Archives of annotated formalin-fixed paraffin-embedded tissues (FFPET) are available as a potential source for retrospective studies. Methods are needed to profile these FFPET samples that are linked to clinical outcomes to generate hypotheses that could lead to classifiers for clinical applications.We developed a two-color microarray-based profiling platform by optimizing target amplification, experimental design, quality control, and microarray content and applied it to the profiling of FFPET samples. We profiled a set of 50 fresh frozen (FF) breast cancer samples and assigned class labels according to the signature and method by van 't Veer et al  and then-7). When applied to the matched FF samples, the FFPET-derived classifier was able to assign FF samples to the correct class labels with 96% accuracy. The single misclassification was attributed to poor sample quality, as measured by qPCR on total RNA, which emphasizes the need for sample quality control before profiling.When a classifier developed with matched FF samples was applied to FFPET data to assign samples to either "good" or "poor" outcome class labels, the classifier was able to assign the FFPET samples to the correct class label with an average error rate = 12% to 16%, respectively, with an Odds Ratio = 36.4 to 60.4, respectively. A classifier derived from FFPET data was able to predict the class label in FFPET samples  with an error rate of ~14% (p-value = 3.7 × 10We have optimized a platform for expression analyses and have shown that our profiling platform is able to accurately sort FFPET samples into class labels derived from FF classifiers. Furthermore, using this platform, a classifier derived from FFPET samples can reliably provide the same sorting power as a classifier derived from matched FF samples. We anticipate that these techniques could be used to generate hypotheses from archives of FFPET samples, and thus may lead to prognostic and predictive classifiers that could be used, for example, to segregate patients for clinical trial enrollment or to guide patient treatment. Standard extraction and amplification microarray protocols  total RNA, few discovery platforms have reliably been applied to formalin-fixed paraffin-embedded tissue (FFPET) samples. Some approaches that assay fewer transcripts  are promising, but do not allow for unbiased discovery of diagnostic signatures, which requires a genome-wide profiling method ,3. For et System  and NuGEt System ) and arrt System ,8) have t System -9, and it System . While st System , similarThe importance of expression-based classification of human tumors to predict treatment response or disease outcome is highlighted by recent publications ,12. van'In the clinical diagnosis of patients with cancer, it is routine to obtain a FFPET sample, but generally rare to obtain a FF sample. Consequently, the requirement of FF samples has limited expression profiling to patients treated at specialized research centers. The ability to use FFPET samples would make this technology available for virtually all cancer patients both in the context of retrospective analyses of banked samples and clinical trials seeking to identify molecular tumor characteristics associated with patient outcomes to treatment. Being able to do such analyses from FFPET samples would simplify sample biopsy collection requirements and enable retrospective studies to develop and test hypotheses for prognostic classifiers for other cancers. As additional tests are developed and as molecular profiling methods mature, health-care providers will come to rely more on such classifiers in the risk-management of disease.In this report, our primary focus was to develop sample processing and classification methods with archived FFPET samples for hypothesis generation. To this end, we optimized a microarray platform and applied it to the profiling of FFPET samples. We demonstrated that FFPET samples can be accurately assigned to class labels using a classifier developed from fresh frozen samples, and we show that a classifier derived from FFPET samples that performs well in classifying FF samples can be developed.Matched pairs of Fresh Frozen and Formalin-Fixed Paraffin-Embedded breast cancer samples were obtained from Genomics Collaborative  and Cytomyx . Colon carcinoma tissues were also from Cytomyx. RNA extraction reagents for FFPET samples were obtained from Epicentre Biotechnologies . Jurkat total RNA and amplification reagents used in this study were from Ambion . Matched FF and FFPET liver and muscle RNAs were obtained from MPI Research . The Universal Human Reference (UHR) total RNA was obtained from Stratagene . Cy-dye reagents were from GE Health sciences . Quantitative PCR reagents were purchased from Applied Biosystems . Microarrays were designed at Rosetta and manufactured by Agilent Technologies .Total RNA from FF samples was extracted by the vendor  immediately prior to shipment. For FFPET samples, the extraction protocol is adopted from MasterPure RNA