity in the use of CAM interventions in the United States. Existing data suggest that CAM use is lower among African Americans compared to non-Hispanic Whites and Asian Americans . In thisThere are multiple research implications resulting from this study. An interesting and important area for a future focus is to examine if a booster (s) between 8 and 12 weeks would continue to help improve the stress levels of the group receiving GI as boosters have been found to sustain and/or improve the effectiveness of health behavior interventions . A largeOur results must be balanced with study limitations. In terms of adherence to the intervention, the majority of participants reported daily use of the GI intervention. However, self-reported measures are dependent on the participant's accurate reporting. It is possible that participants under- or overreported information or did not complete the form in a timely manner, which are all potential threats to validity. Future studies could address this limitation through the use of expanding mobile technology that can facilitate self-report data collection and validate intervention use. In some instances, participants' health history information was self-reported and the recruitment site was unable to verify the information. This is a potential limitation when trying to control for confounding factors. For example, fatigue during pregnancy is known to be influenced by coexisting factors such as anemia, excessive weight gain, diet, and physical activity . Thus, tThis study investigated the effects of a GI intervention on maternal stress and associated symptoms of anxiety and fatigue in pregnant African American women beginning in the second trimester. The findings demonstrated significant effects of the GI intervention on perceived stress, anxiety, and fatigue. Within the GI group, the intervention had an immediate effect as evidenced by significant differences in pre- and postintervention averaged daily scores and the GI group reported significantly lower perceived stress scores compared to UC group at 8 weeks. In addition, the GI group reported significantly less trait anxiety and fatigue compared to UC group at 8 weeks.This study also provides direction for consideration in future studies. For example, the addition of appropriate booster sessions may strengthen or sustain the effects of the intervention and the use of mobile technology may facilitate self-report data collection and potentially enhance the evaluation of treatment fidelity, which is often missing in CAM mind-body intervention research. In addition, when examining measures of stress and associated symptoms in pregnant women, consideration should be given for the addition of pregnancy anxiety measures.The results of this study also support the feasibility and acceptability of this intervention. Of those who met the inclusion criteria, 97% of women agreed to participate and, of those, 83% completed the 12-week study. The majority of participants (85%) reported at the beginning of the study that they were not using any stress management technique. Given the negative health outcomes associated with stress during pregnancy, it is imperative that effective, low-cost, and easy to use interventions be available to this population. GI, a CAM mind-body intervention, is a promising intervention to meet that need.


Although liver function tests (LFTs) are routinely measured in primary care, raised levels in patients with no obvious liver disease may trigger a range of subsequent expensive and unnecessary management plans. The aim of this study was to develop and validate a prediction model to guide decision-making by general practitioners, which estimates risk of one year all-cause mortality in patients with no obvious liver disease.In this population-based historical cohort study, biochemistry data from patients in Tayside, Scotland, with LFTs performed in primary care were record-linked to secondary care and prescription databases to ascertain baseline characteristics, and to mortality data. Using this derivation cohort a survival model was developed to predict mortality. The model was assessed for calibration, discrimination (using the C-statistic) and performance, and validated using a separate cohort of Scottish primary care practices.From the derivation cohort (n = 95 977), 2.7% died within one year. Predictors of mortality included: age; male gender; social deprivation; history of cancer, renal disease, stroke, ischaemic heart disease or respiratory disease; statin use; and LFTs . The C-statistic for the final model was 0.82 (95% CI 0.80–0.84), and was similar in the validation cohort (n = 11 653) 0.86 (0.79–0.90). As an example of performance, for a 10% predicted probability cut-off, sensitivity = 52.8%, specificity = 94.0%, PPV = 21.0%, NPV = 98.5%. For the model without LFTs the respective values were 43.8%, 92.8%, 15.6%, 98.1%.The Algorithm for Liver Function Investigations (ALFI) is the first model to successfully estimate the probability of all-cause mortality in patients with no apparent liver disease having LFTs in primary care. While LFTs added to the model's discrimination and sensitivity, the clinical utility of ALFI remains to be established since LFTs