Circulating tumor cell (CTC) count has prognostic significance in metastatic breast cancer, but the predictive utility of CTCs is uncertain. Molecular studies on CTCs have often been limited by a low number of CTCs isolated from a high background of leukocytes. Improved enrichment techniques are now allowing molecular characterisation of single CTCs, whereby molecular markers on single CTCs may provide a real-time assessment of tumor biomarker status from a blood test or “liquid biopsy”, potentially negating the need for a more invasive tissue biopsy. The predictive ability of CTC biomarker analysis has predominantly been assessed in relation to HER2, with variable and inconclusive results. Limited data exist for other biomarkers, such as the estrogen receptor. In addition to the need to define and validate the most accurate and reproducible method for CTC molecular analysis, the clinical relevance of biomarkers, including gain of HER2 on CTC after HER2 negative primary breast cancer, remains uncertain. This review summarises the currently available data relating to biomarker evaluation on CTCs and its role in directing management in metastatic breast cancer, discusses limitations, and outlines measures that may enable future development of this approach. Despite disease , continuCTCs are tumor cells that have escaped from the primary (or metastatic) tumor into the blood. As this is a critical step in the ability for cancers to metastasise, CTCs are considered potential precursors of metastatic disease  or “metaet al. [i.e., isolated tumor cells detectable in bone marrow), appear to be a better prognostic marker in advanced breast cancer [The prognostic role of CTCs in MBC is now well established. In a landmark study from Cristofanilli et al. , women wet al. ,9,10. Fut cancer . While dt cancer ,13. etal., CTC count after the 1st cycle of chemotherapy was the only independent factor significantly associated with OS or PFS [NCT00382018), which aims to enrol 651 patients with MBC, and is addressing the question of whether or not a treatment regimen should be altered early in its course based on lack of CTC response. Results from this trial may allow refinement of response evaluation and, in particular, prevent unnecessarily prolonged treatment with an ineffective agent. Although CTCs can provide prognostic information in breast cancer, their utility as predictive markers is less certain. CTC count has been suggested as a means of monitoring therapeutic response to treatment. Patients with an elevated CTC count prior to commencing therapy, which subsequently does not decrease following treatment, have been shown to have poorer outcomes, which might therefore predict treatment resistance ,15. In aS or PFS . Based oRather than using CTC count alone, assessment of the molecular characteristics of CTCs might be useful. Theoretically this could enable improved treatment selection and better prediction of response to a specific therapy. Molecular analysis studies of CTCs have previously been hampered by limited ability to detect and enrich for CTCs from a high background of lymphocytes. However, recent advances in isolation and enrichment techniques now allow more detailed assessment of CTCs, including analysis of molecular characteristics or expression of biomarkers, with potential treatment implications.The estrogen receptor (ER), progesterone receptor (PgR) and HER2 receptor are the only three validated biomarkers routinely applied in breast cancer management. ER, PgR and HER2 expression provide prognostic information, and inform on decisions regarding therapy. Endocrine therapy is only effective in the setting of ER and/or PgR positive disease, while anti-HER2 therapies are effective against those tumors overexpressing HER2. Currently, treatment decisions at the time of MBC relapse are generally made based on the receptor status of the primary breast cancer. However, there has been increased awareness of the possibility of discordance in receptor status between primary tumor and disease recurrence, leading to suggestions that reassessment of receptor status at the time of disease recurrence should be recommended. One of the hallmarks of cancer is genomic instability , indicatDiscordance between primary tumor and MBC tissue biopsies occur at rates of around 10%–30% for ER and 20%–50% for PgR, with losses being more common than gains. HER2 discordance has been reported in around 10% of paired primary and metastatic tumors, with loss and gain occurring with relatively equal frequency . While tInterventional radiology and imaging now allow the option of obtaining a biopsy from the vast majority of metastatic sites. While tissue biopsies are relatively cheap, well validated, and generally easy to obtain, they are not benign procedures and can cause pain, minor bleeding or infrequently, more severe complications such as pneumothorax or haemorrhage. Analysis of receptor status on CTC is therefore an attractive option, as it might allow “real-time” analysis of ER, PR, HER2 plus other clinically relevant biomarkers to direct therapy from a simple blood test, that is, a “liquid biopsy” see . Further®  [® is a semiautomated method that isolates CTCs by firstly applying an antibody for the epithelial cell adhesion molecule, EpCAM, which is often present on carcinoma cells including breast cancer, but not present on normal blood components. EpCAM antibodies are attached to microscopic iron particles, thus when a magnetic field is applied across the blood sample, EpCAM positive cells are isolated. CTCs are then identified by expression of cytokeratin 8, 18, 19 and lack of expression of CD45, the latter being a lymphocyte marker. Finally the isolated cells are assessed morphologically for large nuclei and size as well as other characteristics of malignancy [A major challenge in the investigation of CTCs is that they are rare, and thus their detection is particularly challenging. The ratio of CTC to lymphocytes is in the order of 1 to 1,000,000, while it is around one in a billion for CTCs compared with erythrocytes. Given their rarity, the initial step in CTC analysis is their isolation, or enrichment, from other normal blood cell components. There are numerous technologies in development for this step, with the most widely used being CellSearchNJ, USA) ,22. Celllignancy . ® , MACS (Magnetic Activated Cell Sorting system) [This principle of an antibody (most frequently EpCAM) conjugated to magnetic particles is an immunomagnetic method, and forms the basis of CTC enrichment in other technologies including AdnaTest system) , and Mag system) . For ins system) .Importantly, there is increasing evidence that CTCs are a heterogeneous population of cells and that the most invasive and aggressive phenotype is associated with an epithelial to mesenchymal transition (EMT) and stem cell-like portrait. In this context, an enrichment technique that relies on EpCAM might be ineffective at selecting cells undergoing EMT progression, whereby EpCAM expression is lost. Consequently, some researchers are now evaluating the use of EMT markers to select CTCs ,27.®  [® [Other enrichment methods separate CTCs from normal blood components based on physical properties, for example differing density-gradients  ), or difmany) [® ). Densitmany) [® , Herringmany) [® ) can allmany) [® . A potenmany) [® .et al. [® compared with AdnaTest® in a prospective trial of MBC patients, while Punnoose et al. found that CellSearch® and CTC-Chip performed similarly as detection methods for CTCs in cell lines and whole blood samples from healthy donors, breast, and lung cancer patients [® is, at present, the only method adequately validated to receive FDA approval. Few studies have directly compared enrichment methods. Müller et al.  demonstrpatients . Developpatients , and whi® Analyzer digital microscope of the CellSearch® System [Just as there are various methods for isolating CTC from whole blood, assessment of biomarkers on CTCs can be achieved using different techniques: through evaluation of protein expression, mRNA expression, or chromosomal abnormalities. To evaluate protein expression on CTCs, the most well validated assay is the immunofluorescence (IF) staining for HER2 on single CTCs through the fourth filter on the CellTracks® System ,37,38,39® System . in situ hybridization (FISH) to assess for chromosomal abnormalities [–), an absolute HER2 gene copy number between four and six or HER2/CEP17 ratio between 1.8 and 2.2 is considered HER2 equivocal, and an absolute HER2 gene copy number greater than six or HER2/CEP17 ratio >2.2 is considered HER2 positive (HER2+) [et al. recently reported that, from a small case series, seven of 49 MBC patients had polysomy 17 detected on CTCs, although no CTCs with polysomy 17 were also HER2 amplified on FISH analysis [Alternatively, after enrichment isolated CTCs can be transferred onto a coated slide and stained with an antibody of interest, or analysed using fluorescence malities ,38,41. Fmalities ,43. The  (HER2+) . Importaanalysis . ® system, and after a process of nucleic acid enrichment, mRNA expression can be evaluated quantitatively [®, after CTC enrichment and lysis, mRNA is isolated and retrotranscribed in cDNA, cDNA can be tested for ERBB2 (HER2) mRNA [Regarding the expression of mRNA on CTCs, this is assessed using real-time reverse transcription polymerase chain reaction (RT-PCR) ,46. Folltatively . MoreoveR2) mRNA , “stemneR2) mRNA . As the ® are being developed that allow mRNA assessment. For instance, after HB Chip CTC enrichment, Yu et al. was able to evaluate EMT markers on these CTCs using QuantiGene View RNA in situ hybridization . Furthermore, they demonstrated that the presence of CTCs with EMT characteristics correlated with disease progression [Technologies other than AdnaTestgression .