Purification kit . Briefly, three 10 μm sections were subjected to paraffin solubilization with xylene. Tissue was pelleted from solution by centrifugation and residual xylene was removed by two ethanol rinses. The tissue pellets were then air dried and digested overnight in a lysis buffer with Proteinase K. Digested protein and other cellular components were removed by ammonium acetate precipitation and centrifugation followed by a DNase I treatment of the resulting supernatant. A second ammonium acetate precipitation to remove any residual protein was then performed prior to ethanol precipitation and nuclease-free water rehydration of the purified total RNA.For measuring the relative quality of total RNA from FFPET samples, primer pairs for two house-keeping genes, GAPDH and ribosomal protein L13a, were used to estimate the integrity of the total RNA . The original protocol employs oligo-dT priming for first strand cDNA synthesis, in which an oligo-dT primer incorporates a T7 RNA polymerase promoter, necessary for subsequent in vitro transcription of RNA , and co-purified with the same mass of cRNA from the UHR pool labelled with Cy5. Hybridizations were done in fluor-reversed pairs as described . LabelinC) exists in two array-based ratio profiles, if hybridization 1 consists of the ratio C versus A (A/C), and hybridization 2 consists of the ratio C versus B (B/C), re-ratioing of A/C and B/C creates a new ratio experiment B versus A (A/B).Since we are mostly interested in ratio profiles between FFPET samples, we developed methods to recover such information by re-ratioing the ratio profiles derived from each array experiment between the FFPET samples and the corresponding hybridizations with UHR as the reference. Re-ratio of the ratio profiles effectively cancels out the UHR profile while accounting for dye labelling biases and leaving only the FFPET profiles of interest. In other words, given a typical configuration in which the common reference  was initially designed to evaluate several oligonucleotide selection criteria rule sets . The cross-hybridization score reflects the smallest difference between the dG value of self binding and the largest dG value of the probe binding to any other molecular species. However, despite making the cross hybridization filters more lenient, we noticed that the cross hybridization can only be minimized since certain probes still contain relatively higher GC content compared to the majority of probes on the array in order to meet the 3' distance requirement. Selection of the genes for the HumFFPET 44 k v2.0 array was kept as close as possible to those on the current default Human 44 k v1.1 array. The human 44 k v1.1 and HumFFPET v2.0 array share 20,327 probes in common while the HumFFPET v2.0 array has 19,231 unique probes that are specifically designed for FFPET samples. Documentation for the HumFFPET 44 k v2.0 array will be available in the Gene Expression Omnibus (GEO) website in support of this publication, and the HumFFPET 44 k v2.0 array pattern will be publicly available through the Agilent eArray ordering system.Formalin-preserved samples present multiple challenges to whole-genome RNA profiling methodologies. To overcome the extensive degradation, contaminants from the formalin treatment and limited RNA mass availability, we sought to improve 1) robustness of the amplification protocol, 2) quality control assessment of FFPET samples, 3) microarray performance through probe selection and 4) experimental design by validating the use of UHR as the reference channel in two-color hybridization experiments.We started with a commercially available two-round RT-IVT protocol for the target cRNA preparation for non-FFPET samples . We used a mass imbalance of intact Jurkat RNA to assess the potential impact of degradation and chemical modifications that would vary across FFPET samples. While evaluating imbalanced FFPET samples directly was more appealing, we reasoned that the primary impact from the degradation and chemical modifications of the fixation and embedding procedures would be loss of amplifiable mRNA, which the Jurkat RNA experiment adequately approximates.To quantify the degree to which the amplification steps are susceptible to mass imbalance, we performed titrations of input mass for the first round amplification with 25 to 200 ng of Jurkat total RNA sample to model the impact of mass imbalance on microarrray data quality. Following the first round amplification using different mass inputs, a fixed amount (500 ng) of cRNA derived from each input mass titration in the first round amplification was used for the second round amplification. The resulting cRNA from the second round amplification was labelled and hybridized in fluor-reversed pair that was formed to reflect the initial first round mass imbalance between the reference input of 100 ng and the other mass inputs which were originally titrated in the first round amplification. The same experiments were done to titrate input mass for the