did not improve an already high NPV for short term mortality and only modestly improved a very low PPV. Liver function tests (LFTs) are frequently requested and often difficult to interpret in primary care. The results obtained may lead to further invasive and expensive investigations which may be unnecessary. There are a wide variety of reasons for testing liver function including: routine health checks; investigation of non-specific symptoms such as fatigue or nausea; presence of risk factors for liver diseases such as alcohol misuse and/or clinical diagnosis of liver disease; suspected gallbladder or pancreatic problems, and monitoring of drugs such as statins.Despite increasing numbers of LFTs being performed in the UK Clinical prediction models enable accurate probabilities of specific outcomes to be calculated based on characteristics related to the patient, disease or treatment and are a key component of stratified medicine. They are a prognostic strategy often used in primary care This population-based historical cohort study followed-up patients living in Tayside, Scotland with no clinically recognised liver disease who initially had LFTs undertaken in primary care Separate populations were used to develop the prognostic model (derivation cohort) and then externally validate it  The study population was derived from a population laboratory database which contains all electronically available LFT results from patients within Tayside, Scotland, UK during the fifteen year period from January 1989 to December 2003. Tayside is a mixed urban/rural region characteristic of the UK with a population of approximately 410000 Patients aged 16 and above with no obvious or reported clinical signs of liver disease upon presentation to a general practitioner, with at least 2 different LFTs requested from the index appointment, between 1989 and 2003 were eligible for inclusion. The following exclusion criteria ensured that the study population of patients had no clinically recognised liver disease at presentation in primary care:Patients whose bilirubin result was greater than 35 µmol/L in their initial batch of tests, i.e. clinically jaundiced.Patients who had a complication of severe liver disease within 6 weeks of their first LFTs (identified from the ELDIT database detailed in Patients with a history of liver disease before the study period (identified from the ELDIT database).In Tayside, all individuals registered with a general practitioner have a unique identifier, the Community Health Index (CHI) Ethical approval was obtained from the Tayside Committee for Medical Research Ethics in February 2005. Written informed consent from patients was waived by the Tayside Committee for Medical Research Ethics because the databases were anonymised so that no patient identifiable information was accessible. The databases relevant to this study see  covered The databases  identifiThe primary outcome was all-cause mortality during the follow-up year. Patients who died were identified from the Scottish National Death Registry. Underlying causes of death were tabulated for the derivation population and categorised using versions 9 and 10 of the International Classification of Diseases.st December 2003, date of emigration, or death, whichever was earlier. All patients whose endpoint was not death were censored.Parametric survival regression models were fitted to investigate the effect of baseline characteristics on time to all-cause mortality. The starting point was taken as the date of the initial LFT test and the endpoint was one year later, 31The continuous baseline characteristics of age, albumin, ALP, GGT, and transaminase were assessed for their functional form by plotting them against the Martingale residuals and appropriate transformations were carried out, where necessary. The Weibull accelerated failure time model was used for model building, which was conducted in a manual stepwise manner. A multiple imputation procedure was conducted to impute missing baseline data The final model was assessed for predictive ability to examine its ability at discriminating high from low risk using the C-statistic The validation cohort contained all patients registered with 19 practices from across Scotland out with Tayside. The practices were participating in the Practice Team Information project operated by the Information Services Division of the National Health Service National Services Scotland, and contributing data to the Primary Care Clinical Informatics Unit, University of Aberdeen The final model was fitted to the validation cohort using the same parameter estimates derived from the study population. The C-statistic was calculated and the calibration plot drawn as for the study population to assess the performance of the model on the external validation cohort. A further model was fitted to the validation cohort using the same covariates as for the final model and its C-statistic computed. The resulting parameter estimates were compared to the final model's parameter estimates using the z-test to test for equality Analyses were performed using SAS (v9.2) .After exclusions our derivation cohort contained 95977 patients with incident initial LFTs taken in primary care and