Recently it has been revealed that lung adenocarcinomas with distinct gene mutations or fusions are associated with particular histopathological entities. For example, epidermal growth factor receptor (EGFR) gene mutations are often associated with well differentiated adenocarcinoma of the lung with bronchioloalveolar pattern. On the other hand, echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene in a subset of lung adenocarcinoma is related to mucinous cribriform histology.Reported herein is a case of synchronous EML4-ALK positive lung adenocarcinoma and adenocarcinoma in situ in the bilateral lungs of a 55-year-old Japanese woman. The woman had EML4-ALK positive lung adenocarcinoma in the right lower lung while adenocarcinoma in situ in the left upper lung, which was EML4-ALK negative.To our knowledge, this is the first report of synchronous, bilateral lung adenocarcinomas composed of EML4-ALK positive and negative ones. Adenocarcinomas of the lung comprise a group of diseases with heterogeneous clinicopathological characteristics ,2. HistoRecent advances in molecular genomic analyses of lung adenocarcinoma specimens have revealed recurrent association of distinct gene mutations or fusions with particular clinicopathological entities . For exaFollowing this breakthrough, a number of kinase gene fusions have been identified in subsets of adenocarcinoma of the lung, the examples of which include RET or ROS1 fusions -9. Most Since the activities of these fused kinases and mutated kinases are shown to be tumorigenic in cell culture systems and/or transgenic mice, these kinases are promising candidates for therapeutic targets. In fact, small molecule tyrosine kinase inhibitors have shown dramatic therapeutic effects on subsets of lung adenocarcinoma, once oncogenic tyrosine kinases of the cancers are identified. Examples include gefitinib for EGFR mutation-positive lung adenocarcinoma and crizotinib for EML4-ALK positive lung adenocarcinoma.With regard to these advances in molecular phenotyping, identification of particular histopathological subtypes of lung adenocarcinomas, including bronchioloalveolar pattern or mucinous cribriform pattern, will give important clues for therapeutically tractable genomic changes, such as EGFR gene mutations or EML4-ALK fusion gene.Reported herein is a case of synchronous EML4-ALK positive lung adenocarcinoma and adenocarcinoma in situ in the bilateral lungs of a 55-year-old Japanese woman. The woman had EML4-ALK positive lung adenocarcinoma in the right lower lung while adenocarcinoma in situ in the left upper lung.A 55-year-old Japanese woman was referred to Yodogawa Christian Hospital because of incidental finding of an abnormal shadow in the chest X-ray. She was a never-smoker. Chest computerized tomography (CT) examination revealed a 38×12 mm-sized stellate-shaped mass in the right lower lobe . Immunohistochemistry was performed on representative sections with appropriate antigen retrieval. A primary antibody against ALK was purchased from NICHIREI  and the immunostained signal was visualized using I-VIEW DAB universal kit . For mutational analysis, genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue sections using Genomic DNA Extraction Kit . DNA sequencing for EGFR gene mutation was performed as described .Microscopic examination of the resected right lower lobe revealed a heterogenous adenocarcinoma composed of a mucinous cribriform tumor Figure a and lepOn the other hand, the resected left upper lobe specimen contained a adenocarcinoma in situ Figure e. This tAs an adjuvant therapy, the patient has taken orally tegafur-uracil 300 mg/day since 3 months after the resection operation. Until now, she has not shown any signs of relapse or adverse effects. Eleven months after the operation, neither chest CT, nor bone scintigraphy, nor brain magnetic resonance imaging did show any signs of relapse or metastasis.In this report, we described a case of synchronous bilateral lung cancers with EML4-ALK positive adenocarcinoma in the right lung and adenocarcinoma in situ in the left, which was EML4-ALK negative. To our knowledge, this is the first report of a case of synchronous lung adenocarcinomas of this combination.A number of cases of synchronous multiple lung adenocarcinomas were reported previously. The most frequent components in those cases were well differentiated adenocarcinoma with mixed bronchioloalveolar pattern . GrazianIn our case, although the bilateral adenocarcinomas have bronchioloalveolar pattern in common, the right lung cancer was EML4-ALK positive while the left one was not. Furthermore, EGFR gene mutations were not detected in the exons 18 to 21 in “pure” bronchioloalveolar adenocarcinoma in the left lung. Over 90% of EGFR mutations were reported to be localized in these 4 exons . HoweverThe fact that the tumor of the right lung and that of the left lung harbor different genetic alterations will be useful for the follow-up of this patient. The adenocarcinoma in situ of the left lung was 15×10 mm of size and pathologically at stage 0 (TisN0M0). Therefore, the possibility of presenting metastasis in the following five years is as low as nearly 0%. On the other hand, the EML4-ALK-positive tumor of the right lung was an invasive cancer. Thus, if any metastasis or relapse occurs in the future in this patient, it is more likely that the relapse derives from EML4-ALK-positive cancer of the right lung. After the confirmation that the metastasis or relapse harbor EML4-ALK translocation, ALK inhibitor such as crizotinib will be the first choice. EML4-ALK fusion gene occurred around 3% of “non-smoker” adenocarcinomas of the lung . In addiWritten informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written informed consent is available for review by the Editor-in-Chief of this journal.EGFR: Epidermal growth factor receptor; EML4: Echinoderm microtubule-associated protein like 4; ALK: Anaplastic lymphoma kinase; CT: Computerized tomography; HE: Hematoxylin and eosin; FISH: Fluorescent in situ hybridization.All the authors state no competing interests.IM, Kengo Takeuchi, KU, Kazuhiro Teramura, and SH participated in the pathological final diagnosis of the case, and prepared and edited the manuscript. In particular, Kengo Takeuchi performed the FISH analysis. SM and MK were responsible for the preoperative endoscopic examination and the operations, and helped IM and SH in preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/25/prepub


Isoflurane, a volatile anesthetic widely used clinically, has been implicated to be both neuroprotective and neurotoxic. The claim about isoflurane causing neural apoptosis remains controversial. In this study, we investigated the effects of isoflurane exposures on apoptotic and anti-apoptotic signals, cell proliferation and neurogenesis, and astroglial processes in young adult mouse brains.Sixty 6-week-old mice were randomly assigned to four anesthetic concentration groups  with four recovery times  after 2-h isoflurane exposure. Immunohistochemistry measurements of activated caspase-3 and Bcl-xl for apoptotic and anti-apoptotic signals, respectively, glial fibrillary acidic protein (GFAP) and vimentin for reactive astrocytosis, doublecortin (Dcx) for neurogenesis, and BrdU for cell proliferation were performed.Contrary to the previous conclusion derived from studies with neonatal rodents, we found no evidence of isoflurane-induced apoptosis in the adult mouse brain. Neurogenesis in the subgranule zone of the dentate gyrus was not affected by isoflurane. However, there is a tendency of reduced cell proliferation after 2% isoflurane exposure. VIM and GFAP staining showed that isoflurane exposure caused a delayed reduction of astroglial processes in the hippocampus and dentate gyrus.Two-hour exposure to isoflurane did not cause neuroapoptosis in adult brains. The delayed reduction in astroglial processes after isoflurane exposure may explain why some volatile anesthetics can confer neuroprotection after experimental stroke because reduced glial scarring facilitates better long-term neuronal recoveries. Volatile anesthetics have been implicated to be both neuroprotective and neurotoxic. Several studies have shown that volatile anesthetics can reduce neurological damage from ischemia by reducing glutamate release, blocking glutamate receptors, enhancing hyperpolarization by GABA, increasing anti-apoptotic bcl-2 levels, activating p38 mitogen-activated protein kinases, and inducing neurogenesis -3. ConveIt has been proposed that a prolonged exposure to volatile anesthetics is intrinsically neurotoxic and the observed protective effects are due to preconditioning, in which endogenous neuroprotective mechanisms are initiated by a limited exposure . FurtherTo investigate the mechanisms of isoflurane-induced neurological changes, degrees of neurogenesis, apoptosis, and reactive astrocytosis were studied in adult mice after exposure to various concentrations of isoflurane and recovery for different lengths of time after exposure. Similar to the results in a recent study , we founAnimal protocol was approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Sixty 6-week-old male CD-1 mice weighing 35 ± 5 g were used for the experiments. We chose to use six-week-old mice because synaptogenesis has completed in these animals . The use2 as the carrier gas and the desired percentage of isoflurane at a flow rate of ~2 L/min through a plastic tubing for 2 h. A calibrated Datex Capnomac Ultima Gas Analyzer  was used to ensure that the isoflurane concentration inside the cage was maintained at 0 (control), 0.6%, 1.3%, or 2%. During anesthesia, a pulse oximeter  was clipped onto the hindlegs of the animals to monitor the oxygen saturation, heart rate, and respiratory rate. A temperature probe was placed under the ventral surfaces of the animals to ensure that the body temperatures were maintained at 35-37 °C. After anesthesia, mice were returned to their own cages and given 2 h, 1, 6, or 14 d to recover. To avoid any potential complications from invasive manipulations, the blood gases and end tidal CO2 of individual animals were not measured. However, oxygen saturation in the extremity (hindlegs) was maintained above 95%. A previous study in mice using the same anesthesia protocol and the same O2 flow rate showed that ventilation depression was insignificant with the isoflurane concentrations used in the present study [All mice (including the controls) were placed in a standard 9.6-liter mouse cage with a steady flow of Ont study .After the recovery periods, mice were anesthetized with isoflurane and a transcardial perfusion was performed with buffered 10% formalin phosphate, as described previously -16. The 1 anti-GFAP (1:250) and Rabbit anti-Vimentin (1:200) were incubated at room temperature for 2 hours. Secondary antibodies used include: Alexa Fluor 594 donkey anti-rabbit (1:500) and Alexa Fluor 488 goat anti-mouse IgG1 (1:500). Rabbit anti-activated caspase-3 (1:200) and mouse anti-Bcl-xl (1:500) were incubated at 4°C overnight. Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit (1:500) and Alexa Fluor 594 goat anti mouse IgG2a. Goat anti-Dcx (1:250) was incubated at 4°C overnight. The secondary antibody was Alexa Fluor 488 donkey anti-goat (1:500). BrdU staining was performed by first performing antigen retrieval by boiling tissue slices in sodium citrate buffer. Rat anti-BrdU antibody (1:200) was used as the primary antibody, and Alexa Fluor 488 donkey anti-rat (1:500) was used as the secondary antibody. Nuclear DNA was stained with 4'-6-diamino-2-phenylindole (DAPI), 1 mg/ml, in phosphate-buffered saline.Standard immunohistochemistry techniques were used for GFAP, VIM, activated caspase-3, Bcl-xl, doublecortin (Dcx), and BrdU staining. Mouse IgG+ cell soma and then normalized by the area within which the counting was performed. The percent positive areas for GFAP+ and VIM+ cells in the dentate gyrus granular layer, hilus, and the hippocampal SLM layer were determined by the well-established thresholding segmentation method implemented in the Image-Pro processing package. To minimize thresholding bias, all tissue sections were processed, stained, and imaged at the same time using the same batch of staining media and the same imaging acquisition setting. Two experimentalists without knowledge of the resulting positive percentage areas first determined the cut-off background values, which were then averaged for each given staining and used in the final thresholding segmentation measurements of that staining. In few cases when the background intensity of a given image was more than 1.5 standard deviations outside the averaged background intensity, the image's own background threshold was used.Images were acquired using Image-Pro Plus 7.0 software controlling an Olympus IX81 microscope  with a Prior motorized stage, a Sutter Lambda xenon exciter light source, and an ORCA-ER digital camera. All images were tiled using Adobe Photoshop and post-processed using Image-Pro AMS . Doublecortin positive cell densities in the dentate gyrus subgranular zone were determined by counting all Dcxp value of <0.05 was considered significant for all tests.Statistical analysis was performed using the SPSS program . Multivariate analyses of immunohistochemical staining were performed with respect to BrdU injection, anesthetic concentration, and days of recovery. A simple contrast test was performed between animals with and without BrdU injection, and between the control group and various groups with different isoflurane concentrations. Post hoc tests were carried out for between-group comparison using either Tukey's honestly significant difference (HSD) test, Fisher's least significant difference (LSD) test, or Dunnett t test (2-sided against the control). The results are reported as mean ± standard errors except otherwise noted. For cell counts and area-averaged cell density, comparison among different isoflurane concentration groups, including the controls, was made using the nonparametric Kruskal-Wallis test. A One of the 12 mice in the 2% isoflurane group, which was pre-assigned to 6-day recovery, died unexpectedly due to unknown causes and was excluded from the analysis. To determine if 2-h exposure to clinically relevant concentrations of isoflurane can induce apoptosis in young adult mouse brains, we measured activated caspase-3 as well as the anti-apoptosis regulator Bcl-xL using immunohistochemical staining. The controls for positive staining were obtained from brain tissues of adult mice that underwent 20 min of hypoxia with 5% oxygen, which is known to cause hypoxic tissue damage. No measureable apoptotic or anti-apoptotic response was detected in the isoflurane-exposed mice, as evidenced by the absence of any positive immunoreactivity in the staining of activated caspase-3 and Bcl-xL . When all animals with isoflurane exposure were combined as one group (irrespective of concentrations and days of recovery) and compared to the control animals as another group, the nonparametric independent-samples tests, including the Kruskal-Wallis test and median test, showed that the number of BrdU+ cells in the subgranular zone of the dentate gyrus is significantly different between the isoflurane-exposed animals and the controls . The median and the 25th and 75th percentiles of BrdU+ cell counts in the subgranular zone were 8, 2, and 12, respectively. Seven out of eight control animals had a BrdU+ cell count greater than the median, whereas 22 out of 35 isoflurane-exposed animals had a count less than or equal to the median. A closer examination of the data indicated that exposure to 0.6% and 2% of isoflurane seemed to reduce the total number of BrdU+ cells in the subgranular zone of the dentate gyrus 6 and 14 d after the exposure.In animals that received BrdU injections before and after isoflurane exposure, we determined the isoflurane effect on cell proliferation. Figure + cell counts (p = 0.055) or Dcx+ cell densities (p = 0.096) in the subgranular zone of the dentate gyrus between animals in the control group and those exposed to isoflurane . We then studied the effects of anesthetic concentration and recovery time after anesthetic exposure on GFAP and VIM expression Figure . MultivaIn this study, we investigated the effects of the volatile anesthetic isoflurane on possible mechanisms of anesthetic-induced neuroprotection and neurotoxicity in adult mouse brains. The main conclusions of this study are that isoflurane does not alter neurogenesis, nor does it cause neuronal cell death, in various formations of the hippocampus, which is known to be selectively vulnerable to insults. Instead, the clinical concentrations of isoflurane resulted in a decrease in GFAP and VIM immunoreactivity after anesthetic exposure, suggesting that glial cells and microglia may be involved in the neurological changes seen with isoflurane anesthesia.A receptors and inhibit the excitatory action of NMDA receptors, are known to interfere with synaptic functions, including the establishment of new synapses. Thus, it is an anticipated result of the natural and protective pruning process that prolonged exposure to isoflurane will lead to the appearance of increased apoptosis in the neonatal brains undergoing synaptogenesis. The results in this study indicate that in young adult mice after the synaptogenesis period, isoflurane does not cause neuronal apoptosis. Thus, extrapolation of rodent studies to pediatric anesthesia should be approached with caution. In rodents, synaptogenesis starts to accelerate on postnatal day 7 (P7) and completes on P14 [Our results are in agreement with the recent findings of Stratmann et al , but difs on P14 , whereass on P14 . Althougs on P14  during tA recent study in C57/BL6 mice found that a 2-h exposure to isoflurane induced caspase activation in a narrow time window . No casp-/-VIM-/-double knockout mice [+ and VIM+ astroglia after anesthetic exposure as observed in this study may offer an alternative explanation of neuroprotection seen in other experimental studies of brain injuries involving the use of isoflurane anesthesia.Our results, however, did show some delayed effects from isoflurane exposure on glial cells, particularly astrocytes. Since astrocytes are the supporting cells of the central nervous system, it seems plausible that they could play a significant role in mediating neuronal survival versus neuronal death in response to anesthetics. Reactive astrocytosis is known to be protective early in neuronal injury as the astrocytes regulate the external chemical environment of the neurons. When the hyperplasia and hypertrophy of astrocytes are prolonged, however, injury occurs as a result of glial scarring, which creates a barrier to axonal regeneration and synaptogenesis . In ratsout mice  can reduSeveral studies have focused on the neuronal responses to isoflurane anesthesia. In a study of traumatic brain injury (TBI) , CA3 neu+ and VIM+ areas, which are proportional to the density of GFAP+ and VIM+ immunoreactivity, were significantly reduced. Future studies are needed to determine if anesthetic-induced hypotrophy of glial processes will be beneficial in preventing glial scarring in the presence of other insults such as ischemia and traumatic injuries.In conclusion, exposure to isoflurane for two hours did not cause neural apoptosis in the adult mouse brain. Without additional insults, isoflurane exposure alone did not lead to enhanced neurogenesis in the dentate gyrus, one of the two neurogenesis regions in the adult brain. Two weeks after isoflurane exposure, the astrocytic processes, as measured by the GFAPThe authors declare that they have no competing interests.RMD participated in the design of the study, carried out the experiments, acquired and analyzed the data, and participated in the writing of the manuscript. JDB carried out the experiments, acquired and processed the data, and participated in the data analyses. YX conceived and designed the study, secured funding for the study, provided research materials, analyzed and interpreted the data, and wrote the paper. All authors read and approved the final manuscript.