® , a novel semiautomated system comprising of a chip with microelectrode that create electric cages in which single CTCs are effectively trapped [®. Following isolation of single pure CTCs, the cells’ DNA is amplified and sequenced, and genetic markers of interest assessed. As the CTCs collected are pure, downstream analyses are much less likely to be confounded with contaminants. A further analysis technique worth mentioning is that of DEPArray trapped . IdeallyIn a previous study from our group that compared techniques for determining HER2 status on CTCs, good concordance between IF and FISH was demonstrated. From 25 evaluable MBC patients with both FISH and IF analysis of CTCs, 19 of 20 patients with HER2 negative CTCs on FISH were also IF negative, while four of five patients with HER2 amplification had concordant IF results . This coThe majority of studies assessing biomarker expression on CTCs have been focused on HER2, with limited data on hormone receptors or other putative biomarkers. The HER2 status has therapeutic implications, being a strong predictive marker for clinical benefit from HER2-targeted therapies such as trastuzumab and lapatinib, in both the metastatic and adjuvant breast cancer settings ,54,55,56+ CTCs or the efficacy of anti-HER2 therapy.Of the studies that have assessed HER2 expression in CTCs, the majority have evaluated discordance rates between CTC HER2 expression and that of the primary breast cancer, with few assessing the prognostic role of HER2+ breast cancers typified by aggressive clinical course and early relapse. Much less is known about the prognostic implications of HER2+ CTCs. In a study of 76 patients with MBC from Munzone et al. [+ CTCs at baseline after HER2- primary breast cancer had significantly shorter PFS than those who retained HER2− status on CTCs, and patients with no CTCs  [et al. [+ CTCs, compared with 43 MBC patients without HER2+ CTCs. In a multivariate analysis, the presence of HER2+ CTCs was an independent prognostic factor along with the number of prior therapies, while CTC count, although not HER2+ CTCs specifically, was an independent prognostic factor for OS [+ CTCs, as defined by expression of HER2 mRNA derived from CTCs, was also associated with shorter disease free survival (DFS) in a cohort of early breast cancer patients, although this did not remain an independent prognostic factor in a multivariate analysis, nor was there any significant association with OS [+ CTCs was associated with worse DFS and OS. In a multivariate analysis HER2+ CTCs and T stage were the only independent prognostic factors, although importantly, CTC count per se was not included as a covariate [HER2 positivity is a known poor prognostic factor ,58,59 wie et al. , those wctively) . Similar [et al. , PFS andr for OS . Identif with OS . Similarovariate . + CTCs is the potential influence of anti-HER2 therapy. Giordano et al. recently reported that, in a cohort of over 500 MBC patients, CTC count retained prognostic significance in ER+ and triple negative subtypes, but this was lost in patients with HER2+ disease after trastuzumab treatment [et al. similarly found that a CTC count of 0 per 7.5 mL of whole blood was associated with better prognosis in all subtypes except HER2+, further suggesting this may have been due to the impact of targeted therapy, though no data on anti-HER2 therapy received was reported [An additional confounding factor regarding the prognostic role of HER2reatment . Munzonereported . et al. defined HER2 positivity as the presence of ≥1 CTC expressing HER2 with IF staining intensity of at least 2.5 times background [et al. used threshold of at least five CTCs detected, of which ≥75% had to be HER2 positive [et al. [+ CTCs to total CTCs of >10% compared with HER2+ to total CTC ratio of >50% resulted in the proportion of patients being classed “HER2 positive” of 64% and 26%, respectively [® or other RT-PCR based techniques do not allow quantification of the number of HER2+ CTCs, hence any HER2 mRNA detected could be considered positive. Discordance between primary breast cancer and HER2 expression on CTCs in the setting of disease recurrence has been reported at variable rates, with rate of gain of HER2 from 9% to over 60% see . Importackground . In contpositive . In a pr [et al. , whetherectively . It is w+ CTCs in a patient with HER2− disease is effective. As yet, there are minimal data addressing this. Meng et al. [− primary tumor, that four of nine patients with HER2+ CTCs at the time of metastatic disease received trastuzumab. Of these, one had rapid remission of symptoms and complete response on imaging, two patients had partial responses and one no response [Arguably the most important factor relating to HER2 analysis on CTCs is whether targeted treatment of HER2g et al.  reportedresponse .et al. In this prospective feasibility study, patients with recurrent disease after previous HER2− primary breast cancer were screened for the presence of CTCs. From 139 screened patients, 96 had at least five detectable CTCs per 7.5 mL whole blood, and seven (7%) were HER2 positive, with HER2 positivity defined as ≥50% of CTCs staining HER2+ on CellSearch® IF assay. These seven patients were commenced on lapatinib therapy, 1500 mg/day. There were no observed responses, while one patient has stable disease lasting 8.5 months [Conversely, minimal activity from anti-HER2 therapy was evident in an open label phase II trial from Pestrin 5 months . Of noteet al. [+ CTC pool was observed in four patients on treatment, this did not appear to result in a meaningful clinical effect. The small number of included patients limits interpretation of the results from both these trials, highlighting the need for additional prospective studies.In a parallel trial from Stebbing et al. , the feaet al. . While det al. also reported reduction in the pool of EGFR+ CTCs with lapatinib treatment in a case report of a woman with chemotherapy-refractory HER2+ MBC. Decreased EGFR+ CTC count coincided with treatment response, while at the time of disease progression, increased EGFR negative and HER2 negative CTCs were evident [Interestingly, Liu  evident . NCT01619111) was launched by the same group in early 2012. This multicentre study compares standard therapy with or without lapatinib in patients with MBC after HER2− primary tumor but with HER2+ CTCs [et al. being published. The results of this larger trial will be of interest, particularly in light of the negative results from the phase II trial. A further trial in development evaluating anti-HER2 therapy efficacy in the setting of HER2+ CTCs is the provisionally named CirCè XXX1 trial [Based on the results from previous studies demonstrating alteration, in particular gain, in HER2 status during progression to MBC ,67, the R2+ CTCs  . The trial aims to determine if, using trastuzumab to facilitate antibody dependent cell-mediated cytotoxicity (ADCC), HER2+ CTCs can be eradicated, leading to improved clinical outcomes , although the clinical implications of mutations in this gene are unclear. While dysfunction in PI3K pathway can increase tumorogenesis, retrospective data suggest that outcomes might be more favourable in patients with ER+ breast cancer expressing a PIK3CA mutation [Activation of the PI3K/Akt/mTOR signalling pathway can lead to increased cell proliferation and dysfunction of tumor suppression function . Mutatiomutation . PIK3CA mutations, this gene is of significant interest given both the prevalence of reported mutations, and the existence of a number of targeted therapies already in clinical development against this pathway. Thus, evaluation of PIK3CA mutations on CTCs might have clinical relevance. Following enrichment of blood samples from 44 MBC patients with CellSearch®, Schenk et al. were able to isolate, amplify and analyse genomic DNA for PIK3CA mutations in the known mutations hotspots of exon 9 and 22. Mutations were detected in 7 out of 44 patients (16%), although the frequently reported E542K, E545G and E545A variants were not detected [Despite the uncertainty regarding the prognostic impact of detected .PIK3CA mutations, in MBC. Utilization of CellSearch® followed by DEPArray™ enabled recovery of single pure CTCs in patients with MBC. Using whole genome amplification (WGA) and sequencing reactions on single CTC DNA samples, mutations in the PIK3CA gene were identified in 4 out of 13 evaluable patients [PIK3CA gene. While significantly limited in numbers at present, this pilot study demonstrates that isolation and molecular analysis for PIK3CA mutation on single CTCs in MBC patients is feasible. Ongoing planned research includes further validation of this method and evaluation of PIK3CA mutational status defined on CTC compared with that of tumor tissue from primary or metastatic disease. While this approach demonstrated feasibility, ultimately prospective evaluation of targeted agents against aberrant PI3K signalling pathway in patients with PIK3CA mutation positive CTCs would be required to clarify the clinical utility of this approach. Our group has recently reported an alternative feasible method for the molecular assessment of CTCs, in particular patients . In an eChemotherapy is effective in the treatment of MBC, however, not all those patients treated will respond, although all will be subjected to the toxic side effects. Results from neoadjuvant chemotherapy trials suggest tumor biology is an important factor in determining response to chemotherapy, whereby luminal A breast cancer is relatively chemoresistant, with other subtypes are chemosensitive . Otherwiet al. reported that detection of MUC-1 mRNA from CTCs after the first cycle of chemotherapy was significantly associated with poorer response rate compared with patients with undetectable MUC-1 mRNA, while there was a trend towards increased PFS in MUC-1 mRNA negative patients. MUC-1 mRNA prior to commencement of chemotherapy showed no correlation with efficacy of chemotherapy [There are preliminary data suggesting that CTCs might be useful in chemotherapy response prediction both at baseline and after treatment commencement. CTC count has been suggested as a marker of chemotherapy response in MBC ,15. Furtotherapy .et al. [An extension of the concept of evaluation of chemosensitivity in general is that of evaluation of sensitivity to specific chemotherapeutic agents, of which there are currently no known validated biomarkers. Conflicting and inconclusive data exist for the putative biomarker TOP2A and response to anthracyclines ,83,84, wet al.  investiget al. . These data are very preliminary and limited by low patient numbers, yet intriguing, and further research in this area would be of particular interest to improve efficacy and reduce unnecessary toxicities from chemotherapy in treatment of MBC. ®, along with several other CTC enrichment techniques, relies on the presence of epithelial cell markers, CTCs that do not express EpCAM, such as those that have undergone EMT may be missed [+ compared with higher risk tumors has been reported [As CellSearche missed ,89. EMT reported ,64. Althreported ,91,92.et al. recently reported development of a mesenchymal-based method of isolating CTCs in men with metastatic prostate cancer based on surface expression of OB-cadherin [There is a need to improve CTC detection techniques, in particular focusing on methods for identifying CTCs even in the setting of EMT. One such approach might be through use of antibodies against Twist1, Akt2 or other EMT markers . For inscadherin . The uticadherin . CD146 icadherin . Finallycadherin . While there is only one FDA approved method for CTC enrichment, many others are in development and show promising preliminary results. Studies comparing different technologies might be useful to suggest the relative sensitivity and specificity of each. In order for CTC molecular analysis to enter routine clinical practice in the future, enrichment of CTCs and the subsequent method of CTC analysis should ideally be standardised, so that results obtained might be applied more easily across patient populations.In particular, differences in rates of HER2 positivity (or other molecular markers) can be seen depending on which detection and analytical method is applied . This ma+ disease based on CTC HER2 status. While the assays to detect HER2 on CTCs are themselves reliable, interpretation of these results and their utility in a clinical context are not. Given the known heterogeneity of HER2 overexpression within single tumors, not all CTCs would be expected to be HER2+, but the optimal cut-off for the proportion of HER2+ CTCs to define a patients CTC-HER2 positive is yet to be determined. Standardisation of the definition of HER2 positivity can only be achieved through evaluation in a prospective clinical trial setting. Employing a definition of HER2 positivity that results in no response to anti-HER2 therapy has little clinical relevance. It might be possible to evaluate this issue within clinical trials of anti-HER2 therapies by selecting a lower threshold for HER2 positivity, with evaluation of response in subgroups based on number and proportion of HER2+ CTCs.Since formulation of the ASCO and