The Democratic Republic of Congo (DRC) is one of the two most leading contributors to the global burden of disease due to malaria. This paper describes the malaria testing and treatment market in the nation’s capital province of Kinshasa, including availability of malaria testing and treatment and relative anti-malarial market share for the public and private sector.A malaria medicine outlet survey was conducted in Kinshasa province in 2013. Stratified multi-staged sampling was used to select areas for the survey. Within sampled areas, all outlets with the potential to sell or distribute anti-malarials in the public and private sector were screened for eligibility. Among outlets with anti-malarials or malaria rapid diagnostic tests (RDT) in stock, a full audit of all available products was conducted. Information collected included product information , amount reportedly distributed to patients in the past week, and retail price.In total, 3364 outlets were screened for inclusion across Kinshasa and 1118 outlets were eligible for the study. Among all screened outlets in the private sector only about one in ten (12.1%) were stocking quality-assured Artemisinin-based Combination Therapy (ACT) medicines. Among all screened public sector facilities, 24.5% had both confirmatory testing and quality-assured ACT available, and 20.2% had sulfadoxine-pyrimethamine (SP) available for intermittent preventive therapy during pregnancy (IPTp). The private sector distributed the majority of anti-malarials in Kinshasa (96.7%), typically through drug stores . Non-artemisinin therapies were the most commonly distributed anti-malarial , followed by non quality-assured ACT medicines (38.5%). The median price of an adult quality-assured ACT was $6.59, and more expensive than non quality-assured ACT ($3.71) and SP ($0.44). Confirmatory testing was largely not available in the private sector (1.1%).While the vast majority of anti-malarial medicines distributed to patients in Kinshasa province are sold within the private sector, availability of malaria testing and appropriate treatment for malaria is alarmingly low. There is a critical need to improve access to confirmatory testing and quality-assured ACT in the private sector. Widespread availability and distribution of non quality-assured ACT and non-artemisinin therapies must be addressed to ensure effective malaria case management.The online version of this article (doi:10.1186/s12936-016-1659-x) contains supplementary material, which is available to authorized users. The Democratic Republic of Congo (DRC) is the second largest contributor to the global burden of disease due to malaria  price in drug shops for one adult equivalent treatment dose of quality-assured ACT was $6.59 [$2.75–$9.89], compared to $3.85 [$3.30–$4.94] for non quality-assured ACT. The median quality-assured ACT price in private for-profit facilities was $0.00, while non quality-assured ACT was on average sold at $3.71 [$2.75–$4.94]. SP was the cheapest tablet anti-malarial in the private for-profit sector ($0.44 [$0.33–$0.55] for one AETD).Table Public sector readiness for malaria case management was overall low with only one in four public sector outlets stocking both malaria testing and quality-assured ACT (24.5%). While availability of confirmatory testing was high (86.8%), availability of quality-assured ACT was much lower (28.7%). Availability of SP for ITPp was also low; only one in five public facilities had SP available on the day of the survey (20.2%).In the private sector, only 1.1% of outlets had both confirmatory testing and quality-assured ACT available. Readiness was higher among private for-profit facilities (10.7%) as compared with drug stores (0.3%).Data from a 2013 representative medicine outlet survey in Kinshasa confirm the importance of the private sector as a source of anti-malarial treatment, particularly drug stores. Results suggest urgent need to address malaria case management in the private sector, remove non quality-assured ACT medicines and inefective nonartemisinintherapies from the market, and address gaps in public sector readiness for appropriate case management.The distribution of anti-malarials in Kinshasa in 2013 was heavily dominated by the private sector, particularly drug stores which comprised almost 90% of the anti-malarial service delivery points. Drug stores in Kinshasa are not registered or regulated by a government authority, and are not authorized to test for malaria using RDTs. However, the vast majority of people receiving anti-malarial treatment in Kinshasa are receiving medicines from these unregistered and unregulated points of care where confirmatory testing is not permitted and quality-assured ACT treatment and malaria diagnostic testing is generally not available. With the vast majority of the antimalarial distribution moving through drug stores, these outlets most commonly stock and distribute non quality-assured ACT medicines and non-artemisinin therapies.The finding that most of the anti-malarial medicine market share is comprised of drug stores in Kinshasa may provide an opportunity to increase access to affordable, quality-assured ACT medicines, as evidenced in other countries where these facilities are a common source of treatment –13. The Several countries have implemented successful interventions aimed at specifically strengthening drug stores for malaria case management. In Nigeria, Tanzania and Uganda, registration or accreditation programmes have been implemented in recent years to ensure that drug store providers are trained to assess and treat suspected malaria. For example, since 2006 the Tanzanian government has been in the process of accrediting drug dispensing outlets (ADDOs) to sell a limited range of prescriptions only medicines, including quality-assured ACT, after participation in a 35-day training programme. More recently, pilot initiatives have introduced RDTs into ADDOs with relative success, demonstrating patient willingness to pay for an RDT and an increased likelihood to purchase an ACT medicine . Other rIn 2009, the vast majority of anti-malarial-stocking outlets in Kinshasa were stocking oral artemisinin monotherapy. By 2013, oral AMT had disappeared from the market, demonstrating the success of the drug ban which had been implemented in 2009 .Despite this achievement, multiple threats to effective malaria case management persist in the anti-malarial market in 2013. First is the widespread availability of non quality-assured ACT medicines. These are combination therapies that are not manufactured according to global standards set by the World Health Organization  and inclThe availability and distribution of non quality-assured ACT medicines is a growing problem documented in other countries by the ACTwatch project, including Kenya and Nigeria , 21. HowThe 2013 outlet survey also documented ongoing high levels of availability and distribution of non-artemisinin therapies, which are no longer recommended for uncomplicated malaria case management in the DRC. Non-artemisinin therapies accounted for half of all anti-malarials distributed in Kinshasa. These include oral quinine and SP. Although SP may be used for IPTp, availability and distribution through drug stores as opposed to health facilities, as well as product packaging and patient instructions on SP products promoting use for malaria case management for people of all ages, suggests that SP is being used as an inexpensive treatment for malaria . The resThe DRC was one of the first countries to adopt the second edition of the World Health Organization’s malaria treatment guidelines , for whiKinshasa’s readiness for appropriate malaria case management was extremely low in the private sector, and in particular among drug stores where most patients receive anti-malarial treatment. In drug stores, there was negligible availability of diagnostic tests and quality-assured ACT, pointing to the need to reinforce and promote the necessity of diagnostic testing among both providers and patients, while ensuring commodities are available and affordable within this sector as well as the public sector. Low availability of confirmatory testing in particular threatens appropriate management of suspected cases, spurring the potential for presumptive anti-malarial treatment. The low availability of confirmatory testing also merits a revisit of national policy to permit testing within these outlet types and provision of quality-assured treatment. As discussed previously, several initiatives in other countries have introduced RDTs into drug stores with relative success , 16, demGiven significant gaps in readiness for malaria case management documented in 2013, several measures were taken by the National Malaria Control Programme (PNLP), and with support from the Department For International Development (DFID), GFTAM, Association de Santé Familiale (ASF) and Population Services International (PSI), to transform the private sector anti-malarial market to improve quality malaria case management in Kinshasa . This haThe data presented in this paper are representative for Kinshasa province only. Given the importance of the DRC with respect to global malaria burden, a key limitation is the lack of information on malaria markets in other areas of the DRC. Other limitations to the ACTwatch survey methodology have been described elsewhere in detail .While incredible progress has been made in recent years in improving access to malaria testing and quality-assured first-line treatment in many countries in sub-Saharan Africa , progres


Gestational trophoblastic neoplasia (GTN) is a highly treatable disease, most often affecting young women of childbearing age. This study reviewed patients managed for GTN at the Butaro Cancer Center of Excellence (BCCOE) in Rwanda to determine initial program outcomes.A retrospective medical record review was performed for 35 patients with GTN assessed or treated between May 1, 2012, and November 30, 2014. Stage, risk score, and low or high GTN risk category were based on International Federation of Gynecology and Obstetrics staging and the WHO scoring system and determined by beta human chorionic gonadotropin level, chest x-ray, and ultrasound per protocol guidelines for resource-limited settings. Pathology reports and computed tomography scans were assessed when possible. Treatment was based on a predetermined protocol stratified by risk status.Of the 35 patients , 26 (74%) had high-risk and nine (26%) had low-risk disease. Nineteen patients (54%) had undergone dilation and curettage and 11 (31%) had undergone hysterectomy before evaluation at BCCOE. Pathology reports were available in 48% of the molar pregnancy surgical cases. Systemic chemotherapy was initiated in 30 of the initial 35 patients: 13 (43%) received single-agent oral methotrexate, 15 (50%) received EMACO , and two (7%) received alternate regimens. Of the 13 patients initiating methotrexate, three had their treatment intensified to EMACO. Four patients experienced treatment delays because of medication stockouts. At a median follow-up of 7.8 months, the survival probability for low-risk patients was 1.00; for high-risk patients, it was 0.63.This experience demonstrates the feasibility of GTN treatment in rural, resource-limited settings. GTN is a curable disease and can be treated following the BCCOE model of cancer care. The establishment of referral cancer centers in high-income countries has led to centralization of GTN care, facilitating the conduct of clinical trials and development of expertise despite low patient volume in any one center.5 Standard-of-care diagnosis of molar pregnancy is often dependent on the use of ultrasonography in the evaluation of pregnancy-associated bleeding and accurate testing for beta human chorionic gonadotropin (β-hCG), a tumor marker for GTN.8 Suction dilation and evacuation is an accepted procedure for the management and diagnosis of gestational trophoblastic disease, and accurate and timely pathology supports preliminary clinical diagnosis.9 In low-resource settings, manual vacuum suction is often practiced in the management of abortions. Access to ultrasonography in the rural setting can be limited, and pathology analysis is often not performed or available to facilitate early diagnosis of molar pregnancy. Furthermore, delays (both patient and system) often result in a longer time interval between the antecedent pregnancy and diagnosis of GTN, correlating with higher burden of disease, higher GTN risk scores, and possibly poorer outcomes.11However, challenges to GTN diagnosis and treatment exist in resource-limited settings. Approximately 50% of GTN cases arise from hydatidiform molar pregnancy, 25% from miscarriage or tubal pregnancy, and 25% from term pregnancy.12 The center serves as a model of accessibility, where the cost of care is offset for the poor.13 GTN is a priority cancer because of its high cure rate, target population of healthy young women of reproductive age, and manageable subset of chemotherapeutic drugs. Treatment paradigms for GTN were adapted to available resources and designed for care delivery by nononcologists with remote clinical guidance by medical experts, given the limited national oncology capacity.12The Butaro Cancer Center of Excellence (BCCOE), located in the rural northern Burera district in Rwanda, is a pioneer in rural-based cancer treatment in low-resource settings.This article aims to describe the management of GTN in resource-limited countries, report on treatment outcomes among patients referred to and treated at BCCOE in Rwanda, and identify factors associated with the potential for improved outcomes among patients with GTN. This is the first study to our knowledge to report on the GTN experience from a rural, resource-limited cancer center in Rwanda, paving the way for future adaptation of treatment models for this type of setting.BCCOE was established in 2012 by the Rwandan Ministry of Health with support from its collaborators, the nonprofit organization Partners In Health  and the Dana-Farber/Brigham and Women’s Cancer Center . The center serves the Rwandan rural-based population in the Northern Province and has become a national referral center for select cancer care, evaluating 