Chromosomal instability (CIN) is a hallmark feature of cancer cells. In this study, Schukken and colleagues screen for compounds that selectively target CIN cells and identify an inhibitor of Src kinase to be selectively toxic for CIN cells. Chromosomal instability (CIN) and aneuploidy are hallmarks of cancer. As most cancers are aneuploid, targeting aneuploidy or CIN may be an effective way to target a broad spectrum of cancers. Here, we perform two small molecule compound screens to identify drugs that selectively target cells that are aneuploid or exhibit a CIN phenotype. We find that aneuploid cells are much more sensitive to the energy metabolism regulating drug ZLN005 than their euploid counterparts. Furthermore, cells with an ongoing CIN phenotype, induced by spindle assembly checkpoint (SAC) alleviation, are significantly more sensitive to the Src kinase inhibitor SKI606. We show that inhibiting Src kinase increases microtubule polymerization rates and, more generally, that deregulating microtubule polymerization rates is particularly toxic to cells with a defective SAC. Our findings, therefore, suggest that tumors with a dysfunctional SAC are particularly sensitive to microtubule poisons and, vice versa, that compounds alleviating the SAC provide a powerful means to treat tumors with deregulated microtubule dynamics. Chromosomal INstability (CIN) is the process through which chromosomes mis-segregate during mitosis. CIN leads to cells with an abnormal DNA content, a state known as aneuploidy. As three of four cancers are aneuploid , CIN is To protect from CIN, cells have mechanisms in place that maintain proper chromosome inheritance. The Spindle Assembly Checkpoint (SAC) is one such mechanism preventing CIN by inhibiting the onset of anaphase until all chromosomes are properly attached to the two opposing spindle poles, reviewed in detail by Although SAC impairment is rare in human cancer , many caAs CIN and aneuploidy discriminate cancer cells from healthy cells, both make for attractive targets for cancer therapy. To reveal potential general vulnerabilities of aneuploid cells, Although CIN and aneuploidy are intimately related, CIN has additional effects on cell physiology and growth in addition to those imposed by the resulting aneuploidy . BecauseIn this study, we, therefore, performed two small-scale drug screens, one to identify small molecule compounds that target aneuploid cells and another to find compounds that are more toxic to CIN cells than to chromosomally stable cells. For this purpose, we selected a collection of drug-like molecules from a list of drugs already being used in the clinic or in advanced-stage clinical trials. Compounds were further selected for their potential role in targeting CIN or aneuploid cells, such as targeting cell survival , prolifeCIN and the resulting aneuploidy lead to a deregulated transcriptome and proteome , 2014 anWe first selected 95 drug-like molecules from a drug library composed of drugs that target processes that aneuploid or CIN cells might rely on and are already being used in the clinic or being tested in clinical trials (Table S1). Next, we determined the initial drug concentration for each drug to be used in the screen. For this, we exposed wild-type RPE1 cells  to decrList of compounds used in the drug screen to identify compounds that selectively target aneuploid cells.Table S1 Supplemental Data 1) using an IncuCyte high content imager. 2 > 0; P < 0.05) or less sensitive , we only found one compound  for which the effect was significant after Bonferroni multiple testing correction  5 and 12 . IndeedSupplemental Data 1.Growth curves of aneuploid RPE1 cells exposed to small molecule compounds. Raw data used to produce cKD) RPE1 cells. Mad2 knockdown efficiency was quantified by quantitative PCR , which we corrected for in our downstream analyses in each of our experiments  and compared the maximum proliferation rate and cumulative cell number between Mad2cKD RPE1 cells and control RPE1 cells using the same setup as for the aneuploidy screen described above. To assess both short-term and longer term effects of the drugs, we quantified proliferation and cumulative cell number over the first 4 d and over days 5–8 separately  during the first 4 d of the screen, but became fully toxic to both control and Mad2cKD cells from day 5 onward . Conversely, we found that the Src inhibitor SKI606 (compound #1407) at 0.1 μM acted synergistically with CIN  during the second half of the screen  and less so during the first half of the screen. Note that the observed effects were not related to the doxycycline treatment required to induce Mad2 shRNA, as doxycycline alone had no effect on proliferation  as SKI606  off-target effects, we also wanted to confirm the synergy between Src inhibition and an impaired SAC at the genetic level. For this, we designed three inducible shRNA constructs for Src of which one (shRNA3) yielded a significant knockdown of Src protein levels in RPE1 cells  displayGiven our results and a known role for Src in spindle orientation  and MT nVideo 1Download videoRPE1 cells expressing EB3-GFP to track MT polymerization rates quantified in Video 2Download videoRPE1 cells expressing EB3-GFP treated with SKI606 to track MT polymerization rates, quantified in Video 3Download videoHCT116 cells expressing EB3-GFP to track MT polymerization rates quantified in Video 4Download videoHCT116 cells expressing EB3-GFP treated with SKI606 to track MT polymerization rates quantified in Video 5Download videoHT29 cells expressing EB3-GFP to track MT polymerization rates quantified in Video 6Download videoHT29 cells expressing EB3-GFP treated with SKI606 to track MT polymerization rates quantified in Video 7Download videoSW620 cells expressing EB3-GFP to track MT polymerization rates quantified in Video 8Download videoSW620 cells expressing EB3-GFP treated with SKI606 to track MT polymerization rates quantified in cKD RPE1 cells, confirming the synthetic lethality between SAC inhibition and deregulating MT polymerization rates . Also in this setting, the observed synergy between low doses of nocodazole and SAC inhibition coincided with increased chromosome missegregation rates: whereas 33 nM nocodazole provoked mitotic abnormalities in only 6% of control RPE1 cells, 83% of nocodazole-exposed Mad2cKD RPE1 suffered from defective mitoses, compared with 31% in the absence of nocodazole . Alternatively, as ongoing CIN and aneuploidy trigger  different responses in cells (Preprint), they might also display differential vulnerabilities.Notably, ZLN005 did not emerge as a compound selectively targeting cells with an ongoing CIN phenotype (Mad2in cells PreprintcKD RPE1 cells). This screen identified SKI606, a Src inhibitor as a compound that selectively impairs accumulation of cells with an alleviated SAC. Importantly, we validated the phenotype with another Src inhibitor and confirmed that the effect of the inhibitors is caused by Src inhibition because genetic perturbation of Src by shRNA with SAC inhibition yields the same phenotype as inhibitor treatment with SAC inhibition. Of note, we only succeeded in reducing Src protein levels by approximately twofold using shRNA and failed to engineer Src knockout cell lines using CRISPR engineering (data not shown), which suggests that cells critically rely on some remaining Src kinase activity for their survival. Therefore, to phenocopy the selective targeting of CIN cells in vivo in future studies, it will be important to titrate drug concentrations well.In addition to screening for compounds that selectively prevent accumulation of aneuploid cells, we also screened for compounds that selectively target cells with a CIN phenotype. For the latter, we engineered RPE1 cells in which we could alleviate the SAC in an inducible fashion .RPE1 and MCF7 (American Type Culture Collection) cells were grown in DMEM supplemented with 10% FBS and 100 units/ml penicillin and 100 μg/ml streptomycin. HT29 cells were growth in McCoy media supplemented with 10% FBS and Pen/Strep as above. Aneuploid cells RPE1 with trisomy 12 and trisomy 5 (Ts12 Ts5) and RPE1 hTert cells were kindly provided by the Storchova laboratory . Most drSupplemental Data 3.Drug titration curves used to determine starting drug concentrations for aneuploidy and CIN screens as described in the Materials and Methods section.cKD RPE1 cells were generated by transducing RPE1 cells with a lentiviral construct targeting human Mad2l1 (5′-GGAAAGAATCAAGGAGG-3′) in a pTRIPZ backbone . The cells were selected in 2 μg/ml puromycin for 48 h and single-cell clones picked. Knockdown efficiency was determined for several clones by Western blot and the clone with the largest Mad2 reduction was used for further experiments. SrccKD RPE cells were generated by transducing lentiviral shRNA constructs into wild-type RPE1 cells. For this purpose, Src shRNA constructs were engineered into a doxycycline-inducible pLKO backbone that also contains the Tet operator sequence to allow for conditional activation of the shRNA . Protein quantification was performed using Quick Start Bradford assay, the Quick Start BSA standard kit (Bio-Rad Inc.), or the MultiScan Go (Thermo Fisher Scientific). Antibodies used were Actin , Tubulin , Src , and Mad2 . Secondary antibodies were Anti-Mouse IRDye 680RD  and Anti-Rabbit IRDye 800CW . Blots were visualized and quantified using the Odyssey imaging system (Li-Cor).PRJEB33217.For single-cell sequencing, the cells were harvested, nuclei isolated, and stained with Hoechst using nuclear isolation buffer. Single nuclei were then sorted into 96-well plates using a FACSJazz sorter (BD Bioscience). Single-cell DNA libraries were prepared and sequenced  with ∼1% genomic DNA coverage as described previously . SequencCells were cultured with 100 ng/ml Colcemid for 3 h, harvested, incubated in 75 mM KCl for 15 min and fixated in 3:1 methanol: acetic acid. Fixated cells were dropped on glass slides and nuclei visualized using DAPI staining. Metaphase figures were inspected on an Olympus BX43 microscope using a 63× lens. A minimum of 50 karyotyping spreads were counted per condition.cKD cells were treated with 1 μg/ml doxycycline for a minimum of 3 d before the start of any screen and sorted into wells with 1 μg/ml doxycycline. Each well contained media with drugs at the concentrations listed, and all measurements were performed with technical triplicates for each plate. Cell growth was monitored every 2 h using an IncuCyte Zoom live-cell analysis system (Essen BioScience Ltd.). Drug-containing media were refreshed every 4 d, and for the CIN screen, the cells were passaged 1:8 on day 4. Cell density was quantified using IncuCyte ZOOM 2018A software. Cell confluency of control and CIN cells with drugs were normalized to DMSO-treated cells  and calculated using two different approaches: AUC and slope analysis. As for the screens, multiple drugs were tested per plate, the same DMSO control was used (at least one per plate) to compare the effects of the drug-treated cells. The figure legends indicate in which panels the same DMSO control was used.For aneuploid drug screens, 200 RPE1 cells were sorted into each well of 96-well plates by flow cytometry . For the CIN screen and follow-up screens, 1,600 cells were seeded per well in a 96-well plate. For the latter, RPE1 Mad2cKD cells were all analyzed as different cell lines so that the relative effect of each perturbation could be compared per cell type. These relative AUC values were then compared between cell lines per drug using a two-sided t test. P-values in the screen were corrected for multiple testing using Bonferroni correction , and RPE1 Mad2rrection .t test. The modified IncucyteDRC R package for screen slope analysis is available upon request.An existing R script to analyze IncuCyte data  was modidrug-treated cells/AUCcontrol cells). Next, we calculated the expected inhibitions, assuming drugs and CIN/aneuploid conditions were additive, using the Bliss independence equation: expected inhibition = Fa + Fb − (Fa*Fb), where Fa is fractional growth inhibition of drug A and Fb was the fractional growth inhibition of either CIN or aneuploid cell conditions. The expected inhibition was compared with the actual growth inhibition; the P-value was determined using a t test, and the greater-than-additive toxicity was found by taking the difference between expected and actual fractional inhibition.To validate that the observed effects in the compound screens were synergistic and not only additive, we made use of the Bliss independence test. For this, we calculated the fractional growth inhibition, defined as 1-/Tcutoff. This was divided by the average slope of the cell line control to compensate for cell line growth differences. The replicates of the relative slope values were compared between cell lines per condition.Growth curves were plotted on a log scale and the logarithmic growth cutoff point was estimated manually for each condition. To calculate the slope, the negative log of the confluency at that time-point was divided by the cutoff day: −log. Interphase phenotypes were analyzed by quantifying nuclear morphology. Mitotic abnormalities were manually quantified from overnight live cell imaging movies. Measurements include at least three biological replicates, and numbers of cells quantified are indicated in the text. A chi-squared test was used to test whether differences between conditions were significant. Mitotic time was analyzed by calculating the time-points between the first sign of DNA condensation to the last point before anaphase, and from the first anaphase time-point to the time-point at complete DNA de-condensation from time-lapse imaging data.RPE1, Mad2t tests.To quantify cell motility, time-lapse movies were analyzed using FIJI TrackMate . A minimt test.MT plus end assembly rates were determined by tracking EB3-GFP protein (vector kindly provided by Linda Wordeman) in live cell microscopy experiments as in