CAP guidelines for defining HER2 positivity, there is reasonable reliability of HER2 results obtained from tissue samples. Molecular analysis on CTCs is a relatively new field and as yet, it is not known how best to define a patient as having HER2CTC analysis remains a relatively costly research tool at present, primarily due to the cost of the analytical equipment. In addition, expertise is required to perform CTC analysis, limiting access of this technology to dedicated research laboratories. Ongoing expansion of this field however, will ideally see a decrease in the costs related to access to CTC technologies, as well as an increase in the number of skilled researchers with expertise in interpretation of CTC molecular analyses.Studies evaluating CTC molecular characterisation are all relatively limited in terms of number of included patients. As such, results across all studies should be interpreted with caution and no firm conclusions be drawn. Unlike the prognostic utility of CTCs in MBC, which has been well established and validated in prospective trials, predictive biomarkers on CTCs lack adequate prospective validation and therefore are not yet ready to be applied in a clinical setting.CTCs are rare, and analysis of biomarker expression, such as HER2, on CTCs provides markedly less comprehensive assessment of the entire tumor than might a tissue biopsy, where hundreds of cells can be analysed. There is minimal data available on heterogeneity of biomarkers on CTCs, raising the question of whether molecular analysis results are fully representative of the metastatic tumor. Furthermore, tumor heterogeneity in tissue samples is well recognised, increasing the probability that CTCs expressing differing molecular profiles might be seeded into the bloodstream. Research into the impact of heterogeneity is crucial, and specifically if, in the setting of discordance with the primary tumor, treatment should be targeted against the most aggressive characteristic found, or, alternatively, the most common. Indeed, it will not be possible to reliably interpret CTC biomarker results in a clinical context until our understanding of CTC biomarker heterogeneity improves.Biomarker analysis of single CTCs is now becoming a feasible approach analytically, although, critically, the clinical relevance of biomarker expression on CTCs is unknown. In particular, there is no evidence that treatment of patients’ metastatic disease based on the CTC molecular profile, rather than the primary tumor, improves clinical outcomes. Studies that have assessed HER2 discordance between primary breast cancer and tissue biopsy of metastatic disease report alterations in treatment administered due to rebiopsy, with some suggesting treatment response to anti-HER2 therapy with HER2 gain, however these data are very limited . In ordeMolecular analyses on CTCs are feasible, although there are many questions that must be addressed in terms of reproducibility, assay sensitivity, reliability of results and clinical relevance before this approach can be considered suitable for routinely guiding management in MBC. Although the existing challenges that must be met in order to translate CTC biomarker analysis into a technique that can improve outcomes in MBC patients are substantial, the potential benefits are at least as, if not more, substantial. Such benefits include improved prognostication, increased understanding of breast cancer disease processes, feasible disease monitoring on therapy, and adjustment of treatment based on real-time biological changes in cancers. With ongoing development of this field, CTC analysis might provide us in the future with a relatively simple, repeatable and reproducible assay to help tailor treatment for individual MBC patients, leading ideally to improved outcomes in this disease.


Synchronous tumors can be independent primary tumors or a primary-metastatic  pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC) mutations/ # total HC mutations).Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas.We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs  and 283 known tumor clonal pairs , across renal cell, testicular, and colorectal cancer. The CLS test correctly classified all validation samples but one, which it appears may have been incorrectly classified in the published data. As proof-of-concept we then applied the CLS test to two new cases of invasive synchronous bilateral breast cancer at our institution, each with one hormone receptor positive (ER+/PR+/HER2-) lobular and one triple negative ductal carcinoma. High confidence mutations were identified by exome sequencing and results were validated using deep targeted sequencing. The first tumor pair had CLS of 81% , supporting clonality. In the second pair, no common mutations of 184 variants were validated , supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair.We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility. Synchronous bilateral breast cancer (SBBC), in which separate tumors are diagnosed simultaneously in each breast, occurs in 1–3% of all breast cancer patients . The twoPrior studies aiming to distinguish clonal from independently arising SBBC tumors have been limited in their ability to detect clonal status. They have used presence of concordant histological features and systemic metastases  or been We investigated whether mutational profiling from whole exome sequencing can distinguish between clonal and independently arising tumors in SBBC and several other cancer types. The Clonal Likelihood Score (CLS) test statistic was computed as the percentage of high-confidence (HC) mutations shared by both tumors, out of the total number of HC mutations identified in the pair. A formal statistical test was developed and recommended parameters were defined using tumor pairs (mainly breast cancer) of known clonal status in The Cancer Genome Atlas (TCGA) database. We validated the test using recommended parameters on five independent datasets with known or putative clonal status from TCGA and the literature, including renal cell carcinoma, testicular cancer, and colorectal cancer. We then applied the CLS test to whole-exome sequencing data from two SBBC cases of unknown clonal status at our institution, and validated our conclusions with targeted deep sequencing. We also evaluated the biological evidence for our resulting call of metastatic SBBC. We developed recommendations for carrying out the CLS test on tumor mutational profiling data.Tissue specimens were collected by the Moores Cancer Center Biorepository from consented patients under a University of California, San Diego Human Research Protections Program Institutional Review Board approved protocol (HRPP#050887 then 090401). Biorepository subjects provide a written consent which is maintained in the Biorepository archives. Detailed sequencing methods are in We called single nucleotide variants (SNVs) in each tumor and germline sample using GATK  with defn denote the total number of genomic loci where a mutation was called in either tumor, and let X denote the number of such loci where the mutation is shared. The CLS test is based on the proportion of shared mutations p out of n total mutations identified in a tumor pair, that is, p = X/n. Since n is known and can be considered fixed, X can be modeled as Binomial with parameters n and p.Let 0: p<p0, against the alternative hypothesis Ha: p>p0. Here, p0 is the maximum rate of shared mutations expected to be called among independent tumor pairs, either from chance occurrence of true shared mutations, such as driver mutations, or from false positive (FP) calls from a given sequencing technology. In order for the CLS to have acceptable specificity and control of Type I error, p0 must be set at an appropriate empirically determined rate, as discussed below. The CLS test rejects the hypothesis of independence in favor of clonal status if CLS > c/n, where c is the critical value from an exact one-sided binomial test at the 5% significance level with parameters n and p0 . Thus, the proposed CLS test should be applicable to any variant calling assay which has a FP rate for shared mutations below 3%-4%.To establish the parameters of the CLS, we computed the empirical distribution of the CLS from independent unrelated tumors in the TCGA (June 2012 release) . We down3% to 4% . Settingpa = 15% or 20% as a lower bound for the rate of shared mutations expected to occur in clonal tumors appears to conservatively allow for adequate power. The number of mutations needed for the CLS test to achieve 80% power under reasonable assumptions is given in p0 = 4%, if a shared mutation rate of 15% or greater is expected in clonal tumors, at least 44 called HC mutations are needed to have 80% power to detect clonal status, using the CLS test. If we consider that a 20% rate of shared mutations can be expected in clonal tumors, then only 21 total mutations observed for the tumor pair are needed to achieve 80% power. Any variant calling method which attains sensitivity to detect shared mutations set by pa would have adequate power for the CLS test.We also investigated the distribution of the CLS in known clonal tumor pairs. In total we found fifteen primary and metastatic tumor pairs with genome wide mutation profiling data in TCGA (July 2015 release) across ten tumor types  by the clustering analysis of SCNAs, however the tumor pair had 31 shared mutations out of 59 total mutations with a CLS of 53%, making it extremely unlikely to be of independent origin. Lee et al. noticed that patient 353 had the fewest number of SCNA (in particular loss of heterozygosity) among all the patients [Next, we applied our test to the validation dataset that had the strongest similarity to our proposed setting of clinical testing for synchronous tumors. Lee et al. characterized fifteen pairs of synchronous primary and metastatic colorectal tumors at the time of diagnosis by both SNP array and exome sequencing . Using upatients . Howeverpatients . Furtherpatients . TherefoWe obtained germline DNA and fresh frozen tumor samples from two women who presented with synchronous bilateral node-negative invasive breast cancers, each with one tumor ER+/PR+/HER2- (HR+) lobular and the other triple negative (TN) ductal . PatientWe identified 50 to 100 HC mutations per tumor . For patH0: p<0.04 against a one sided alternative, with X = 50 successes in n = 62 trials, yields p<1 x 10−15, rejecting the null hypothesis of independence. An exact Clopper-Pearson 95% confidence interval for the CLS is 69% to 90%. This establishes strong evidence in favor of clonal status for patient 1.For patient 1, an exact binomial test of 0: p<0.04 against a one sided alternative, with X = 2 successes in n = 184 trials, yields p >0.99, failing to reject the hypothesis of independence. The 95% Clopper-Pearson confidence interval is 0.1% to 4%, consistent with independence, and well below shared mutation rates observed in TCGA.For patient 2, an exact binomial test of HWe used an independent targeted deep sequencing assay to validate the called shared mutations. For patient 2, we re-sequenced the two called HC shared mutations. We also re-sequenced all loci  with an alternate allele prevalence > 5% in both tumors (n = 8), as these appeared to be the most likely candidates for a false negative call. All ten mutations sent were confirmed as sequencing artifacts, with homozygous reference genotype in the germline and two tumor DNA samples. Thus, there were no confirmed