New genetic markers for adult acute lymphoblastic leukemia (ALL) have been found to have prognostic impact, such as the lymphoid transcription factor gene IKZF1 alterations, which are associated with a high rate of leukemic relapse in B-ALL. Although complete remission rates by induction chemotherapy in ALL are now high, the long-term survival is still disappointing. Improvements in the survival outcome of ALL have been observed in young adults as a result of the use of pediatric inspired regimens and the broadening of the number of patients who are eligible for allogeneic SCT. Development of new and less toxic agents also provide promise to improve the outcome in adult ALL, such as tyrosine kinase inhibitors in Ph-positive ALL, rituximab in CD20-positive disease, blinatumomab in precursor B-ALL and nelarabine in T-lineage ALL. Challenges for the future are to implement genomic profiling into the clinical setting to guide risk stratification and providing novel targets for tailored therapies. Acute lymphoblastic leukemia (ALL) is a biologically and clinically heterogeneous neoplasm of lymphoid progenitors, with approximately 85% of cases being of B-cell lineage and 15% of T-cell lineage. In contrast to childhood ALL, in whom approximately 90% are now cured , even wiThe inherent heterogeneity of ALL requires an accurate assessment of risk to aid treatment decisions. In the past, the classic prognostic factors were age, presenting white blood cell (WBC) counts, cytogenetic abnormalities and upfront response to induction therapy. One of the strongest adverse prognostic features is the presence of the Ph chromosome t. Recently, a retrospective analysis of the Mayo Clinic leukemia database has identified high risk cytogenetics , very high risk cytogenetics categories  , low pla], very h;19, t, transcriptional regulation ETV6, ERG), lymphoid signaling , cell-cycle regulation and tumor suppression , and drug responsiveness (the glucocorticoid receptor NR3C1) [, ERG, lyIKZF1 encodes IKAROS, a zinc finger transcription factor that is required for the development of all lymphoid lineages . During In addition to IKZF1, other genetic alterations were also identified to have some prognostic impact on ALL and incorporated into risk stratification algorithms Table . For exaPhiladelphia chromosome/BCR-ABL–positive ALL has long been recognized as a high-risk subset of adult ALL with the most unfavorable prognosis. Published data show a disappointing long-term survival of 20% or less with chemotherapy alone . IntroduIt is well established that TKI-based treatment, followed by allo-SCT in CR1, is the gold standard therapy and provides curative potential for adults with Ph + ALL. Studies from several cooperative groups, have showed 3-year survival probabilities of 55–70% in young adults receiving IM-containing induction followed by myeloablative allo-SCT ,28. And Of note, there is increasing reluctance to offer allo-SCT to younger patients in the TKI era, because younger individuals usually have a better outcome to chemotherapy/TKI alone and have more to lose by risking the long-term adverse consequences of SCT. In a previous study, Schulz et al. found that intensive chemotherapy plus continuous IM improved survival rate for children and adolescents, with minimal toxicities . And theNevertheless, interpretation of these findings is limited by the small patient numbers and the relative short follow-up. Thus, it is difficult to answer whether IM/intensive chemotherapy could replace allo-HSCT for younger patients with Ph + ALL. However, these provocative data open the way for further discussion. Future studies will focus on development of independent prognostic algorism, such as MRD at relevant time-points, to identify a subgroup of good risk younger patients, eligible for receiving treatment with chemotherapy and TKI only, and accordingly sparing SCT.Ph + ALL accounts for 40 – 50% of ALL in patients over 50 years, many of whom cannot tolerate intensive chemotherapy and are not eligible for SCT. The question of whether minimization of therapy-related toxicity by combining TKIs with less intensive chemotherapy yielded equivalent results were proposed in this subgroup of patients. Several studies showed IM combined with minimal chemotherapy was able to yield a higher CR rate and fewer deaths without major toxicity in elderly patients than observed in historical controls. However, a main problem of this less intensive approach is the very high relapse rate, yielding an OS probability of less than 20% in the studies with long-term follow-up . DasatinInterferon as treatment for Ph + leukemia has attracted renewed interest, fueled by preclinical observations that IFN may recruit dormant CML stem cells into the cell cycle, and enhance their susceptibility to eradication by TKIs. More available clinical data come from the treatment of CML. Recently, an open-label phase II study was designed to investigate the combination of low-dose IFN-alfa with IM as maintenance therapy (MT) in the elderly with Ph + ALL patients . MaintenMinimal residual disease after allo-SCT for Ph + ALL is predictive of relapse. Imatinib administration subsequent to SCT may prevent relapse, but to date there is no large-scale prospective randomized control trial to provide sufficient evidence to conclude that IM or other TKIs should be given to all patients after SCT, even though small studies suggested efficacy ,39. A si9/L and platelet counts were >50.0 × 109/L, or if they displayed either elevated BCR-ABL transcript levels in two consecutive tests, or a BCR-ABL transcript level ≥ 10-2 after initial engraftment [The optimal time for initiating IM treatment following SCT is not well established. The CSTIBES02 trial reported that IM was poorly tolerated following myeloablative SCT: only 62% were able to start at a median of 3.9 months after SCT, and many patients required discontinuation or dose reduction due to complications such as GVHD . The GMAraftment . In thisExperience on the use of dasatinib after HSCT is very limited. A study by Caocci and colleagues suggested that administration of dasatinib 120 days after SCT is highly effective in preventing Ph + ALL relapse after both auto- and allo-SCT . HoweverDespite high remission rate induced by TKI-containing regimens, fewer than 50% of these patients sustain molecular remission after initial therapy, and overt clinical relapses are frequent, indicating that TKI resistance is a substantial obstacle in Ph + ALL. The emergence of BCR–ABL mutations is the most important mechanism of resistance to TKI. Mutations can occur within the A (activation)-loop, the P (ATP-binding)-loop region or at the so-called “gatekeeper” residue, threonine 315. Given that the prognosis of patients with Ph + ALL with T315I mutation is poor, many new agents have been clinically investigated in the setting of T315I and other mutations. Ponatinib is a pan-BCR-ABL inhibitor with activity against all IM-resistant mutants, including the T315I mutation. Ninety four patients with CML lymphoid blast phase or Ph + ALL were included in the pivotal phase 2 study, who were resistant or intolerant to other TKIs . With a Although cure rates in pediatric ALL are now around 90%, there is still room for improvement in the management of adult patients with Ph- ALL, where little progress has been made over the past two decades and long-term cure rates usually do not exceed 40%, even with allo-SCT . Given tOne of promising approaches in treating young patients with Ph- ALL is adopting pediatric protocols - the so called “pediatric inspired” regimens, especially for those adolescent and young adult (AYA) aged 15 to 39 years. Retrospective studies have shown that AYA with ALL treated with pediatric-inspired regimens have better outcomes than similarly aged patients with adult protocols, and improved the event-free survival rate of newly diagnosed Ph- ALL to 60% or higher . This coClassic treatment regimens using combination chemotherapy are associated with substantial toxicities that limit further dose escalation. Therefore, development of new, targeted and less toxic agents, such as nucleoside analogues and monoclonal antibodies (mAbs), offer promise for improving the outcome of Ph- ALL.As mentioned above, young adults with ALL adopted pediatric regimens, with prolonged use of ASP, mostly E. coli, had better outcomes than historically reported. Considering its potential higher toxicity, however, ASP is either not used or given for much shorter duration, particularly in patients aged > 45 years. Some new types of ASP are emerging. The pegylated E. coli derived form, PEG-ASP, is most commonly used, because it shows less immune response and a prolonged half-life of 5.7 days compared to 1.3 days for native ASP. The incorporation of PEG-ASP in induction and especially during consolidation therapy seems to improve the outcome with a manageable and reversible toxicity ,54 as shRecently, the development of monoclonal antibodies has provided hope that tumor-targeted therapy would one day play a role in the treatment of ALL with less systemic toxicity profile . To date2 IV once every 3 to 4 weeks in 49 patients with R/R B-lineage ALL, the overall response rate was 57% and the median survival of all patients was 4.5 months. Given the heavily pretreated patients, the response rate in R/R ALL was promising and twenty-two of the 49 patients were able to proceed to subsequent allo-SCT. The most common adverse effect was liver function abnormalities that were severe in 31% of the patients [Another actively investigated MoAb is anti-CD22. Epratuzumab, the “naked” humanized MoAb against CD22, was first studied in children with relapsed ALL showing minimal single-agent activity . For adupatients . Recentlpatients .2/day in week 1 and 15 μg/m2/day for week 2–4 was selected for further investigation based on safety/efficacy considerations [Bi-specific MoAbs constitute a new generation of compounds with a promising future. In a recent phase II trial, blinatumomab, a bi-specific MoAb with a CD3-binding site for the T cells and a CD19 site for the target B cells , has beeerations .In addition to mAbs, other potential new agents are mammalian target of rapamycin (mTOR) inhibitors, JAK inhibitors  and the Allogeneic SCT is the most potent postremission anti-leukemic therapy in ph- adult ALL, with OS rates exceeding 50%. Several older randomized trials and meta-analyses concluded that high-risk, not standard-risk, patients benefited from allogeneic SCT in CR1 . Modern The increasing use of unrelated donors, umbilical cord blood (UCB), haploidentical donors, and reduced intensity SCT have expanded the opportunity for more patients to undergo SCT. The OS rate of unrelated donor (URD)-SCT are currently close to those for related donors; the higher transplant-related mortality of the former is counterbalanced by the lower relapse rate . DevelopDespite outstanding advances in the knowledge of the molecular basis of adult ALL, the treatment of this disease remains challenging. Improvements in the outcome of ALL have been observed in young adults as a result of the use of inspired regimens and the broadening of the number of patients who are eligible for allogeneic SCT, whereas the prognosis of the elderly remains poor. Development of new and less toxic agents provide promise to improve the outcome in adult ALL, such as tyrosine kinase inhibitors in Ph-positive ALL, rituximab in CD20-positive disease, blinatumomab in precursor B-ALL and nelarabine in T-lineage ALL. From the biological prospective, genetic profiling approaches will provide a comprehensive view of the complexity of genetic alterations in ALL. Identifying miRNAs, genes, and pathways relevant for the pathogenesis of the leukemia and the biologic determinants of treatment failure may provide novel insights on novel targets . HoweverThe authors declare that they have no competing interests.GW participated in concept design and critically revising the manuscript. YZ participated in data collection and drafting the manuscript. HH participated in reviewing and revising the manuscript. All authors have read and approved the final manuscript.