The discovery of circulating tumour DNA molecules created a paradigm shift in tumour biomarkers as predictors of recurrence. Non-invasive prenatal testing (NIPT) to detect circulating cell-free foetal DNA in maternal plasma is increasingly recognised as a valuable substitute to perceive foetal copy number variation (CNV). This study aimed to determine whether the copy number detection in plasma samples using NIPT platform could be used as a prognostic biomarker in patients with gynaecological cancer. We conducted a prospective study using samples containing preoperative plasma from 100 women with gynaecological cancers. Samples were randomly rearranged and blindly sequenced using a low-coverage whole-genome sequencing plasma DNA, NIPT platform. The NIPT pipeline identified copy number alterations (CNAs) were counted in plasma as a gain or loss if they exceeded 10 Mb from the expected diploid coverage. Progression-free survival (PFS) and overall survival (OS) were analysed according to the presence of CNA in plasma using Kaplan–Meier analyses. The NIPT pipeline detected 19/100 cases of all gynaecological cancers, including 6/36 ovarian cancers, 3/11 cervical cancers, and 10/53 endometrial cancers. Patients with CNA in plasma had a significantly poorer prognosis in all stages concerning PFS and OS. Therefore, low-coverage sequencing NIPT platform could serve as a predictive marker of patient outcome. One such example involves a patient who was diagnosed with metastatic small cell carcinoma of the vagina that was suggested to account for aneuploidies of chromosome 18 and 13 identified using NIPT2. In other reports, cell-free DNA discordances were determined using MPS for NIPT, and two patients with Hodgkin’s Disease4 were identified. In a recent study, use of a clinical NIPT platform detected early-stage ovarian cancer5. As potential biological explanations for cell-free DNA discordance include confined malignancy, this suggests that genomic profiling by the NIPT platform, which is broadly used for testing foetal aneuploidies, may also represent a practical approach for clinical neoplasm management.In recent years, cell-free DNA has been broadly studied using circulating tumour DNA (ctDNA) as a liquid biopsy, including the detection of minimal residue, early detection of resistance to therapy, early detection of disease and assessment of molecular heterogeneity. Occult maternal malignancies can be detected via non-invasive prenatal testing (NIPT) using massively parallel sequencing (MPS) of cell-free DNA from the maternal plasma for prenatal screening of common foetal autosomal aneuploidies and trisomies 21, 18 and 13. In many cases, the cell-free DNA in the plasma of pregnant women is a mixture of placental and maternal DNA. Follow-up studies have demonstrated that some cell-free DNA events are discordant with the direct foetal karyotype and may detect asymptomatic neoplasms in the mothers8. Cell-free DNA released from apoptotic cells is shortened to 185–200 bp-fragments. DNA fragments are released into the bloodstream from dying cells during cell turnover or from apoptotic and necrotic cells9. Under normal physiological circumstances, apoptotic and necrotic cells are cleared by infiltrating phagocytes, and cell-free DNA levels are relatively low. In solid tumours, cell-free DNA is also released via necrosis, autophagy, apoptosis and other physiological events induced by micro-environmental stress and treatment pressure10. This phenomenon suggests that ctDNA may be more likely to originate from genomic regions with an increased euchromatic DNA structure resulting in observed differential fragment length distribution in coverage relative to somatic cell-free DNA. Recent improvements in the analysis of blood samples for circulating tumour cells or ctDNA has provided rapid, cost-effective and non-invasive liquid biopsy surrogates, which provide valuable complementary information on therapeutic targets and drug resistance mechanisms in cancer patients12. Tumour heterogeneity introduces significant challenges in designing effective treatment strategies13. CNV is amplified or deleted in regions of the genome that are recognised as a primary source of average human genome viability and contribute significantly to phenotype variation. One crucial feature arising from previous studies is the observation that tumour DNA carries genomic alterations corresponding to CNA14. CNA plays a significant role in carcinogenesis in many cancers, such as ovarian cancer15, hepatocellular carcinoma16, and colorectal carcinoma17. Several studies have verified that somatic CNVs in ctDNA match those present in the primary tumour18.Several studies have revealed the presence of tumour-derived DNA in the plasma of cancer patients20. These methods require deep sequencing that significantly increases the cost and difficulty to use in clinical practice. Chromosomal instability analysis in cell-free DNA by low-coverage whole-genome sequencing was used for the primary diagnosis of ovarian cancer21. In prenatal testing, several studies have demonstrated the possibility of using whole-genome sequencing-based NIPT to detect fetal CNV23. Recently, several studies using MPS have also reported that personalised analysis of rearranged ends was developed to detect unselected genetic events that span across the whole genome in cancer patients25. These findings demonstrate the performance of cancer genome scanning through MPS of plasma DNA.Genome-wide detection of CNA can be characterised in ctDNA, acting as tumour biomarkers with excellent sensitivity and specificity26. The critical advantage of MPS technologies is the reduced cost and time required to sequence a sample. This method allows for more examples to be investigated than previously possible, and an increase in available information and statistical power has resulted in the identification of many new genes thought to be involved in cancer biology. We hypothesised that CNA in plasma derived from tumours would be detected in patients with gynaecological cancer before primary surgery and would predict prognosis. This study aimed to determine whether the use of an NIPT platform for CNA in plasma from patients with gynaecological cancer could serve as a predictive marker of patient outcome.Several prototype studies also evaluated the low-coverage sequencing method using MPS for the detection of foetal CNVs. Recently, detection of CNA using MPS was reviewedWe enrolled 100 patients with gynaecological cancer in the study and analysed plasma samples from those with ovarian cancer (n = 36), cervical cancer (n = 11) and endometrial cancer (n = 53). We detected CNA in 1/21 early-stage ovarian cancers, 5/15 advanced stage ovarian cancers, 3/11 early-stage cervical cancer cases, 5/41 early stage endometrial cancer cases and 5/12 advanced stage endometrial cancer cases using the NIPT platform . CNA detection by high throughput sequencing still faces analytical challenges due to the rampant biases and artefacts that are introduced during library preparation and sequencing. Studies are gradually producing more robust detection of CNAs, particularly in targeted sequencing panels using hybridisation capture approaches. Targeted sequencing panels focus on individual genes or specific regions of interest. This method supports the detection of identified variants within targeted regions and, but it needs previous knowledge of related regions of the genome, and the variability in efficiency of amplification during library preparation leads to jagged amplicon coverage from one experiment to another. Although several targeted sequencing methods also allow the detection of CNAs, WGS presents an additional advantage of unbiased sequencing and cost33.The present study used the SeqFF method34. Numerous patient cohorts have enabled the precise characterisation of CNAs that predict clinical outcomes in high-grade serous ovarian cancer36. Moreover, in cell model study, CNAsdiffered between matched highly and minimally invasive/migratory subclones of ovarian cancer37. Also, in endometrial cancer, copy number high in a tumour have shown the poorest disease-free survival27. In other cancers, low‐pass whole‐genome sequencing and evaluated CNA in cell-free DNA demonstrates progressive CNA accumulation from stage I to IV and significant association of specific genomic loci with overall survival or metasis39. We have shown that CNAs, as a global measure of the level of CNA across cell-free DNA in plasma, is associated with progression-free survival and overall survival of gynaecological cancer and moreover especially in the advanced stage of ovarian cancer and endometrial cancer. This study further confirmed potential clinical applications of cell-free DNA based CNAs as a promising biomarker for cancer prognosis, especially for advanced stage cancers.In general, increased detection rates can be achieved with a higher input cell-free DNA and higher cancer stages40. Trastuzumab, an antibody to ERBB2 used in breast cancer therapy, provides an excellent example of an amplified cancer gene as a specific therapeutic target41. Some high-level amplifications have been highlighted as predictive biomarkers, including CCNE1, RB1, MYC, ERBB2, PIK3CA, EVI1, AKT2, NOTCH3 and FGFR1 in ovarian cancer42. Frequent increases in DNA copy number at chromosomal region 8q24.3, which contains cancer-related genes such as PTP4A3, have been reported to serve as a prognostic marker in ovarian carcinomas43. This region is also frequently amplified in endometrial cancer44. Another study and analysis of the cancer genome atlas data suggested that chromosomal gains in endometrial endometrioid adenocarcinoma were observed with relatively high frequencies in 1p36–p31 and 1q12–q4445. Our data reveal that three cases of endometrial endometrioid adenocarcinoma had amplification of 1p36–p31 and 1q12–q44 or 8q24.3. Carcinosarcoma, serous adenocarcinoma and leiomyosarcoma cases expressed CNA in many regions.Genomic alterations such as CNAs are known to harbour drivers of carcinogenesis. Several known CNA drivers in cancers include receptor tyrosine kinases, which are targets for drug therapies5. Third, the sample numbers were too small to perform analyses stratified by histological subtypes within the tumour site groups, which could be confounding the survival analysis. Further studies preferably using prospective CNA samples are required for clinical validation of the method and confirmation of the origin of the CNA. The costs and feasibility of sequencing