2,326 patients in its first 2 years.A retrospective medical record review was performed for all patients assessed or treated for GTN between May 1, 2012, and November 30, 2014, at BCCOE in Rwanda. Thirty-five patients were identified for inclusion in this study. The study protocol was reviewed and approved by the Rwandan National Ethics Committee.Patients were treated for GTN on the basis of a protocol adapted to regionally available resources. Clinical suspicion combined with persistently elevated or rising β-hCG levels longer than 3 weeks after evacuation of a molar pregnancy prompted further work-up. On the basis of prespecified protocol guidelines, minimal diagnostic work-up included quantitative serum β-hCG level, physical examination, abdominal and pelvic ultrasound, and chest x-ray. Preferred evaluation when available included computed tomography (CT) scans of chest and abdomen; if positive, a CT scan of the head was thereafter included.15 Any laboratories or imaging reports not included in a patient’s medical record were assumed not to have been performed. Furthermore, given that protocols were adapted to resource availability, staging and risk scores were calculated on the basis of best available data. β-hCG values were obtained from three different laboratories in the country; units and reporting styles (exact result v range) varied among reports.Stage, risk score, and risk category were based on International Federation of Gynecology and Obstetrics staging and modified WHO risk scoring system.17 In response to challenges in chemotherapeutic drug storage, patient compliance, and cost limitations, oral MTX was used as first-line treatment of low-risk GTN.17 Oral MTX was delivered at a dose of 0.4 mg/kg  once a day for 5 days every 2 weeks. Patients with stage IV or high-risk disease (WHO risk score ≥ 7) were received the combination chemotherapy regimen EMACO .19 Quantitative β-hCG levels were recommended on day 1 of every cycle, and chemotherapy continued until normal β-hCG levels were reached. In line with principles of consolidation therapy in GTN management, patients received two additional cycles of chemotherapy after normalization of quantitative β-hCG values. Once therapy commenced, all β-hCG values were sent to the same laboratory.Before initiation of therapy, all patients were counseled on contraception. Chemotherapy was based on staging and risk classification. Patients with stage I or low-risk stage II or III disease (WHO risk score ≤ 6) were treated with single-agent methotrexate (MTX).Once a patient achieved a complete response, follow-up was recommended every month for 3 months and included complete clinical examination and quantitative β-hCG. Thereafter, surveillance examinations continued at an interval of every 3 months for a year. For patients whose β-hCG levels did not fall within the respective half-lives, persisted, or rose, a multidisciplinary call was organized with the appropriate surgical and medical consultants for individual and optimal care.Data extraction was performed by two independent researchers . Where questions or discrepancies existed, the opinion of a clinician was requested . Medical record review was supplemented by the existing electronic medical records. Data were analyzed using STATA software . The Kaplan-Meier plot was stratified by low- and high-risk GTN categories.Patient characteristics at diagnosis are listed in The majority of patients had an antecedent molar pregnancy . Patients also presented with an antecedent pregnancy with abortion or antecedent term pregnancy. Of the 35 patients, 19 (54%) had undergone evacuation and 11 (31%) hysterectomy. The three other prior surgical interventions performed included a cesarean section and two laparotomies. In total, 28 patients  had received some form of surgical evaluation related to gestational trophoblastic condition before arrival at BCCOE. Most patients (n = 29 [83%]) had not received any form of chemotherapy before presentation at BCCOE.International Federation of Gynecology and Obstetrics staging and work-up results are listed in Less than half  of patients received a full work-up as per protocol. The protocol for complete work-up at BCCOE includes intake β-hCG, chest x-ray, and liver ultrasound. Most patients (89%) had a β-hCG value available at intake, and 80% received a chest x-ray. Less than half (45%) of the patients received an abdominal or pelvic ultrasound, and only eight (23%) received any form of CT scan as part of their initial staging work-up.With these data, 10 patients (29%) were determined to have a localized tumor, and 19 (54%) were found to have tumor metastasis. Among high-risk patients, disease had metastasized to the lungs in 16 (62%), to the liver in one (4%), to the brain in one (4%), intra-abdominally in two (8%), vaginally in two (8%), and to other sites in two (8%); 12 (46%) had two or more verified sites of disease.The availability of pathology reports from any surgical interventions was reviewed. Of the 27 patients with antecedent molar pregnancy, seven had undergone hysterectomy, 14 had undergone evacuation, two had undergone both hysterectomy and evacuation, one had undergone laparotomy, and three had had no prior surgical intervention . PatholoTreatment course variables are listed in There were few reports of chemotherapy toxicity events with single-agent oral MTX . Of the There were six treatment delays experienced by five patients receiving single-agent MTX. Three of the MTX delays resulted from medication nonavailability and three from missed appointments. Of the total 18 patients receiving EMACO, five did not experience any delays, whereas 13 experienced 38 documented delays. The causes for these treatment delays included neutropenia and associated complications on 22 occasions (average of two events per patient), infection not related to neutropenia in one patient, elevated liver function tests in two patients, one missed appointment each in six patients, medication stockout in one patient, and other reasons in six patients.Of the 35 patients diagnosed at BCCOE, 30 initiated systemic chemotherapy. Five patients did not receive chemotherapy treatment at BCCOE; two of these were lost to follow-up after the initial consult, one died as a result of disease before therapy, and two were found to be disease free posthysterectomy for presumed invasive mole .Outcomes for those patients treated (n = 30) are summarized in Overall, of the 30 patients receiving systemic therapy, 12 (41.4%) were alive and disease free, seven (24.1%) continued to receive therapy, six (20.7%) died, and five (13.8%) were lost to follow-up. For the remaining patients, the average length of follow-up time was 15 months for low-risk patients and 6 months for high-risk patients. When comparing risk category and outcome, the challenges of high-risk and late-stage presentation of patients become especially apparent. All patients who died while receiving treatment were high risk.Finally, a Kaplan-Meier survival curve that includes estimated 1-year survival probability from initiation of treatment at BCCOE is included in Cancer is one of the more challenging noncommunicable conditions to address in resource-limited settings because of its multidisciplinary nature, the high cost of care, and the need for skilled professionals. However, the BCCOE model serves as a step toward comprehensive cancer care provided by specialists. The protocol-driven treatment provided by nononcologists with guidance from specialists remotely has preliminarily led to successful treatment of GTN. Remote monitoring has included electronic consultation of individual patient cases and telemedicine oncology care conferences for complex cases. Despite many of the limitations in diagnosis and treatment of GTN, 100% survival for low-risk GTN and 63% for high-risk GTN, albeit with early follow-up data, in this study demonstrate the capacity of a rural low-resource oncology program to deliver much-needed cancer care.22 However, the Kaplan-Meier curves were highly influenced by the relatively short follow-up time. More-accurate comparisons may be made in the future with additional follow-up and an expanded patient population.Compared with those in other studies, survival outcomes for high-risk GTN at BCCOE fell below expected treatment outcomes in established GTN treatment centers.23 Led by the Rwandan Ministry of Health, care for all patients, including those with GTN, has been integrated into the existing health system, allowing for sustainable growth with early support from collaborators. Furthermore, given patient referral from across the country, communication among all national referral centers has been critical. Along with national and international collaborations, social service supports have led to higher patient retention rates than expected, given the socioeconomic landscape among our patients.Successful treatment of patients at BCCOE has in part been based on foundational collaborations and an emphasis on the rural poor population the program serves.25 As a result of limited resources, CT imaging was not mandated in the initial protocol. Consequently, only 30% of high-risk patients received chest and abdominal CTs, likely leading to understaging in many patients. Patients with MTX resistance received EMACO as salvage chemotherapy. Single-agent dactinomycin can be used as second-line chemotherapy, but because of concern surrounding understaging of patients and risk of loss to follow-up, escalation to EMACO in lieu of single-agent dactinomycin was incorporated into the GTN BCCOE treatment protocol.5 Even when health services were available, as among patients who were surgically treated for antecedent molar pregnancy, only 52% of patients had pathology reports available. Both incomplete information and incomplete documentation affect appropriate risk stratification, a challenge in GTN management that has been noted in cancer centers in low-resource settings.26 Additional study limitations include the short duration of follow-up and information only on patients with GTN at BCCOE, providing a partial representation of the country.Although preliminary results are promising, a great limitation to the study is incomplete information, commonly encountered in clinical care and program evaluation in similar settings.Moving forward, continuing to bolster health infrastructure will improve care of patients with GTN. At diagnosis, additional imaging for metastatic work-up will more accurately stage disease and guide appropriate therapy. Specific to GTN, available and timely β-hCG levels direct patients’ clinical course. Ideally, levels would be available at the start of each new chemotherapy cycle; however, only a few laboratories in the country evaluate β-hCG levels, many using differing measurement scales, leading to confusion in clinical care and evaluation. Increased or decentralized access to β-hCG testing along with standardized measurement could lead to earlier diagnoses and more-accurate treatment monitoring.Programs treating GTN should also ensure supply of necessary chemotherapeutic agents and consider supplemental medications that support treatment response. Relatively consistent access to critical chemotherapy agents was achieved, with an average of 4.7 cycles of MTX and 6.7 cycles of EMACO provided to patients to achieve complete response.9 Although still costly despite recently becoming available in generic formulary, granulocyte colony-stimulating factor could be considered for neutropenic patients in subsequent cycles.Nevertheless, two patients’ chemotherapy was delayed by stockouts of EMACO agents, one of whom then received an alternate regimen and later died. Although a majority of deaths were attributed to disease, EMACO is a myelosuppressive regimen that led to grade 3 or 4 neutropenia in 72% (13 of 18) of patients thus treated. Clinical guidelines include the use of granulocyte colony-stimulating factor to reduce risk of treatment delays resulting from neutropenia; however, because of cost, these agents were not available to our patients.27 Such treatment modifications in select patients could improve overall outcomes.The current protocol is tailored to resource-limited settings; however, nuanced modifications could enhance care of high-risk patients with GTN. Using the current protocol, all deaths in the GTN cohort occurred early in the course of treatment, reflecting high disease burden. Other studies have modified chemotherapy regimens for patients with high disease burden to reduce early death (< 4 weeks after initiation of treatment).Longer-term studies can present a more accurate portrait of patient outcomes and barriers to care in GTN in rural resource-limited settings. Both at BCCOE and at decentralized health facilities, understanding of barriers to care and disease awareness among care providers will help identify areas to enhance care delivery. Need for cancer care is anticipated to grow in Rwanda, and data that allow for scaling up of cancer care that is both effective and cost efficient are lacking. In addition, system-based barriers, including lack of pathology analysis at the local health facility, must be addressed with innovative solutions. Finally, although social support has been beneficial to patients both anecdotally and in other studies, more careful analyses of benefits and gaps in services may further increase patient retention. These lessons will help optimize rural-based cancer models for GTN.In conclusion, our study supports GTN as a priority disease, given that patients tend to be young, the disease is highly curable, and few medications with manageable toxicities are required. The BCCOE model involving protocol-based cancer care by mentored nononcologists has successfully increased access to GTN treatment in rural settings. Preliminary outcomes support the importance of integrated care by oncologists and local nononcologists, prioritizing diagnostic and management tools, protocol modifications, integrated monitoring and evaluation, and social support in resource-limited settings. Optimal models for resource allocation in cancer care can best be developed in the context of resource-limited health systems similar to BCCOE yet applicable in all settings.