MicroRNAs are regulators of central cellular processes and are implicated in the pathogenesis and prognosis of human cancers. MicroRNAs also modulate responses to anti-cancer therapy. In the context of radiation oncology microRNAs were found to modulate cell death and proliferation after irradiation. However, changes in microRNA expression profiles in response to irradiation have not been comprehensively analyzed so far. The present study's intend is to present a broad screen of changes in microRNA expression following irradiation of different malignant cell lines.1100 microRNAs (Sanger miRBase release version 14.0) were analyzed in six malignant cell lines following irradiation with clinically relevant doses of 2.0 Gy. MicroRNA levels 6 hours after irradiation were compared to microRNA levels in non-irradiated cells using the "Geniom Biochip MPEA homo sapiens".Hierarchical clustering analysis revealed a pattern, which significantly (p = 0.014) discerned irradiated from non-irradiated cells. The expression levels of a number of microRNAs known to be involved in the regulation of cellular processes like apoptosis, proliferation, invasion, local immune response and radioresistance  displayed 2 - 3-fold changes after irradiation. Moreover, several microRNAs previously not known to be radiation-responsive were discovered.Ionizing radiation induced significant changes in microRNA expression profiles in 3 glioma and 3 squamous cell carcinoma cell lines. The functional relevance of these changes is not addressed but should by analyzed by future work especially focusing on clinically relevant endpoints like radiation induced cell death, proliferation, migration and metastasis. MicroRNAs are small non-coding RNAs of typically 20 - 22 base pairs length. They are involved in gene regulation at the post-transcriptional level by silencing mRNA translation. To date more than 1000 microRNAs have been discovered. MicroRNAs are involved in the regulation of diverse cellular processes, including programmed cell death, proliferation, differentiation, metabolism, migration and stress responses . We describe alterations in the abundance of 1100 microRNAs in six malignant cell lines following irradiation. To our knowledge this represents the broadest analysis of irradiation induced changes in microRNA patterns to date.The response of cancer cells to ionizing radiation has been extensively studied resulting in the discovery of central regulators of radiosensitivity. However, irradiation-induced alterations in microRNA profiles have hitherto not been analyzed. This stimulated us to perform the present study: a microarray based analysis of irradiation-induced changes in all microRNAs published in the Sanger miRBase release version 14.0  and cell lines from brain tumors . SCC-4, SCC-25 and CAL-27 were purchased from the "Deutsche Sammlung von Mikroorganismen und Zellkulturen" , T98G and U-87 MG were purchased from the European Collection of Cell Cultures . LN229 was a gift from the Department of Neurosurgery, University of Munich. SCC-4 and SCC-25 were grown in Dulbecco's MEM/Ham's F-12 medium supplemented with 20% fetal bovine serum (FBS) and Hydrocortisone (40 ng/ml) and Sodium Pyruvate (1 mM), respectively. CAL-27 were grown in Dulbecco's MEM, supplemented with 10% FBS. LN229, T98G, U-87 MG were grown in Earles buffered Salt Solution (EBSS), supplemented with 10% FBS, Sodium Glutamine (2 mM), Non Essential Amino Acids (1x) and Sodium Pyruvate (1 mM). All media and supplements were purchased from Biochrom, Germany.Cells were seeded in culture flasks and grown for 5 - 10 passages. For the experiments, cells were grown to a confluency of ~70%. Cells were then irradiated with 6 MeV Photons at a dose rate of 3 Gy per minute using a linear accelerator (Siemens Mevatron) to a total dose of 2 Gy. After irradiation cells were incubated at 37°C for 6 hours before extraction of the total RNA. Non-irradiated cells were used as controls.mirVana™ microRNA Isolation Kit (Ambion) according to the manufacturer's instructions. Quantity and quality of the extracted RNA was analyzed with the Agilent 2100 Bioanalyzer (Agilent Technologies), using the company's RNA 6000 Nano Kit according to the manufacturer's instructions. RNA extracts were stored at - 20°C.Total RNA was extracted using the The RNAs patterns were analyzed by Febit (Heidelberg) using the company's "Geniom Biochip MPEA homo sapiens", generating five data points for each microRNA measured. To adjust for a systematic spatial variability on each microarray, the intensities of black probes were used for background correction. To test the hybridization process as well as positioning features additional hybridization controls were added to the array template (data not shown).Hierarchical cluster analysis (bottom-up complete linkage clustering using Euclidean distance as a measure) was performed using the normalized data of the 65 most deregulated microRNAs to identify differentially expressed microRNAs following irradiation. The correlation between two dichotomous variables was assessed using the chi-square test. A two-tailed p-value < 0.05 was considered significant. MicroRNA changes in single cell lines were not analyzed because the statistical power was not sufficient.Hierarchical cluster analysis revealed two clusters of microRNAs which significantly p = 0,014) differentiated between irradiated and non-irradiated cells. Figure 4 differeAlthough statistical interpretation of the data is difficult since commonly used p-values of 0.05 might lead to false positive results when analyzing more than 1000 microRNAs, the microRNAs displaying the most striking up- or downregulation after irradiation are presented in Table When comparing irradiated cells with their non-irradiated counterparts, the levels of several microRNAs that target central regulators of cancer cells were found to be susceptible to ionizing radiation. Of note, miR-1285, which negatively regulates the expression of the crucial tumor suppressor p53 , was uprOn the contrary, miR-24-1, a member of the miR23b cluster  that intMoreover, our screen identified different other microRNAs with so far unknown function to be up- or downregulated in response to irradiation. For many of them this is first time that they are reported to be susceptible to irradiation, like for instance, miR-144*, which was upregulated approximately 3-fold . A detailed comparison of the levels of all 1100 microRNAs before and after irradiation can be found in the additional file The data presented here show that ionizing radiation with clinically relevant doses of 2 Gy stimulate significant changes in microRNA expression patterns in different cancer cell lines. These changes might well influence the clinical outcome, since the expression of microRNAs, which are known to regulate apoptosis, migration and proliferation was altered in response to irradiation.Although the functional role of single microRNAs can not be established from the data presented here, several hypotheses can by generated which should be addressed by future experiments.In this context, the up-regulation of the Epidermal Growth Factor Receptor (EGF-R) following ionizing radiation might serve as a paradigm. Irradiation-induced EGF-R expression leads to increased radioresistance of malignant cells in subsequent treatment sessions during the course of fractionated radiotherapy . SimilarAnother candidate for regulating responsiveness to anticancer therapy is the let-7 family, although certain members of the let-7 family had different effects on radiation sensitivity in A549 lung cancer cells . Let-7 fAnother mechanism, through which ionizing radiation exerts its effects, involves changes in the tumor microenvironment. Interestingly, miR-24-1 levels were increased following irradiation and miR-24-1 might influence angiogenesis, invasion and local immune response through down-regulation of TGFβ .In summary, the present study revealed altered expression levels of microRNAs known to influence apoptosis, migration and proliferation, angiogenesis and local immune response in response to irradiation. Moreover, a number of microRNAs with unknown functions were found to be radiation-responsive.The power of the present study is based upon the huge number of investigated microRNAs (>1000) and the combined analysis of different malignant cells lines. Although adjusted p-values of changes in the expression levels of single microRNAs were not significant because of the huge number of microRNAs analyzed, the changes observed allow the generation of hypotheses and the design of further experiments validating the initial findings presented here and investigating the functional relevance of microRNA level alterations in the context of radiation oncology.The authors declare that they have no competing interests.OMN: RNA-Isolation and Purification, writing Manuscript, MN: Statistics and critical revision of the manuscript, SC: Cell culture and critical revision of the manuscript, FZ: Organization and Negotiation with Febit and critical revision of the manuscript, ML: Irradiation and critical revision of the manuscript, KL: Support concerning all technical questions, planning of experiments and critical revision of the manuscript.CB: Development of the concept and critical revision of the manuscript.All authors read and approved the final manuscript.Changes in 1100 microRNAs of six malignant cell lines following irradiation. Raw data of the levels of all 1100 microRNAs before and after irradiation.Click here for file


Over 10,000 long intergenic non-coding RNAs (lincRNAs) have been identified in the human genome. Some have been well characterized and known to participate in various stages of gene regulation. In the post-transcriptional process, another class of well-known small non-coding RNA, or microRNA (miRNA), is very active in inhibiting mRNA. Though similar features between mRNA and lincRNA have been revealed in several recent studies, and a few isolated miRNA-lincRNA relationships have been observed. Despite these advances, the comprehensive miRNA regulation pattern of lincRNA has not been clarified.ab initio method to annotate lincRNAs expressed in a group of normal breast tissues and breast tumors.In this study, we investigated the possible interaction between the two classes of non-coding RNAs. Instead of using the existing long non-coding database, we employed an Approximately 90 lincRNAs show strong reverse expression correlation with miRNAs, which have at least one predicted target site presented. These target sites are statistically more conserved than their neighboring genetic regions and other predicted target sites. Several miRNAs that target to these lincRNAs are known to play an essential role in breast cancer.Similar to inhibiting mRNAs, miRNAs show potential in promoting the degeneration of lincRNAs. Breast-cancer-related miRNAs may influence their target lincRNAs resulting in differential expression in normal and malignant breast tissues. This implies the miRNA regulation of lincRNAs may be involved in the regulatory process in tumor cells. Deep sequencing data from the Encyclopedia of DNA Elements Consortium (ENCODE) suggests that 70-80% of the human genome can be transcribed, and non-protein-coding RNAs (ncRNA) exceed the number of protein-coding genes . The recIn two recent studies, over 10,000 lincRNA regions were identified in human and mouse genomes. These regions were discovered based on epigenetic marks, where the promoter mark H3K4me3 is followed by the transcript mark H3K36me3. Comparative genomics analysis suggests that a significant portion of these regions do not have strong protein coding potentials, and do not have sequence homology with other known proteins . The K4-MicroRNA (miRNA) is another type of noncoding RNA whose biological functions have been extensively studied. MiRNAs are ~22nt small non-coding RNAs  that proDespite the extensive investigation on the roles of miRNAs in regulating protein-coding genes, only a few isolated non-coding transcripts (CDR1 antisense and MEG3) are reported to be regulated by miRNA ,32. Thouab initio approach is usually employed to reconstruct the annotation of lincRNAs from the RNA-seq data [GENCODE project and NONCODE database have both collected and integrated long noncoding RNA annotations,37. Baseseq data ,3,39. CoRNA-seq experiments sequence millions to billions of short RNA fragments in a single experiment, by which it measures the expression levels of the whole transcriptome, including noncoding RNAs, without relying on detailed annotation. To explore the potential miRNA-lincRNA expression relationship, we analyzed RNA-seq data from a group of normal and malignant breast tissues. Random primers were used in the reverse transcription step, and the strands of the transcripts were preserved to aid in identifying non-coding RNAs that are not poly-adenylated. Due to their small size (~22nt), the mature miRNAs were not included in the sequencing libraries. However, our data suggested that we can detect precursor miRNAs (80-120nt) from these RNA-seq data sets. For lincRNAs, the sequence fragments across two exons of the transcripts (junction reads) are able to reveal exon boundaries, especially novel ones. This feature helps us to understand the splicing patterns of unannotated lincRNAs. Several tools have been developed for mapping junction reads , detectiIn this study, for each putative miRNA-lincRNA pair, (1) we required reverse correlation between the expression levels of pre-miRNA and lincRNA within the 20 examined tissue samples. This is due to the well-established roles of miRNAs in facilitating RNA degradation on protein coding genes; and (2) we also required seed sequence of the miRNA (positions 2-7 on the miRNA) should be present in the exon regions of identified lincRNA, and the surrounding sequences on the lincRNA should also favor miRNA targeting conditions. We further examined the evolutionary conservation of the identified miRNA seeds in lincRNAs.® Tissue Bank at the IU Simon Cancer Center. As shown in Figure In order to investigate the potential relationships between miRNA and lincRNA, we used RNA-seq data from 20 samples, including 10 breast tumor samples, and 10 breast tissues from healthy pre-menopausal volunteers with no history of disease; these normal tissues were obtained from the Susan G. Komen for the CureBased on the RNA-seq data, the discovery of lincRNAs in breast tissue includes the following steps: (1) Epigenetic marks and comparative genetics. As reported previously, thousands of lincRNAs have been discovered by searching for the genomic regions with enhanced promoter marks  following the transcript marks . Our analysis started from 2,513 intergenic K4-K36 regions in the human genome (hg18) and 1,665 intergenic K4-K36 regions in the mouse genome (mm9) from previous studies . LiftOve(2) Evidence of transcriptional activity in other breast cancer cell lines. We have previously reported genome-wide binding patterns of RNA polymerase II (RPolII) in MCF7 cell lines , derived(3) Coding potential. We evaluated coding potentials of the identified lincRNA regions using the methods presented in ,50. By mWe used Scripture  for recoWe employed TargetScan  and PITAWe quantified the expression levels of mRNA, lincRNA and precursor miRNAs using RPKM measures (reads per kilo-base exon model per million mappable reads). In total, there are 15,381 genes, 303 lincRNAs and 286 miRNAs expressed more than 0.5 RPKM in more than 15 samples. Our further analysis focuses on these sets of genes whose expression levels are detectable.A generalized linear model (GLM) was used for modeling the potential effects of miRNA in down-regulating the expression levels of genes and lincRNAs in tumor and normal breast samples. Among the expressed mRNA, lincRNAs and miRNAs, 38,828 pairs are predicted to have regulatory relationships by miRNA target prediction algorithms. Among these potential target pairs, 1,742 and 213 mRNA-miRNA and lincRNA-miRNA pairs also showed significant reverse correlation, as derived from the GLM model (FDR<0.2).Among the 213 lincRNA-miRNA pairs, there are 315 predicted miRNA target sites  show differences in expression relationships with a significant p-value for the GLM interaction factor (p < 0.05), including 121 (~57%) lincRNA-miRNA pairs  were used to quantify transcription activity. One potential drawback of this strategy is that the RPolII data is from MCF7 cells due to the lack of measurement for the breast and tumor samples. This may cause bias in estimation. To ensure the RPolII data is representative of the transcript activity in breast, we surveyed the correlation between RPolII signal of MCF7 cell line and the RNA-seq levels of the breast tissues. We observed a very strong correlation  between RPolII signal in MCF7 cells and RNA-seq data from normal breast samples Figure .MiRNAs regulate degradation of its target mRNA through binding on the 3'-un-translated regions. Since the entire noncoding RNA is un-translated, the whole mature transcript can serve as potential miRNA target sites. We therefore predicted miRNA binding sites on the entire exonic regions of the identified lincRNAs.One advantage of RNA-seq data is that it can simultaneously measure the expression level of both mRNAs/lincRNAs and precursor miRNAs in the same sample. This provides opportunity for revealing the relationship of the mRNA/lincRNA-miRNA in their expression levels. As a precursor stage of mature miRNA, pre-miRNA expression levels can be different from their final functional form. Therefore, major conclusions from this analysis are subject to further experimental validation.Among 30,069 mRNA-miRNA pairs whose relationships were established based on target prediction alone, 31 pairs were experimentally validated, based on the records documented in the miR2Disease database and TarBase database ,55. Ten cDNA libraries from 10 normal breast tissues from the Susan G. Komen Tissue Bank at the IU Simon Cancer Center and 10 triple-negative breast cancer tumors were sequenced on an Applied Biosystems (ABI) SOLiD3 sequencer. We used a customized pipeline for RNA-seq data analysis, which includes three steps, QC filtering, sequence alignment, and gene expression quantification. 1) QC filtering. We first used SOLiD™ Instrument Control Software (ICS) and SOLiD™ Experiment Tracking System (SETS) software for the read quality recalibration. Sequences containing more than two 'N' or wildcards were discarded. Each sequence was scanned for low quality regions, and if a 5 base sliding window had an average quality score less than 20, the read would be truncated at that position. Any read with a length of less than 35 bases was discarded. Our experience suggests that such strategy effectively eliminates low quality reads while retaining high quality regions. 2) Alignment. We used BFAST  as our pIn potential lincRNA regions, we systematically searched regions with short read enrichment as putative lincRNA exons. A putative exon must contain at least 8 reads. If the distance between two exons was less than 10 bases, the two exons would be merged together. To define the exact exon boundaries, we searched for potential splicing sites around the putative exons. Splicing donor/acceptor (GT-AG or CT-AC for RNAs on Watson or Crick strand respectively) sites within 25 bases from the 5'- and 3'- ends of two putative exons would be considered as possible splicing sites. A lincRNA junction library was constructed by connecting all the possible donor and acceptor sites within the 100,000 bases span. The minimum intronic length is set to be 70 bases. After aligning the remaining unmapped reads to the new lincRNA junction library, we used Scripture  for predWe calculated the conservation score across placental mammals using Siphy  on vario mammals . The inpWe used the Codon Substantial Frequency (CSF) score to measure the coding potential of candidate lincRNA regions ,50,63. WThe mature and precursor miRNA annotations were extracted from miRBase . The tarWe used a generalized linear model (GLM) to characterize the relationship between the expression levels of genes, lincRNAs and miRNAs, by treating gene/lincRNA and microRNA as dependent and independent variables, respectively. Assuming the number of the reads falling into a genomic region follows a Poisson distribution, a natural logarithm function was chosen as the link function:In which Poisson distribution. For the significance tests on the global gene/lincRNA-miRNA expression correlation, we employed a t-test on the miRNA expression coefficient To allow over-dispersion, in practice, we employed a quasi-http://www.r-project.org/.To correct for multiple hypothesis testing, false discovery rate (FDR) was calculated based on Benjamini-Hochberg correction . All staThe authors declare that they have no competing interests.LJ, YW, and YL contributed to the design of the study. LJ and YL designed and performed the computational modeling and drafted the manuscript. GW, SC and YW participated in coordination, discussions related to result interpretation and revision of the manuscript. MR, BS and SC contributed to the experiment performing and data collection. All the authors read and approved the final manuscript.Genomic coordinates of 525 lincRNAs showed expression evidence in breast tissues.Click here for fileGenomic coordinates of the 315 predicted microRNA binding sites on lincRNAs.Click here for file


In the absence of stand-alone infrastructures for delivering cervical cancer screening services, efforts are underway in sub-Saharan Africa to dovetail screening with ongoing vertical health initiatives like HIV/AIDS care programs. Yet, evidence demonstrating the utilization of cervical cancer prevention services in such integrated programs by women of the general population is lacking.We analyzed program operations data from the Cervical Cancer Prevention Program in Zambia (CCPPZ), the largest public sector programs of its kind in sub-Saharan Africa. We evaluated patterns of utilization of screening services by HIV serostatus, examined contemporaneous trends in screening outcomes, and used multivariable modeling to identify factors associated with screening test positivity.Between January 2006 and April 2011, CCPPZ services were utilized by 56,247 women who underwent cervical cancer screening with visual inspection with acetic acid (VIA), aided by digital cervicography. The proportion of women accessing these services who were HIV-seropositive declined from 54% to 23% between 2006–2010, which coincided with increasing proportions of HIV-seronegative women (from 22% to 38%) and women whose HIV serostatus was unknown (from 24% to 39%) . The rates of VIA screening positivity declined from 47% to 17% during the same period (p-for trend <0.001), and this decline was consistent across all HIV serostatus categories. After adjusting for demographic and sexual/reproductive factors, HIV-seropositive women were more than twice as likely  to screen VIA-positive than HIV-seronegative women.This is the first ‘real world’ demonstration in a public sector implementation program in a sub-Saharan African setting that with successful program scale-up efforts, nurse-led cervical cancer screening programs targeting women with HIV can expand and serve all women, regardless of HIV serostatus. Screening program performance can improve with adequate emphasis on training, quality control, and telemedicine-support for nurse-providers in clinical decision