with no obvious liver disease Only 8388 (8.7%) patients were tested for all five liver enzymes. The percentage of complete data for each LFT was as follows: ALP (99.2%), albumin (99.2%), bilirubin (93.6%), transaminases (76.5%), and GGT (10.9%). There were more males with complete data  than females (54.6% versus 45.4%) . The groA total of 2613 patients (2.7%) died of any cause during one year follow-up. The commonest underlying cause of death was cancer (39.3%) . Of thesThe final model is presented in The C-statistic for discriminatory ability of the prediction model for risk of one year mortality was 0.82 . With LFTs excluded the C-statistic was lower with a value of 0.79  in the derivation cohort, and demonstrates that LFTs add some discriminatory ability to the model. For a model without LFTs the performance measures were similar to the full model for the median cut-off see . For exaThe external cohort contained 11653 patients . The proThe C-statistic for the final model applied to the external cohort was 0.86 (95% CI 0.79 to 0.90). The calibration slope test was borderline significant meaning that the model required a small amount of recalibrating which is usual in external validation , that can predict mortality within one-year following liver function testing in primary care. ALFI performs better at lower cut-offs of predicted probabilities. The NPV and specificity measures were excellent but decreased little when LFTs were removed from the model . However, whilst LFTs did improve sensitivity and moderately improved an already low PPV, the clinical utility needs to be established, e.g. through the use of a web-based tool to see the impact of ALFI on clinical decision making, further investigations, patient outcomes, and costs to the health service. The majority of deaths were from previously undiagnosed cancer and cardiac disease which may warrant further investigation.th percentile (3.84% for the model without LFTs and 2.78% for the model with LFTs) the sensitivity improved from 78.1% without LFTs to 84.8% with LFTs. Even with LFTs included the PPV never gets as high as the NPV and as it increases the sensitivity decreases dramatically. For example, if we used the model to refer those with a very high mortality risk of greater than 60%, the GP would over-investigate 26.4% patients but correctly investigate 73.6% patients. However, at this cut-off the sensitivity is extremely poor meaning that ALFI would fail to detect 95.4% of those who did die within the year. With such small numbers at high cut-offs and with poor sensitivity, it is clear that ALFI performs best at low cut-offs of predicted risk but at the expense of low PPV.ALFI will give an individualised prognosis based on the patient's characteristics, LFTs, comorbidity history and statin use. The resulting probability estimate combined with other clinical information  at the GP's disposal might facilitate their decision-making with regards to further investigations, referral or watchful waiting. With its extremely high NPVs, ALFI has an excellent ability to accurately assign a low probability of short term mortality. For example, if we take 0.61% as our predicted probability cut-off, the model can accurately identify those patients with a very low probability of poor prognosis and the GP can confidently use this with any other contextual information not in the model e.g. body mass index, smoking status, alcohol intake, to inform their decision making. Using the model alone, with such a cut-off the GP would under investigate 83/47989 (0.2%) patients but correctly rule-out one-year mortality in 47906/47989 (99.8%) patients. Furthermore, at this cut-off the sensitivity is very high since ALFI detected 97% of all actual deaths within the year. However, the PPV was low meaning that 94.3% of patients detected over the cut-off would be over-investigated. However patient outcomes beyond one year may be improved by investigation. The use of clinical parameters without LFTs in the model had high enough sensitivity to give very similar NPVs given the low overall risk of mortality. However, the PPV increased at a lesser rate and sensitivity reduced at a greater rate as the cut-offs increased meaning that LFTs contributed more to the proportion of true positives than to true negatives. For example, for a predicted probability cut-off at the 75One prediction model involving liver disease is the Model for End-Stage Liver Disease (MELD) Similarly, the ALFI model could be used to prioritise referrals to secondary care. However, it would not tell the user who to refer to as this would require additional contextual information and clinical judgement. ALFI's main use could be to identify those who have a very low probability of death and do not need further investigation for severe life threatening conditions. It would not, however, detect those who have important underlying conditions that might reduce survival in the longer term or non fatal conditions than can impair quality of life and still need a diagnosis and appropriate treatment.This is the first successfully derived and externally validated population risk prediction model for one year all-cause mortality in primary care patients with LFTs using a large