The IBC phenotype was an independent prognostic feature. Similarly, IBC patients displayed shorter 5-year loco-regional relapse-free survival than LABC patients (86% versus 95%). Conclusions: Similar pCR rates to chemotherapy were found in IBC and LABC, suggesting that IBC is not less chemosensitive than LABC. Survival was shorter in IBC, suggesting that the corresponding poorer prognosis is more due to a higher metastatic risk and/or other feature(s) than to a lesser chemosensitivity.Inflammatory breast cancer (IBC) is a very aggressive form of breast cancer, as compared to locally advanced breast cancer (LABC). Neoadjuvant chemotherapy followed by surgery is the standard treatment in both cases. Whether IBC is less chemosensitive than LABC remains unclear. We retrospectively compared the rate of pathological complete response (pCR) to neoadjuvant chemotherapy in IBC and LABC. Methods: Patients with IBC or LABC treated with neoadjuvant anthracycline-based chemotherapy followed by surgery were selected from our institutional database. The primary endpoint was the pCR rate, defined as absence of invasive tumor in breast and axillary lymph nodes. Results: A total of 450 patients were included, 144 with IBC and 306 with LABC. The pCR rate was similar between the two groups, in the whole population (31%) and in each molecular subtype separately. Univariate analyses for pCR in IBC and LABC separately identified the same predictive variables, except the pathological type that was associated with pCR in LABC only, but not in IBC. IBC patients displayed shorter 5-year metastasis-free survival and overall survival than LABC patients in the whole population (57% and 69%  Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. IBC is classified as cT4d according to the AJCC TNM staging system . Before The model of neoadjuvant chemotherapy provides opportunity for assessing the tumor chemosensitivity in non-metastatic patients , 8. Pativersus non-IBC with respect to the response to chemotherapy. Here, we retrospectively compared the pCR rate to neoadjuvant chemotherapy in a large series of patients with IBC and LABC treated in our institution.Because IBC is clinically and biologically different from operable or locally advanced non-IBC, patients with IBC have often been excluded from neoadjuvant clinical trials dedicated to non-IBC. Furthermore, few prospective trials have been dedicated to IBC because of the low incidence of disease. Thus, prospective data on IBC are rare in the literature, as well as the direct comparison between IBC vs 82%), and had more frequently dermal emboli (46% vs 6%), and grade 3 tumors (62% vs 50%). No significant difference was observed regarding age, menopausal status, pathological type, and molecular subtypes. The molecular subtype of tumors was defined as follows: HR+ for ER+ and/or PR+ and HER2- tumors, HER2+ for HER2+ tumors, and triple negative (TN) for ER-, PR- and HER2- tumors.A total of 450 non-metastatic breast cancer patients treated in our institution between 1992 and 2015 with anthracycline-based neoadjuvant chemotherapy were included, comprising 144 patients with IBC and 306 with LABC (Table vs 89%), and less neoadjuvant/adjuvant trastuzumab (17% vs 30%). All patients had definitive surgery, with more frequent mastectomy than lumpectomy in patients with IBC, compared with LABC patients (97% vs 66%). The proportion of patients treated with adjuvant radiotherapy was similar between the two groups (97%), as was the proportion of patients treated with adjuvant hormone therapy (55% in IBC vs 63% in LABC).Regarding the treatment, and compared with patients with LABC, patients with IBC had received less anthracycline and taxane-based neoadjuvant chemotherapy (58% vs lobular or other), the HER2+ and TN molecular subtypes (vs HR+ subtype), the delivery of neoadjuvant trastuzumab (vs no delivery), and the delivery of anthracycline and taxane-based regimen of neoadjuvant chemotherapy (vs anthracycline without taxane) were associated with higher pCR rate ). The pCR rate was similar between patients with IBC ) and those with LABC ; p=1, Fisher’s exact test; Table 7-36). Thper molecular subtype. In the HR+ subtype, the pCR rate was 17% in patients with IBC versus 14% in patients with LABC ; p=0.585, Wald’s test). In the HER2+ subtype, the respective pCR rates were 43% versus 49% ; p=0.538, Wald’s test). In the TN subtype, they were 40% versus 43% respectively ; p=0.784, Wald’s test). Thus, like in the whole population, the pCR rate was not different between IBC and LABC in each molecular subtype.Similar comparative analyses were repeated In order to further compare IBC and LABC in term of pCR, we did univariate analyses for pCR in each of them separately. The results were similar for all tested variables, except the pathological type . The ageversus 76 patients with LABC . The 5-year metastasis-free survival (MFS), calculated from the date of diagnosis until the date of first distant metastasis, was 68% (95%CI (63–73)) in the whole population, and 57% (95%CI (49–67)) in patients with IBC versus 74% (95%CI (68-79)) in patients with LABC , 140 out of 450 patients (31%) experienced metastatic relapse, including 64 patients with IBC (45%) e Figure . Of noteversus 95% (95%CI (92-97)) in patients with LABC . Thirty-three patients experienced loco-regional relapse (7%), including 18 with IBC (13%) and 15 with LABC . The 5-year LRRFS, calculated from the date of diagnosis until the date of first local and/or regional relapse, was 92% (95%CI (89-95)) in the whole population, and 86% (95%CI (80-93)) in patients with IBC s Figure .versus 88% (95%CI (83-92)) in patients with LABC , including 49 with IBC (34%) and 32 with LABC . The 5-year overall survival (OS), from the date of diagnosis until the date of death whatever its cause, was 82% (95%CI (78-86)) in the whole population, and 69% (95%CI (62-78)) in patients with IBC e Figure . Here toIBC is the most aggressive form of breast cancer. Compared to LABC, survival is shorter, whereas the treatment is similarly based on neoadjuvant anthracycline-based chemotherapy followed by surgery. Better understanding the causes of its poor prognosis is crucial. Today, it remains unclear whether IBC is less chemosensitive than LABC. In this retrospective study of 450 patients treated in our institution, including 144 with IBC and 306 with LABC, we showed that the pCR rate after anthracycline-based neoadjuvant chemotherapy was similar between IBC and LABC, although the MFS, LRRFS, and OS were different. To our knowledge, this is the largest unicentric series comparing both the pCR rate to neoadjuvant chemotherapy and the survival rates between patients with IBC and with LABC.per molecular subtype.The retrospective nature of our study was justified by the scarcity of prospective data in this field, due to at least two reasons: the scarcity of IBC and dedicated clinical trials, and its differences with non-IBC leading in most of the cases to its exclusion from breast cancer clinical trials. IBC and LABC patients were defined according to clinical criteria in the AJCC/TNM staging. For IBC, the definition is consensual . For LABOur two groups (IBC and LABC) were balanced in terms of patients’ age, menopausal status, pathological type, and molecular subtypes. As expected, there were more tumors with emboli in dermal lymphatic vessels in the IBC group, since emboli represent the pathological hallmark of disease . The numversus weekly doxorubicin and daily oral cyclophosphamide administered with granulocyte colony-stimulating factor support for 15 weeks (continuous arm). All patients had subsequent weekly paclitaxel for 12 weeks before surgery. The pCR rate (ypT0/Tis-ypN0) was not different between IBC (19.8%) and LABC , although the 5-year DFS and OS were longer in patients with LABC. In the SWOG S0800 trial [During the last ten years, several prospective, large-scale, randomized trials of neoadjuvant chemotherapy in LABC have been reported and included some patients with IBC. However, in most of them -33, the 00 trial , patient00 trial  that tesTP53 mutations. However, in our present series, the IBC and non-IBC groups were well balanced with respect to these features, and the pCR rates were identical in IBC and LABC not only in the whole cohort, but also in each molecular subtype. Similarly, the secondary analysis of the GeparTrio trial [Altogether, these results, including ours, suggest that the poorer survival of IBC patients, compared to LABC patients, is more due to the higher metastatic risk of disease and/or other feature(s) than to a lesser chemosensitivity. This peculiar metastatic potential of IBC was already demonstrated several years ago by the very low 5-year survival rates (<5%) observed when the patients were treated with surgery and/or radiotherapy only . It is aio trial  showed tio trial , in whicio trial , more pCio trial , which sIn conclusion, our data show that anthracycline-based neoadjuvant chemotherapy provides similar patterns of pathological response in IBC and in LABC, suggesting that IBC is not less sensitive to chemotherapy than LABC. However, the long-term survival is shorter in patients with IBC. The strength of our study lies in its originality and the size of our IBC series. To our knowledge, it is the largest unicentric in term of IBC (more than 140 cases) that compares both the pCR and survival rates in patients with IBC and with LABC. Its main limitation lies in its retrospective nature and associated biases. However, our population was more likely representative of a “real-life” cohort, with some patients not receiving the complete treatment or ideal care because of their own choice, advanced age, or comorbidities. Furthermore, no direct comparison planned in a prospective trial combining pCR and survival data and concerning more than one hundred IBC patients has been published to date and it is very likely that there will never be such randomized prospective trial in the future. In this context, a meta-analysis of prospective clinical trials of neoadjuvant chemotherapy including both IBC and non-IBC is warranted for helping to answer this issue.in situ hybridization), the existence of neoplastic emboli in dermal lymphatic vessels, the treatment received , and the clinical outcome in terms of pCR and survival. Identification of molecular subtypes is most precise using gene expression profiling [We retrospectively selected the patients with a diagnosis of breast cancer included in our institutional database. The study was approved by our Institutional Review Board. The main inclusion criteria included the pathological diagnosis of invasive primary carcinoma of the breast based on tumor biopsy, non-metastatic at time of diagnosis  as metastasis(ses)), treated with neoadjuvant anthracycline-based chemotherapy followed by surgery (lumpectomy or mastectomy and axillary lymph node dissection), and documentation of pathological response. The variables collected and analyzed included the patients’ age at diagnosis, the menopausal status, the cT and cN classes, the pathological type and grade, the ER and PR immunohistochemistry (IHC) status (with a 10% of stained tumor cells as threshold of positivity), the HER2 status (determined according to French guidelines by IHC +/- fluorescent rofiling . But sucrofiling . When neth to 7th editions) concerned by the two-decade period covered by our series. These four editions have changed over this period regarding the N2 and N3 classes and the M1 class. Thus, to avoid any bias related to the use of different staging editions, we excluded from analyses the patients with ipsilateral supraclavicular lymph node(s) at diagnosis, and we pooled the N2 and N3 tumors in the cN1-3 class in statistical analyses. IBC were defined by inflammatory clinical signs