and CNA analysis are continually decreasing. However, the costs are still high to use the genomic data in a clinic. Our data showed the commercial method for non-invasive CNA analysis could apply for using publicly available genomic data to provide the information of the recurrence of cancer in gynaecological cancer patients before surgery.There are several limitations of this study. First, plasma sequencing data was not compared to the tumour DNA and this made it difficult to confirm the tumour origins of the CNA. Second, we only used commercially based techniques for our calculations and did not evaluate the association between the amount of cell-free DNA and size distribution. The principle that tumour DNA is detectable in plasma using NIPT sequencing platforms has been previously reportedIn summary, we have demonstrated that CNA in patients with gynaecological cancer detected using a commercially based NIPT platform could predict recurrence after initial surgery, particularly in advanced stages. The ability of the NIPT platform to identify CNA in plasma has numerous potential clinical applications and provides the opportunity to detect potentially aggressive cancers in pregnant women. As sequencing techniques develop and become more affordable, non-invasive and longitudinal surveillance may become a valuable tool available to clinical oncologists. We focused on the application of CNA in plasma and believed that this method has a broader scope for genetic diagnoses, such as the analysis of ctDNA to detect cancer and predict prognosis, although its clinical utility must be further studied. The software could be used to perform one or more steps in the processes and, regardless of aetiology, detection of ctDNA augmented for cell-free DNA fragment lengths may lead to non-invasive diagnosis of malignancy, improved detection of tumour recurrence, and better monitoring of response to therapy.We performed a case-control study of patients with gynaecological cancer recruited from Showa University Hospital. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the ethical committee of the Show University Hospital . Written informed consent about this study was obtained from all patients before undergoing surgery at our institution. We enrolled 116 participants including 100 gynaecological cancer patients between January 2016 and June 2017 Fig. . Blood sg for 15 min at 25 °C, and the plasma fraction was collected and centrifuged for a second time at 2500 × g for 10 min at 25 °C. After the second spin, the plasma was transferred into barcoded tubes and immediately stored at ≤70 °C until DNA was extracted. Cell-free DNA was extracted from 4 mL of patient plasma using the QIAamp Circulating Nucleic Acid Kit .A sample of 10 mL of whole blood was collected in Cell-Free DNA BCT tubes , centrifuged at 1600 × 46. Automated library preparation was performed on the Zephyr liquid handler  using 96 unique index barcodes, enabling up to 96 sample multiplexing. Libraries were quantified on the LabChip GX . All libraries were normalised to 1.6 nM, multiplexed and sequenced on HiSeq. 2500 with 27 sequencing cycles of the cell-free DNA insert and an additional eight sequencing cycles for the index barcodes.A volume of 40 mL of extracted cell-free DNA was used to prepare libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina , using custom index adapters modified to create 96 unique molecular barcodes with a minimum of three base edit distance47. Reads mapped to each chromosome were aggregated using nonoverlapping 50 kbp-genomic segments. Regions were excluded from analysis based upon high variance, low capability, or a high percentage of repetitive elements as described by Jensen et al.48. Sequencing reads corresponding to the remaining 50 kbp-genomic segments were adjusted for sequencing biases as described in detail by Zhao et al.49. Briefly, sequence reads were first processed with a LOESS-based, sample-specific adjustment, and a principal component analysis-based smoothing was utilised to remove higher order artefacts with a population-based correction.Sequencing reads were aligned to the human reference genome (hg19) using Bowtie 2et al.49. CADET incorporates z-statistics for a CBS-detected CNA to assess the statistical significance and a log odds ratio to provide a measure of the likelihood of an event being real, based on an observed fraction of DNA across the genome49. To further improve the specificity of CNA detection, bootstrap analysis was performed as an additional measure of the confidence of the candidate CNA. The within-sample read count was compared with a standard population and quantified by bootstrap confidence level (BCL). To assess within-sample variability, bootstrap resampling (described below) was applied to every candidate CNA50. For each identified segment within the CNA, the median shift of segment fraction from the average level across the chromosome was calculated. This median change was then corrected to create a read count baseline for bootstrapping. Next, a bootstrapped segment of the same segment length as the candidate CNA was randomly sampled with replacement from the baseline read counts. The median shift was then applied to this bootstrapped fragment and calculated as follows:A multivariate model was derived to predict the tumour fraction from the regional autosome read depth coverage from single-end sequencing to calculate tumour DNA fraction. The amount of CNV can also be used to estimate the tumour DNA fraction, and bins located on chromosomes 13, 18, 21, X and Y are excluded from this method. To determine the association between the response and predictor variables, i.e. the model coefficients, various statistical modelling methods can be employed. Detailed bioinformatics analysis of the previously sequenced DNA sample was performed, and mapped sections of the human genome were analysed using circular binary segmentation (CBS) to identify CNA. The measured Z-scores form part of an enhanced version of Chromosomal Aberration DEcision Tree (CADET) previously described in detail by Zhao 50. A chromosome was classified as having an amplification or a deletion if:This process was repeated 1000 times to generate a bootstrap distribution of segment fractions for an affected population. The median chromosome fraction was calculated specific to each flow cell while the median absolute deviation was a constant value derived from a static median absolute deviation. A threshold was then calculated as the segment fraction that was at least 3.95 median absolute deviations away from the median segment fraction of the reference distribution. Lastly, the BCL was calculated as the proportion of bootstrap segments whose fractions had absolute z-statistics above the significance threshold50; therefore, this value was used in the present study. Using this scale, we counted identified CNA as gains or losses if they exceeded 10 Mb from the expected diploid coverage. These parameters were only used for visual interpretation of the data and were not intended to identify cancer signatures.The comparison U test . We compared progression-free survival (PFS) and overall survival (OS) between patients with CNA present in plasma using the log-rank test for univariate analyses and the Cox proportional hazards for multivariate analyses. Computer analyses were performed using Prism 7 (Graphic Pad Software Inc.). Statistical significance was defined as p < 0.05.Various methods were used to determine significance. Differences in means of unpaired samples were tested using Mann–Whitney Dataset 1


Retrospective studies show improved outcomes in colorectal cancer patients if taking statins, including overall survival, pathological response of rectal cancer to preoperative chemoradiotherapy (pCRT), and reduced acute and late toxicities of pelvic radiation. Major tumour regression following pCRT has strong prognostic significance and can be assessed in vivo using MRI-based tumour regression grading (mrTRG) or after surgery using pathological TRG (pathTRG).A double-blind phase 2 trial will randomise 222 patients planned to receive long-course fluoropyrimidine-based pCRT for rectal adenocarcinoma at 18+ sites in New Zealand and Australia. Patients will receive simvastatin 40 mg or placebo daily for 90 days starting 1 week prior to standard pCRT. Pelvic MRI 6 weeks after pCRT will assess mrTRG grading prior to surgery. The primary objective is rates of favourable (grades 1–2) mrTRG following pCRT with simvastatin compared to placebo, considering mrTRG in 4 ordered categories . Secondary objectives include comparison of: rates of favourable pathTRG in resected tumours; incidence of toxicity; compliance with intended pCRT and trial medication; proportion of patients undergoing surgical resection; cancer outcomes and pathological scores for radiation colitis. Tertiary objectives include: association between mrTRG and pathTRG grouping; inter-observer agreement on mrTRG scoring and pathTRG scoring; studies of T-cell infiltrates in diagnostic biopsies and irradiated resected normal and malignant tissue; and the effect of simvastatin on markers of systemic inflammation (modified Glasgow prognostic score and the neutrophil-lymphocyte ratio). Trial recruitment commenced April 2018.When completed this study will be able to observe meaningful differences in measurable tumour outcome parameters and/or toxicity from simvastatin. A positive result will require a larger RCT to confirm and validate the merit of statins in the preoperative management of rectal cancer. Such a finding could also lead to studies of statins in conjunction with chemoradiation in a range of other malignancies, as well as further exploration of possible mechanisms of action and interaction of statins with both radiation and chemotherapy. The translational substudies undertaken with this trial will provisionally explore some of these possible mechanisms, and the tissue and data can be made available for further investigations.ACTRN12617001087347.ANZ Clinical Trials Register www.anzctr.org.au, registered 26/7/2017)(Protocol Version: 1.1 (June 2017). Colorectal cancer (CRC) is common: 3016 cases were diagnosed in New Zealand (NZ) in 2012 with 1283 deaths . In addiUnfortunately, the majority of patients (about 60%) with high-risk tumours have poor responses of their tumour to pCRT, and this group have double the risk of relapse compared to good responders . Furtherp < 0.001) [Statins offer the opportunity to improve outcomes in the treatment of rectal cancer. A Danish population study of 295,925 cancer cases of all types revealed that the use of statins significantly improved overall survival (OS) and specifically in those with CRC  [< 0.001) . More re< 0.001) .generation of pro-apoptotic, growth-inhibitory and pro-differentiation responses in tumours;inhibition of angiogenesis, invasion and metastasis;reducing inflammation and inhibiting radiation (RT)-induced gut and skin toxicities while radiosensitising tumour cells and maintaining tumour control compared to RT alone –20.Preclinical studies have elaborated effects on cell signalling pathways that may contribute to better cancer outcomes with statins, many of which are independent of cholesterol metabolism :generatp = 0.004  [in 407 patients at the Cleveland Clinic, favourable pathological regression was seen in 65.7% of statin users vs. 48.7% of others, 33–3.82) .p = 0.044) [multivariate analysis of 891 Canadian patients from multiple centres showed a significantly higher pathological CR rate in statin users  ;p = 0.003) [in 349 patients from Memorial Sloan-Kettering Cancer Center pathological CR was higher in statin users (30% vs. 17%), with multivariate OR 4.2  .This correlates with retrospective clinical studies in which patients taking statins during RT or chemo-RT for rectal, bladder or prostate cancer had significantly higher rates of pathological complete response (CR), local control and progression-free survival, respectively –25. The p = 0.04) and 1 year later [Furthermore, in a prospective observational study of 308 patients treated with radical pelvic RT at the Royal Marsden Hospital, London, those taking statins had significantly reduced RT-induced bowel toxicity, both during treatment , is a well-known