population dataset. The ability of the model to discriminate between patients at high and low risk was excellent (c = 0.82) and the calibration curve showed reasonable accuracy of the probability estimates from the prediction model across the range of values of predicted risk. The discrimination of ALFI when applied to the external cohort was also excellent (c = 0.86). In comparison, the Framingham equation c-statistic ranged from 0.63 to 0.83 for its external validation on six different cohorts ALFI was derived from unselected “real-world” observations in a geographically defined population: an approach being encouraged by the National Institutes of Health LFTs are non-specific markers of illness i.e. either disease processes in the liver, local involvement from disorders of other organs, or as a marker of systemic disease. The inflammatory or infective liver diseases tend to produce abnormality of the transaminases and may produce mortality rises in the long term (decades) but the effects of other organ disorders (e.g. aggressive cancers or heart disease) are more likely to be manifest in the short term, so a one-year follow-up period was chosen for this study. The algorithm is not a diagnostic one but an aid to clinical decision making and all-cause mortality was deemed the most appropriate to ‘catch all’ patients with very poor or very good prognosis. Clearly, a model for mortality over a longer follow-up period would have a poorer predictive ability following one set of LFTs since other events would intervene. In contradistinction the use of LFTs as a predictor of outcome in chronic liver disease would need a much longer follow-up period as the natural history of most primary liver diseases is decades. The model including LFTs had better discriminatory ability and higher PPV than the model without. Furthermore, albumin and ALP explained the second and third most variation of all the predictors in the model proving that LFTs are valuable prognostic factors for short term mortality.m imputations relative to one obtained from an infinite number of imputations GGT was missing for a large proportion of patients in the derivation cohort but less so in the validation cohort. However the appropriate guidelines for handling this problem were followed Out of 95977 patients from the derivation cohort, 2613 died (2.7%) within one year. Almost 40% of the 2613 deaths were caused by cancer. A Korean study prospectively followed up 142055 men and women who had a transaminase test for a maximum period of 8 years and they also found that out of 3786 deaths, 46.2% were from cancer External validation of prediction models is extremely important in order to support their application and transportability to different geographical or temporal populations. Since our external cohort came from 19 primary care practices across nine different regions of Scotland during a different time period than the population used to develop the model, we have shown that ALFI predicts well with regards to both aspects. The fact that the external cohort had patients with greater rates of deprivation and comorbidity than the derivation cohort shows that the model is robust to changes in baseline characteristics. The median LFT values for both cohorts were very similar, as were the proportions of patients who died within one-year. The next step is to convert ALFI into a web-based decision aid to assess its impact on GP management of these patients. Using the sensitivity and PPV results, suggested management plans can be determined for different risk cut-offs. A feasibility study of its implementation, focused on general practitioner acceptability and impact on decision making and costs might then usefully follow.This study has developed and externally validated a novel risk prediction model for one-year mortality in patients, with no clinically obvious liver disease, having their LFTs taken in primary care. All five LFTs were predictive of mortality and improved the discriminatory ability, sensitivity and PPV of the model. ALFI performs best at lower cut-offs of predicted probabilities of mortality with excellent sensitivity and NPV and good specificity. However, low PPV may mean over-investigation of some patients. However, the addition of LFTs did not improve NPV for short term mortality in these types of patient and only modestly improved the PPV. Therefore the clinical utility of ALFI as a decision tool in primary care needs to be established in terms of further testing, patient outcomes and health service costs. The utility of liver function testing per se needs to be further examined using other outcomes such as impact on the detection of modifiable diseases such as chronic liver disease.Appendix S1Further information on statistical methods.(DOC)Click here for additional data file.Appendix S2Complete case analyses.(DOC)Click here for additional data file.Appendix S3Calibration of the final model.(DOC)Click here for additional data file.Appendix S4Discrimination and calibration of the model applied to the external cohort.(DOC)Click here for additional data file.Appendix S5How to calculate the predicted probability of mortality within T days (where 1≤T≤365).(DOC)Click here for additional data file.