involving more than one-third of the breast (cT4d classification). LABC were defined as non-IBC with a tumor diameter superior to 5 cm (cT3), or a tumor involving the skin or muscle (cT4a-c), or a tumor diameter between 2 and 5 cm (cT2) associated with metastases to ipsilateral regional lymph nodes .The patients were divided in two groups (IBC and LABC) based on the clinical criteria of the four successive AJCC/TNM cancer staging editions  after neoadjuvant chemotherapy. Post-chemotherapy mastectomy or lumpectomy and axillary lymph node dissections were examined by pathologists to describe the pathological response. pCR was defined as absence of invasive tumor in breast and axillary lymph nodes (ypT0/Tis-ypN0). The secondary endpoints included the metastasis-free survival (MFS), calculated from the date of diagnosis until the date of first distant metastasis, the loco-regional relapse-free survival (LRRFS), calculated from the date of diagnosis until the date of first local and/or regional relapse, and overall survival (OS), from the date of diagnosis until the date of death whatever its cause. The follow-up was measured from the date of diagnosis to the date of last news by using the reverse Kaplan-Meier method. Data concerning patients without death or metastatic relapse at last follow-up were censored.Patients’ characteristics were compared with the Fisher’s exact test and the Mann-Whitney test. Survivals were estimated using the Kaplan-Meier method and curves were compared with the log-rank test. Univariate and multivariate pCR analyses were done using a logistic regression analysis. Univariate and multivariate survival analyses were done using Cox regression analysis. Variables tested in univariate analyses included age at diagnosis, clinical axillary lymph node status, pathological type and grade, ER, PR and HER2 status, molecular subtype, neoadjuvant chemotherapy regimen, neoadjuvant/adjuvant trastuzumab. Additional variables for univariate survival analyses included adjuvant radiotherapy and hormone therapy, and pathological response. Multivariate analyses were conducted by including variables significant in univariate analysis and the IBC/LABC type and were limited to patients for which all variables tested were specified. All statistical tests were two-sided at the 5% level of significance. Analyses were done in the R software and associated packages (version 2.15.2).


C. elegans, miRNAs are genetic biomarkers of aging. Similarly, multiple miRNAs are differentially expressed between younger and older persons, suggesting that miRNA-regulated biological mechanisms affecting aging are evolutionarily conserved. Previous human studies have not considered participants' lifespans, a key factor in identifying biomarkers of aging. Using PCR arrays, we measured miRNA levels from serum samples obtained longitudinally at ages 50, 55, and 60 from 16 non-Hispanic males who had documented lifespans from 58 to 92. Numerous miRNAs showed significant changes in expression levels. At age 50, 24 miRNAs were significantly upregulated, and 73 were significantly downregulated in the long-lived subgroup (76-92 years) as compared with the short-lived subgroup (58-75 years). In long-lived participants, the most upregulated was miR-373-5p, while the most downregulated was miR-15b-5p. Longitudinally, significant Pearson correlations were observed between lifespan and expression of nine miRNAs . Six of these nine miRNAs  were also significantly up- or downregulated when comparing long-lived and short-lived participants. Twenty-four validated targets of these miRNAs encoded aging-associated proteins, including PARP1, IGF1R, and IGF2R. We propose that the expression profiles of the six miRNAs  may be useful biomarkers of aging.In  Biomarkers of aging are biological parameters that change in a predictable direction with aging in most individuals and, when assessed early in life, may predict subsequent longevity better than chronological age alone. Beyond their prognostic utility, the discovery of biomarkers of aging is attractive because they may shed light into the intrinsic mechanism of aging as a biological process . IdentifmiRNAs have emerged as important regulators of biological mechanisms that are relevant for aging. miRNAs are short non-coding RNAs that regulate gene expression generally by triggering mRNA decay and/or translational repression . With ovProfiles of miRNAs found in plasma and serum have been linked to numerous cancers –8, cogniThese studies vary in the types of samples used, groups of participants, methods of profiling miRNA expression, and number of miRNAs profiled. Perhaps because of these differences, there is no overlap in the identified miRNAs that are up- or downregulated in the older vs. younger participants. More importantly, because these studies were based on case-control or cross-sectional designs, the lifespans of participants were not known and longitudinal blood samples were not analyzed.To address these limitations, we used miRNA PCR arrays to measure miRNA levels in serum samples obtained longitudinally at ages 50, 55, and 60 from 16 participants of the Baltimore Longitudinal Study of Aging (BLSA) who had documented lifespans. We compared miRNA expression changes not only across  but also within participants . In accordance with recent research that found a strong association between circulating miRNAs and human aging , our stuAs shown in Table We compared the age 50 samples between the long-lived and short-lived subgroups by using methodology described in We focused our analysis on comparing the longer-lived to the shorter-lived subgroup , as this2 values rounded up to two decimal places). miR-211-5p, miR-5095, and miR-1225-3p all had positive correlations and were similarly upregulated in the longer-lived subgroup but downregulated in the shorter lived subgroup. miR-374a-5p, miR-340-3p, and miR-376c-3p all had negative correlations and were similarly downregulated in the longer-lived subgroup but upregulated in the shorter lived subgroup. To illustrate these findings, However, only six miRNAs  Polymerase 1], was found in the target lists of both miR-374a and miR-1225.After confirming that the six miRNAs of interest were found on the miRandola circulating miRNA database , we explWe conducted a pilot study of miRNAs as biomarkers of aging by analyzing miRNA expression in serum samples from a longitudinal human aging study. We found that the expression of six circulating miRNAs in mid-adulthood significantly correlates with subsequent longevity, suggesting that these miRNAs may be useful biomarkers of human aging. As far as we know, this is the first study that directly correlates miRNA with human longevity using data from a longitudinal study.Many interesting expression profiles were observed between study participants with different lifespans. For example, when comparing samples analyzed at age 50 between the long-lived and short-lived subgroups, we identified the 10 most differentially higher and lower expressed miRNAs  may serve as useful biomarkers, as each of the six miRNAs were correlated with lifespan and were significantly up- or down-regulated. Future studies can identify how examining expression of multiple miRNAs simultaneously versus one or a few miRNAs individually would affect these correlations. While some miRNA biomarker or disease-association studies have found significant correlations only by analyzing a profile of expression of multiple miRNAs , our stuMost of the 16 participants died from either heart disease or cancer Table , and 15 PARP1 mRNA, found in target lists of both miR-374a and miR-1225, encodes PARP1, a protein known for its role in repairing single-strand breaks during DNA replication [IGF1R mRNA (a miR-376c target) and IGF2R mRNA (a miR-211 target) encode receptors of the insulin signaling pathway , which has been tightly linked to variation in human longevity[We identified 24 aging-associated mRNAs that are also validated targets of the five miRNAs . Notably, lication , 30. PARlication . Additiolication . PARP aclication , suggestlication . Additiolongevity, 36. By Calculating Pearson correlations and separating participants into longer-lived vs. shorter-lived subgroups were only possible because we had obtained serum samples from a longitudinal study. This is a unique strength of the current study relative to prior miRNA profiling studies that have used blood samples from a single point in time among aged participants Table . HoweverOur study has implemented a novel approach to identify human aging biomarkers in an attempt to translate basic scientific discoveries in model organisms to human aging. Our results suggest that the expression profiles of six miRNAs may be useful biomarkers of aging. Although preliminary, these results provide a basis for investigating miRNAs as potential predictors of future longevity, and they highlight the potential roles of select miRNAs in regulating aging processes, thus warranting further validation and mechanistic explorations.The Baltimore Longitudinal Study of Aging (BLSA) is a longitudinal study of human aging that began in 1958 with more than 1400 volunteers, ranging in age from 20s to 90s . A detaiLongitudinal serum samples for this pilot study were obtained for 16 BLSA participants. The inclusion criteria were: male, availability of blood samples at around age 50 with at least three samples between ages 50 and 60, at least 800 μl of available serum, all fasting and non-hemolyzed samples (preventing introduction of miRNAs from red blood cells ), havingC. elegans miR-39 spike-in) for each of the 48 serum samples. A Roche LightCycler 480 machine was used to perform qPCR. Using the miScript miRNA PCR Array Handbook, the handbook protocol determined the same baseline and threshold across all amplification plots, in which the earliest amplification was manually set to cycle 15 and the threshold was set to 0.1. To confirm qRT-PCR results from the first RNA isolation, RNA was isolated a second time from serum in separate tubes from the same participants. In this second experiment, miScript Primer Assays were ordered to separately confirm expression of six identified miRNAs of interest , along with spike-in control (miR-39) and four stable miRNAs  by following standard protocols.The Serum/Plasma miRNA isolation kit, miScript II RT Kit (using HiSpec buffer), and miScript SYBR Green PCR Kit (Qiagen) were used to prepare samples for profiling following standard protocols. The miScript Human Serum & Plasma 384HC miRNA PCR Arrays (Qiagen) were used to profile 372 miRNAs and 12 controls . The four miRNAs  with stable Ct values across all 48 serum samples were chosen from a list of stable miRNAs used for normalization by previous miRNA profiling studies using blood samples from aged participants , 42. TheThe method for calculating fold regulation for a given miRNA (repeated for each miRNA) among different age groups is described in detail in By using both miRTarBase and miRWalk, we identified validated targets of the candidate biomarker miRNAs. We then checked for overlap with a collection of 300 genes (300 mRNAs) listed in GenAge: The Ageing Gene Database from Human Ageing Genomic Resources , 43 to d