and widely available HMG-CoA reductase inhibitor commonly used in the treatment of hypercholesterolaemia and ischaemic heart disease. In retrospective studies it reduces recurrence in breast cancer patients  and precMRI-based tumour regression grading (mrTRG), a 5-point system validated by the MERCURY group , is usedp < 0.001) and OS  [The rate of favourable (grades 1–2) mrTRG is the primary endpoint for the SPAR trial, based on three recently-published pCRT rectal cancer trials , 41. In While the mrTRG training provided in SPAR has been shown to achieve moderate-to-excellent agreement between expert and training radiologists , it is iWhile pathological CR with pCRT is associated with the best clinical outcomes, and is commonly considered the “gold standard”, the spectrum of response on standardised pathological tumour regression grading (pathTRG) systems correlates with DFS and overall survival (OS), and informs prognosis in the full spectrum of patients, not just the small minority with pathological CR. Thus a 4-tier system has been widely adopted, including in Australasia , but a 3SPAR will provide independent validation of the correlation of mrTRG with pathTRG and cancer outcomes, as well as evaluating the reproducibility of mrTRG assessment by NZ and Australian radiologists (not yet commonly used) and pathTRG by pathologists (standard practice).clinicaltrials.gov No. NCT02704520).Early surrogates for tumour response to pCRT are being investigated in clinical trials, particularly for their potential to modify the extent of surgery, or possibly avoidance of surgery in those who achieve a radiological CR . The impIn the SPAR study, the second MRI is scheduled at 6–8 weeks after pCRT to allow for surgery at 7–12 weeks. Surgery is now commonly delayed to 10–12 weeks after completion of pCRT in anticipation of improved tumour regression with additional time after pCRT . There aThis trial is an important opportunity to identify whether assessment of systemic inflammation  and the local inflammatory response (through characterising infiltrating lymphocytes) could be an important translational research component of a subsequent phase III trial of statins in rectal cancer patients. Additionally, it will allow us to evaluate the impact of SIM on the relationship between specific T-cell infiltrates in pre-pCRT biopsies and pathTRG in the resected tumours, and with normal tissue inflammation post-pCRT.It is long-recognised that a local inflammatory response with infiltration of T-lymphocytes into CRC carries a better prognosis, independent of tumour stage , 51. ConThe Immunoscore is a recently-validated test that characterises T-cell subsets infiltrating into the centre of the tumour and at the invasive margin of CRC by immunohistochemistry (IHC) for CD3 and CD8; higher scores correlate with a lower risk of relapse and improved DFS and OS, independently of stage , 55. WhiStatins have complex effects on T-cell biology, including induction of regulatory T-cells (Tregs), their migration into tumours and inhibition of the induction of Th1 and Th17 cells . Tregs, The overall purpose of this trial is to determine the effect of SIM on outcomes of pCRT for rectal cancer. Outcomes will be evaluated both by tumour regression as well as the tolerability of pCRT, estimating the size of the benefit by MRI-based assessment as well as pathological assessment of tumour regression. In addition, we will examine the biological mechanisms involved. The study will also assess the reproducibility of assessing mrTRG and pathTRG by radiologists and pathologists in Australia and New Zealand. Furthermore, the information provided by the post-CRT mrTRG could influence the intended surgical plan and optimise timing of surgery depending on tumour response to pCRT.A positive outcome, either improved tumour regression or reduced toxicity from pCRT or both, would lead to a larger phase III trial to confirm these findings. It may also lead to evaluation of statins in prospective trials in many other settings where radiation is used in cancer treatment. This trial is an important opportunity to identify whether the addition of SIM to pCRT significantly modulates systemic inflammation (reflected in the NLR and mGPS) and the local inflammatory response (through characterising infiltrating immune cells). If so, it would inform the inclusion of these translational research components in a subsequent phase III trial of statins in rectal cancer patients. It will also allow us to evaluate the impact of SIM on the correlation of T-cell infiltrates in pre-pCRT biopsies with pathTRG in the resected tumours, and with normal tissue inflammation after pCRT.SPAR is a randomised phase 2 study with the overall aim to evaluate the effect of SIM on efficacy and toxicity of pCRT in rectal cancer patients, and on systemic and local inflammatory responses. Recruitment of 222 patients is required to address the primary objective. The study will recruit patients from participating AGITG hospitals/institutions in Australia and New Zealand.Primary Objective:The primary objective is to compare rates of favourable (grades 1–2) mrTRG  following pCRT with SIM versus with placebo, considering mrTRG in 4 ordered categories: 1, 2, 3, 4–5 (proportion of patients with favourable mrTRG in SIM and placebo groups). mrTRG will be assessed by comparison of the MR scan taken after pCRT with the scan before pCRT, which will be performed using the MERCURY protocol 38), and images analysed as described [, and imaSecondary Objectives:> grade 2 acute GI and non-GI adverse events, assessed using CTCAE version 4.03; 3) The incidence of late GI adverse events; 4) Compliance with intended pCRT (proportion of patients completing > 90% of planned pCRT without dose reductions or delays); 5) Compliance with trial medication ; 6) The proportion of patients undergoing surgical resection post-pCRT; 7) 3-year local recurrence (LR) rate, disease-free survival (DFS) and cancer-specific survival (CSS) ; 8) the pathological scores determined by the central pathologist for radiation colitis in irradiated rectum in the resected specimen (radiation colitis scores).Secondary objectives are to compare between the SIM and placebo groups treated with pCRT: 1) The rate of favourable (grades 1–2) pathTRG in resected tumours by central review ; 2) The incidence of Translational Science Objectives:A number of correlative/translational objectives are intended. We aim to determine in the total trial population: 1) The association between mrTRG and pathTRG grouping (association between mrTRG and pathTRG grouping); 2) The inter-observer agreement between site radiologists and a central radiologist on mrTRG scoring (inter-observer agreement on mrTRG scoring); 3) The inter-observer agreement between site pathologists and a central pathologist on pathTRG scoring (inter-observer agreement on pathTRG scoring). In addition we aim to compare between the SIM and placebo groups treated with pCRT: 1) The association between CD3+ and/or CD8+ T-cell infiltrates in the tumour in the pre-pCRT diagnostic biopsies and pathTRG (association between T cell infiltrates and pathTRG); 2) the intensity and distribution of subsets of infiltrating T-cells in irradiated normal and malignant tissue in the resected specimen; 3) The influence of SIM on systemic inflammation, assessed with the mGPS and the NLR (mGPS and NLR).SPAR is a randomised, double-blind, placebo-controlled, multicentre phase II trial. Eligible patients will be allocated to one of two treatment groups (SIM or placebo) in a 1:1 ratio AJCC clinical N stage (< 2 vs. 2)the presence of either mesorectal fascia involvement (tumour margin within 1 mm of the fascia) or lymphovascular space invasion  on MRITreatment allocation will be balanced using minimisation for the following characteristics:The target population is adult patients with biopsy-proven rectal adenocarcinoma  planned for concurrent long-course pCRT using a standard fluoropyrimidine-based chemotherapy schedule.Males or females with biopsy proven rectal adenocarcinoma, or high-grade dysplasia with radiological evidence of invasive tumour.Distal border of the tumour is below the peritoneal reflection as assessed by MRI scan.Age ≥ 18 years.Clinical TNM tumour staging is T2–4 N0–2 M0 after staging investigations including CT scan of chest, abdomen and pelvis and pelvic MRI scan. Patients with resectable M1 (e.g. oligometastatic liver or lung) disease who are being treated with curative intent can be eligible, following approval from the SPAR Study Chair.Planned for concurrent long-course pCRT using fluoropyrimidine-based chemotherapyRadiologically measurable disease on baseline pelvic MRI scan.9/L, neutrophils > 1.5 × 109/L, ALT/AST < 3 x ULN, bilirubin < 1.5 x ULN, estimated creatinine clearance > 50 ml/min).Adequate bone marrow, liver and renal function  as part of pCRTTaking statins in the 6 weeks before planned start of pCRTPredicted life expectancy of less than 3 yearsPrior pelvic or rectal radiotherapyHistory of another malignancy within 5 years prior to registration . Patients with a history of other malignancies are eligible if they have been continuously disease free for at least 5 years after definitive primary treatmentConcurrent illness, including severe infection that may jeopardise the ability of the patient to undergo the procedures outlined in this protocol with reasonable safetySerious medical or psychiatric conditions that might limit the ability of the patient to comply with the protocolPregnancy, lactation, or inadequate contraception.Simvastatin or placebo is the trial intervention. Concurrent preoperative chemoradiation (pCRT) using fluoropyrimidine-based chemotherapy and radiotherapy is required standard concomitant treatment Fig. .One simvastatin 40 mg capsule or one matching placebo capsule taken orally each evening, will commence 7 (+/− 3) days prior to pCRT and continued for 90 consecutive days. Subject compliance with the trial treatment will be determined at protocol-specified assessments by questioning the participant and a formal count of the capsules returned at completion of study treatment (6 weeks after pCRT).Trial treatment (SIM or placebo) will be permanently discontinued if progressive disease (PD) is documented, unacceptable treatment-related toxicity occurs, a delay of pCRT of > 28 days occurs, the clinician believes that continuation of trial treatment is not in the patient’s best interest, or the patients fails to comply or declines further treatment.www.eviq.org.au).It is recommended to follow eviQ Rectal (Neoadjuvant EBRT Chemoradiation Pre-operative Long Course) Guidelines version 2 or later  is defined as per the recent international consensus guidelines . The PlaClinical Target Volume B (CTV B) includes the mesorectum and pre-sacral region at involved levels with a 1–2 cm margin cephalad and 1 to 2 cm on gross tumour within the rectum. The PTV B margin should be 0.7 to 1.0 cm, except at skin, where planning system requirements mandate it be trimmed to 2–5 mm within the skin surface.For 3-dimensional conformal radiation therapy (3DCRT), a tumour boost of 5.4Gy at 1.8Gy per fraction to PTV B (50.4Gy cumulative including contribution from PTV A) is required for patients with T3 tumours. A boost dose up to 10.8Gy at 1.8Gy per fraction  is allowed for patients with T4 fixed cancer and high-risk T3 tumours.For intensity modulated radiation therapy (IMRT) or volumetric modulated arc therapy (VMAT), simultaneous boost technique is recommended. Total cumulative dose to PTV B is to be 50 Gy.Permission for other radiation techniques aiming to deliver equivalent radiation dose should be obtained in advance from Trial Management Committee.2/day PO BID 5 days a week on days of RT administrationcapecitabine 825 mg/m2/day PO BID 7 days a week for the duration of RTcapecitabine 825 mg/m2/day via continuous venous infusion for the duration of RT5-fluorouracil 225 mg/mOther therapies and/or dosing regimen that have been accepted as standard of care in Australia and New Zealand may be allowed following agreement from the Study ChairThe accepted chemotherapy and dosing regimens  are one 2 is recommended for dosing of fluoropyrimidine-based chemotherapy. Clinicians must pre-specify which schedule they