Polydora cornuta, to determine if modifications could be detected immunohistochemically and if there were characteristic changes in modifications throughout ontogeny (surveyed at representative stages from oocyte to adult). We found a common time of onset for three histone modifications in early cleavage , some differences in the distribution of modifications among germ layers, differences in epifluorescence intensity in specific cell lineages suggesting that hyperacetylation (H3K14ac) and hypermethylation (H3K9me) occur during differentiation, and an overall decrease in the distribution of modifications from larvae to adults. Although preliminary, these results suggest that histone modifications are involved in activating early development and differentiation in a marine invertebrate.Histone modifications are widely recognized for their fundamental importance in regulating gene expression in embryonic development in a wide range of eukaryotes, but they have received relatively little attention in the development of marine invertebrates. We surveyed histone modifications throughout the development of a marine annelid,  One of the central questions in biology is how differences in gene expression during development lead to the generation of form. Epigenetic mechanisms such as histone modifications activate or silence gene expression and thereby provide rapid, reversible mechanisms that regulate gene expression in embryonic development. The importance of histone modifications in development has been extensively studied in model systems. As this approach is gradually extended to nonmodel species, histone modifications are being discovered as mechanisms that are highly conserved in a wide variety of eukaryotes and critically important in regulating fundamental developmental processes, including meiosis , cell diPolydora cornuta Bosc, 1802  for this study. P. cornuta is a small, opportunistic detritivore that is common in intertidal mudflats and has a wide distribution in temperate and subtropical coastal areas [P. cornuta is internal, and females deposit zygotes in a string of egg capsules that they brood in their mud tubes. Larval development for this species has been described by several authors and is strongly influenced by the presence of nurse eggs in the egg capsules [P. cornuta, the present study focuses on epigenetic similarities between morphs. Our goal is to establish a foundational understanding of changes in the epigenome throughout development, as the first step in a larger project that investigates the potential for histone modifications to influence plasticity in larval development in this species. Despite the clearly established importance of histone modifications in the development of many eukaryotes, they have received almost no attention in the development of benthic marine invertebrates. Benthic marine invertebrates represent an exciting group for epigenetic research as they not only are morphologically diverse as adults, but their larvae are morphologically and behaviorally distinct from adults and form the basis for an impressive diversity of life-history patterns. Our objectives are to determine if histone modifications can be detected in a marine worm using immunohistochemistry, if modifications differ among differentiating tissues, and if changes in modifications correlate with ontogenetic transitions. We chose the worm al areas , 8. Fertcapsules . Some brPolydora cornuta, and also because of the overall complexity of the epigenome, we follow the advice of Turner [Polydora cornuta. Similarities in the distribution of three histone modifications suggest that certain phases of development  represent transition points during which widespread changes in histone modifications occur. We surveyed histone modifications throughout ontogeny using immunohistochemistry. The survey included oocytes, embryos, early larvae, and adults. This allowed us to correlate histone modifications with specific developmental events  and life history stages . We focused on histone modifications as histones are among the most highly conserved proteins in eukaryotes . Their mf Turner , 21 and Polydora cornuta were collected from intertidal mudflats at West Marsh, Halifax Co., Nova Scotia  in early summer (May to July) of 2010 and 2011. Adults were cultured in 250 mL Pyrex crystallizing dishes which contained enough sand to cover the bottom. Each dish contained approximately 10–16 worms, including some males to ensure sperm availability. Cultures were immersed in seawater at approximately 14-15°C, provided with continuous aeration, and maintained on a 15 : 9 LD photoperiod. Adult P. cornuta is poecilogonous [P-brood to refer to broods in which there are few or no nurse eggs , most eggs develop (approximately 80 embryos/capsule) and young hatch as small (3 to 5 segments), planktotrophic larvae. The term A-brood is used for broods in which most eggs (>90%) are nurse eggs, few young develop (approximately 