shared mutations out of 184 HC candidates, consistent with independence. The estimated false positive rate for this putative independent pair was about 1% (2/184 = 0.011). The validated CLS was 100 x 0/182 = 0%.In our own data, the FP rate for shared mutations was about 1% of total called mutations in the putative independent SBBC pair; detailed analysis of false positive and false negative rates from targeted deep sequencing is in ESR1 is located and observed large-scale loss-of-heterozygosity (LOH) common to both tumors  in gene NF1 and a deleterious missense mutation (E1436A) in gene NCOR1 [MYC and NF1 alterations are potentially actionable [In addition, we identified several known shared driver alterations including chromosome 8 amplifications affecting ne NCOR1 . Among ttionable , 35, demWe have developed a carefully defined and effective approach using mutational profiling from next generation sequencing data to distinguish between clonal and independently arising tumors in SBBC, and potentially in other cancer types. The Clonal Likelihood Score (CLS) is computed as the percentage of High Confidence mutations which are shared by both tumors, out of the total number of distinct genomic loci with High Confidence mutations identified in the pair. The CLS test is implemented as a one sample binomial test of proportions, with null hypothesis of independence, and recommended test parameters determined empirically from TCGA data. Importantly, the data suggest that up to 2% of tumors from different patients may have at least one shared mutation, arising from common driver mutations, technical artifacts, and shared passenger mutations arising by chance, and the rate is higher, up to 4%, for tumors arising within the same patient. Thus, careful implementation of the CLS test such as we propose here is needed to control error rates. Using data from TCGA and the literature, we have identified conditions under which the CLS test can be expected to have at most 5% Type I error rate, , and also adequate power (80%) to correctly detect a clonal tumor pair, using high confidence somatic mutation calls.The CLS test has a null hypothesis of CLS ≤ 4% against a one-sided alternative; the test achieves 80% power to detect a CLS of 15% if there are at least 44 HC mutations called in the tumor pair. We validated the CLS test in five independent data sets containing both clonal and independent tumors, with various tumor types: renal cell carcinoma, testicular cancer and colorectal cancer. In these validation data, the CLS test correctly called all but one of 15,758 putative independent tumor pairs as independent, with maximum CLS of 4.1% and minimum p-value of 0.48. We believe the one discordant result (CLS = 0.52) was likely a clonal pair which was missed due to low power of the copy number based analysis which was used to classify this pair as independent in the published paper from which we drew these data. The CLS test also correctly called all 283 clonal pairs in the validation data as clonal, with minimum CLS 13%  and maximum p-value p< 0.01.We then applied the formal CLS test to exome-wide sequencing data on two cases of invasive SBBC of unknown clonal status, each with one HR+ lobular and TN ductal carcinoma. In one SBBC patient, the CLS was 81% 95% CI: 69%, 90%) shared out of 62 total HC mutations called (p< 0.0001), establishing clonal origin of the two tumors. In the other patient, the CLS was 1.1%  shared of 184 total HC mutations called (p = 0.99), consistent with independent origin. For the clonal SBBC case, we were able to determine the likely primary tumor by investigation of copy number alterations and to identify a plausible mechanism of the change from ER+ to ER- during metastasis. We also discovered a novel potential driver alteration (e.g. 6q25.1-6q27 deletion) associated with metastasis but not needed to adapt to a new physiological environment outside the breast, highlighting the unique biological information available in SBBC. It is also important to note that two clinical measures currently used to identify clonal SBBC are concordant histological features and presence of systemic metastases [9%, 90% sFor adequate control of Type I error in the CLS test, the mutational profiling pipeline should maintain a call rate of shared mutations of 4% or less in independent tumors. To achieve this, we recommend using a moderately sensitive, but highly specific, variant caller and stringent filtering rules. We used GATK with default parameters and required adequate information across all three tissues at the same locus (germline and two tumor samples). We used targeted deep sequencing (8000x) to validate our results, and our validation data support the use of this approach Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×103 CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2–3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management. Xanthomonas fragariae is a bacterium that causes angular leaf spot (ALS) of strawberry. It is an important disease in strawberry production regions worldwide, particularly in nursery production [oduction –3. The bX. fragariae, and production sites be documented free from ALS for the past five growing seasons [ALS significantly affects the commercial value of the crop in several ways. In nursery production, losses occur when plant batches are rejected by inspectors due to violations of quarantine regulations. For example, to export strawberry plants to Europe, the European and Mediterranean Plant Protection Organization (EPPO) mandates that nurseries maintain phytosanitary standards that includes planting material to be derived from mother plants certified free of  seasons , 3. In dX. fragariae can survive at low densities on plants without producing symptoms, thus complicating our ability to identify infected plants and remove them from the production chain. To address this issue, it is necessary to develop an user-friendly and reliable assay capable of detecting low quantities of X. fragariae contamination in nursery stock before they go into planting. To this end, several molecular detection tools have been developed for X. fragariae over the past twenty years. These include an enzyme linked immunosorbent assay (ELISA) [Xanthomonas species [ALS has become increasingly problematic over the past ten years, likely due to a combination of the bacteria becoming entrenched in some production settings, our inability to accurately diagnose infected nursery stock, and the absence of effective disease management strategies. One of the factors that makes management particularly difficult is that  (ELISA) , convent (ELISA) , nested- (ELISA) –8, and T (ELISA) –11. ELIS species , and amoIn recent years, loop-mediated isothermal amplification (LAMP) has emerged as a popular tool for pathogen detection in different phytopathological systems, including several with Xanthomonas species –22. In cOne limitation of many DNA-based detection methods is that plant samples are required to be purified through use of commercial DNA extraction kits which usually allow only small amounts of tissue to be sampled, or the more tedious, technically challenging, and hazardous phenol-chloroform method. Development of a method that allows a greater proportion of tissue to be sampled and increase the likelihood of detection could greatly improve our ability to detect ALS in nursery stock.X. fragariae in strawberry plants. A LAMP assay was developed and was coupled with a new sample preparation procedure that included a sample incubation step and a simple boiling protocol for DNA extraction. The incubation procedure was developed to facilitate detection when low densities of the bacteria are expected. Latent class analysis (LCA) was used to evaluate the performance of the real-time LAMP assay with fluorescent dyes. The performance of the LAMP assay with HNB dye for end product detection was also characterized. The latter method may serve as a more convenient method for on-site diagnosis.The aim of this study was to develop a more cost-effective and user-friendly method to detect X. fragariae isolates were grown on sucrose-peptone agar (SPA) for 3 to 7 days at room temperature, then transferred to sucrose-peptone broth (SPB) on an orbital table shaker to grow for an additional 24–48 h [g for 3 min, re-suspended in 1x phosphate buffered saline (PBS), and adjusted to an optical density (OD620) of 0.1, which is equivalent to ~108 CFU/mL [3, 1×103, 6×102, 4×102, and 1×102 CFU/mL. Aliquots of each dilution (100 μL) were plated on SPA plates and were counted 5 days after plating to confirm bacterial densities. 24–48 h . The bac8 CFU/mL . SamplesX. fragariae in leaf and petiole tissue for situations where the bacterial population is expected to be low and unevenly distributed. Four candidate media were chosen for testing using past experience and a cursory review of the literature as a guide for their selection [2O), SPB, and R2A broth . We use the term media broadly to refer to substances that may or may not contain nutrients. Several experiments were conducted to identify which of these media would yield the best detection results.A simple sample preparation procedure was developed to increase the likelihood of detection of election , 27, 29.2 pieces, respectively, and added separately with a 100 μL aliquot of a suspension of X. fragariae strain Xf100 in PBS (106 CFU/mL) to 10 mL of each candidate media in 50-mL sterilized flasks. Flasks were covered with aluminum foil and placed on an orbital shaker overnight. At 21 h after incubation, 1 mL of the media was collected per sample and processed for DNA extraction and tested by both LAMP and quantitative (q)PCR (both described below) to identify the media best suited for detection. Media containing leaf and petiole tissue without added bacteria served as negative controls. Each sample had three replicates. The experiment was repeated three times.About 1 g of healthy leaf or petiole tissue from strawberry variety Festival were cut with a clean razor blade in ~5 and 2 mm6 CFU/mL water suspension of X. fragariae strain Xf100. Seven days after inoculation (dai), 1 g samples of tissue from asymptomatic leaves  were collected, processed as above, and incubated in 10 mL of each of the four media for 21 h. A 1 mL sample was drawn from each flask after the incubation period and processed for DNA extraction and tested by both LAMP and qPCR (both described below) to identify the media best suited for detection. There were two replicate samples per variety/media and replicates were run over time. Pure media served as the negative controls.Strawberry plants from six varieties  were inoculated with a 10X. fragariae in downstream LAMP and qPCR amplification. First, to examine the effect of incubating tissue alone (without added bacteria) on DNA amplification, 1 g of healthy ‘Festival’ leaf or petiole tissue (processed as above) was incubated in 10 mL of each media in 50-mL sterilized flasks for 21 h at room temperature  to determine the robustness of the procedure. For each combination of strawberry variety and strain of X. fragariae, 100 μL of a 106 CFU/mL bacterial-PBS suspension was added into the incubation flasks containing 10 mL of PBS and 1 g of fresh leaf or petiole tissue processed as described above. Leaf or petiole tissue incubated in PBS buffer alone served as negative controls. Samples were collected at both 3 h and 21 h after incubation to determine if the 21 h incubation period could be shortened. The experiment was