will be using for each participant. Once allocated, the patients must then adhere to the specified schedule throughout the treatment period, unless modified for safety reasons.A maximum BSA of 2.2 mPatients should be managed with pCRT according to institutional protocols, including dose modifications and delays for treatment-related toxicities. Recommendations are provided for guidance on management of toxicities related to pCRT and are defined in the protocol. If pCRT is discontinued, trial treatment should continue unless unacceptable trial treatment toxicity is observed. Other concomitant medications and supportive therapies are permitted, and there is a list of prohibited medications  in the detailed protocol.Patients will undergo resection of their rectal cancer at a time recommended by their surgeon . Patients with an excellent clinical and radiological response may be observed under a “watchful waiting” programme if agreed with their surgeon.Surgery may include open, laparoscopic, robotic or transanal total mesorectal excision approach. This will include both restorative low anterior resection, or abdomino-perineal resection with permanent end-colostomy.Treatment after discontinuation of trial treatment is at the discretion of the patient’s clinician. Adjuvant chemotherapy may be administered on the recommendation of the treating clinician and its use will be recorded.Blood tests will be performed on the first day of pCRT, prior to starting chemotherapy, to assess the impact of trial medication on the mGPS and NLR. Clinical assessments including IBDQ-B questionnaire will be conducted on Trial Weeks 3, 5 and 7 .Week 13 assessments will be performed 6 weeks (+/− 7 days) after completion of pCRT and prior to surgery. An MRI scan for mrTRG must be completed 6–8 weeks after completion of pCRT and must be prior to surgery. The postoperative visit will include assessment of length of hospital stay after surgery and any readmission data. Subsequent follow-up is annually.The study will collect data on: baseline patient and tumour status; treatment delivery; baseline and post-pCRT MRI assessments; clinical and laboratory toxicity assessments; baseline and resection histopathology; followup data for PFS and OS Table .Table 1The primary endpoint of this trial is rates of favourable mrTRG (grades 1 or 2). This will be analysed using ordinal regression with 4 ordered categories of mrTRG  to provide greater sensitivity. Based on published data using MRI 6–8 weeks after pCRT, the expected rate of mrTRG in the control group is 9% grade 1, 39% grade 2, 20% grade 3 and 32% grades 4–5. A 35% relative increase in favourable mrTRG would be a worthwhile difference to inform a phase III trial.Based on an ordinal proportional odds assumption, a sample size of 222 patients (111 treated with SIM and 111 controls) will have > 80% power to detect a change in mrTRG rates to 17% grade 1, 48% grade 2, 16% grade 3 and 19% grades 4–5 with 95% confidence.Intention-to-treat analysis of results will be the primary analysis. In addition, an exploratory per protocol analysis will also be conducted which will exclude ineligible patients, those not taking trial medication when starting pCRT and those who withdraw their consent to participate prior to response evaluation.The primary endpoint and all secondary endpoints expressed as proportions will be estimated, together with corresponding 95% confidence intervals based on exact binomial distributions. Kaplan-Meier curves will be calculated for all time-to-event endpoints. Rates at specific points in time  will be estimated from these Kaplan-Meier curves.> 0.40 is defined as moderate agreement. This will be assessed for mrTRG after recruitment of 35 randomised patients to ensure procedural consistency and again at the conclusion of the trial.Inter-observer agreement is defined as the degree of agreement in mrTRG (or pathTRG) results when reviewed by the central radiologist (or pathologist) and the reporting site radiologists (or pathologists). Results for each of mrTRG and pathTRG will be reported separately in a 3X3 grid comparing favourable, intermediate, and unfavourable scores between central and site radiologists and pathologists respectively. Inter-observer agreement will be assessed using a weighted kappa statistic Adverse events in each arm will be tabulated and graded according the NCI CTCAE version 4.03.For analysis of translational objectives, association between CD3+ and CD8+ T-cell infiltrates in diagnostic rectal cancer biopsies and pathTRG following pCRT will be evaluated using the chi-square test for trend. The effect of SIM on subsets of T-cells in tumours and normal tissue will be evaluated by comparison of distribution of scores for each cell type in SIM and placebo groups using the chi-square test for trend. Changes in the NLR and mGPS over time will be analysed using repeated measures ANOVA.No formal interim analysis for efficacy is planned but review of safety data by the AGITG Independent Safety and Data Monitoring Committee (ISDMC) is planned. No early stopping for larger-than-expected differences in mrTRG rates is planned because this endpoint is a putative surrogate for improved patient outcome. Demonstrating a significant difference in the clinically-important DFS and OS endpoints requires a much larger phase III trial.The study is conducted by the Australasian Gastrointestinal Trials Group, in conjunction with the NHMRC Clinical Trials Centre. Formal study oversight is by a Trial Management Committee and an Independent Data and Safety Monitoring Committee.The study will be conducted according to the Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95) annotated with TGA comments (Therapeutic Goods Administration DSEB July 2000) and in compliance with applicable laws and regulations. The study will be performed in accordance with the NHMRC Statement on Ethical Conduct in Human Research 2007, the NHMRC Australian Code for the Responsible Conduct of Research 2007, and the principles laid down by the World Medical Assembly in the Declaration of Helsinki 2008.The study will be conducted in accordance with applicable Privacy Acts and Regulations. All data generated in this study will remain confidential. All information will be stored securely at the NHMRC Clinical Trials Centre, University of Sydney, and will only be available to people directly involved with the study and who have signed a Confidentiality Agreement.Little progress has been made in the management of T3 rectal cancer in the last 10 years. The findings in several retrospective studies that statin use in patients undergoing pCRT appears to confer higher pathological regression rates, more pathological CR –24 and aIf this study has a positive result, a larger RCT will be needed to confirm and validate the merit of statins in the preoperative management of rectal cancer, especially long-term clinical outcomes. Such a finding could also lead to studies of statins in conjunction with radiation and chemotherapy in a range of other malignancies, as well as further exploration of possible mechanisms of action and interaction of statins with both radiation and chemotherapy. The translational substudies undertaken with this trial will provisionally explore some of these possible mechanisms, and the tissue and data can be made available for further investigations.This trial is an important opportunity to identify whether assessment of systemic inflammation (reflected in the neutrophil-lymphocyte ratio and the mGPS) and the local inflammatory response (through characterising infiltrating immune cells) could be important translational research components of a subsequent phase III trial of statins in rectal cancer patients. Additionally, it will allow us to evaluate the impact of SIM on the interaction of specific T-cell infiltrates in pre-pCRT biopsies with pathTRG in the resected tumours, and with normal tissue inflammation after pCRT.Recent reports on the merits of mrTRG to assess effects of pCRT suggest that it is a valid endpoint to use in clinical trials –41. Thisn = 6397) evaluating time between RT and resection for rectal cancer found that an interval > 60 days was significantly associated with inferior survival and rates of sphincter-preserving surgery and an increased rate of positive surgical margins [Surgery is now commonly delayed to 10–12 weeks after completion of pCRT in anticipation of improved tumour regression with additional time after pCRT. There are conflicting reports as to whether this is the case, based on pathological CR rates –48. Howe


The investigation and management of celiac disease places a high burden on the health care system. Accurate methods to ascertain cases of celiac disease (CD) in population-based administrative data can facilitate epidemiologic and health services research to guide disease management. The study aim was to develop and validate administrative data case definitions for CD to facilitate further studies about the effect of CD on osteoporosis and fracture risk.Population-based data from the Manitoba Bone Mineral Density (BMD) Program registry, which contains medical information on all individuals in the province of Manitoba, Canada who have received BMD testing, was used to define the study cohort. Linked hospital discharge abstracts and physician billing claims were used to ascertain diagnoses of celiac disease in administrative data. A population-based CD serologic registry was used as the validation database. One diagnosis code in hospital discharge abstracts or two or more diagnosis codes in physician billing claims optimized the detection of positive celiac serology with sensitivity of 84% (95% CI 80–88%), specificity of 97% (95% CI 80–88%), PPV of 80% (95% CI 80–88%), and NPV of 97% (95% CI 80–88%). Our administrative data case definition for celiac disease demonstrates good sensitivity and specificity for detecting positive celiac serology. Celiac disease is a common intestinal disorder affecting an estimated 1% of individuals in North America . Given tOne of the disease associations relevant to celiac disease is osteoporosis and increased fracture risk –9. The dThe study cohort was developed from the Manitoba Bone Mineral Density Database. The population-based Manitoba BMD Program registry contains information on all individuals in the province of Manitoba, Canada who have received BMD testing with dual-energy X-ray absorptiometry (DXA). The accuracy and completeness of the BMD registry exceeds 99% and has been well described . In addiAdministrative health databases in Manitoba capture virtually all physician claims and all hospitalizations on provincial residents eligible to receive health services. Physician claims are submitted to the provincial ministry of health by physicians paid on a fee-for-service basis; they capture virtually all outpatient services and contain a single diagnosis code recorded using the International Classification of Diseases, 9th revision, Clinical Modification (ICD-9-CM). Only the first three digits of the ICD-9-CM code are recorded in physician billing claims data, which is insufficient to definitely distinguish celiac disease from other causes of intestinal malabsorption. Hospital discharge abstracts are completed at discharge from acute care facilities and contain up to 16 diagnoses coded using ICD-9-CM up to March 31, 2004 and up to 25 diagnoses coded using the Canadian Adaptation of the 10th revision of ICD  after this date. There is no restriction on the number of digits/characters used to record diagnoses in hospital abstracts; hence, celiac disease can be distinguished from the other causes of intestinal malabsorption in this database.Physician billing claims and hospital discharge abstracts can be linked via an encrypted personal health number to other administrative data sources. As well, they can be linked to the population registry, which contains information about dates of health insurance coverage, demographic characteristics, and location of residence.Repository databases were used to identify the number of physician billing claims (P) and hospital discharge abstracts (H) related to celiac disease and malabsorption disorders from the ICD codes noted above. The index date for each case was defined as the date of the BMD test.Central to the diagnosis of celiac disease is serologic testing . While tCase definitions were developed using hospital discharge abstracts and/or physician billing claims (denoted P). Administrative data was examined up to 3 years prior to the index date, the date of the BMD test. For hospitalizations, we examined coding at the less specific integer level  and coding at the more specific decimal level . Case definitions using different combinations of administrative data contacts were developed. For example, H1P2 denotes 1 hospital discharge abstract (integer level) or 2 physician claims; H1*P3 denotes 1 discharge abstract  or 3 physician claims.The study cohort was described using frequencies, percentages, means, and standard deviations. Chi square tests of independence and t-tests were used to test for differences between seropositive cases and seronegative non-cases on socio-demographic and comorbidity characteristics. Estimates of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy for each case definition were computed, along with 95% confidence intervals (95% CIs).