making. Invasive cervical cancer (ICC) is a leading cause of cancer-related mortality and morbidity among women in the developing world Stand-alone infrastructures for delivering public sector-based cervical cancer screening services are largely unavailable or poorly developed in most low and middle income countries Zambia, a sub-Saharan African nation with a population over 13 million, has the world’s second highest annual cervical cancer incidence and mortality rates We sought to evaluate the utilization of screening services by Zambian women stratified by HIV serostatus and examine the trends and factors associated with screening-test positivity over the program scale-up period, with a goal to inform the roll-out of similar programs being implemented in other resource constrained settings.The operational details of CCPPZ have been reported in previous manuscripts apriori. Missing values of predictors were multiply imputed to prevent case-wise deletion www.r-project.org ) was used for statistical analyses.For this study, for evaluating programmatic outcomes we analyzed de-identified patient records and operations data utilizing unique  visits of women accessing screening. We focused on outcomes and trends of two key program indicators: proportion of women who were HIV-seropositive and proportion of women who were VIA-positive. We compared clinical and demographic characteristics by HIV serostatus utilizing Chi-square statistic for categorical variables and the non-parametric Wilcoxon rank sum test for continuous or ordinal variables This outcomes evaluation study of anonymized patient records of a public sector implementation program was deemed exempt from human subjects review or the need for informed consent by the Research Ethics Committee of the University of Zambia.CCPPZ provided screening services for a total of 56427 women in the period between January 2006 and April 2011 . A total of 17 clinics were operated by 17 nurses and an outpatient surgery care center housing a Gynecologic Cancer Prevention Clinic. A total of 15081 (26.7%) women were HIV-seropositive, 21322 (37.8%) were HIV-seronegative, and 20024 (35.5%) reported not knowing their HIV serostatus at the time of enrollment .The median age of screened women was 32 years . Over two-fifths (42.8%) of women with recorded educational status reported receiving less than a high school education. About a third (37.1%) of the screened population was married and living with spouses while the rest reported as being unmarried, widowed, or separated. Just over a fifth of the screened women (21.1%) reported being employed in the formal sector while over a third (35.7%) were housewives. A majority (53.3%) lived on a family income of less than 500,000 Zambian Kwacha per month . The median number of pregnancies reported was 3 (IQR: 2–5), the median number of lifetime sexual partners was 2 (IQR: 1–4) and just over a third (35.7%) reported ever use of a condom with a regular partner .HIV-seropositive women were more likely to be older than HIV-seronegative women . Higher proportion of HIV-seropositive women were likely to be married , have less education , and have family incomes lower than 500,000 Zambian Kwacha per month  than HIV-seronegative women . On averThe relative proportion of women who were HIV-seropositive as a share of the total women who utilized the screening services declined over five years from 54% in 2006 to 23% in 2010. Consequently, the proportion of HIV-seronegative women increased in the same duration from 22% to 30%, while that of women with unknown HIV serostatus went up from 24% in 2006 to 39% in 2010. All these trends were statistically significant (p<0.001). The crude probability of the HIV serostatus of women utilizing the screening services is graphically depicted in Cumulatively between January 2006 and April 2011, out of the total of 50,355 visual screening results available for analysis, 13,978 (27.7%) women were detected as VIA positive while 35,409 (70.3%) were VIA negative. Only a small minority  were classified as VIA-uncertain/indeterminate on initial screening. The VIA positivity rates declined over time from 47% in 2006 to 17% in 2010, and rates of VIA negativity increased from 52% in 2006 to 82% in 2010. This decline in VIA positivity, while directly coinciding with the increase in program scale-up activities, was consistent regardless of changes in utilization patterns by HIV serostatus, i.e., the crude probability of being detected as VIA positive , Y-axis Overall, HIV-seropositive women had higher rates of VIA positivity (40.9%) than HIV-seronegative women (20.3%), while a quarter (25.2%) of women with unknown HIV serostatus were VIA positive. After covariate-adjustment in a multivariable logistic regression model, HIV-seropositive women had 2.62 times higher odds of being detected as VIA positive [adjusted odds ratio (AOR) 2.62 ] than HIV-seronegative women . FactorsVIA-based screening is increasingly being adopted for cervical cancer prevention interventions in the developing world. CCPPZ is the first and largest PEPFAR-supported VIA-cryotherapy based ‘screen-and-treat’ cervical cancer prevention initiative linked to HIV/AIDS care and support programs in the developing world HIV/AIDS care and treatment programs provide very highly suitable platforms for launching ‘screen-and-treat’ cervical cancer prevention services, a very critical and life-saving intervention for all eligible women, regardless of their HIV serostatus. As we have demonstrated through this study, the increasing utilization of program services by women other than who are HIV-seropositive has coincided with program expansion in response to increased demand, and greater acceptance and uptake of screening services in the general population. Sustainability of the screening program has been achieved through community involvement and continued training of non-physician community mobilizers and care givers The successful scale up of cervical cancer screening activities was aided by expansion from the initial 2 clinics in 2006 to 17 clinics by 2011 and from 5 nurses in 2006 to 17 nurses in 2011. CCPPZ program nurses form the backbone of the ‘screen-and-treat’-based program operations. They function as the primary care givers independently performing screening and same-visit cryotherapy procedures, and refer patients needing histologic evaluation to tertiary care facilities. The training of nurses has been a vital component, largely achieved through a train-the-trainer model, where nurses serve as educators for their peers. This model has also proven successful in training nurses from 12 other African countries who have received on-site clinically-mentored training within CCPPZ-supported government clinics as part of a global cervical cancer training program.One of the most important factors in the improvement of VIA performance is the use of digital camera enhancement of the cervix , a low-cost adaptation for the Zambian setting. This is modeled after colposcopy, yet does not require a costly colposcope or prolonged training In the wake of projections of a startling rise in new cancer cases and deaths in Africa over the next 20 years


Next generation sequencing technology has revolutionized the study of cancers. Through matched normal-tumor pairs, it is now possible to identify genome-wide germline and somatic mutations. The generation and analysis of the data requires rigorous quality checks and filtering, and the current analytical pipeline is constantly undergoing improvements. We noted however that in analyzing matched pairs, there is an implicit assumption that the sequenced data are matched, without any quality check such as those implemented in association studies. There are serious implications in this assumption as identification of germline and rare somatic variants depend on the normal sample being the matched pair. Using a genetics concept on measuring relatedness between individuals, we demonstrate that the matchedness of tumor pairs can be quantified and should be included as part of a quality protocol in analysis of sequenced data. Despite the mutation changes in cancer samples, matched tumor-normal pairs are still relatively similar in sequence compared to non-matched pairs. We demonstrate that the approach can be used to assess the mutation landscape between individuals. Mutations are hallmark of cancers and identification of the mutations is imperative in our understanding of the disease. The advance in next generation sequencing (NGS) has transformed the way to identify mutations, just like microarray did a decade ago. Currently, the technology offers a powerful and yet relatively cost effective approach to characterize genome-wide mutations that occur in diseases. It enables identification of somatic mutations, including base substitutions, indels, chromosomal rearrangements, copy number alterations, and transcriptional aberrations. The rapid increase in NGS publications recently illustrated the potential of the technology, reporting rare mutations in various cancers, many previously undetected We propose here, a simple method using the concept of identity-by-state (IBS), an allele sharing approach used in genetics study to verify matched pairs. IBS is often used in single nucleotide polymorphism (SNP) data to ascertain relationship between individuals in the absence of pedigree structure Two datasets were used in the study; 82 matched pairs on SNP6 and 7 pairs sequenced using NGS see . Five maFrom both the SNP6 and NGS data, we observed that although there is differential clustering between the matched and unmatched pairs, the relative position in both data differed slightly. In the ‘NGS & SNP6 matched’ cluster , SNP6 damv score (IBSmean/IBSvar) and ranked it against tumor content assessed by pathologist  Seven matched pairs (5 from the 82 samples in SNP6) were sequenced using array-based sequence capture (Agilent SureSelect) and Illumina GAIIx sequencer, targeting mostly the exons. The data was put through our sequencing pipeline consisting of base calling using the Illumina Pipeline, mapping and alignment with BWA http://www.chromosomechronicles.com/2009/10/22/identity-by-state-snp-analysis-find-relatives-test-paternity-and-determine-allele-sharing/). The R code for IBS computation is available at http://research.duke-nus.edu.sg/papers/IBS.zip.Any two individuals (or samples), whether related or not, can share 0, 1, or 2 alleles, denoted as IBS-0 , IBS-1 , and IBS-2  respectively. Alleles in the dataset were coded using A and B and pairwise IBS was computed between all samples. See Figure S1IBS landscape of 7 matched pairs in NGS.(TIF)Click here for additional data file.Figure S2Comparison of IBS for 20K, 25K, and 30K SNPs using our algorithm (left) and GRR (right). Note that GRR shows standard deviation (y-axis) instead of variance. Clustering in both plots is similar regardless of the number of SNPs indicating that there is no bias and a smaller set of SNPs would suffice for assessing matched tumor-normal pairs.(TIF)Click here for additional data file.Figure S3IBS plot of all 868155 SNPs. The clustering is similar to the datasets of 20K, 25K, and 30K SNPs indicating that the clustering is not bias by number of SNPs. Note that this computation was not possible with GRR and IBS computation for entire SNP dataset can be intensive even with our algorithm.(TIF)Click here for additional data file.Figure S4IBS plot using GRR showed similar clustering. Note that y-axis is standard deviation instead of variance in the manuscript.(TIF)Click here for additional data file.Table S1*Sample 76629543 was sequenced (NGS) and profiled for SNP6.IBS scores and tumor content for the 22 samples in SNP6 data. (XLSX)Click here for additional data file.