conducted twice with leaf tissue and once with petiole tissue. There were two replicated samples for each variety/strain combination in each of the three experiments. DNA was extracted and tested by both LAMP and qPCR as described below.PBS was selected as the optimal medium based on results obtained from the experiments outlined above see . The fin4, 103, or 102 CFU/mL water suspension of X. fragariae strain Xf100; nine plants per inoculum concentration. At 20, 30, and 50 dai, 18, 27, and 33 leaf and petiole samples were collected from these plants, respectively. Each sample was processed with both the QIAGEN DNeasy Plant Mini Kit –generally our standard method for processing samples–and through the sample preparation procedure developed in this study.Twenty-seven ‘Festival’ plants were spray-inoculated with either a 10X. fragariae.For standard processing , 100 mg of tissue was macerated with two nickel plated steel balls  in 600 μL of AP1 buffer mixed with 4% of PVP-40  using a Mini Beadbeater-96 . After incubation at 65°C for 10 min, samples were mixed with 180 μL of P3 buffer, processed following manufacturer’s protocol, and then eluted in 100 μL of AE buffer. For samples processed through the sample preparation procedure, DNA was extracted via the Triton-boiling approach (described below) and was suspended in 100 μL of 1% Triton X-100. A 1 μL aliquot of DNA from each method was tested by LAMP and qPCR (both described below) to compare which of these methods was best suited for detection of g for 5 min. After discarding the supernatant, the pellet was re-suspended in 100 μL of 1% Triton X-100 by vortex. Cell suspensions were then boiled for 10 min, cooled at room temperature for 5 min, and frozen at -20°C for 10 min. Frozen samples were thawed at room temperature for 5 min and centrifuged at 12,000 g for 3 min. The supernatant contained the DNA template used in downstream LAMP and qPCR reactions.The method of DNA extraction for the sample preparation procedure was a modified version of the Triton-boiling approach developed by Sowmya et al. . In the X. fragariae (GenBank accession No. EU176816.1) [X. fragariae detection and it was highly conserved among X. fragariae strains tested in previous studies and this current study [X. fragariae detection [http://primerexplorer.jp/e/), and then searched in Genbank databases by BLAST for specificity (LAMP primers were designed to target the genomic sequence of RAPD marker 295 of 76816.1) . The marnt study . The Taqetection . Primer cificity .® Green I and SYTO®9, Invitrogen, Grand Island, NY), adjusting the concentration of dNTPs, primers, MgSO4, and PCR additives (DMSO and Betaine). The final 20 μL LAMP reaction consisted of 0.2 μM each of outer primers (F3 and B3), 1.6 μM each of inner primers (FIP and BIP), and 0.8 μM each of loop primers (LF and LB), 2 μM of SYTO®9 or 125 μM of hydroxynaphthol blue , 1.2 mM of dNTPs, 6 mM of MgSO4, 1x isothermal amplification buffer, 8 U of Bst2.0 WarmStart DNA polymerase , and 1 μL of template DNA. The real-time LAMP reactions with SYTO®9 were conducted in an Eppendorf Realplex4 master cycler  at 65°C for 45 min. Melting curve analysis was conducted at 95°C for 15 s, followed by an increment from 78 to 98°C at 0.04°C /s. To view the LAMP products in agarose gel, 4 μL of the real-time LAMP amplicons were run in 1.5% of agarose gel with a 100 bp DNA ladder. The gel was stained with ethidium bromide (EtBr) for visual detection with a UV illuminator. The LAMP reactions with HNB dye were observed for color change from violet to sky blue for positive samples at 35 min after incubation at 65°C. Non-template controls were included in each run. Each sample was run in three technical replicates.LAMP reactions were optimized through testing of different fluorescent dyes  was extracted using the modified Triton boiling method described above, while fungal DNA was extracted using QIAGEN’s DNeasy Plant Mini Kit  following manufacturer’s protocol.Both LAMP and qPCR primers were tested with 40 er hosts . DNA froX. fragariae in pure culture, a serial dilution of X. fragariae strain Xf100 suspension in 1% triton was prepared to achieve 108, 107, 106, 105, 104, 6×103, 3×103, 2×103, and 1×103 CFU/mL. DNA from 100 μL of each cell suspension was extracted by adding 1 μL of 100% Triton X-100 and following the modified Triton-boiling method described above, starting with the boiling step. The experiment was repeated three times, with three biological replicates per experiment.To determine the minimum detectable concentration of X. fragariae strain Xf100 suspension in PBS was added into 10 mL PBS at concentrations of 1×106, 1×105, 6×104, 3×104, 2×104, 1×104, and 6×103 CFU/mL with 1 g of healthy leaf or petiole tissue from ‘Festival’. Healthy leaf or petiole tissues in PBS alone were included in each experiment as negative controls. Samples (1 mL each) were collected at 21 h after incubation. There were three replicates for each sample and the experiment was conducted three times. DNA samples were tested by both LAMP and qPCR.To determine the detection limit from samples processed through the sample preparation procedure, 100 μL of X. fragariae by both LAMP and qPCR as described above. A total of 293 test results were obtained (one petiole sample was lost). Of these, 123 samples were from the commercial operation, and 150 and 20 samples were collected from the experimental plots at USHRL and GCREC, respectively. Results were used for determining the sensitivity and specificity of the LAMP and qPCR assays.Strawberry leaf and petiole samples were collected from: a) commercial strawberry fields located in Balm, FL with widespread, visible symptoms of ALS; b) experimental field plots at the University of Florida’s Gulf Coast Research (GCREC) and Education Center in Wimauma, FL with very low incidence of ALS; and c) field plots without detectable ALS located at the US Horticultural Research Laboratory (USHRL) in Fort Pierce, FL. Samples from commercial fields were collected from the variety Radiance; however, fields differed in that plants were purchased from different nurseries in North America, i.e., Canadian, California, and North Carolina. Samples from the experimental field plots were collected from ‘Festival’ and ‘Ventana’. Samples were collected to have approximate even representation of leaves and petioles from symptomatic and asymptomatic plots. Samples from symptomatic plots contained leaves/petioles with and without visible ALS symptoms. A total of 147 matching leaf and petiole samples were processed through the sample preparation procedure and tested for t, threshold cycle) and LAMP  were analyzed in separate general linear mixed models (GLMM) using the SAS procedure GLIMMIX. Media and tissue type were treated as crossed fixed effects, and replication, experiment, and experiment’s interactions with the fixed effects were treated as random effects. An identity link function and Gaussian  error distribution were specified. Treatment differences were obtained using the LSMEANS statement with the LINES option. For the experiment based on inoculated plant tissues, the response variables Ct and Tt were analyzed in a general linear model (GLM) treating media and variety as crossed fixed effects, specifying an identity link function and Gaussian  error distribution, using the SAS procedure GLIMMIX. Treatment differences were obtained using the LSMEANS statement with the LINES option.For the experiment based on bacteria-spiked tissue samples, the response variables for qPCR  error distribution were specified. To evaluate the effect of the incubation step on detection in each of the media, the differences in Ct or Tt values between samples incubated with and without bacteria were obtained and analyzed by paired t-tests.DNA amplification from tissue samples incubated for 21 h with and without added bacteria  were chat and Tt were analyzed in GLMMs using the SAS procedure GLIMMIX, treating variety and Xanthomonas strain as crossed fixed effects, and replication, experiment, and experiment’s interactions with the fixed effects as random effects. An identity link function and Gaussian  error distribution were specified. Treatment differences were obtained using the LSMEANS statement with the LINES option. The 3 h and 21 h results were analyzed separately for both Ct and Tt. Paired t-tests were used to evaluate the difference in Ct or Tt values between the 3 h and 21 h results combined across all factor levels.The response variables Ct and Tt were analyzed in a GLM treating the independent variables procedure, tissue, sampling time (dai) and inoculum concentration as fixed, categorical effects. An identity link function and Gaussian  error distribution were specified. Treatment differences were obtained using the LSMEANS statement with the LINES option. The analysis was done using the SAS procedure GLIMMIX.The response variables CLatent class analysis (LCA) was used to estimate the sensitivities and specificities of the LAMP and qPCR assays based on results obtained from testing of the field samples, although it is the characteristics of the LAMP assay that is of immediate interest. The sensitivity of a test is defined as the conditional probability of a positive test result given the sample is truly positive, while the specificity is the conditional probability of a negative test result given the sample is truly negative. The latent class approach is distinguished from other methodologies used to evaluate diagnostic tests in that it does not require results from a gold standard test  as a reference. Instead, LCA exploits the cross-classified results from two or more tests and uses a maximum likelihood approach to designate individual test results into two mutually exclusive categories (+ or −) and then uses this information to estimate each test’s characteristics. A detailed description of the procedure in a plant pathology setting can be found in Turechek et al. .t and Tt are continuous variables. For example, if Ct = 35 was selected as a cutoff, then samples with Ct > 35 would be classified as pathogen free and samples with Ct ≤ 35 would be classified as positive for X. fragariae. However, because threshold selection is subjective and dependent upon the application, test performances were characterized initially for all combinations of qPCR Ct values between 30 and 40 cycles and LAMP Tt values between 22 and 32 min, in increments of 1 and 2 min, respectively. A narrower range of threshold values in smaller increments (0.1 and 0.2) was investigated between cutoff values where qPCR sensitivity and specificity were equal to one. A categorical variable was necessarily created to increase the models degrees of freedom to allow estimation of two sensitivities, two specificities, and the incidence of disease in the two groups created by the categorical variable [The LCA model was constructed to assume that both tests produced a binary outcome; pathogen presence or absence. Therefore, cutoff or threshold values were constructed to produce a binary result even though both Cvariable . Sample The latent class analysis was conducted using the SAS procedure LCA , 33 and X. fragariae at 21 h after incubation was significant according to qPCR and LAMP (p ≤ 0.0007); tissue type  did not have an effect on detection  and LAMP (p < 0.0001) (2O demonstrating relatively greater inhibitory effects on amplification compared to samples in R2A and PBS. The effect of media on X. fragariae detection in bacteria-spiked strawberry samples