second round amplification by holding the mass of the first round constant at 100 ng and varying the second round from 250 to 2,000 ng, with the input of 500 ng used as the baseline to form the reference for the different fluor-reversed pairs.In these mass imbalance experiments, hybridizations of fluor-reversed pairs that are formed between different mass input are still defined as 'same-vs-same' hybridizations since the exactly same mRNA-containing total RNA are used in the amplification whether the input varied in the first or the second round. If there is no amplification bias resulting from the initial mass inputs, either for the first round or the second round, then these same-vs-same hybridizations should have shown no signatures of differential expression beyond background level. Same-vs-same hybridization data are presented as a heat map in Figure The extent and nature of RNA degradation in FFPET blocks depends on FFPET preparation method, length of storage and storage conditions ,17,18. WFirst, we noted that mean log ratio of some samples displayed a dependence on the distance of the probe to the 3' end of the message. We reasoned that a non-zero slope of this plot indicates a bias in the data quality , and that the 3' slope of mean log ratio could be used as a key quality metric for microarray hybridization. In fact, the 3' slope metric is analogous to the RNA Degradation metric  on AffymThen, we developed a qPCR-based assay to measure the relative abundance of transcripts in the total RNA and the amplified cRNA to quantify the relative quality of FFPET-derived total RNA as a way to relate the RNA quality to the subsequent microarray hybridization quality. Then, as we had done with FF samples, we started by selecting a set of FFPET samples that had been previously profiled and that cover a range of microarray data quality, and then assayed several housekeeping transcripts by qPCR. Figure In summary, the hybridization data of a sample of ideal quality should show no correlation between the Ct count of the total RNA or the amplified cRNA and the detected expression pattern, either positive or negative. Since the measured Ct count from the total RNA and amplified cRNA correlate with the quality of microarray hybridization Figure , the qPCStandard microarrays with probes approximately 500 nucleotides or greater from the 3' end have been found to be ill-suited for FFPET profiling. Therefore, we designed an array with probe content more suited to nature of FFPET samples and less susceptible to cross-hybridization by optimizing probe distance to the 3' end of the transcript, the probe base composition and the OSD cross-hybridization filter. While we had preliminary success at obtaining gene expression data from FFPET samples on a 44 K array (Human 44 K) designed for FF samples, the raw intensity of signals measured on the array drops off more precipitously for FFPET samples than FF samples as the probes' distance from the 3' end is increased. This is due to the extensive degradation of total RNA and the use of 3' poly-A priming amplification approach. One way to overcome the reduced intensity of hybridizations with cRNA from the 3' poly-A priming method is to optimize the probe content and composition on the array to reflect the proximity of amplified cRNA sequence of each transcript to its 3' end from degraded FFPET samples. To this end, we designed an array specifically suited to profiling FFPET samples with probe sequences within the first 400 nucleotides of the 3' end of each transcript , a set of matched FF and FFPET liver and muscle samples were used to measure the sensitivity and specificity of the HumFFPET 44 k 2.0 array in comparison with our standard Human 44 k array (see methods for details). To compare the overall performance of the FFPET and standard array designs, we used a ROC curve analysis  to show 4 k 2.0, Using the HumFFPET 44 k 2.0 array with a set of FF and FFPET samples, we found that the measured expression ratio correlation between FF and FFPET samples is also improved on the HumFFPET 44 k 2.0 array (r = 0.80) over the Human 44 k array  versus a FFPET-based self-reference pool. UHR RNA is composed of total RNA from 10 human cell lines and has been used widely as a reference in gene-profiling experiments, microarray platform evaluations, and cross-platform comparisons . Using UBefore applying the two-color microarray platform for classification, we validated the optimized platform measuring the correlation of gene expression profiles from the matched fresh frozen and FFPET tumor samples. As shown in Figure A number of papers have recently been published utilizing microarray technology to identify prognostic and diagnostic biomarkers as a tool to predict treatment response or disease outcome ,23. SuchTo answer the first question, we chose a well established biomarker, the breast cancer prognostic signature identified by van 't Veer et al. . In the Using the prognostic scores from 50 FF samples, we assigned 25 "good