Lupus anticoagulant (LAC) testing is challenging. Most data are derived from a well-controlled study environment with potential alterations to daily routines. The aim of this retrospective cohort study was to assess the capacity of various LAC screening tests and derived mixing tests to predict a positive result in subsequent confirmation tests in a large cohort of patients.screen, aPTT-FS and dRVVTscreen and of their derived mixing tests in detecting a positive confirmation test result within the same blood specimen. The group differences, degree of correlation and the predictive accuracy of LAC coagulation tests were analysed using the Mann-Whitney U test, the Spearman-rank-correlation and by area under the receiver operating characteristic curve (ROC-AUC) analysis. ROC-AUCs were compared with the Venkatraman´s permutation test.In 5832 individuals, we retrospectively evaluated the accuracy of the aPTT-A, aPTT-LAscreen showed the best diagnostic accuracy with a ROC-AUC of 0.84 (95% CI: 0.82 – 0.86). No clear advantage of the dRVVT-derived mixing test was detectable when compared to the dRVVTscreen (P = 0.829). Usage of the index of circulating anticoagulant (ICA) did not improve the diagnostic power of respective mixing tests.The pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LAscreen and derived mixing test parameters were the most accurate tests. In our study cohort, neither other mixing test nor the ICA presented any further advantage in LAC diagnostics.Among the parameters evaluated, aPTT-LA The aPTT-LAscreen appears to display higher sensitivity and the dRVVT, described as being the most robust assay for LAC testing, might possess a higher specificity for detecting LAC (The detection of lupus anticoagulants (LAC) is based on interference testing of the coagulation cascade and therefore testing it proves challenging (There is no agreement between the guidelines regarding LAC testing for patients taking vitamin K antagonists (VKA) or heparin. Generally, factor deficiency in patients on VKA with an international normalized ratio (INR) above 1.5 can affect LAC testing and the results have to be taken with caution. While CLSI and ISTH suggest caution with respect to the results of LAC testing of patients under heparin therapy, the BCSH recommends no LAC testing of such samples at all.Due to high demands on laboratory facilities, the incurred costs and the wide availability of integrated confirmation testing, the need for mixing tests has to be carefully evaluated. However, there is hardly any data for a suspected LAC patient cohort with representative pre-test probability of having LAC. Hence we conducted a retrospective cohort study to evaluate the predictive capacity of various LAC screening tests and derived mixing tests in detecting a positive confirmation test result within the same blood sample in 5832 patients without any factor deficiency or anticoagulation therapy (dataset A), in patients with heparin therapy (dataset B) and in patients on VKA therapy (dataset C). Whether or not positive results of confirmation testing could be confirmed at a later time point  was not the goal of this assessment.This retrospective cohort study was performed between 2010 and 2014 at the Vienna General Hospital, Medical University of Vienna, Austria, a tertiary teaching hospital with 2145 beds. The study was approved by the local ethics committee (EC-Number: 1197/2013) and was conducted in accordance with the ethical principles of the Declaration of Helsinki.screen or dRVVTscreen, and one derived mixing test as well as the aPTT-LA- and dRVVT- confirmation tests (see Supplementary figure 1). No patients were included more than once, as only the first LAC request for a patient was included during the study period.Due to the retrospective design of the study, informed consent was not obtained from study participants. All consecutive patients older than 18 years of age with clinical suspicion of having LAC from whom the treating physician requested LAC testing were included. Before requesting coagulation testing, clinicians were obligated to document any anticoagulant therapy. Based on this information, INR measurements (for patients on VKA) or anti-Xa-measurements (for patients on heparin therapy) were performed. Patients without a complete LAC testing panel, patients with known coagulation interfering antibodies other than LAC or known haemophilia A, B or contact factor deficiency, patients under direct oral anticoagulants or heparin therapy with anti-Xa activities above 1 IU/mL were excluded. A complete LAC panel included the following assessment: prothrombin time (PT) according to Owren and Quick, thrombin clotting time (TCT), fibrinogen, LAC screening tests including aPTT-A (using STA–PTTA reagent), aPTT-FS (using Actin FS reagent), aPTT-LATo increase the within-group homogeneity and to address the discrepancy between available literature and recommendations with the clinical daily practice, three datasets  were estBetween January 2010 and December 2014, LAC testing was conducted in 9328 patients. In 597 patients, no full LAC testing panel was available. In further 178 patients, the anti-Xa activity for monitoring heparin therapy was above 1 IU/mL . For dat9/L). Except LAC confirmatory testing, all coagulation analyses were conducted by using freshly drawn samples within 4 hours after blood taking. Thawed plasma specimens, stored below - 70°C upon analysis, were used for LAC confirmatory testing. Reagents, analysers and established reference ranges are summarized in Supplementary table 1 and quality control materials as well as their coefficient of variation are presented in Supplementary table 2. All test procedures were conducted according to the manufacturer´s recommendations. Using a coagulometric method, PT Owren , PT Quick , TCT , fibrinogen , activated partial thromboplastin time , aPTT-LAscreen , factor sensitive activated partial thromboplastin time , dRVVT  and dRVVT confirmation test  were assessed on the STA-R Evolution . The aPTT-LA confirmation test  was applied on the MC10 PLUS .Blood samples were collected in siliconized tubes  with 0.023 M pre-filled sodium citrate . In accordance to the H-60-A CLSI guideline, all samples were first centrifuged at 2500xg for 15 minutes at 15 °C , transferred to a secondary polypropylene tube  and again centrifuged under the same conditions to obtain platelet-poor plasma  analyser, the coagulation factors VIII (FVIII), IX (FIX), XI (FXI) and XII (FXII) were determined by one-stage coagulometric methods in a multidilution procedure using Actin FS reagent  and appropriate factor deficient plasmas . For the LAC screening procedure the aPTT-A, aPTT-FS, aPTT-LAconfirm  assessment, patient plasma is mixed with reagent buffer 1 (tube 1) or with reagent buffer 2  and incubated for 9 minutes at 37 °C. Afterwards human plasma (reagent 3) was added to both tubes, and incubated for one minute and finally lyophilized PTT-LS reagent (reagent 4) was added and incubated for another five minutes. CTs were measured after adding CaCl2 . The dRVVTconfirm  assessment was again carried out in two separate reactions. Patient plasma was mixed either with DRVV-Screen reagent or with the DRVV-Confirm reagent containing excess phospholipid concentration . After adding the pre-incubated starting reagent, both CTs were measured and the ratio between CTscreen and CTconfirm was calculated.For the LAconfirm decreased CT (CTtube1 – CTtube2) for more than 3 s or the ratio of dRVVTscreen to the dRVVTconfirm was above 1.25 within the same sample. The LAC confirmatory cut-offs were established by in-house evaluations. According to our standard operation procedures, the dRVVTscreen was not assessed in patients with hereditary or acquired FX deficiency. All analyses were performed under standardized conditions at the Department of Laboratory Medicine, which maintains a certified (according to ISO 9001:2008) and accredited (according to ISO 15189:2008) quality management system.Both confirmation tests, which are widely used, were applied in all patients. Confirmation testing and occurrence of LAC was considered positive when either LAMetric data are given as median  and occurrence of normal distribution was analysed using the Shapiro-Wilk test. Group-differences of metric data were compared by applying Mann-Whitney U tests. The diagnostic performance of parameters was analysed using the area under the receiver operating characteristics curve (ROC-AUC). Since no perfect LAC screening test exists and the majority of relevant guidelines recommend the utilization of at least two screening tests, the correlation between LAC screening parameters was assessed by the Spearman correlation coefficients and graphically presented as a correlogram . Calculation of ICA significantly decreased the performance of the ∆Mix–PNP parameters in the aPTT-A, aPTT-LA, and dRVVT . The mixing test was performed only in the test system showing the largest absolute deviance from the upper reference range, which are presented as subgroups in In 1390 patients of dataset B (LAC positivity rate: 16.3%), 319 patients presented with heparin therapy (up to 1 IU/mL anti-Xa activity). In these patients, a tendency for lower ROC-AUCs to detect LAC was found in all aPTT-A- and aPTT-LA-derived parameters (see Supplementary table 3). Due to the considerably low sample size of patients with heparin therapy, statistical significance was achieved in none of the ROC-AUC differences with the exception of the aPTT-FS .range < 0.001 to 0.969).In patients with VKA , the LAC positivity rate ranged between 11.1% and 16.7% (see Supplementary table 3). While the accuracy of the aPTT-A mixing test was lower in patients with INR > 3.51, this effect was not seen in the aPTT-LA mixing test or their derived parameters. Patients with VKA therapy were statistically compared to those patients without evidence of factor deficiency and anticoagulation therapy  , the LAC positivity rate was 36%. Within dataset A patients, the highest ROC-AUCs for detecting LAC were found by using the aPTT-LAscreen was higher than those ROC-AUCs of dRVVT-derived mixing parameters. This effect was not observed in aPTT-LA derived parameters. Although these differences were not statistically significant, the use of the dRVVT mixing test should be carefully evaluated. A clear benefit of the dRVVT mixing test was not observed in this cohort. This finding adds to the controversial discussion regarding the clinical need for the mixing test, especially in the context of integrated test systems  whose difference to the reference value was the highest in the aPTT-FS among the LAC screening parameters. Typically this occurred in patients with factor deficiency. These patients were presented in the results section, since their deletion would have altered the pre-test probability of a positive confirmation test, and since it is important to make clear the inappropriateness of such an algorithm. Even with an optimal threshold of only 1.75 s, the aPTT-FS ∆Mix–PNP presented with only 66.7% sensitivity, which clearly restricts its usefulness for LAC testing. To prevent a potential impact of the inappropriate aPTT-Actin FS on the overall study results, the diagnostic accuracy analysis of screening tests were split and statistical comparison of aPTT-Actin FS to LAC-sensitive screening tests was avoided.Interestingly, the FVIII, FIX, FXI, FXII activities and fibrinogen concentrations had some predictive capacity for identifying patients with LAC. For all five parameters, patients with LAC had significantly higher levels than patients without evidence of LAC. This might be based on an acute phase reaction or an inflammatory response reaction, which might be accompanied with the expression of LAC or be based on an infectious genesis of the expression of LAC  was not evaluated, since NOACs were hardly used in Austria during the study period. According to the manufacturer´s instruction sheet  the test is insensitive to heparin up to 1 IU/mL due to a heparin inhibitor and no false positive result was found in patients under VKA therapy. In the case of dRVVT confirmation test, no precise information is given in the manufacturer´s instruction sheet on testing patients with heparin or with VKA (#140 0312A). According to our data, LAC testing in patients taking VKA was not statistically impaired in comparison to those patients without