> 50 years), sex , area of residence , calendar year (before 2006 vs after 2006), level of comorbidity and income quintile group. Comorbidity was defined using Aggregated Diagnosis Groups™ (ADGs™) codes from the Johns Hopkins Adjusted Clinical Group® (ACG®) Case-Mix System version 9. Low comorbidity was denoted by an ADG score <6 whereas high comorbidity was denoted by an ADG score of 6 or greater [We conducted sensitivity analyses stratified by age (< 50 years vs  greater . Mean ho greater .There were 250 seropositive cases 14%) and 1588 seronegative non-cases (86%). The median time between dates of serologic and BMD testing was 1.6 years (interquartile range 0.7–2.9 years). Table 4% and 15Clinical registries in Manitoba have been used to validate administrative case definitions for inflammatory bowel disease, diabetes mellitus and multiple sclerosis –5. TheseEstablishing a case definition of celiac disease has some inherent challenges. The three-digit ICD-9-CM code present in physician billing claims and used in most provinces in Canada  for manyAlthough our study did not validate the case definition of celiac disease with biopsy positive celiac disease as biopsy data is not part of the database, recent pooled sensitivity and specificity data demonstrate very high sensitivities (92.8%) and specificities (97.9%) of TTG antibodies in predicting biopsy proven celiac disease . EMA antIn conclusion, this study demonstrates that an administrative definition for celiac disease based on ICD correlates well with positive celiac serology. Given the high sensitivity and specificity of celiac serology in predicting celiac disease, this definition could be used in population-based epidemiologic and health services research on this condition.The current study utilized celiac serology results linked to health claims data in a comprehensive BMD database to establish an administrative definition of celiac disease. There is potential for selection bias as this provincial bone mineral density database included a disproportionate number of elderly, post-menopausal females. This is evident in Table


However, it is frequently associated with enamel damage that include chipping, demineralisation, and white spot formation. So far, current bonding systems that can maintain shear bond strengths (SBS) suitable for clinical performance are unable to limit enamel demineralisation, adhesive remnants and damage caused on removal of brackets after treatment. This study reports a novel “safe enamel etch” clinically viable procedure that was accomplished via application of novel etchant pastes developed with β-tricalcium phosphate and monocalcium phosphate monohydrate powders mixed with citric acid (5 M) or phosphoric acid (37% PA) to yield BCA and BPA etchants respectively. Although enamel etched with clinically used PA gel yielded higher SBS than the BCA/BPA etchants, it exhibited greater adhesive remnants with evidence of enamel damage. In contrast, the experimental etchants resulted in unblemished enamel surfaces with zero or minimal adhesive residue and clinically acceptable SBS. Furthermore, the BPA etchant caused lower enamel decalcification with extensive calcium-phosphate precipitation. The study conclusively showed that BPA facilitated  Orthodontic treatment using fixed appliances is widely used to correct irregular teeth and/or jaw discrepancies and improve aesthetic appearance of the face. Attachment of a fixed appliance involves bonding of metal or ceramic brackets onto the enamel surface of teeth and the use of special orthodontic wires/elastics to deliver forces to align the irregular teeth. The brackets are removed by the orthodontist using bracket debonding pliers at the completion of treatment . However, the treatment outcomes are frequently associated with a wide range of enamel damage including demineralisation or white spot formation that occur during the prolonged treatment, whilst enamel chipping or cracking very often ensues at the time of appliance removal due to the high debonding force required to remove the brackets2, which also results in some of the adhesive being left on the enamel surface. The removal of these adhesive remnants is required at the completion of treatment, which causes further trauma to the enamel3. These problems are commonly encountered with metal orthodontic brackets and have been exacerbated following the advent and popular use of ceramic brackets due to their rigidity and lack of deformation during removal, which has led to more frequent incidence of enamel damage4.Malocclusion of teeth is common in different age groups  and can have negative influence on dentofacial development, contributing to impaired orofacial function, patient’s psychological well-being and quality of life. A prevalence rate of orthodontic treatment needs of 21.3–39.5% has been reported in different European countries according to the official index for assessment of need for subsidized orthodontic treatment in a country of interest 5. There is consensus that an etch-and-rinse approach using phosphoric acid remains the preferred choice for enamel conditioning, since it not only guarantees a durable bond to enamel but also seals and thus protects against degradation7. The tooth enamel surface is known for its low-energy and hydrophobic properties, with its outermost layer being rich in fluoride. Enamel etching with phosphoric acid (PA) removes the fluoride-rich layer, dissolves enamel mineral, creates micro-pores, and renders a high-energy and hydrophilic enamel surface to permit wetting by an adhesive; yet at the expense of increased surface porosity, susceptibility to staining, caries, more remnant adhesive and enamel loss8. Alternative techniques to the conventional acid-etching have been sought to decrease the adhesive remnants and enamel damage such as the use of self-etch adhesives and laser application, which result in acceptable bond strengths10. However, these methods are not able to eliminate the remnant adhesive left on enamel after bracket removal that exhibit comparable etch-patterns to the etch-and-rinse approach in terms of cracking and roughening of enamel surface; hence enamel damage is not eliminated12. On the other hand, methods to prevent and manage enamel demineralisation or promote remineralisation have been attempted through application of topical fluoride around orthodontic brackets13, incorporation of calcium-phosphates (CaP) in bonding resins for remineralisation14, and antibacterial agents in orthodontic cements15. However, these strategies only partially address the clinical concerns and although a slight regression of white spot lesion is reported16, improvements in terms of long-term ion release, adhesive remnants and/or enamel damage elimination are non-significant17.Fixation of brackets to teeth involves joining of two different solid substrates or adherents through an intervening adhesive layer. Bracket bases are currently provided with mesh designs that ensure robust mechanical interlocking between the bracket and adhesive Fig. , whilst 18. The overall goal of this study is to develop a novel enamel conditioning technique that aims to yield clinically acceptable bond strengths, induce CaP re-precipitation, and upon bracket removal leave no or minimal remnant adhesive on teeth and eliminate enamel damage. The rationale is premised on enamel etching exploiting a low-pH CaP paste capable of simultaneously dissolving surface enamel and precipitating a CaP phase on the enamel. The hypothesis is that the use of CaP precursors, which on mixing with an acid produces a CaP etchant capable of overcoming the detrimental effects of a strong acid alone on enamel damage, without compromising the bracket bond strength.In contrast to restorative dentistry where adhesive materials are generally bonded to teeth permanently, orthodontic treatment entails bonding of attachments (mostly brackets) for limited periods of time (1–2 years) to serve as means of application of force to teeth. Thus, enabling “in function” retention of both metal and ceramic brackets, minimising enamel demineralisation, and the ease of bracket debonding with minimal remnant adhesive and limited risk of permanent damage to the enamel surface are critical in orthodontics, yet challenging to achieveA commercial PA gel was used as the control group whilst the experimental etchants comprised of equimolar amounts of β-TCP and MCPM in presence of either phosphoric (BPA) or citric acid (BCA). The experimental etchants had a higher pH of 1.4 in comparison to the pH of the PA gel at 0.8. The X-ray diffraction (XRD) spectrum analyses indicated that monocalcium phosphate monohydrate (MCPM) is the predominant phase of the BCA and BPA formulations Fig. , whose up < 0.001) in SBS and ARI among the 3 subgroups  and Mann-Whitney (for ARI) multiple comparisons demonstrated that the control subgroups showed significantly higher mean SBS and ARI scores than the CaP subgroups, which exhibited non-significant differences between them in ARI scores only. The BPA subgroups exhibited significantly higher SBS than BCA subgroups.The ARI scores obtained on enamel etching with PA gel mainly yielded scores of 2 and 3 for both metal and ceramic brackets. The SEM results showed retention of adhesive and enamel damage encountered following both 24 h water storage (WS) and thermocycling procedure  survived the rigorous artificial ageing tests, yielding satisfactory bond strengths to both metal and ceramic brackets, whilst upon bracket removal reducing the amount of remnant adhesive on teeth and eliminating the risk of enamel damage. Hence, the interaction of the BPA etchant paste with enamel surface was thoroughly investigated both before bracket bonding and after bracket removal.Flat molar buccal surfaces were obtained by grinding and polishing, which clearly resulted in a scaly appearance of enamel prisms oriented in different directions Fig. . The etcFigure . In contAccording to the enamel damage index (EDI), enamel etching with 37% PA gel yielded a mixture of grades 2 and 3  following both metal and ceramic bracket debonding Fig. . On the v1-PO at 958 cm−1), doubly degenerate O-P-O bending modes (v2-PO at 439 cm−1), triply degenerate asymmetric P-O stretching modes (v3-PO at 1040 cm−1), triply degenerate modes of mainly O-P-O bending character (v4-PO at 594 cm−1), and the B-type carbonate at 1075 cm−1.The flat-surface enamel specimens obtained from molar teeth were used to compare the Raman spectra of untreated and etched enamel surfaces. Each specimen was divided into three areas and used to record the spectra of untreated enamel area and two enamel areas conditioned with either of the two etching protocols (PA gel and BPA paste). All enamel surfaces showed the 5 characteristic peaks of enamel hydroxyapatite (HA) Fig. : symmetr−1. The potential demineralisation/remineralisation effect induced by the two etching protocols was examined depending on the phosphate peak ratio (PPR) of v1/v4. The greatest drop in peaks intensity and v1/v4 PPR manifested in the 37% PA group, whilst the BPA group showed values closer to the untreated enamel. One-way ANOVA showed a statistically