prognosis" patients and 25 "poor prognosis" patients according to their signature patterns. Figure -7), with errors mostly coming from poor quality samples flagged by qPCR. If poor quality samples as indicated by qPCR are excluded, the error rate will be 10% or less, mostly caused by samples originally very close to the threshold. More importantly, the classifier derived from the FFPET samples is confirmed by the FF sample data  process to select "prognostic" genes based only on FFPET profiling data. The FFPET-derived classifier prediction accuracy was then evaluated against the FF samples. As shown in Figure Enabling personalized medicine in the near future will rely to a large extent on extracting data from well-annotated archived samples for which the three-to-five year outcome of the subject is known. We consider this approach as a bridging strategy for patient stratification and enrollment, during which time the hypotheses are tested and confirmed. Part of this strategy includes integrating FDA guidelines relating to retrospective sample and data mining; key factors in this regard are (1) storage conditions; (2) samples are representative of intended use; (3) samples meet inclusion/exclusion criteria; and (4) performance is comparable to that expected from a prospective study . The FDAWe demonstrated the ability to derive gene expression-based classifiers from FFPET data that sort patient samples into class labels that recapitulates the sorting of FF samples. The method involved the development of an optimized microarray platform with two-round RT-IVT amplification using 100 ng of total RNA input for the target preparation, a quantitative PCR method for assessing the relative quality of FFPET samples and a custom microarray with content and probe features specifically optimized for FFPET profiling. We found this microarray platform reliably and reproducibly measured differential gene expression of FFPET samples with a good correlation to corresponding FF samples. FFPET samples were correctly assigned to class labels developed from FF-derived classifiers. Although we cannot directly attribute the success of the classifiers to the optimizations performed on the profiling platform, further study with FFPET samples also showed our platform is of sufficient quality to enable hypothesis generation that could be validated with FF or FFPET samples in controlled, well-designed clinical trials.The authors declare that they have no competing interests.SD, RS, JG carried RNA extraction optimization, amplification, and hybridization. SD, SL, HD, TF, GT, BF and MM contributed to the design of the studies. MZ and MM drafted the manuscript. AK and JC designed the FFPET array. HD and YW developed the classifier and led the interpretation of the data. All authors read and approved the final manuscript.Two-round amplification workflow. Diagram of the Two-round amplification work flow used for amplification of total RNAClick here for fileFF to FFPET correlation. The correlation between the FF and FFPET samples is increased significantly on the HumFFPET 44 k array 2.0 when compared to the Human 44 k v1.1 array.Click here for file


To clarify the relationship between presenting clinical condition and blood alcohol concentration (BAC) among adult patients admitted to a resuscitation room (RR) of an emergency department (ED) in order to help guide clinical practice.Single-site prospective cohort study of all patients admitted to the RR of an inner-city hospital over a one-year period. The study sample comprised all those aged 16 years and over from whom a blood sample was taken, with BAC (results not known to ED staff), pathology by International Classification of Diseases (ICD) version 10 coding, injury severity score for trauma, return visit to hospital and mortality during the subsequent 6-month period, being recorded.291 (15%) of 1908 presentations had a positive BAC  ranging from 11 to 574 mg/100 ml, of which almost 40% were over 240 mg/100 ml (ICD-10 code Y90.8). In addition to collapse from alcohol/drugs, almost half of those presenting following self-harm or assault had a positive BAC. Those with a positive BAC had a higher rate of ED re-attendance in the following 6 months. 10% of all presentations were due to trauma.The following five presentations to the RR are associated with a positive BAC: collapse from alcohol/drugs, self-harm, trauma, gastrointestinal bleeding (ICD-10 code K92.2) and non-cardiac chest pain (ICD-10 code R07). Patients with a positive BAC demonstrate a very wide range of pathology, some with severe levels of misuse. This highlights the opportunity for prompt feedback when sober, to ensure all is done to encourage patients to contemplate change in order to reduce re-attendance. This work follows directly on from our first paper: “Use of blood alcohol concentrations in resuscitation room patients”,5–11Identification of patients with alcohol problems in the ED may be by history (PAT), clinical examination or blood alcohol concentration (BAC). PAT application is usually not possible for patients who are potentially critically ill and the need is for urgent assessment and treatment. Clinical examination for possible alcohol use is limited for the obtunded patient and has variable concordance with BAC.8The extent of concurrent alcohol use among all categories of patients treated specifically in the resuscitation room (RR) of an ED has not previously been reported; every ED in the UK has a specific RR, usually between two and six bays, for potentially critically ill patients.The study took place in the four-bayed adult RR of the ED of an acute hospital serving a metropolitan inner-city population of over 500 000 adults, seeing 65 000 new adult (16 years and over) ED visits each year. We measured BAC over a 12-month period from all blood samples taken from patients initially presenting to the RR, with the exception of direct admissions to specialists and transfers from other hospitals . The spectrum of disease of those patients presenting to the RR was broad see . All hadBlood samples were collected into a fluoride-oxalate bottle (as for blood sugar estimation); BAC was measured by an automated enzymatic method—DRI ethyl alcohol assay  on an Olympus AU2700 analyser . BAC are reported in mg/100 ml; less or equal to 10 approximated to BAC negative and more than 10 was BAC positive.Electronic and written ED and hospital records were subsequently examined by researchers masked to the BAC results. For those patients transferred to seven other hospitals, records were similarly examined for length of stay and outcome. In addition to basic demographic information, the time, date and reason for presentation to the RR were recorded, together with Glasgow coma scale (GCS) scores, information about immediate management (intubation/whether they received a computed tomography scan), final primary diagnosis  version 10 codesTrauma severity was rated by an independent team from the UK trauma audit and research network, commissioned for this study. All trauma cases were assessed using the injury severity score. This method uses the abbreviated injury scale scores based on the severity of the injury, which is applied to the regions of the body.Each blood sample was assigned an identifying number to log the BAC results into an Excel spreadsheet that was not accessible by the treatment team. All data were then entered onto a database and analysed using SPSS (version 14.0). Univariate and multivariate tests were used to compare the characteristics and outcomes of those with positive and negative BAC. The impact of a positive BAC on management and outcomes was examined using binary logistic regression. For rates and proportions, 95% CI were calculated.There were 2326 direct patient presentations to the RR during the course of the study, 22 August 2005 to 31 August 2006. Of these, 2137 patients were eligible to participate, but 229 of these were not tested . The chaOf the 1908 with BAC results, 1617 had BAC scores of less than 11 mg/100 ml (BAC negative). The remaining 291 (15%) had a positive BAC, ranging from 11 to 574 mg/100 ml. The characteristics of those BAC negative are compared with those with a positive test in BAC were divided into ICD-10 diagnoses  and Y90.In our study, collapse due to alcohol misuse was classified under mental and behavioural disorders, F10 .Using binary logistic regression, the 196 incidents involving trauma  were found to be more likely to be BAC positive than other presentations , apart from collapse due to alcohol/drugs. Those with a high trauma score were no more likely to be BAC positive than those with a low score: the mean BAC in those with a score below 12 was 68.1 compared with 76.6 in those with a trauma score above 12 . Nearly half (46%) of patients with head and neck trauma were BAC positive, which demonstrated a trend towards significance .Running a binary logistic regression, those BAC positive were found to be more likely to have been intubated while in the resuscitation room , even after taking into account the confounding effect of their presenting condition. However, they were not more likely to have been sent for a computed tomography scan . Of the 1858 (97.4%) who had a GCS score documented in their notes, the median score was lower when BAC positive . A total of 1412 (74%) of the 1908 presentations to the RR resulted in admission to hospital.Regarding 6-month outcomes, of the 1753 patients (155 were return visits) who presented to our RR and had a BAC measured, 494 (28%) subsequently visited our ED during the following 6 months. It is notable that 219 (12%) died within 6 months, according to computer data held by our hospital (which records later patient death for those previously attending our ED). When taking into account the confounding effects of age and presenting condition, a positive BAC was associated with an increased likelihood of a return visit to the ED within 6 months . The likelihood of admission to a ward on first attendance to the