There is evidence that traumatic birth experiences are associated with psychological impairments. This study aimed to estimate the prevalence of childbirth-related post-traumatic stress symptoms and its obstetric and perinatal risk factors among a sample of Iranian women.This was a cross-sectional study carried out in Bushehr, Iran during a 3-months period from July to September 2009. Data were collected from all women attending eleven healthcare centers for postnatal care 6 to 8 weeks after childbirth. Those who had a traumatic delivery were identified and entered into the study. In order to assess childbirth-related post-traumatic stress, the Post-traumatic Symptom Scale-Interview (PSS-I) was administered. Data on demographic, obstetric and perinatal characteristics also were collected. Multivariate logistic regression was performed to examine the association between childbirth-related post-traumatic stress and demographic and obstetric and perinatal variables.In all, 400 women were initially evaluated. Of these, 218 women (54.5%) had a traumatic delivery and overall, 80 women (20%) were found to be suffering from post-partum post-traumatic stress disorder (PTSD). Multiple logistic regression analysis revealed that post-partum PTSD was associated with educational level, gestational age at delivery, number of prenatal care visits, pregnancy complications, pregnancy intervals, labor duration, and mode of delivery.The findings indicated that the prevalence of traumatic birth experiences and post-partum PTSD were relatively high among Iranian women. The findings also indicated that obstetric and perinatal variables were independently the most significant contributing factors to women’s post-partum PTSD. It seems that a better perinatal care and supportive childbirth might help to reduce the burden of post-partum PTSD among this population. The post-traumatic stress disorder (PTSD) after childbirth was initially described by Bydlowski and Raoul-Duval. SeveralThe PTSD is an anxiety disorder that can occur secondary to exposure to extreme events (stressors) that are at outside of the usual range of human experience. ChildbiThere is evidence that traumatic birth experiences are associated with post-traumatic stress disorder symptoms-12. StudEvery year about 1,363,542 women in Iran give birth. Of thesThis was a cross sectional study carried out in Bushehr, Iran during a 3-months period from July to September 2009. Data were collected from all women attending eleven healthcare centers for postnatal care 6 to 8 weeks after childbirth. They were interviewed to indicate those who experienced a traumatic birth. One of us (SA) carried out all interviews in a private setting. Women who did not experience a traumatic delivery and those with stillbirth or neonatal deaths were excluded. Subsequently, the following questionnaires were completed for women who experienced a traumatic childbirth.1. A study specific questionnaire in order to collect data on demographic, obstetric and perinatal information.2. The Post-traumatic Symptom Scale-Interview (PSS-I): This is a 17-items questionnaire that assesses PTSD symptoms based on the Diagnostic and Statistical Manual of Mental Disorders-Revision 4 (DSM-IV). It contParticipants were classified as with and without post-partum PTSD based on their PSS-I diagnosis. Descriptive statistics including chi-square were used to explore the data. Forward conditional multiple logistic regression analysis was performed to examine the association between dependent (post-partum PTSD) and independent variables. The level of significance was set at 5%. The SPSS version 16 was used to analyze the data.The ethics committee of Tehran University of Medical Sciences approved the study. We obtained written informed consent from participants after comprehensive explanation of procedure involved.In all 400 women were studied. Of these, 218 women (54.5%) experienced a traumatic delivery. The mean age of participants (n = 218) was 26.9 (SD = 4.83) years. The mean number of children was 1.77 (SD = 0.86), and 119 women (54.6%) were experiencing their first childbirth. Thirty-four women (15.6%) completed their formal education at higher level. Forty-nine women (22.5%) had unwanted pregnancy; and 15 women (6.9%) had less than 8 times prenatal care visits (lower than the routine care). There were 139 women (63.8%) who had complicated pregnancy and 13 women (6.0%) had hospital admissions due to these complications. The duration of labor was lower than 3 hours in 32 women (14.7%), 67 women (30.7%) had emergency cesarean section, 60 (27.5%) had elective cesarean section, and 91 (41.7%) had normal vaginal delivery. Eighteen women (8.3%) had preterm deliveries and 14 women (6.4%) have delivered low birth weight babies (<2.5 kg). Overall, 80 women (20%) met the criteria for post-partum PTSD. Further analysis of the data showed that 131 women (60.1%) were suffering from re-experiencing, 91 (41.7%) from avoidance, and 120 (55.0%) from increased arousal. The detailed results are shown in TableThe relationship between post-partum PTSD and independent variables was examined by performing forward conditional multiple logistic regression analysis. The diagnosis of post-partum PTSD was considered as outcome variable and all significant findings in univariate analyses were considered as independent factors. As shown in TableThe findings from present study indicated that more than half of women experienced a traumatic delivery. In addition we found that post-partum PTSD was associated with low educational level, premature labor, inadequate prenatal care visits, having complications due to pregnancy, pregnancy intervals less than 2 years, labor duration, and emergency cesarean section.There are many studies that investigated about post-partum PTSD,26-29. FIt has been shown that emergency cesarean section compared to elective cesarean section and normal vaginal delivery was more unpleasant birth experience and also was a risk factor for development of post-partum PTSD,30. In fUnlike the study by Adewuya et al. that did not find any association between socio-demographic factors and post-partum PTSD, the finDuration of labor and complications of pregnancy were found to be associated with post-partum PTSD. Studies showed that the increased medical interventions during labor, more intense pain in the first stage of labor and prolonged labor duration and perinatal complications similarly increase the likelihood of unfavorable birth experiences and consequently resulting in developing post-traumatic stress symptoms,34.The finding from this study showed that preterm labor was associated with post-partum PTSD. Similarly, studies have reported that the birth of a premature infant was a predictor for the development of post-traumatic stress symptoms.Prenatal care is an essential reproductive health service, and demonstrated that women who regularly see their health care providers during their pregnancy are less likely to expereince serious pregnancy complications. The finAlthough unwanted pregnancy was significant factor in univariate analysis (see TableIn general, both fathers and mothers might be affected by a traumatic childbirth, but mothers are more vulnerable to PTSD, especially those who are unprivileged. Unfortunately in developing countries, there are few supporting services for women who exposed to a traumatic childbirth, althougThis study had some limitations. Firstly, this was a cross-sectional study and thus the results should be interpreted with caution. Secondly, we only included demographic and obstetric and perinatal factors in the analysis, while post-partum PTSD symptoms may occur among women who have experienced a history of trauma regarding other factors including locus of control, perceptions of the mother-child relationship, partner attachment and perception of partner support. Also we obtained our sample from public health services, thereby excluding women who did not use any healthcare facilities. Finally, since women from different cultural backgrounds might show different patterns for post-partum PTSD, the results from this study could not be generalized to western women or even all women from Iran.The findings indicated that the prevalence of traumatic birth experiences and post-partum PTSD were relatively high among Iranian women. The findings also indicated that obstetric and perinatal variables were independently the most significant contributing factors to women’s post-partum PTSD. It seems that a better care, supportive childbirth, and attention paid to human rights for women might help to reduce the burden of post-partum PTSD among this population.The authors declare that they have no competing interests.MM supervised the study. SA was the main investigator, designed the study, and collected the data. PR helped the main investigator to analyze the data and wrote the first draft. AM contributed to the analysis, critically evaluated the paper, and provided the final draft. All authors read and approved the final revision of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/12/88/prepub


Few data exist on survival or health-related quality of life (QOL) related to herbal remedy use among long-term breast cancer survivors. The objective of this report is to examine whether herbal remedy use is associated with survival or the health-related QOL of these long-term breast cancer survivors.In 1999-2000, we collected the information of herbal remedy use and QOL during a telephone interview with 371 Los Angeles Non-Hispanic/Hispanic white women who had survived more than 10 years after breast cancer diagnosis. QOL was measured using the Medical Outcomes Study Short Form-36 (SF-36) questionnaire. Patients were followed for mortality from the baseline interview through 2007. 299 surviving patients completed a second telephone interview on QOL in 2002-2004. We used multivariable Cox proportional hazards methods to estimate relative risks (RR) and 95% confidence intervals (CI) for mortality and applied multivariable linear regression models to compare average SF-36 change scores (follow-up - baseline) between herbal remedy users and non-users.P = 0.16), but herbal remedy users' physical component summary scores decreased more than those of non-users .Fifty-nine percent of participants were herbal remedy users at baseline. The most commonly used herbal remedies were echinacea, herbal teas, and ginko biloba. Herbal remedy use was associated with non-statistically significant increases in the risks for all-cause  and breast cancer  mortality. Both herbal remedy users' and non-users' mental component summary scores on the SF-36 increased similarly from the first survey to the second survey (Our data provide some evidence that herbal remedy use is associated with poorer survival and a poorer physical component score for health-related QOL among women who have survived breast cancer for at least 10 years. These conclusions are based on exploratory analyses of data from a prospective study using two-sided statistical tests with no correction for multiple testing and are limited by few deaths for mortality analysis and lack of information on when herbal remedy use was initiated or duration of or reasons for use. While the use of complementary and alternative medicine , has increased among the general population . CAM useP = 0.056) during the follow-up period than non-CAM users [The relationship between CAM or herbal remedy use and cancer patients' survival has rarely been studied. One Norwegian study following 252 patients with a mix of different cancers for 5 years reported that CAM use had no impact on mortality . AnotherAM users . NeitherMost previous studies that evaluated CAM or herbal remedy use by patients in relation to QOL were conducted within 5 years of their initial diagnosis -14. ReceWe previously reported that, among women who were long-term (> 10 years) breast cancer survivors and aged 40 years or younger when initially diagnosed, herbal remedy users had a lower mental component summary score on the Medical Outcomes Study Short Form-36 (SF-36) questionnaire than non-users, but herbal remedy users and non-users did not differ on the SF-36 physical component summary scores . In our in situ or invasive breast cancer between July 1, 1983, and January 1, 1989, who were 40 years of age or younger at diagnosis, and who were born in the United States, Canada, or Europe, were eligible for participation in the parent study. Nine hundred and sixty nine eligible breast cancer patients were identified by the Los Angeles County Cancer Surveillance Program (LA-CSP), a population-based SEER registry. Of the 969 eligible women with incident breast cancer, 225 were not interviewed for the following reasons: death (n = 20), physician refusal (n = 54), serious mental or physical illness (n = 7), patient refusal (n = 111), no longer living in Los Angeles County (n = 12), and inability to be located (n = 21). Interviews were completed with 744 breast cancer patients .Participants were breast cancer patients enrolled in a Los Angeles County population-based epidemiologic case-control study of breast cancer among Non-Hispanic/Hispanic white women who were 40 years or younger at diagnosis (referred to as the "parent study"). Details of the parent study have been published . BrieflyThe 744 participants were traced in 1998 and interviewed in 1999 and 2000 by telephone to collect the detailed information regarding herbal remedy use and concurrent QOL. Of the 744 women, 276 were known to have died by the time the baseline telephone interview for herbal remedy use was initiated; 78 women were not located; 6 subjects refused participation; and 10 women were interested but unable to schedule an interview during the study period. Three hundred seventy-four women were interviewed. The interviews were conducted, on average, 13 years after diagnosis. The cross-sectional relationship between herbal remedy use and QOL has been reported elsewhere [The 374 women who participated in our baseline telephone interview for herbal remedy use were followed for survival through December 31, 2007 by linking with the LA-CSP and the National