at 21 h after incubation (p = 0.0012) and LAMP (p = 0.0052). Tissue type did not have a significant effect on DNA amplification in either treatment. Based on the differences of Ct or Tt values between samples incubated with and without added bacteria, the 21 h incubation of bacteria-spiked strawberry tissues in PBS and SPB enhanced detection by Ct’s of 1.2 and 1.4 , respectively, and Tt’s of 1.2 and 6.2, respectively . Despite highest average differences in Ct or Tt values, the statistical outcome for samples incubated in SPB was affected by the greater variability among replicates.The effect of incubating strawberry tissue alone ( 0.0001) , with saectively . Howeverp > 0.05). However, paired t-tests showed significantly lower Ct and Tt values for 21 h versus 3 h measurements (p <0.0001). The mean difference was -1.05 and -1.87, for Ct and Tt values, respectively.The effects of strawberry variety (note: this is a different set of varieties than used in the experiment describe in (i) above) and bacterial strain had no significant effect on detection at the 3 h or 21 h measurement periods according to qPCR and LAMP results (p > 0.05).Bacterial concentrations were statistically equivalent for inoculated strawberry samples processed through the new sample preparation procedure or the QIAGEN kit based on quantification from LAMP and qPCR, and results were not affected by sampling time (dai), tissue, or inoculum concentration  and qPCR (Ct = 35 cycles), LAMP had lower detection limit  than qPCR   (The detection limit of LAMP and qPCR was tested with pure 3 CFU/mL . At the samples) . Increassamples) .X. fragariae cells to healthy strawberry leaf or petiole tissue in PBS for the 21 h incubation period , LAMP and qPCR detected 300 and 600 CFU/mL of starting X. fragariae in 94.4% of the samples, respectively  and enhanced detection of X. fragariae , was the least effective of the four media tested. When the same concentration of  and R2A . This inX. fragariae using LAMP was 200 CFU/mL, which is equivalent to 200 CFU per 100 mg of strawberry tissue for qPCR with Ct = 37. This limit is more sensitive than those reported previously for qPCR assays with similar Ct cut points for X. fragariae detection by Weller et al. [With the sample preparation procedure, the detection limit of r et al. , Turecher et al. , which rX. fragariae. Moreover, since the procedure allows sampling of a much larger amount of tissue (at least 10×), use of the procedure should increase the chance of detection for those real-life samples where bacteria are  heterogeneously distributed.One of the motivations for developing the sample preparation procedure was to allow sampling of a greater proportion of tissue than what is common in many routine DNA extraction kits. The current sample preparation procedure was developed to accommodate sampling in the ratio of 1 g of strawberry tissue per 10 mL of PBS. Thus, the amount of tissue can be proportionally adjusted with the PBS volume to accommodate many different sampling scenarios. This ratio was determined to provide accurate results with minimal interference from PCR inhibitors. For spray-inoculated strawberry samples, LAMP and qPCR results were similar irrespective of whether the DNA was processed through the sample preparation procedure or through a commercial kit; in our case we mainly used the QIAGEN DNeasy Plant Mini Kit. This indicated that the sample preparation procedure performed as well as samples processed through a standard commercial kit for samples with uniformly distributed X. fragariae isolates tested and exhibited excellent specificity by failing to amplify any of the other bacteria and fungi tested within the acceptable Tt threshold, including some closely-related Xanthomonas species and microbes associated with strawberry. Primers were not tested on X. arboricola pv. fragariae because it was not part of our available collection. Nevertheless, this pathogen is known only to occur in Europe and is currently not considered an important pathogen in U.S. strawberry production [®9 dye yielded higher amplification efficiency than SYBR Green I, and this has been reported for different organisms in other studies [™, Diagenetix Inc., Honolulu, HI) for field diagnosis; see reference [For LAMP reactions, the LAMP primers efficiently amplified all 40 oduction . For rea studies . The opt studies , 16–18. eference  for an eOn the other hand, LAMP with end point detection is simpler and less expensive to implement than real-time detection, making it more attractive to a wider range of users. In our assay, HNB dye was added prior to LAMP reactions to avoid cross-contamination among samples and eliminate the use of a UV illuminator. This contrasts to LAMP assays developed for other Xanthomonas species where fluorescent dyes were added to samples after the reaction , 17, 19.t value of 37 or a Tt value of 30) would accomplish the goal but at a cost of a greater false positive rate. Hence, the analysis conducted offers the reader the opportunity to analyze the outcome over a range of combinations. In addition, the selection of multiple thresholds was done to alleviate some of the issues associated with the dichotomization of a continuous variable [The sensitivity and specificity of the LAMP assay was measured through a latent class analysis. As mentioned above, LCA exploits the cross-classified results from two or more tests and uses a maximum likelihood approach to designate individual test results into two mutually exclusive categories (+ or −) and then uses this information to estimate each test’s characteristics. Inherent in this approach is the assumption that each of the two tests produce a binary outcome. This was not the case for either LAMP or qPCR. To overcome this problem, a factorial-like combination of cutoff points for both tests was used to evaluate their performances. For tests that produce a quantitative output, the selection of a cutoff can be somewhat arbitrary and is based on the type of error one is trying to minimize. For example, to avoid false negative decisions, selecting a threshold with a high sensitivity , LAMP had detection limits 2–3 times lower than qPCR for detecting both pure bacterial cells and from bacteria-spiked strawberry samples. When the Tt and Ct thresholds were increased to 30 and 37, respectively, qPCR’s detection limit improved and was equivalent to the LAMP assay, which was unchanged, for pure bacterial cells at 2×103 CFU/mL. This is 2.5–5 fold lower than those reported in previous qPCR studies with Ct thresholds of ~37: 104 CFU/mL in Turechek et al. [3 CFU/mL in Vandroemme et al. [Using the Te et al. . With the et al. –11.8 CFU/mL of X. fragariae using LAMP with SYTO®9 dye could be detected within 8 min from the start of the reaction, compared to 25 min for qPCR. LAMP reactions are conducted at a constant temperature, thus it does not require stringent thermal cycling nor an expensive qPCR machine, making it more cost-effective and applicable. Moreover, LAMP reactions generate large amounts of pyrophosphate ion byproduct which interacts with magnesium ions in the reaction mixture to allow colorimetric end-point detection using a metal-ion detector, such as HNB dye, to further reduce the cost [R2 of 0.910 for LAMP vs. 0.998 for qPCR for their linear standard curves–of which similar observation has been reported [In addition to being more sensitive than qPCR at select thresholds, LAMP is also easier to perform and can be done more quickly than qPCR. For example, amplification of DNA from 10reported , 18. If X. fragariae in strawberry nursery stock. Detection at the early stages of production is essential in order to apply effective disease management strategies–usually plant removal–in efforts to reduce economic losses associated with quarantine violations.In conclusion, the LAMP assay with the sample incubation and crude DNA extraction procedure could serve as a sensitive and cost-effective method for detection of low quantities of


The false negative rate of sentinel lymph node biopsy (SLNB) is 5-10%, and results in improper patient management. The study was to assess the value of ultrasound-suspicious axillary lymph node biopsy (USALNB) in patients with early breast cancer, and to compare SLNB combined with USALNB (SLNB + USALNB) with SLNB alone.From January 2010 to July 2013, 216 patients with early breast cancer were enrolled consecutively at the Department of Breast and Thyroid Surgery, Qianfoshan Hospital, Shandong University. All patients underwent wire localization of the suspicious node by color Doppler ultrasonography, followed by SLNB 2–3 hours later, suspicious node lymphadenectomy, and level ≥ II axillary dissection (as the gold standard). The predictive values of node status between SLNB + USALNB and SLNB alone were compared.The success rate of SLNB was 99.1% (214/216). After axillary dissection, 71 patients were confirmed with axillary lymph node metastases by pathological examinations. Eight false negatives were observed using SLNB alone, resulting in sensitivity of 88.7%, specificity of 100%, false negative rate of 11.3%, and false positive rate of 0% in predicting the axillary node status. SLNB + USALNB resulted in sensitivity of 97.2%, specificity of 100%, false negative rate of 2.8%, and false positive rate of 0%. The false negative rate of SLNB + USALNB was significantly different from that of SLNB alone (P = 0.031).SLNB + USALNB seems to be a low-risk procedure that might be useful in reducing the false negative rate of SLNB, improving the accuracy of axillary nodes evaluation in early breast cancer. The presence or absence of axillary lymph node involvement represents one of the most important prognostic indicators of long-term patient outcome for breast cancer. In that same regard, accurate axillary staging is extremely important for guiding the surgical management and for directing the appropriate selection of adjuvant therapies for breast cancer. Sentinel lymph node biopsy (SLNB) is usually performed first because it is less morbid than axillary lymph node dissection (ALND). Therefore, patients with negative SLNB may avoid unnecessary ALND and its complications such as upper extremity edema and shoulder joint movement disorders . HoweverIdentification and sampling of suspicious axillary lymph nodes (SALN) using ultrasound has been proposed in order to improve axillary staging without the necessity to perform an ALND. Indeed, a number of recent studies used ultrasound to identify SALN and to sample them, either using fine needle aspiration (FNA) and/or core needle biopsy, and showed relatively good predictive value for axillary status -7. HowevIt is a well-accepted concept that the lymphatic spread of tumor cells from the primary breast cancer site occurs through defined regional lymphatic pathways leading to the axillary region and to other regional lymphatic chains. Therefore, tumor cells from the primary breast cancer site will first be recognized within a defined lymph node or defined group of lymph nodes which we designate as the sentinel lymph nodes (SLNs) . The abiTherefore, in the present study, we evaluated a new approach for axillary staging using both SLNB and USALNB by wire localization. In our study design, we attempted to evaluate this new approach for predicting the axillary status by comparing SLNB alone technique to that of a combined SLNB + USALNB technique, and utilized concomitant ALND as the gold standard.Between January 2010 and July 2013, 216 consecutive patients