5/capsule), and most young are adelphophagic while in the egg capsule (data from MacKay and Gibson) [After spawning, broods were removed from the females' tubes and cultured. As logonous , broods  Gibson) . IndividForty-eight A-broods and sixteen P-broods were examined. Unequal sample sizes reflect the fact that P-broods were relatively uncommon in the West Marsh population. Approximately eight egg capsules were fixed per brood per ontogenetic stage. We processed two to three egg capsules per assay and examined all embryos per capsule for consistency in epifluorescence . A complete examination was done for young from both A- and P-broods at all ontogenetic stages, and observations of histone modifications common to both morphs are presented.Embryos and larvae of both morphs were fixed and labeled with commercially available primary antibodies for histones and histone modifications. Egg capsules were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 45 minutes on ice, rinsed in PBS, dehydrated to absolute methanol, and stored at −20°C for up to two months. Immunohistochemistry was performed using a procedure modified from Sagawa et al.  and kindn = 8 females) or in paraffin section (n = 5). Whole-mounted segments were processed as for embryos. For paraffin sectioning, females were placed in filtered seawater for 24 h to allow the gut to void of sand and then fixed and dehydrated as described for egg capsules. After females were embedded in paraffin, they were sectioned at 10 μm and 5-6 sections from each female placed on poly-L-lysine coated slides. Sections were deparaffinized in xylene, rehydrated to PT, and processed as described for egg capsules. Sections were ringed with a liquid blocker pen to avoid loss of reagents, and the immunohistochemistry protocol was done in a humid, sealed chamber.Gravid females were examined in whole mounts of individual segments . Micrographs were adjusted for size and contrast using Corel PhotoPaint 11.0. Epifluorescence of TRITC-conjugated antihistone indicated that histones were present in the nuclei of all cells throughout development . TRITC eAcetylation of H3K14 was not detected in oocytes located within the coelom of gravid females although it was detected in a few of the follicular cells associated with the oocytes Figures . The earIn gastrulae, H3K14ac was detected as weak epifluorescence throughout the epidermis and as strong epifluorescence in a few cells associated with the mouth (shown below). In trochophores and early larvae, bright epifluorescence was detected in the mouth and also on the ventral surface and pygidium Figures . Older lDrosophila) is the lack of detectable H3K14ac in oocytes and early embryos (2- to 4-cell stages). Histone acetylation is important in oogenesis in mammals [Drosophila and has been shown to vary at different points in the meiotic cycle [P. cornuta, it appears that H3K14ac is important in at least some aspects of meiosis, as it was detected in polar bodies and suggests a potential pathway by which polar bodies may be determined.Histone acetylation is generally associated with transcription and in eukaryotes is common in undifferentiated cells, while differentiated cells often contain hypoacetylated chromatin . Our obs mammals . H3K14acic cycle . Our detThe onset of acetylation of H3K14 occurred in early embryos (8-cell stage), when it was detected in all blastomeres except for the D macromere; this pattern was retained at least through the sixteen-cell stage but was difficult to follow in later development (from 32 cells on) given the techniques used here. In polychaetes, the D macromere gives rise to most of the segmented tissue including ectoderm and mesodermal derivatives . Lack orH3K9me was not detected in oocytes located within the coelom of gravid females Figures . The earIn trochophores and metatrochophores, moderate levels of H3K9me were detected throughout the ectoderm with bright epifluorescence in the head and laterally in the region of the presumptive chaetal sacs. Epifluorescence was also detected in the mesoderm and endoderm (demonstrated below). Early larvae had moderate FITC signal throughout the epidermis, chaetal sacs and mesoderm, and weak epifluorescence in the gut , and H3K9me-positive cells decreased in distribution in adults. Here, we interpret the presence or relative intensity of H3K9me as indicating a change in histone modifications  rather than specifically indicating gene activation or repression, as H3K9me has been associated with both , 30, 31.H3K4me2 was not detected in oocytes located within the coelom of gravid females Figures . H3K4me2P. cornuta, as dimethylation of H3K4 is associated with varying transcriptional activity depending on interactions with other histone modifications as differentiation occurs [These observations suggest that H3K4me2 was similar in distribution to H3K14ac and H3K9me: H3K4me2 was not detected in oocytes, had an onset in early cleavage, was