VKA (dataset A).The aPTT-LA-derived parameters presented with partly better ROC-AUCs in patients with VKA than in the control cohort. This finding might indicate that the presence of LAC is more easily unmasked in patients on VKA than in patients without factor deficiency. However, the pre-test probability for having LAC or the positivity rate of confirmation tests might be altered in these selected patients. Further prospective studies are needed to assess the possibility to evaluated LAC testing in patients with VKA. Alternatively, an additional dilution step of patient’s plasma and PNP in patients with VKA might be considered. However, our results are concordant with the literature, which indicates the possibility of LAC testing in patients with VKA  assessed in all dataset A patients.For this retrospective cohort study approach, several limitations have to be addressed. Although the overall sample size is considerably large, a bigger sample size would be needed for some subgroup analyses. LAC positivity was defined as a positive result of the aPTT-LA or/and dRVVT-based confirmation test in the same plasma sample, thus false positive or false negative confirmation test results could not be ruled out. Finally, in our laboratory the established reference range of the aPTT-LAIn conclusion, the pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LA-derived parameters showed a better diagnostic accuracy for identifying LAC than dRVVT-derived parameters. Usage of the mixing test had no further advantage in the LAC diagnostics and the ICA did not improve the performance of the mixing test. According to the results of this study, no impairment was found in patients with VKA, while patients with heparin presented with considerably lower diagnostic accuracy.Supplementary figure 1. The standard operation procedure for LAC testing in our laboratory.Supplementary table 1. Overview of methods used for assessment of LACSupplementary table 2. Descriptive information about quality control materials and coefficients of variationSupplementary table 3. Coagulation parameters in patients with and without detectable anti-Xa activitiesSupplementary table 4. Coagulation parameters in patients with INR assessment


P-values all <0.03). By univariate analysis, higher Ki-67 scores were associated with a significant reduction in cancer-specific survival (P≤0.05); however, this effect was substantially attenuated in the multivariate model. In conclusion, hot spot scoring of whole sections using Definiens is an optimal method to quantify Ki-67 in endometrial cancer window study specimens. Measured this way, it is a clinically relevant marker, though further work is required to determine whether reductions in Ki-67 in neoadjuvant intervention studies translate into improved patient outcome.Ki-67, a marker of cellular proliferation, is increasingly being used in pre-surgical window studies in endometrial cancer as a primary outcome measure. Unlike in breast cancer, however, there are no guidelines standardizing its measurement and its clinical relevance as a response biomarker is undetermined. It is, therefore, imperative that Ki-67 scoring protocols are optimized and its association with patient survival rigorously evaluated, in order to be able to clinically interpret the results of these studies. Using the International Ki-67 in Breast Cancer Working Group guidelines as a basis, whole slide, hot spot and invasive edge scoring protocols were evaluated using endometrial biopsies and hysterectomy specimens from 179 women. Whole sections and tissue microarrays, manual and semi-automated scoring using Definiens Developer software were additionally compared. Ki-67 scores were related to clinicopathological variables and cancer-specific survival in uni- and multivariate analysis. Against criteria of time efficiency, intra- and inter-observer variability and consistency, semi-automated hot spot scoring was the preferred method. Ki-67 scores positively correlated with grade, stage and depth of myometrial invasion ( Similarly, there is a lack of robust evidence to guide the clinical care of women who are unfit for surgery or who desire fertility-sparing treatment.Despite the rising incidence and corresponding increase in deaths from endometrial cancer, there is a noticeable lack of research into new prevention and treatment strategies.3 Their use allows the rapid screening of new interventions so that time, effort, and financial resources can be directed at treatments that hold the most promise.Clinical trials require large numbers of participants over long follow-up periods to demonstrate the superiority of one treatment over another on clinically important outcomes, such as overall and cancer-free survival. In cancer types that are amenable to diagnostic sampling, novel interventions can be screened for efficiently using the pre-surgical window study design, whereby tissue endpoints are compared before (at diagnosis) and after treatment (at definitive surgery) using biomarkers as surrogates for clinical endpoints. Ideally such biomarkers should have prognostic utility and be able to predict response to adjuvant treatment and longer term outcome.6 Expressed only during the active G1, S, and G2 phases of the cell cycle, its expression is a marker of cellular proliferation and is readily detected by immunohistochemistry.7 The International Ki-67 in Breast Cancer Working Group set standards for the staining, scoring and analysis of Ki-67 in breast cancer to ensure the reproducibility, reliability and accuracy of studies using Ki-67 as their primary outcome measure.8 In brief, these included:Sole use of the MIB-1 antibody with heat-induced epitope retrievalInclusion of positive and negative controls in all batchesScoring at least three high-power fields (× 40 magnification) across whole sections, incorporating the invasive edge of the tumor and hot spotsAssessment of nuclear staining only (intensity of staining not relevant)Counting at least 500 (and preferably 1000) malignant cellsExpressing the Ki-67 score as the percentage of positively stained cells among the total number of malignant cells assessedWindow studies in endometrial cancer are hampered by the lack of validated biomarkers meeting these criteria. In breast cancer, the nuclear protein Ki-67 is an established prognostic and predictive biomarker.13 Although Ki-67 expression has been shown to positively correlate with tumor grade,16 there is a lack of consensus as to whether it has prognostic value.18 Heterogeneity of staining, scoring, and analysis protocols, including the use of study-specific cut-off values, have also hampered the validation of findings in other cohorts and, by extension, hindered the clinical interpretation of results from the aforementioned window studies. Furthermore, most previous studies were published over 10 years ago, using the now superseded FIGO 1988 staging criteria, limiting their applicability to modern clinical research.18Despite ambiguity in the literature about the value of Ki-67 as a biomarker in endometrial cancer, pre-surgical window studies using a change in Ki-67 as their primary endpoint have begun in earnest.8 to determine the correlation between Ki-67 and known pathological prognostic variables; and to investigate whether higher Ki-67 expression is associated with a shorter cancer-specific survival and, therefore, has clinical value as a biomarker in endometrial cancer trials.The aims of this study were three-fold: to identify the most reliable, reproducible and time efficient method of Ki-67 scoring using the recommendations of the International Ki-67 in Breast Cancer Working Group as a guide;19 Tumor tissues from 179 patients undergoing hysterectomy for endometrial cancer were retrospectively selected. This included 128 consecutive patients who had donated tissue for research to the Manchester BRC Biobank from 2009 to 2014. Due to a preponderance of low grade and stage disease in this cohort, an additional 51 high-risk patients, for whom tissue and clinical follow-up data were available, were included from partner institutions of the TransPORTEC consortium  to ensure a representative population. This latter group included patients who had undergone primary surgery between 1991 and 2010. All grades, stages, and histological subtypes of endometrial cancer were included. All patients underwent surgery. Patients with intermediate or high-risk disease were given adjuvant treatment according to local protocols.The study was designed, analyzed, and reported in accordance with the REMARK guidelines for tumor marker prognostic studies.μm thick sections were cut from representative paraffin blocks using a cryostat and mounted onto a histological glass slide. Slides were either stained immediately or stored at +4 °C pending immunohistochemistry. Whole hematoxylin and eosin-stained slides were reviewed by experienced gynaecological histopathologists  to confirm FIGO (2009) stage, histological subtype, grade, depth of myometrial invasion and the presence or absence of lymphovascular space invasion. Tissue microarrays were created by the study histopathologists from hysterectomy specimens for a subset of the Manchester patients and the transPORTEC cohort using triplicate tumor cores. This allowed the effect of slide preparation technique to be determined by comparing Ki-67 scores from whole sections and tissue microarrays obtained from the same tumor.Tumor from hysterectomy specimens and, for a subset of tumors, corresponding endometrial biopsies taken immediately before the start of surgery, were formalin fixed and paraffin embedded, and stored at room temperature for up to 24 years. Four-8 Antigen retrieval was undertaken at pH 9 for 20 min. A casein block of 30 min duration was carried out to reduce non-specific antibody binding. Slides were incubated at room temperature for 1 h with the MIB-1 antibody , at a dilution of 1:100. Primary antibody detection was undertaken using the Refine Detection Kit (Leica Biosystems), which contains a rabbit anti-mouse IgG secondary antibody and anti-rabbit poly-HRP IgG antibody and utilizes 3,3′-diaminobenzidine as a chromogen. Slides were counterstained with hematoxylin. Negative (isotype control) and positive (tonsil) controls were used for quality assurance.Immunohistochemistry was performed using the Leica Bond Max  with heat-induced epitope retrieval. This fully automated system is routinely used in many hospitals and ensures consistent staining across runs. Staining was performed using the optimized protocol recommended by the International Ki-67 in Breast Cancer Working Group.Slides were digitized using the Leica SCN400 Slide Scanner . A semi-automated score was obtained by applying a computerized algorithm (Definiens Developer) to the malignant glands . Manual Individual tumor cores and full sections were scored three times  and the final Ki-67 score was calculated as the mean value of the three repeats. For the tissue microarrays, the final Ki-67 score for each tumor was the average of nine measurements; three cores from each tumor scored on three separate occasions. Discordant results of >10% (between SK and VS) were settled by consensus. The time to score individual slides was measured using a stopwatch.Demographic, pathology, and follow-up data were obtained from electronic and hard copy patient records. In Manchester, patients were reviewed in specialist clinics every 4 months for the first two years and six monthly thereafter for a total of five years. The detection of recurrent disease was by way of symptom enquiry and clinical examination, with imaging as required. Cause of death was determined from primary care and mortuary records. For the transPORTEC patients, clinical follow-up data were provided by individual clinicians and stored in a secure