significant difference in v1/v4 PPR among the three groups , whilst Tukey Post Hoc multiple comparisons revealed that 37% PA group had significantly lower PPR than the control (p < 0.001) and BPA (p = 0.010) groups, and a non-significant difference between untreated enamel and BPA group (p = 0.312).The four phosphate peaks were observed within untreated and treated enamel spectra with no difference in the Raman peaks; however, enamel conditioning with the two etching protocols resulted in marked reductions in the mean intensity of the 4 phosphate peaks as compared to those of untreated enamel area Table . The str5. This new bioactive orthodontic etchant can alleviate subsurface enamel demineralisation in comparison with the traditional phosphoric acid etchant and the pre-clinical viability assessment using in vitro studies show exemplary advantages. The new low-pH CaP paste etchant application is a clinically viable bracket bonding method with steps similar to PA application7 that yielded adequate bond strength with the advantages of minimal adhesive remnants and no enamel chipping or cracking on debonding of the bracket. The mechanism of bonding to enamel in the conventional etch-and-rinse approach is essentially based on the loss of superficial enamel and preferential dissolution of the underlying enamel on phosphoric acid treatment  that create asperities on the surface to enable ingress of resin monomers that upon polymerisation become micro-mechanically interlocked5. Etching enamel with phosphoric acid leads to dissolution of the enamel hydroxyapatite releasing calcium, which to a small extent is able to buffer if the enamel surface is not agitated by resisting changes in hydrogen ion concentration. However, the inclusion of a low pH calcium phosphate in the etchant becomes more effective in buffering, limiting enamel decalcification and, instead, enhancing CaP re-precipitation without adversely affecting the etching process.Orthodontic treatment is aimed to align mal-positioned teeth and correct malocclusions. In order to enable movement of teeth, force is applied through use of bonding attachments (mostly brackets) that are bonded to enamel. It is a very successful treatment, however, alterations to the enamel surface occur during and at the bracket removal stage, which are induced by the iatrogenic manipulation. Enamel acid-etching is one of the most fundamental methods of facilitating adhesion and the acid acts by decalcification that causes an irregularity on the surface of enamel. Phosphoric acid liquid or gel is widely used to etch enamel, and conventional bracket bonding procedure is based on enamel pre-conditioning with PA gel for 30 s, water rinsing of the gel, then air-drying the etched surface before attaching the bracket using resin adhesives21 and remains the standard reference value for the vast majority of in vitro bond strength studies23. On the other hand, adhesion forces should not be too strong in order to avoid enamel loss after debonding (40–50 MPa)24. Therefore, the ideal orthodontic biomaterial should have bonding forces included in the interval of 5–50 MPa, even if these limits are mostly theoretical25. Literature findings on enamel etched with 37% PA using the conventional etch-and-rinse approach report an average bracket bond strength ranging from 9–35 MPa, with usually higher values attained for ceramic than metal brackets7. The SBS results of most metal and ceramic subgroups in this study are within the confines of this range. Enamel etching with conventional 37% PA gel (pH = 0.8) before metal or ceramic bracket bonding resulted in significantly higher mean SBS than etching with the CaP pastes prepared from comparably strong acids. Mixing of CaP powders with 37% PA and 5 M CA imposed a buffering effect by raising the pH of these acids in the resulting paste from 0.8 to 1.4 and from 1.0 to 1.4 respectively, hence yielding less aggressive etchants. Typically, when equimolar amounts of β-TCP and MCPM are mixed in aqueous conditions, brushite forming cements are formed; however, different end-products, such as stoichiometric or calcium-deficient hydroxyapatite, calcium octaphosphate or brushite may result by an acid-base or hydrolysis reaction, depending on the pH26. In addition, acid-etching of enamel creates surface micro-pores by dissolving calcium and phosphate ions, which re-precipitate as brushite or MCPM depending on the type and concentration of the etchant used. Formation of brushite has been reported to follow enamel conditioning with PA of concentrations less than 27%, whilst MCPM is formed when using concentrations exceeding 27%28. The latter finding is supported in this study as the XRD data revealed MCPM as the predominant phase of the BPA paste (made of β-TCP and MCPM powders in equimolar amount) prepared by mixing in presence of 37% PA. The milder etching effect and diminished resin infiltration due to the CaP salt precipitation led to the lower SBS values attained by the CaP BPA etchant compared to the PA gel; however, the mean SBS obtained from BPA paste subgroups ranged between 18.2–25.4 MPa, which were well above the lower limit for acceptable clinical performance. The stagnation of CaP crystals and resistance to water irrigation due to the low solubility and the volume of crystallites29 remain the main factors that impede the complete infiltration of the resin into the micro-pores, hence adversely affect the bond strength.Although there is no general consensus on the optimal bond strength required for successful clinical performance of bonded brackets, a minimum range of 6–10 MPa has been suggested as suitable for bracket bonding to achieve acceptable clinical performance31.The citric acid based etchant (BCA) pastes, however, were unable to sustain the thermocycling regime and resulted in statistically significant low SBS values. Despite the use of a high CA concentration  to prepare BCA pastes, the buffering effect induced by mixing with the CaP powders led to an enamel-adhesive interface of inadequate resistance to prolonged thermal fluctuations as indicated by all failures at the interface. A finding in literature which supports this experimental observation, albeit for a different application, is that the addition of calcium to CA minimised enamel erosion and reduced its erosive and demineralisation effects significantly32. The debonding of the brackets also tend to leave adhesive remnants on the enamel surface, hence enamel clean-up and polishing is required to remove them  resulting in increased chair time and inevitable enamel scratching33. The results of the adhesive remnant index when enamel etching was carried out with 37% PA gel were in agreement with previously reported data and as expected resulted in significantly greater amount of remnant adhesives after bracket removal with six samples exhibiting cracked enamel and 11 with chipping. In contrast, the application of CaP etchant pastes to effect bonding left minimal or no adhesive with no signs of enamel damage. Thus far no study or other bonding systems have been able to achieve this level of unperturbed enamel surface without significantly compromising the bond strength. The unprecedented combined advantages of this approach are expected to impact orthodontic treatment leading to less enamel damage, thereby alleviating long term problems associated with it. The ARI scores depend on the interplay of many factors including the bracket base design, etching technique, adhesive type, and location of the debonding force; therefore, higher ARI scores are not necessarily only concomitant with higher bond strengths35. However, this means that attaining bond strengths exceeding the minimum requirement for clinical performance whilst maintaining minimal remnant adhesive on the enamel post bracket debonding is still possible. Manipulating the etching chemistry to weaken the enamel-adhesive interface was achieved in this study through the employment of acidic CaP pastes with potential enhancement of CaP precipitation, hence facilitating the preferable debonding failure at enamel-adhesive interface and saving chair-side time and cost at the post debonding clean-up stage.Etching of enamel with 37% PA generates high bracket bond strengths, however this excessive bond strength has been a point of contention as debonding these brackets often lead to enamel cracks and fractures37. To examine the enamel surfaces after treatment with the control and the experimental etchant paste, characterisation of the resulting surfaces by different techniques is essential; hence flat enamel specimens were used to enable CLSM and Raman spectroscopy analyses. The CLSM can provide evidence about the enamel etching effects induced by acids and the enamel remineralisation potential with remineralising agents depending on the degree of image fluorescence, which increases with the greater loss of organic matrix and exposure of enamel prisms38. This is achieved through the use of fluorescent dyes (e.g. Rhodamine B dye) which promote certain emission wavelengths when they are excited by laser with specific wavelengths, hence can be traced at their locations in a material and/or tissue at dilute concentrations40. The CLSM images revealed that the etched surfaces showed a clearly visible increase in fluorescence, evidenced by the more apparent honeycomb morphology in areas of enamel etched with 37% PA gel, the etched enamel prisms clearly exposed and a better defined honeycomb morphology with an apparently greater loss of organic matrix  as the main and strongest phosphate peak within hydroxyapatite49. Since Raman peak intensity is proportional to the number of molecules within the volume of the scanned area50, monitoring the intensity of this peak has been used to assess the degree of demineralisation within carious enamel52. Furthermore, it has been recently used to assess potential increase in phosphate content within white spot lesions following remineralisation treatments using bioactive glass powders53, and to examine the remineralisation potential of artificial carious lesions with high-frequency microwave energy therapy54. Following a similar principle, a comparison of the 4 phosphate peaks between the untreated and etched enamel showed that areas etched with 37% PA gel or the experimental BPA paste exhibited marked reductions in their intensities, with the main phosphate peak (v1) showing the greatest, and peak v4 showing the lowest decrease in intensity  and the strong peak v1, were found to be distinctive for HA and reliable in differentiating between HA and other calcium phosphates55. Accordingly, to avoid the potential of biased demineralisation/remineralisation information solely extrapolated from the main phosphate peak (v1), peak v4 was used as a reference in this study to calculate the v1/v4 phosphate peak ratio (PPR). The outcomes of PPR comparisons confirmed the remineralisation potential following enamel etching with BPA paste, exhibiting a non-significant difference in comparison with the untreated enamel group and a significantly higher PPR than enamel with the conventional 37% PA gel etching. The PPR of BPA group was close to that of untreated enamel, confirming the SEM findings in terms of both the mild etch pattern produced and the considerable CaP precipitation. To enable the efficacy of a bonding system, an ideal scenario is an accurate simulation of the oral environment, however an extremely difficult goal compounded by patient related variables. It is also extremely difficult to clinically distinguish the adhesive potential of a specific bonding system independent of the numerous variables that can influence either the quality or the longevity of bracket bonding to enamel23. However, mimicking the extreme conditions that may be experienced in the oral environment can reflect on clinical performance. In addition, in vitro investigations allow for more standardized procedures for testing a specific bonding system and are valuable for at least the initial screening and selection of materials. The outcomes of this study serve as an in vitro robust screening of the orthodontic etchant system towards a controlled clinical investigation as the results obtained following rigorous testing are promising for a