broadly distributed in embryos and larvae, and was detected in adults in specific tissues only. The major difference between H3K4me2 and the modifications described above was the lack of hypermethylation in specific cell lineages. These observations suggest that widespread changes in H3K4me2 occur at roughly the eight-cell stage  and possibly also with metamorphosis . As with H3K9me, the specific functional implications of H3K4me2 are not yet known for n occurs , 32.We attempted to detect H4K20me2 in female tissue , in trochophores, and in three-chaetiger larvae. H4K20me2 was not detected at any of these ontogenetic stages, and therefore, our search for it was discontinued.In many metazoans, histone modifications are re-programmed during meiosis and embryos gradually acquire modifications during differentiation . Our resHydroides elegans and the sea urchin Strongylocentrotus purpuratus [P. cornuta. Additionally, histone modifications are associated with differentiation as they were also detected in specific larval and adult tissues. Collectively, these observations suggest that global changes in gene expression occur at about the eight-cell stage with the onset of three modifications that affect gene transcription . The onset of histone modifications was consistent but not uniform among blastomeres; for example, the onset of H3K14ac was delayed in the D blastomere relative to other cells of the same embryo. While the importance of histone modifications in the early development of marine invertebrates has not received much attention, the importance of histone variants has been demonstrated by Arenas-Mena and colleagues for the polychaete rpuratus . Both sprpuratus . Our obsThe potential for histone modifications to be associated with tissue differentiation is supported by the presence of hyperacetylation of H3K14 in cells of the mouth and pygidium of larvae, of hypermethylation of H3K9 in cells of the presumptive head of embryos (blastulae and gastrulae), and of the restriction of some modifications to specific organs . Thus, lineage-specific modifications also occur and suggest that histone modifications may influence not only early specification of cell fate but also cell differentiation as tissues (such as the gut) specialize and become functional in larvae.All three histone modifications had patterns of distribution that differed between larvae and adults. In larvae, H3K14ac was broadly distributed throughout the epidermis, and H3K9me and H3K4me2 were detected throughout derivatives of ectoderm, mesoderm, and endoderm. In contrast, the distribution of all three modifications was restricted in adults, in terms of being detected in relatively few cells within a tissue  or no longer being present at detectable levels . This suggests that a transition in the histone code may occur as larvae undergo metamorphosis. Metamorphosis was not a focus of this study, but this general pattern suggests two hypotheses. One is that histone modifications affect a change in gene expression that correlates with changes in growth from rapidly growing larvae to more slowly growing adults. The other hypothesis is that changes in histone modifications correlate with a developmental reprogramming at metamorphosis. Both hypotheses have merit. Most larval tissues contribute directly to adult tissues in spionid polychaetes, suggesting that the first hypothesis may be more valid, but settlement involves widespread behavioural and morphological changes; thus, the potential for a global reprogramming of gene expression at metamorphosis is also to be considered. Polydora cornuta, using immunohistochemistry. We found that three of the four tested primary antibodies for histone modifications appeared to recognize and bind to antigens of this species. H3K14ac, H3K9me, and H3K4me2 colocalized with DAPI and were consistently detected throughout development. The fourth primary antibody, H4K20me2, did not react with the tissue. The three detected modifications collectively suggest that these histone modifications are first present in early cleavage, are widely distributed throughout larval development, and also are found in some adult tissues but with a more restricted distribution. The observed common onset in histone modifications suggests that a global change or activation of gene expression occurs in early embryos. Two modifications showed a generally low level of epifluorescence in most cells but a very strong signal in a few cell lineages, indicating a role in tissue differentiation. Finally, differences in the distribution of three modifications between larvae and adults suggest a second transition in histone modifications may occur at metamorphosis. Although preliminary, this research indicates that histone modifications are present in a marine invertebrate and show characteristic changes with tissue differentiation and also with specific life stages.We surveyed histone modifications in the development of a polychaete,