were enrolled at the Department of Breast and Thyroid Surgery, Qianfoshan Hospital, Shandong University. Inclusion criteria were: 1) women with clinical stage I or II breast cancer according to the AJCC TNM version 6.0 ; 2) a siThe study was approved by the Medical Ethics Committee of the Qianfoshan Hospital affiliated to Shandong University (20110133), and written informed consent was obtained from each participant.This was a cohort study aiming to determine the predictive value of the axillary status in early breast cancer using SLNB alone and SLNB+ USALNB compared with axillary dissection as the gold standard. All patients underwent, in order: ultrasound-guided wire localization of the suspicious lymph node, SLNB, USALNB, and ALND.B-mode ultrasound  was conducted to determine the axillary lymph node that was the most likely to arbor a breast cancer metastasis Figure . BecauseTwo radiologists including one chief and one senior attending doctor were asked to identify the suspicious lymph nodes. Disagreements were discussed and resolved by consensus.SNLB was performed 2–3 hours after ultrasound. A subcutaneous injection of methylthioninium chloride  was performed at the surface of the tumor. Methylene blue was used instead of isosulfan or isotopes because of the limited availability of these products. The skin and subcutaneous tissues were incised 10 min later, and skin flaps were isolated routinely. Adipose connective tissues were incised parallel to the outer edge of the pectoralis major muscle. After identification of the blue-stained lymphatic vessels, dissection of the tissues was performed along the lymphatic vessels to find and resect blue-stained lymph nodes. The resected blue-stained lymph nodes were labeled as the sentinel lymph nodes for pathological examinations  staining, and estrogen receptor, progesterone receptor and HER2 immunohistochemistry. Cytokeratins were not evaluated in the lymph nodes due to the limited amount of available tissues. H&E staining were considered as the gold standard to assess sensitivity and false negative rate.Pathologists were blind regarding the origin of the lymph nodes. All researchers were blind to the pathological results.All patients were followed up after treatments were completed. Follow-up was performed every 6 months by plain X-ray of the chest, bone scanning, ultrasound examination of the liver, thoracic wall and breasts, and axillary ultrasound examination, according to the NCCN guidelines.2 or the Fisher exact test, as appropriate. SPSS 16.0  was used for statistical analysis. P-values <0.05 were considered statistically significant.Data are presented as mean ± standard deviation (SD), absolute numbers and percentages. Variables within the contingency tables were analyzed using the χAge ranged from 26 to 68 years old. All patients were without clinically palpable nodes before surgery. There were 76 patients with clinical stage I breast cancer and 140 patients with clinical stage II breast cancer . Tumor was found in the upper outer quadrant in 96 cases, in the lower outer quadrant in 54 cases, in the upper inner quadrant in 12 cases, in the lower inner quadrant in 10 cases, and in the central quadrant in 44 cases. Of these cases, 196 underwent modified radical mastectomy, and 20 underwent breast-conserving surgery. Most tumors were invasive ductal carcinomas (85.2%). Tumor grade was I in 10.2% of cases, II in 83.3% and III in 6.5%. Tumors were hormone receptor-positive in 66.2% of cases, HER2-positive in 19.4%, and 68.1% showed a Ki-67 index >14% (Table SLNB using the methylene blue dye technique was successful in 99.1% of patients. A mean of 2.3 (range: 1–5) SLNs were excised, with 1 SLN in 54 patients, 2 SLNs in 78 patients, 3 SLNs in 60 patients, and >3 SLNs in 22 patients. A mean of 1.3 (range 1–2) SALN were identified by ultrasound and excised, including 1 SALN in 150 patients and 2 SALNs in 64 patients , only two patients were noted to develop distant metastatic disease.The first patient was a 65-year-old female patient with grade II invasive ductal carcinoma . She underwent a level II ALND, and all sampled lymph nodes were negative. Tumor immunohistochemistry revealed a triple-negative breast cancer, P53-positive, and the Ki-67 index was <40%. She received four cycles of docetaxel and cyclophosphamide. Pulmonary and bone metastases were found one year after surgery.The second patient was a 31-year-old female patient with grade II invasive ductal carcinoma . She underwent level II ALND and all sampled lymph nodes were negative. She received epirubicine, 5-fluorouracil and cyclophosphamide for 6 cycles, followed by 2 months of oral toremifene. The patient stopped her hormonal therapy by herself, and thoracic vertebral metastases were found two years after surgery.Axillary lymph node status is one of the most important prognostic factors in patients with breast cancer, and is used for the selection of the appropriate adjuvant therapy. ALND was previously the standard management of the axilla in breast cancer, but ALND failed to add benefits to node-negative patients. In addition, ALND results in certain complications, such as upper limb edema and shoulder joint movement disorders. Recent studies using large randomized samples, such as the ALMANAC  and NSABTherefore, the aim of the present study was to assess the value of USALNB in patients with early breast cancer, and to compare SLNB + USALNB with SLNB alone. Results showed that SLNB + USALNB resulted in a significantly better false negative rate (2.8%) compared with SLNB alone (11.3%).Preoperative ultrasound can be used to make a preliminary assessment of the status of the axillary lymph nodes ,17. UltrThe factors contributing the occurrence of false negative results during SLNB can be multifactorial, including variability in lymphatic drainage pathway, variability in SLNB techniques utilized, varying levels of surgeon experience, and patient selection issues. All current methodologies for SLNB, including the blue dye technique alone, the isotope technique alone, or the combined blue dye/isotope technique, rely on the lymphatic drainage pathways for successful outcome. Lymphatic drainage pathways depends on the integrity and physiology of the lymphatic channels and the corresponding lymph nodes . Once thIn many countries, SLNB is performed using an isotope (such as 99 m-Tc-colloid) in combination with a blue dye (such as methylene blue or isosulfan blue). However, in China, secondary to isotope shortage and governmental regulations, blue dye  is generally used alone for axillary staging for breast cancer, as in accordance with the breast cancer treatment guidelines and norms (2011 edition) from the Chinese Anti-Cancer Association. These established guidelines state that methylene blue may be used alone for SNLB, and that the results are reliable. Nevertheless, if isotope, such as 99 m-Tc-colloid, was more readily available to medical centers throughout China, then our current analysis would have been able to undertake a comparison of SLNB alone (by the methylene blue and isotope technique) versus SLNB (by the methylene blue and isotope technique) plus USALNB, which would be more applicable to the approach for axillary staging which is practiced internationally in other developed countries outside of China. Nevertheless, previous studies have demonstrated that the results from the use of a blue dye alone were comparable to the combined use of blue dye and isotope ,32. FurtThe present study is not without limitations. First, the sample size was not very large, and the study was not randomized. In addition, the follow-up was short, and longer follow-up is necessary to correctly assess recurrences. Multicenter randomized studies should be performed to correctly assess the predictive value of SLNB + USALNB.The present study showed that USALNB + SLNB was better than SLNB alone. Moreover, this method was simple to use, and has potential clinical applications. However, the quantitative indicators of suspicious axillary lymph nodes need further investigation.


Timely diagnosis of pneumonia in intubated critically ill patients is rather challenging. Pentraxin 3 (PTX3) is an acute-phase mediator produced by various cell types in the lungs. Animal studies have shown that, during pneumonia, PTX3 participates in fine-tuning of inflammation . We previously described an association between alveolar PTX3 and lung infection in a small group of intubated patients. The aim of the present study was to determine a threshold level of alveolar PTX3 with elevated sensitivity and specificity for microbiologically confirmed pneumonia.We recruited 82 intubated patients from two intensive care units  undergoing bronchoalveolar lavage (BAL) as per clinical decision. We collected BAL fluid and plasma samples, together with relevant clinical and microbiological data. We assayed PTX3 and soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in BAL fluid and PTX3, sTREM-1, C-reactive protein (CRP) and procalcitonin (PCT) in plasma. Two blinded independent physicians reviewed patient data to confirm pneumonia. We determined the PTX3 threshold in BAL fluid for pneumonia and compared it to other biomarkers.n =12), viral (n =4) or fungal (n =8) etiology was diagnosed in 24 patients (29%). PTX3 levels in BAL fluid predicted pneumonia with an area under the receiving operator curve of 0.815 , whereas none of the other biomarkers were effective. In particular, PTX3 levels ≥1 ng/ml in BAL fluid predicted pneumonia in univariate analysis  with elevated sensitivity (92%), specificity (60%) and negative predictive value (95%). Net reclassification index PTX3 values ≥1 ng/ml in BAL fluid for pneumonia indicated gain in sensitivity and/or specificity vs. all other mediators. These results did not change when we limited our analyses only to confirmed cases of bacterial pneumonia. Moreover, when we considered only the 70 patients who fulfilled the clinical criteria for the diagnosis of pneumonia at BAL fluid sampling, the diagnostic accuracy of PTX levels was confirmed in univariate and ROC curve analysis.Microbiologically confirmed pneumonia of bacterial  contains supplementary material, which is available to authorized users. Timely and accurate diagnosis of pneumonia is a challenging task for intensive care unit (ICU) physicians . AccordiPentraxin 3 (PTX3) is an acute-phase inflammatory mediator produced at the site of infection that can be assayed in a few hours . In the Given the sound pathophysiological background and the experimental data in animal models and patients, we conducted the present two-center international study to identify the threshold PTX3 level in BAL fluid for accurate diagnosis of microbiologically confirmed pneumonia and compare the diagnostic accuracy of alveolar PTX3 levels with those of other biomarkers.We performed a prospective, threshold-finding observational study with patients admitted to two university-affiliated ICUs . The inclusion criteria were age ≥18 years, intubation and mechanical ventilation and BAL fluid obtained as per clinical decision. The exclusion criteria were insufficient quantity of recovered BAL fluid, BAL performed in a patient previously enrolled in the same study, organizational reasons  and refusal to give consent. The ethics committee of the San Gerardo Hospital and the Partners Human Research Committee of the Massachusetts General Hospital approved the research protocol. Informed consent was obtained either from the patients or, when deemed incompetent to provide consent, from their next of kin.n =6, 20 ml each) of 0.9% sterile saline solution were instilled into the aforementioned lobe, and each aliquot was gently aspirated. Each BAL procedure took 10 to 20 minutes to perform. The first recovered BAL fluid portion was discarded. The remainder was mixed and sent in part to the microbiology department , cultures were obtained only when clinically suspected ) and in part to the cytology department for semiquantitative leukocyte counts (ranging from no leukocytes (−) to few (+) or many (++ or +++)). The remaining BAL fluid was centrifuged, and the supernatant was stored at −80°C, along with one plasma sample. Within 1 month after fluid and plasma collection, biological samples were transferred in dry ice to the Department of Inflammation and Immunology, Humanitas Clinical and Research Center, Rozzano (MI), Italy.BAL fluid was obtained using a standard technique . BrieflyUpon arrival of the fluid samples, biomarkers were assayed in duplicate using the following procedures. 