successful in vivo performance. Other variables that have been demonstrated to have significant effects on shear bond strength values and ARI scores of dental and orthodontic materials are the etching period and type of adhesive used and enamel contamination, however the present study reports the result of a systematic in vitro study using one bonding agent with a specified etching period and a specific enamel cleaning regimen. Hence the results may change if a different bonding agent is used but the efficacy of the etchant has been clearly established with the recommended enamel treatment.Micro-Raman spectroscopy is used both as a quantitative and qualitative chemical assessment methodology for biological samples. In this study, the Raman spectra of all untreated and etched enamel surfaces demonstrated the 5 characteristic peaks of enamel similar to previous findingsIn contrast to the conventional paradigm of incorporating remineralising agents in the adhesives to release calcium and phosphate ions, which only partially address the clinical concerns with non-significant enamel damage limitation, our strategy of using a two-in-one acidic CaP etchant paste successfully resulted in less aggressive etching of the enamel and creating a “CaP-built-in” enamel surface simultaneously. This new strategy of enhanced CaP re-precipitation promotes safer enamel etching and bracket debonding and may serve as an adjunct to contemporary attempts of enhancing enamel resistance to demineralisation during orthodontic treatment.in vitro bonding procedures after acquiring ethical approval from the National Research Ethics Service Committee London-Riverside (REC Reference 14/LO/0123). Informed consent for study participation was obtained from each participant, and from a parent and/or legal guardian for participants under the age of 18 years and all methods were performed in accordance with the relevant guidelines and regulations. After extraction, the teeth were cleaned in running water to remove any blood and soft tissue debris, then stored in a 1% chloramine-T trihydrate bacteriostatic/bactericidal solution for a maximum of one week and thereafter stored in distilled water (ISO/TS 11405:2015)56. Criteria for tooth selection included: intact buccal enamel surface without cracks and caries (examined under stereomicroscope x10 magnification), with no history of previous endodontic, orthodontic or bleaching treatments57.Extracted human premolar and molar teeth were collected from adolescent and young patients (10–25 years) to carry out Beta-tricalcium phosphate (β-TCP) and monocalcium phosphate monohydrate (MCPM) powders  were used in equimolar amounts as principal constituents of the solid phase for preparing acidic CaP etchant pastes (capable of forming brushite on enamel) using 37% PA [BPA] and 5 M citric acid  [BCA] as a liquid phase, in a powder-to-liquid ratio of 0.8:1 and 3:1 respectively. The assigned powder and liquid were mixed using a stainless steel spatula on a glass slab for 30 s until a homogenous workable paste was obtained. The 30 s pH of the resulting paste was measured by a flat-end electrode using a digital pH meter . All formulations were prepared under ambient conditions (20–23 °C and 50–60% humidity). A conventional 37% PA gel  was used as the control etchant, whilst Transbond XT light-cure primer and adhesive  were used for all brackets bonding. Two types of pre-adjusted upper premolar brackets were used: metal  and ceramic .Premolar teeth were mounted in acrylic blocks using rubber moulds (14 mm length ×14 mm width ×17 mm depth). First, soft sticky wax was used at the tooth root apex inside the mould to align the tooth in such a way that the middle third of the buccal surface was parallel to the analyzing rod of a surveyor, to ensure a debonding force running parallel to the bonded bracket base. Then self-cure clear acrylic  was poured around the tooth up to about 1 mm apical to the level of cemento-enamel junction. The teeth were subsequently stored in distilled water at lab temperature until bonding.5 was followed to bond all brackets. The buccal surface of each tooth was polished (10 s) with pumice slurry using rotary rubber cups, followed by water irrigation (10 s) and oil-free air dryness (10 s). This was followed by application of the etchant on the middle third of the buccal surface for 30 s, then irrigation with water for 20 s and dryness for 20 s. A thin layer of bonding primer was applied onto the etched enamel and spread by air-jet (3 s). The bracket base was loaded with the adhesive composite and placed onto the middle third of buccal surface with a pressing force of 300 g  for 10 seconds to ensure an even adhesive thickness. Excess composite was removed by a dental probe. LED Light curing  was applied for 20 s  according to the manufacturer instructions.The mounted teeth were randomly allocated into two main groups to be bonded with 120 metal (stainless steel) and 120 ceramic (sapphire) brackets. Each main group was further divided into three subgroups according to the type of etchant used: 37% PA gel  , CaP paste made of 37% PA solution [BPA], and CaP paste made of 5 M CA [BCA]. The conventional etch-and-rinse protocolFollowing bracket bonding, the bonded teeth were stored in distilled water at 37 °C for 24 h. Then half of the samples (n = 20 per subgroup) were debonded, while the other half subjected to a thermo-cycling regimen of 5000 cycles between cold and hot water baths of 5 °C and 55 °C with a dwell time of 30 s in each bath and transfer time of 5 s , followed by bracket debonding.14 of 0.5 mm/min and SBS values were calculated in MPa by dividing the load at failure by the bracket base surface area. The debonded teeth were examined under x10 magnification  for the amount of remnant adhesive left and scored according to the ARI system58. Enamel damage was also examined under x10 magnification, and two random samples of each subgroup were further sputter-coated with gold palladium (15 nm) and examined using SEM machine  at an accelerating voltage of 10 kV.The SBS test was conducted using a chisel on a universal testing machine  with an occluso-gingival load applied vertically at the bracket base at a crosshead speedCalcium phosphate phase determination of BCA and BPA formulations was carried out using XRD. Three pastes of each formulation were prepared, each was kept in a glass vial for 24 h and ground with a mortar and pestle after complete setting to get CaP powder. The powder samples were examined using a Bruker D4 Diffractometer in a Flat-plate geometry using Cu Kα12, 40 kV and 30 mA X-ray radiation.In order to examine the etch-patterns produced, CaP precipitation potential and enamel damage, SEM examination of the etched buccal enamel surface with 37% PA gel and BPA paste was conducted before bracket bonding and after bracket removal.Nine extracted premolars were used in this analysis and divided into three groups: three teeth were kept intact and the others randomly etched with either of the two aforementioned etchants. The crown of each tooth was sectioned mesio-distally through the occlusal central fossae using a diamond wafering blade to obtain nine buccal halves. The etchant was applied on the buccal enamel surface for 30 s, followed by irrigation with water for 20 s and then dried for 20 s. All samples were kept dry at ambient laboratory conditions for 24 h, then sputter-coated with gold palladium (15 nm) and examined using SEM machine  at an accelerating voltage of 10 kV.59:The quality of enamel etch pattern produced was assessed and scored according to the etch-pattern scaleType (1): Ideal etch, preferential dissolution of the prism cores resulting in a honeycomb-like appearance.Type (2): Ideal etch, preferential dissolution of the prism peripheries resulting in a reverse honeycomb or cobblestone-like appearance.Type (3): A mixture of type 1 and 2 patterns.Type (4): Pitted, roughened enamel surface. Structures look like unfinished maps or networks, enamel prisms not evident.Type (5): Flat smooth surface, no apparent etch.60 which includes the following categories:The samples for this analysis were obtained from bracket-bonded premolars previously subjected to metal and ceramic bracket debonding following 24 h water storage at 37 °C and 5000 cycles thermo-cycling. Three metal and three ceramic bracket-debonded premolars of each of the PA gel- and BPA paste-etched subgroups were randomly collected, the crown of each tooth was sectioned mesio-distally through the occlusal central fossae using a diamond wafering blade to obtain the buccal bracket-debonded half, sputter-coated with gold palladium and examined with SEM. The debonded enamel surface was assessed according to the Enamel Damage IndexGrade (0): Smooth surface without scratches, and perikymata might be visible.Grade (1): Acceptable surface, with fine scattered scratches.Grade (2): Rough surface, with numerous coarse scratches or slight grooves visible.Grade (3): Surface with coarse scratches, wide grooves, and enamel damage visible to the naked eye.36 . The buccal enamel halves were mounted face down in cold-cure acrylic resin using silicon molds (8 × 21 × 24 mm). The outer enamel layer was removed using a water-cooled rotating polishing machine  and a sequential polishing protocol with silicon carbide abrasive disks  at a speed of 200 rpm: 600-grit for 10 s, 1200-grit for 20 s, 2500-grit for 30 s, and 4000-grit for 60 s. Each was followed by 1 min of water bath ultra-sonication to remove the smear layer at the enamel surface, excepting the 4000-grit which was followed by 3 min ultra-sonication. This standardized polishing protocol permits the removal of approximately 400 µm from the outer enamel layer and results in a flat, smooth and highly polished enamel surface36 Fig. . The sur36 Fig. . The sam61  was added to the control 37% PA gel and the liquid phase (37% PA solution) of the BPA paste to obtain fluorescent etchants. Three samples were used for CLSM examination. The microscopy examination was performed using a CLSM  equipped with a 63x magnification/1.4 NA  oil-immersion lens, and a laser illumination setting of 568-nm krypton (rhodamine excitation). Representative images of the most common distinguishing characteristics detected in each specimen were captured. All images were further reconstructed with Image J software.CLSM was conducted to compare the etch-patterns produced by the BPA etchant paste and 37% PA gel. Before enamel etching, 0.1 wt.% Rhodamine B dye−1 and a CCD (charge-coupled device) exposure time of 2 s. The microscope was calibrated using an internal silicon sample with a characteristic band at 520 cm−1. For each control and treated area, a Raman map was recorded at the middle part covering an area of 200 × 300 µm2 acquired with a 2.7 µm resolution. Raman maps were exported into an in-house curve-fitting software to fit the spectra and obtain the intensity mean of four phosphate peaks, in addition to the phosphate peak ratio (v1/v4)53.Ten enamel samples were used. A Renishaw inVia Raman microscope  running in a Streamline scanning mode was used to scan each area using a 785-nm diode laser (100% laser power) focused using a 20×/0.45 NA air objective. The signal was acquired using a 600 lines/mm diffraction grating centered at 750 cmp< 0.05. The statistic value of level of significance for multiple Mann-Whitney comparisons, when conducted after Kruskal-Wallis test, was 0.008 instead of 0.05; calculated according to Bonferroni correction.Sample size determination was based on one-way analysis of variance (ANOVA) comparing the SBS of three different subgroups. A sample of 15 specimens per subgroup is required to detect a significant difference with an effect size of 0.35 and 80% power 2-tailed test at 5% level of significance. Sample size calculation was achieved using G-power version 3.1.7 . Analysis was conducted using SPSS statistical software . Data were tested for normality using Histogram/Q-Q plots/Shapiro-Wilk tests. One-way ANOVA was conducted for parametric data analysis , followed by Tukey HSD post hoc multiple comparisons. Kruskal-Wallis and Mann-Whitney tests were carried out for non-parametric data analysis (ARI score). All statistical analyses were conducted at level of significance