database. All cases without events were censored at the last follow-up visit.18 Importantly, this cohort included 26 endometrial cancer-related deaths and 41 recurrences, ensuring that the study was adequately powered to investigate the effect of Ki-67 on endometrial cancer recurrence and survival.19Tumor availability and consent for follow-up data collection limited the sample size to 179 patients; similar numbers to previous studies of Ki-67 in endometrial cancer.U-test for non-parametric data and Spearman rank correlation for continuous and ordinal variables. Kaplan–Meier curves were constructed to estimate cancer-specific survival according to Ki-67 score and the log-rank test for trend used to compare curves. Cancer-specific survival was defined as the time between date of surgery and death from endometrial cancer. Recurrence-free survival was the interval between date of surgery and first documentation of recurrent disease. A Cox proportional hazard regression model was used in uni- and multivariate analyses of cancer-specific and recurrence-free survival, after confirming that the data complied with the proportional hazards assumption using log–log curves. These analyses examined Ki-67 as a continuous variable, using 10% increments to derive hazard ratios. The univariate analysis included previously documented important co-variates; age, body mass index (<30 kg/m2vs ≥30 kg/m2), grade , stage , histological type (endometrioid vs non-endometrioid), lymphovascular space invasion (presence vs absence), depth of myometrial invasion (<50% vs ≥50%), and adjuvant therapy use (yes vs no). The multivariate analysis utilized the significant prognostic variables identified in the univariate analysis. The model was developed using forward stepwise regression and confirmed using backward stepwise regression. Both methods produced identical results. A P-value of ≤0.05 was regarded as being of statistical significance. The statistical analysis was carried out using SPSS version 22 and GraphPad Instat.Ki-67 was measured as a continuous score using the hot spot method and data conformed to a negatively skewed distribution . Intra- vs 7.7 min).The semi-automated platform, combined with whole slide and hot spot scoring methods, demonstrated excellent intra- and inter-observer agreement, comparable to that seen with manual scoring . The intn=17) and 50 of the 51 TransPORTEC patients. In general, there was poor agreement between whole slide and tissue microarray scores for individual patients , respectively.The cohort included 116 endometrioid and 63 non-endometrioid type  cancers, of which 108 were FIGO stage 1 (60%), 22 were stage 2 (12%), 42 were stage 3 (24%), and 6 were stage 4 (3%). The estimated median follow-up time, using the reverse Kaplan–Meier method, was 39.5 months, during which time 41 (23%) patients had local  and/or distant recurrences . There were 47 deaths (26%), of which 26 (15%) were from endometrial cancer. For grade 1/2 endometrioid, grade 3 endometrioid and non-endometrioid type cancers, 5-year cancer-specific survival rates were 93%, 88%, and 43% (P≤0.001). In addition, it was also positively correlated with patient age, stage, depth of myometrial invasion, and adjuvant therapy use . Scores were higher in those tumors with lymphovascular space invasion present and non-endometrioid histology, though these results did not reach statistical significance.The median Ki-67 score in the overall cohort was 40%, with an interquartile range of 24–52%. The relationship between Ki-67 and patient clinicopathological characteristics was investigated . As expeP=0.05).Ki-67 scores were divided into two equal groups using the median score of 40% to denote low and high expression, to explore the relationship between Ki-67 and cancer-specific survival. The Kaplan–Meier curves suggested that greater tumor proliferation was associated with a significant reduction in survival; 5-year cancer-specific survival rates were 58% for those tumors with high Ki-67 expression, compared with 88% for those with tumors with low Ki-67 expression , P=0.05.In a univariate analysis, Ki-67 score, as a continuous variable, as well as age, grade, stage, and histological type of endometrial cancer, presence or absence of lymphovascular space invasion and depth of myometrial invasion, was a prognostic indicator of cancer-specific survival . A 10% iAnalyses were repeated using recurrence-free survival as the outcome of interest and produced similar results.This is the first study to compare semi-automated scoring using Definiens Developer software with manual Ki-67 scoring in endometrial cancer. Although unable to differentiate between malignant glands and stromal tissue, when the areas to be scored were manually selected by observers blinded to outcome, the accuracy and reproducibility of scoring by Definiens was extremely high. Automated scoring was superior to manual scoring in terms of speed and it was reliable across time and between scorers.Compared with whole slide and invasive edge scoring, hot spot scoring was the most reproducible scoring method for Ki-67, with excellent intra- and inter-observer agreement, and the most consistent across different endometrial tumor-sampling techniques. This is of particular importance for window studies using Ki-67 as a primary outcome measure, where an endometrial biopsy taken prior to intervention is frequently compared with the hysterectomy specimen at the end of treatment to determine response.8 A study in ovarian cancer similarly showed that Ki-67 staining of tissue microarray cores may not be representative of the results obtained from whole section immunohistochemistry.20 Appreciation of the heterogeneity of staining seen within whole sections of endometrial tumors is lost when only a small area is sampled in a core, reflected in the poor correlation of scores. This becomes even more evident if there is a time interval between slides being cut and stained; a delay in staining of more than 6 weeks resulted in lower Ki-67 scores. This has previously been described for sections stored under varying conditions; even at 4 °C the resulting hydrolysis negatively impacts on antigenicity.21 The authors, therefore, recommend undertaking staining on freshly cut sections to avoid this problem and limiting assessment to whole sections only. If freshly cut sections are not available, it is important that all slides are cut at the same time and later stained together for accurate comparison within a study, with the caveat that this limits comparability between studies.The scoring of whole slides was found to be superior to that of tissue microarrays in terms of both reproducibility and consistency. There are no published comparisons of Ki-67 assessment by tissue microarray and whole slide scoring in the breast cancer literature for guidance, but the International Ki-67 in Breast Cancer Working Group do note anecdotal evidence for lower scoring on tissue microarrays and advise avoiding their use when establishing quantitative relationships with clinical outcomes.et al,22 Stefansson et al,14 Geisler et al,18 and Liu et al,15 who described Ki-67 as a prognostic biomarker in endometrial cancer, although significance was generally lost after adjusting for important pathological variables like grade of disease and histological subtype. These studies were considerably larger than those of Fanning et al17 and Huvila et al,16 who published conflicting results; the latter studies had fewer disease events, shorter follow-up periods and were fundamentally underpowered to detect a significant effect of Ki-67 on cancer-specific outcomes.Using the optimized methodology of semi-automated hot spot scoring, Ki-67 score was strongly associated with known pathological prognostic variables, including grade, stage, and depth of myometrial invasion. Although not independent of other prognostic factors, high Ki-67 was associated with poor cancer outcomes. These data are consistent with those of Salvesen 19 Meticulous documentation of date of recurrence and cause of death allowed cancer-specific and recurrence-free survival to be calculated, arguably more clinically relevant endpoints than the overall survival used in other studies.18Detailed clinical follow-up and expert pathology review are strengths of this study. The included population was sufficiently large to ensure that the study was adequately powered; 10–25 events are required per prognostic variable under investigation.vs 33–40%),18 who used median Ki-67 to dichotomize tumors into low and high Ki-67 expression. This approach is crude and prevents extrapolation across study populations. Ours is the first study to consider Ki-67 as a continuous variable, equating a 10% increase in Ki-67 expression with a cancer-specific survival hazard ratio of 1.31. This information is important for clinical trials using Ki-67 as a primary endpoint as it provides some degree of clinical context in which to interpret the results. The magnitude of effect seen in this study is similar to that shown in breast cancer studies, where Ki-67 expression is routinely log transformed to normalize the data. In breast cancer, the hazard ratio per 2.7-fold increase in Ki-67 expression was 1.95 for recurrence-free survival.23 Applying the same methodology to our findings for ease of comparison, the hazard ratio for recurrence-free survival in endometrial cancer was 1.94 (95% CI 1.10–3.43).The median Ki-67 score was similar to that of other studies  will be closely associated with disease metastasis and recurrence.These findings are unsurprising, given that cancer is a disorder of unregulated cell proliferation.6 Our data suggest that Ki-67 could be used to stratify patients for entry into endometrial cancer adjuvant drug trials, excluding those whose prognosis is so good that they are unlikely to derive benefit from further therapeutic intervention beyond surgery. This is tentative speculation that requires formal testing. Ideally, this should include testing the same novel therapy before and after surgery and assess changes in pre-surgical Ki-67 score alongside longer-term cancer-specific and recurrence-free survival as outcome measures. Response to treatment could then be stratified according to baseline Ki-67 score. Such data are clearly required for drugs like metformin, which is increasingly being investigated in endometrial cancer window studies, if there is to be sufficient evidence of clinical efficacy for them to be used in routine practice.A limitation of this study was that too few tumors were available to adequately power the assessment of Ki-67 in a multivariate analysis, controlling for all known prognostic clinicopathological variables. The aim of this study, however, was not to ascertain whether Ki-67 could replace pathological prognostic variables, but rather to determine its value as a primary tissue endpoint for use in clinical trials, where the window is of treatment is too short to observe changes in grade and stage of disease. In breast cancer, a drop in Ki-67 following short-term treatment with neoadjuvant chemotherapy predicts long-term response to that drug in the adjuvant setting.In conclusion, these data provide evidence that semi-automated scoring of Ki-67 using Definiens Developer software is reliable, reproducible, and more time efficient than manual scoring and that hot spot methodology should be employed in future clinical trials as it is the most consistent across endometrial biopsies and hysterectomy specimens. When measured using standardized protocols of immunohistochemical staining, Ki-67 is associated with endometrial cancer survival and is, therefore, a clinically relevant endpoint, though further work is required to determine whether it fulfills all of the criteria to be used as a biomarker of treatment response.