1) In one experiment, plasma and alveolar PTX3 was assayed with a specific enzyme-linked immunosorbent assay (ELISA) kit developed in-house . Out-of-range BAL fluid samples were diluted at a 1:2 to 1:18 ratio and plasma specimens at a 1:5 to 1:90 ratio. This assay does not cross-react with C-reactive protein (CRP) or serum amyloid A protein. The time required to perform a single measurement was about 6 to 7 hours. The cost to perform one measurement was €5 per patient . (2) Pla In one eInternal body temperature >38°C,3 and/orWhite blood cells count >12,000 or <4,000 cells/mmPurulent tracheobronchial secretions; as well as with:New and persistent radiographic infiltrates associated with at least two of the following:4 or more colony-forming units per milliliter and/or significant noncontaminant viral load).A positive BAL culture , healthcare-associated pneumonia , hospital-acquired pneumonia , VAP  or no pneumonia ,6,8. ThePneumonia cases of fungal and viral etiology were carefully reviewed and then confirmed if risk factors were present , airway colonization was excluded  and specific antifungal or antiviral therapy was started.Pneumonia was considered absent when clinical signs of pneumonia were not present at the time of BAL fluid sampling and/or when an alternative cause for pulmonary infiltrate was established and there was no significant growth in BAL cultures and/or colonization, together with absence or full recovery from clinical signs of sepsis without any or new antibiotic therapy.The cell pellets of 20 BAL fluid aliquots (24%) from 20 consecutive patients  were resuspended, spun and stained to measure the fraction of cells in BAL fluid positive for intracellular PTX3, as previously described .P-value <0.05 was considered statistically significant. The percentage agreement and Cohen’s κ were calculated to compare the performance of the two independent intensivists’ assessments of the pneumonia diagnoses [t-test or the Mann–Whitney U test as appropriate. For comparison of categorical variables, a χ2 test or Fisher’s exact test  was performed. The correlation coefficient between biomarkers was analyzed using Spearman’s ρ coefficient.Statistical analysis was performed using SAS 9.2 software . A iagnoses . NormallROC) with pneumonia as the outcome of interest. AUCROC values with 95% CIs and P-values for differences from chance are reported. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Youden index [P-values for differences from 0  are reported. The net reclassification index (NRI) was used to compare the performance of the identified cutoff PTX3 levels in BAL fluid vs. other biomarkers and CPIS; that is, we used the cutoff identified by the Youden index value for plasma PTX3 and clinically accepted cutoffs for all other mediators and CPIS. More details on the methods used in this study can be found in Additional file The discriminatory ability of each biomarker was measured as the area under the receiving operating characteristic curve , suspected aspiration pneumonia , suspected malignancies , suspected autoimmune disease , exclusion of significant bacterial growth to start steroids  and undefined . The main characteristics of the patients are listed in Table n =25).We enrolled a convenience sample of 82 patients, including 64 in Monza between April 2009 and July 2011 and 18 in Boston between January and July 2011. Figure Among patients enrolled in the study, data review by two independent physicians blinded to biomarker levels and the initial diagnosis yielded coincident results in 79 cases . The three controversial cases were then jointly reviewed, and consensus was achieved in all of them. Lung infection was diagnosed in 24 cases (29%); among these, 14 were VAP (58%), 7 were HAP (30%) and 3 were CAP (12%). No HCAP case was identified. The etiology of pneumonia was bacterial in 12 cases (50%), fungal in 8 (33%) and viral in 4 (25%)  , whereas BAL fluid levels of sTREM-1, plasma PTX3, sTREM-1, CRP and PCT did not ; all other biomarkers were not effective (Table Net reclassification indexes for PTX3 levels ≥1 ng/ml in BAL fluid were elevated (range =0.38 to 0.55) in comparison to all other mediators and to CPIS with gain in sensitivity, specificity or both Table . In univN =70), PTX3 diagnostic accuracy was confirmed by AUC-ROC analysis ; however, BAL fluid sTREM-1, plasma PTX3, sTREM-1, CRP and PCT levels were not predictive of bacterial pneumonia , whereas all other mediators could not predict bacterial pneumonia.When we compared the 58 negative cases to the 12 patients with proven bacterial pneumonia  and univariate analysis , whereas all other mediators could not predict pneumonia.When we considered only the 70 patients who fulfilled clinical criteria for the diagnosis of pneumonia at the time of BAL fluid sampling, PTX3 diagnostic accuracy was confirmed both by both the AUCn =20, all from the Monza cohort), and the median percentage of cells in BAL fluid positive for intracellular PTX3 was 36% (IQR =32% to 51%) . More results can be found in Additional file Cells retrieved by BAL with intracellular PTX3 were present in all immunostained samples  and, possibly, improved specificity. Moreover, when we restricted our analysis to the subgroup of patients who met clinical criteria for pneumonia at the time of BAL, our results were also confirmed. In this subset of patients, pretest probability was higher and the diagnostic accuracy of a screening test such as PTX3 was expected to be poorer. Another strength of the study is that oxygenation did not differ between infected and uninfected patients. We previously showed that plasma PTX3 level is correlated with the severity of ARDS [Pneumonia represents one of the most widespread infections in intubated ICU patients  and is a of ARDS , but thipost hoc analysis of proven bacterial pneumonia cases.In our study, alveolar PTX3 was an independent marker of pneumonia due to any etiology with elevated NRI values in comparison to other mediators and to CPIS. In 2004, in an observational single-center study, Gibot and colleagues proposed sTREM-1 as an early and accurate marker of pneumonia . HoweverThus, our study indicates that, in intubated critically ill patients, the accuracy of PTX3 levels in BAL fluid for diagnosing pneumonia might be superior to other current biomarkers and clinical markers, especially when nonbacterial and/or non-VAP cases are suspected. Still, the diagnostic value of plasma PTX3 might be markedly increased by serial measurements over time  , which cde novo synthesis of the protein by immune and stromal cells and release of the stored protein from leukocytes.Previous studies showed that neutrophils, which constitutively store PTX3 in specific granules, rapidly release PTX3 in response to microorganisms and many other inflammatory stimuli -20,28. ICytomegalovirus, Candida spp and A. fumigatus pneumonia are difficult to diagnose unless invasive methods not reported in the present study are used. Moreover, the comparison with biomarkers not expected to increase in the presence of viral or fungal infections is a major limitation. Therefore, to increase practical implications, we performed a post hoc analysis limited to proven bacterial infections that confirmed the diagnostic accuracy of PTX3 levels in BAL fluid. (6) In a recent study, researchers showed that receipt of hematopoietic stem cell transplantation from a donor with a homozygous defective expression of PTX3 in neutrophils was associated with an increased risk of invasive aspergillosis [This study has many limitations, and therefore the information regarding alveolar PTX3 should be interpreted with caution. (1) We conducted an observational, hypothesis-generating study with a relatively modest sample size, and the clinical impact of PTX3 levels in BAL fluid-based protocols for the diagnosis of pneumonia and for prescribing (or withholding) of antibiotics remains to be determined in larger, prospective randomized studies. (2) The timing of BAL was not preplanned; instead, we collected BAL fluid whenever the attending physician decided to perform BAL, to the point that 15% of total cases did not fulfill the clinical criteria for pneumonia. In this way, we could evaluate PTX3 diagnostic performance in real-life clinical routine. Moreover, all results were confirmed in the subgroup of cases with clinical criteria for suspected pneumonia. (3) The PTX3 assay used in this study has a relatively narrow dynamic range, thus requiring dilution of biological samples with higher concentrations. This may limit the clinical robustness of the results, as translation into routine practice is far easier with tests involving undiluted samples. However, 60% of the BAL fluid samples, which were the main focus of this study, were tested without any dilution, as they were within the assay dynamic range. Nonetheless, more effort should be put into development of new PTX3 assays with wider dynamic ranges to help bench-to-bedside translation. (4) Immunostaining was performed in only 20 BAL fluid samples (24%); however, the immunostained samples were consecutive and pneumonia incidence and PTX3 levels in BAL fluid in this subgroup closely resembled those of the whole study population. (5) Pneumonia cases of different etiologies were included in the study, which introduced some heterogeneity. gillosis . Thus, aOur study shows that, in a convenience sample of unselected intubated critically ill patients undergoing BAL, alveolar PTX3 level is discriminative for microbiologically confirmed lung infection. In particular, PTX3 levels ≥1 ng/ml in BAL fluid were associated with elevated sensitivity and NPV, which may enable timely and accurate recognition of the vast majority of true negative cases. Future studies designed to validate the PTX3 threshold and further investigate the role of PTX3 levels in BAL fluid to guide antimicrobial therapy seem warranted.Early diagnosis of pneumonia in intubated ICU patients is fundamental but rather challenging, as clinical criteria yield many false-positive results.Pentraxin 3 is an acute-phase inflammatory mediator involved in the local recognition and clearance of bacteria, viruses and fungi.In the present study, Pentraxin 3 proved to be a useful early marker of pneumonia, with high negative predictive value, in intubated ICU patients.A discriminative threshold PTX3 level ≥1 ng/ml in BAL fluid was identified to help translation into clinical practice, but validation is still required.