Death Index. If death occurred, information on date of death and cause of death was collected. Person-days of follow-up for each woman began on the date of baseline telephone interview and ended on her date of death (n = 44 including 33 who died of breast cancer) or December 31, 2007 (n = 330). Among 33 who died from breast cancer, 13 had metastasis of breast cancer; 2 had recurrence of breast cancer; 2 had a contralateral breast cancer; 10 had a combination of these three conditions, and the other 6 women did not have any of these three conditions by the time of our baseline telephone interview. We excluded three women with incomplete questionnaire data who were alive at the end of the follow-up period. Therefore, 371 women were eligible for survival analysis.We conducted a follow-up telephone interview in 2002-2004 to collect additional SF-36 QOL information 2-5 years after the initial collection of herbal remedy and QOL information. Of 374 case patients participating in the baseline telephone interview, 26 were known to have died when we began the follow-up interview. Of the remaining 348 women, 17 women were not located (4.9%); 26 subjects preferred not to participate (7.5%); and 3 women were too ill to participate (0.9%). Three hundred two women were interviewed. After excluding the same three women with incomplete questionnaire data, 299 women remained for analysis of the association between herbal remedy use and QOL. The recruitment results are combined and shown in Figure In the parent study, we obtained data on summary stage at breast cancer diagnosis from the LA-CSP. The baseline telephone interview for herbal remedy use (1999-2000) obtained information on herbal remedy use, SF-36 QOL measures, psychosocial functioning, cancer-related factors including recurrences, breast-cancer surgeries and reconstructive history, treatments received for breast cancer, and comorbidities. We also updated demographic information and information on lifestyle practices after breast cancer diagnosis.Women were given a list of herbal remedies and asked whether they had used any of the listed remedies within the past six months. The remedies listed were bee pollen, licorice root, black cohosh, mother wort, blue cohosh, nux vomica, chaste berries, progesterone topical (vitex agnus cactii), progesterone cream (wild Mexican yam), chickweed tincture, pulsatilla, dong quai (tong Kwai), royal jelly, echinacea, sage tea, evening primrose oil, sarsaparilla, false unicorn, sepia, garlic, St. John's Wort, ginko biloba, valeriana, ginseng, wild yam root, herbal tea used as a remedy, shark cartilage, or lachesis. Use of any of the herbal remedies qualified a woman as an herbal remedy user.The follow-up telephone interview (2002-2004) again measured SF-36 QOL and psychosocial functioning; updated demographic information and lifestyle practices and cancer-related and treatment-related factors including additional surgeries. Subjects were mailed a list of scale responses to the QOL and psychosocial instruments prior to each of their telephone interviews to facilitate the interview process.Health-related QOL was assessed using the SF-36 questionnaire at each telephone interview. This widely used self-report measure contains 36 items in eight subscales  ,23. EachAll participants who had previously signed an informed consent as part of their recruitment for the parent study gave a verbal informed consent prior to the conduct of each telephone interview. The study was approved by the Institutional Review Boards at the University of Southern California and the City of Hope.We compared demographic characteristics between herbal remedy users and non-users using Pearson chi-square tests for differences in frequency distributions.Survival time was calculated as the time from the collection of herbal use at baseline telephone interview to the first of the following events: date of death or the end of follow-up on December 31, 2007. For breast cancer specific survival, we censored women who died from other causes on their dates of death. We used the Kaplan-Meier method to calculate survival rates and applied the log-rank test to test for any difference in survival between herbal remedy users and non-users.Multivariable Cox proportional hazards regression models were fit separately for all-cause and breast cancer mortality to assess the hazard rate ratios of mortality associated with a woman's herbal remedy use within the 6 months prior to the baseline telephone interview. Hazard rate ratios, presented as relative risks (RRs) with 95% confidence intervals (CIs) were estimated using age in days at the start and end of follow-up to define a woman's participation time in the study.Among women who participated in the follow-up telephone interview, we used t-tests to compare the QOL summary scale measures between herbal remedy users and non-users at baseline and follow-up surveys, respectively. We further applied multivariable linear regression models to compare average SF-36 health summary scale change scores (change score = follow-up score - baseline score) between herbal remedy users and non-users.in situ, localized invasive, non-localized invasive), type of non-surgical treatment for initial breast cancer , type of surgery , post-diagnosis cancer-related conditions , comorbidities within six months of baseline interview , interval between diagnosis and initial interview , and annual income  at baseline interview. In addition, when comparing average SF-36 health summary scale change scores between herbal remedy users and non-users, multivariable linear regression models were adjusted for the SF-36 scores at baseline.All multivariable models adjusted for the following factors, selected a priori, as potential confounders: ethnicity , age at diagnosis , stage of cancer at diagnosis  or a 180% or greater increase in risk (RR = 2.8) for overall mortality with 80% statistical power given a 2-sided hypothesis test with a 5% level of statistical significance .P-values of 0.05 or less were considered statistically significant. We did not adjust P values for multiple comparisons as these analyses were considered as exploratory [Two-sided loratory . All anaOf 371 breast cancer patients, 217 (58.5%) reported having used herbal remedies within six months of baseline interview .Participants in the cohort were followed for an average of 7.8 years following their baseline telephone interview. During the follow-up, 44 women died, including 33 who died from breast cancer. The Kaplan-Meier survival curve showed that both overall survival and breast cancer survival of CAM users appeared to be poorer than that of non-CAM users, but the differences were not statistically significant , and breast cancer mortality  Table . FurtherP = 0.05) than women who reported no herbal remedy use, but their mean SF-36 physical component scale summary score was not statistically different from that of non-users (P = 0.37). However, 2-5 years after herbal remedy use information was collected, herbal remedy users' SF-36 mental and physical component summary scores were lower than those for non-users .Univariate analyses showed that at the baseline interview assessment, women reporting herbal remedy use had a lower mean SF-36 mental component summary score (P = 0.16), but herbal remedy users' physical component summary scores decreased more than those of non-users , physical role limitations , bodily pain , and general health  subscales and a smaller increase in the social functioning subscale . No statistically significant difference in change scores was observed for the most commonly used specific types of herbal remedies .Multivariable analyses of change scores between baseline and follow-up survey showed that both herbal remedy users' and non-users' mental component summary scores on the SF-36 increased similarly from baseline to follow-up ; we did not adjust for multiple comparisons as, with the relatively small number of participants, these analyses were consider as exploratory.This study has several important limitations which may affect the results we observed. First, although our results were obtained from a prospective study of young breast cancer patients who had survived more than 10 years, 276 (37%) breast cancer patients had died by the time we initiated this study, so that participants were more likely to be a select group of healthy women who were long-term survivors. Thus, our findings may not be generalizable to all breast cancer patients, but they do provide important information regarding long-term survivors of breast cancer who were diagnosed at a young age. Second, herbal remedy use, as measured in our study, represents use during a six month time period beginning, on average, 12.5 years after breast cancer diagnosis. We did not collect information regarding the timing of initiation of herbal remedy use, duration or frequency of use or reasons for use. Herbal teas were commonly used herbal remedies among our study population, but we did not collect the components of herbal teas or brand of herbal teas or whether any additives had been added to herbal teas to alter color or taste. Third, we relied on self reported information on herbal remedy use. Errors in reporting use of herbal preparations are possible. However, we worded questions carefully, mailed scales and questionnaire items in advance of the telephone interview to enhance comprehension and consistency of reporting, and limited the questions to recent herbal remedy usage (within the past six months of interview). Fourth, among 33 women who died from breast cancer during our follow-up for survival, 27 had post-diagnosis cancer-related conditions including two who had a contralateral breast cancer by the time of our baseline telephone interview. Unfortunately, we were unable to collect this information throughout the follow-up for survival. Therefore, we cannot rule out the possibility that women might have died of a new breast cancer rather than their first primary breast cancer. Fifth, due to the relatively small number of participants, we only assessed the effects for overall herbal remedy use and three commonly used herbal remedies. We were unable to explore the effects for those having a lower frequency of use among our participants. In fact, even for overall herbal remedy use, the small number of deaths led to insufficient statistical power to detect small or moderate differences in mortality risk between herbal remedy users and non-users. Due to the same limitation, our main exposure variable, herbal remedy use, grouped all types of herbal remedies. The definition of herbal remedy use was therefore non-specific. Finally, six of forty Our data show that herbal remedy use tended to be associated with a poor survival among long-term breast cancer survivors although the associations did not reach statistical significance, due to the small number of deaths and further deaths could alter the direction of findings. Our findings are consistent with a Norwegian study which followed 515 patients with a variety of cancers for up to 8 years and showed that CAM use was associated with poorer survival; 79% of CAM users died during follow-up compared with 65% of nonusers . HoweverOur data also showed that herbal remedy users' physical component summary scores decreased more than those of non-users between the first and second surveys. Most previous studies that have evaluated CAM or herbal remedy use in relation to QOL were conducted among patients within 5 years of initial diagnosis -14; howeReasons are unclear for our observation that herbal remedy use was associated with a poorer survival or greater decline in physical health summary scores between our two surveys. A recent published Norwegian study investigating CAM use in 735 cancer patients who had survived 5 years or more after breast cancer diagnosis, found CAM use initiated after diagnosis was associated with a poor prognosis suggesting that CAM might be used by those with less hope of a cure by conventional therapy . AlthougWe also considered that the lower physical QOL scores of the herbal remedy users might result from some of the users opting out of conventional therapy for breast cancer. Therefore, we adjusted for types of initial treatments  in our analyses. Based on previous studies, approximately 5-12% of cancer patients may abandon appropriate therapy and pursue alternative treatments ,34; yet It is possible that herbal remedy use could have interfered with chemotherapy treatment by inhibiting some enzymes in the cytochrome P450 (CYP) family, such as CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 ,36 if thOur data provide some evidence that herbal remedy use is associated with a poorer survival and a poorer physical component score for health-related QOL among long-term (> 10 years) breast cancer survivors. These conclusions are based on the exploratory data from a prospective study using two-sided statistical tests with no correction for multiple testing and are limited by the few deaths for the survival assessment and the lack of information regarding the timing of initiation of herbal remedy use or duration or frequency of, or reasons for herbal remedy use. The potential roles for herbal remedies in breast cancer patients' survival and QOL merit further exploration.CAM: complementary and alternative medicine; CI: confidence interval; LA-CSP: Los Angeles County Cancer Surveillance Program; QOL: quality of life; RR: relative risk; SEER: Surveillance, Epidemiology, and End Results; SF-36: Short Form-36.The authors declare that they have no competing interests.HM obtained funding for the study of herbal remedies, carried out the statistical analysis and drafted the manuscript. LB obtained funding for the original study and the follow-up interviews, designed and implemented the original and follow-up studies, and supervised data collection, data management, and data cleaning. CLC assisted with questionnaire design and participated with JSH in data collection, data management, and data cleaning. All authors participated in the revision of the manuscript and have read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/222/prepub