Mycobacterium avium ss paratuberculosis (MAP) treatment in CD is gaining attention.Fistulizing Crohn's disease (CD) presents a therapeutic challenge as fistulae are notoriously difficult to heal. We evaluated healing of CD fistula(e) using a novel combination therapy.Nine consecutive patients who failed to heal fistulae on conventional treatment including anti-TNF, were treated with at least three doses of infliximab, 18–30 courses of hyperbaric oxygen therapy and anti-MAP antibiotics comprising rifabutin, clarithromycin and clofazimine.All patients achieved complete healing of fistulae by 6–28 weeks and follow-up for mean 18 months.Combining infliximab, hyperbaric oxygen therapy and anti-MAP, seems to enable healing of recalcitrant fistulae and although a small case series, all nine patients achieved complete healing. Lay abstract: Nine consecutive patients with Crohn's disease and fistulae were treated with a special combination of infliximab, numerous hyperbaric oxygen sessions and combined antibiotics including rifabutin, clofazimine and clarithromycin as the base antibiotic combination – called anti-Mycobacterium avium ss paratuberculosis (MAP) therapy. At between 6 weeks and 6 months all fistulae healed – included rectovaginal – so that the skin was dry and there was no discharge and no need to wear a pad. Their Crohn's symptoms of diarrhea, urgency and bleeding also resolved. Continuation with anti-MAP therapy alone maintained healing although one patient who ceased the anti-MAP therapy had a relapse. Due to the transmural nature of Crohn's disease (CD), approximately 20–40% of patients develop perianal fistulae ,2, whichMycobacterium avium ss paratuberculosis (MAP) has been debated vigorously and for a long time. It originated from early observed similarities between CD and Johne's Disease, the enteric MAP infection in cattle. In select institutions worldwide, antibiotic combination (anti-MAP) therapy has been offered as a treatment option for CD, often with striking results [et al. of anti-MAP [A possible causative link between Crohn's disease and  results . Prelimi results . Althouganti-MAP  showed tanti-MAP . IndicatHyperbaric oxygen therapy (HBOT), the inhalation of 100% oxygen at a pressure of >1 ATM, is an established treatment for accelerating healing of chronic nonhealing wounds, for example, diabetic foot ulcers . It presGiven the partial success individually of infliximab, HBOT and anti-MAP the potential of combining all three was explored in CD patients with treatment-refractory fistulae. Some of these patients had reached the stage of being offered colectomy.Infliximab 5 mg/kg ;Continuous anti-MAP therapy (see HBOT concurrently 18–30 daily sessions at 2.0–2.4 ATM (2–2.4 bar) for 90 min (mean: 21.8 sessions) in a monoplace or multiplace chamber.Nine consecutive patients with severely active CD and intractable fistulae were prospectively followed. All were nonresponsive to standard CD treatments, including corticosteroids, 5 amino salicylates (5ASA), thiopurines, methotrexate and biologics. All had documented CD and fistulae and after obtaining signed consent all were treated with a combination of:Response was assessed by a change in clinical symptoms – particularly complete fistula healing with dry, nonoozing skin, colonoscopic findings of healed mucosa and pre- and post-treatment MRI improvement. Given an accuracy of 90%, MRI is considered the preferred imaging modality for detecting and classifying fistulizing perianal disease, and was used in each case to confirm fistulae healing .A 24-year-old male with long-standing CD presented with multiple rectum-to-rectum fistulae and inflammatory changes .MRI confirmed the presence of multiple (>3) perirectal abscesses, the largest of these being 1 cm in diameter. The patient had failed previous numerous treatments. He underwent three infusions of infliximab and 30 sessions of HBOT, while being treated with combination anti-MAP . At 3 moA 32-year-old male presented with a 3-year history of CD and a high left-sided posterior, and right anterior fistulae, which required ‘setons’. Symptoms included diarrhea, urgency and perianal discharge and tenderness. On colonoscopy, inflammation extended throughout the entire colon, with Crohn's ulcerations destroying areas of haustrations. The patient underwent three infliximab infusions, 21 HBOT sessions and anti-MAP therapy . At 6 weA 32-year-old female presented with a 22-year history of CD, complicated by a rectovaginal fistula . The patA 31-year-old male with a 10-year history of CD presented with lower abdominal and rectal pain, bloating, flatulence, fecal urgency and soiling. On colonoscopy, the patient had a posterior anal fissure and an intersphincteric fistula . He had A 40-year-old female presented with CD symptoms of rectal bleeding, urgency, fatigue, anemia and three perianal fistulae. Her fistulae were previously treated with long-term ‘setons’ accompanied by various treatments including infliximab without complete healing. The patient was initiated on anti-MAP therapy before the addition of 21 HBOT sessions and three doses of infliximab. She experienced dramatic improvement permitting removal of ‘setons’ and complete healing of all fistulae. Progressive CD remission occurred within 4–6 weeks. A marked reduction in stool frequency was also observed to one formed stool per day from her baseline of >10 stools per day. There was no associated urgency, pain or tenesmus. She has remained asymptomatic without recurrence of fistulae over the last 7 years on a maintenance dose of anti-MAP therapy alone.A 38-year-old female presented with markedly active CD (CDAI of 416) and an unresolving deep rectolabial fistula that had been present for over a year, despite mesalazine, prednisolone and azathioprine therapy, later infliximab. Colonoscopy revealed extensive ulceration, contact bleeding, pus and inflammation in the left colon. Combination therapy included 21 sessions of HBOT, which led to complete fistula healing, which was confirmed via MRI. The patient also experienced improvement in CD symptoms, reporting two to three soft, formed stools daily, with an absence of blood, fecal urgency and tenesmus. Her CDAI score reduced to 15.in situ due to numerous intractable fistulae and abscesses requiring extensive (nine) surgical intervention intravenous antibiotics, anti-inflammatory drugs and infliximab , combination therapy with infliximab, HBOT and anti-MAP therapy achieved 100% fistulae healing rates in this small cohort. This healing persisted for an average of 18 months with continuation of anti-MAP but on stopping anti-MAP fistulae returned in one patient. This appears to be higher than comparable data for each of the three therapies individually, and indeed any available treatments . Fistuliet al. have shown fistula healing in three out of five patients treated with anti-MAP therapy [In our small study, using the combination of infliximab, HBOT and anti-MAP therapy achieved high healing rates of perianal fistulae, including rectovaginal fistulae. Patients had severe CD and were refractory to a number of therapies, including infliximab and anti-MAP therapy alone. Not only did they experience resolution of fistulae, but all nine patients experienced an improvement in CD symptoms. These patients had failed infliximab with or without thiopurines in the past. Given infliximab also has anti-MAP activity  we decid therapy . Adverseet al. reported a patient with an 8-year refractory CD fistula that healed with one session of HBOT [Due to their location, perianal fistulae are frequently prone to coexisting infection as a result of stool bacterial contamination. Fistulae are proposed to persist due to ongoing ischemia and persistent colonization with anaerobic microorganisms . HBOT is of HBOT . Minimal of HBOT .It is therefore likely that a combination of these diverse mechanisms enabled the profound healing of fistulae seen in these patients, and allowed the subsequent effective maintenance of CD with anti-MAP therapy alone. The effect of the combination therapy generally became evident soon after commencement of the therapy, and such early response could be used to indicate surgery can be avoided. One of the major limitations of this study lies in the use of all three treatments  concurrently. Hence, it is difficult or even impossible to assess which of these treatments is or are particularly responsible for results of such quality. It is possible that only two of the three treatments used together would provide similar results. Also, this is not a prospective study but all-comer consecutive patients are reported, variations existed among patients, for example, severity of the Crohn's disease, duration of each treatment, number of HBOT sessions and antibiotic regimens . This heOur results demonstrate that combining infliximab with anti-MAP therapy and HBOT can achieve a high healing rate of perianal fistulae in Crohn's disease, including rectovaginal fistulae. The use of such combination treatment is a viable option for the treatment of persistent and recurrent fistulae. Future studies using larger sample sizes will enable analysis of fistula characteristics which could predict long-term healing with this combination of therapies.Crohn's disease can be complicated by fistulae development between the rectum and the external skin of the anus, and from the rectum to the vagina in more than 20–40% of patients. This is a distressing and chronic complication with poor response to current therapies.The arrival of the immunomodulator, infliximab (Remicade) has improved healing with initial rates being 60% but in the long-term healing falls to below 40% in spite of administering Remicade.Mycobacterium avium ss paratuberculosis (MAP) have also been shown to heal fistulae but in no more than 30–50% of patients. Combining these partially effective treatments has not been reported.Oxygen therapy in a hyperbaric chamber (hyperbaric oxygen therapy) and special combinations of antibiotics targeting This report describes the use in nine consecutive patients of a combination of infliximab, hyperbaric oxygen and anti-MAP therapy used together, to see if greater healing of fistulae is achieved and if maintenance of healing is possible with the anti-MAP component.Although this study is quite small and future results may see some failures, all first nine patients treated with this method healed their fistulae completely without any discharge and without the need to wearing pads to absorb leakage. This appears to be a combination that is worth working with, perhaps optimizing the use of infliximab and hyperbaric oxygen therapy to further optimize healing rates and improve maintenance of healing with long-term antibiotic usage.