RR  or death within 6 months  were also higher among those with a positive BAC, but these were not statistically significant.This is the first such report assessing BAC taken over one year specifically from RR patients. We show that more than one in seven people treated in our RR over a 12-month period have consumed alcohol before their presentation. Half of all those treated following self-harm or assault will have a positive BAC. Injury to the head and neck (S00–S19) was associated with a positive BAC, being related to assault.Patients presenting with trauma showed no relationship between severity, as defined by trauma scoring using the TRISS methodologyet al17Requesting a BAC should be according to clinical judgement as for any investigation,All ED systems are a reflection of healthcare practice and medical culture; BAC is a universal standard, laboratory facilities permitting.18Our study is confined to RR patients initially attending a single urban ED in a metropolitan area. The extent to which these data can be generalised to other ED is unclear. Although drinking patterns in the area served by our ED are not atypical of those in other densely populated areas in the UK,In our study BAC results were unavailable to the medical staff; BAC were not previously routinely requested from our RR, nor was BAC testing available on site. The results of testing remained confidential, follow-up  not yet organised.10As alcohol is metabolised at approximately 15 mg/100 ml per hour,2It is simple to add BAC to an initial set of RR blood investigations, a fluoride-oxalate bottle being used for both sugar and BAC estimations. BAC provides an important and inexpensive means of identifying the possible role, or not, of alcohol in a patient’s presenting complaint and furthermore, may act as “an alert” to counter possible clinical inertia of the treating doctor.


The genetic basis of postzygotic isolation is a central puzzle in evolutionary biology. Evolutionary forces causing hybrid sterility or inviability act on the responsible genes while they still are polymorphic, thus we have to study these traits as they arise, before isolation is complete.D. mojavensis vary significantly in their production of sterile F1 sons when females are crossed to D. arizonae males. We took advantage of the intraspecific polymorphism, in a novel design, to perform quantitative trait locus (QTL) mapping analyses directly on F1 hybrid male sterility itself. We found that the genetic architecture of the polymorphism for hybrid male sterility (HMS) in the F1 is complex, involving multiple QTL, epistasis, and cytoplasmic effects.Isofemale strains of The role of extensive intraspecific polymorphism, multiple QTL, and epistatic interactions in HMS in this young species pair shows that HMS is arising as a complex trait in this system. Directional selection alone would be unlikely to maintain polymorphism at multiple loci, thus we hypothesize that directional selection is unlikely to be the only evolutionary force influencing postzygotic isolation. During the modern synthesis, Dobzhansky and Mayr proposed the Biological Species Concept that defines species by their ability to exchange genetic material within their group while being prevented from exchanging genetic material between groups Postzygotic isolation exists when a hybrid, or its progeny, experience a reduction in fitness relative to the parental types. Whether the genetic variation needed for postzygotic isolation is segregating within species in the form of epistatic variation Drosophila and interestingly, also for systems where the female is the heterogametic sex, such as birds and LepidopteraThree general patterns of postzygotic isolation are well supported. First, Haldane's rule Lhr and Hmr genes in the Drosophila melanogaster – D. simulans pair have been demonstrated functionally to cause hybrid male lethality The problem of how a trait that decreases the fitness of hybrids could evolve between two populations sharing a common ancestral genome without either population passing through a state of reduced fitness is solved by the Dobzhansky-Muller (DM) model of reproductive isolation Expanding the Dobzhansky-Muller model, Orr 1 phenotype we cannot be certain that the genetic architecture for the incompatibility observed in the F1 hybrid has been fully characterized. Evidence of substantial within species epistasis contributing to postzygotic isolation has been demonstrated in Tribolium by joint-scaling analysis A major lingering question in speciation genetics concerns the actual genetic architecture of postzygotic isolation as it first emerges. Does it have a simple or a complex genetic basis at the earliest stages of the speciation process? A burdensome disadvantage to genetic studies of postzygotic isolation is that the phenotype of interest, sterility or inviability, precludes direct genetic crossing schemes. Many successful studies of species pairs, in which the factors underlying isolation are effectively fixed, utilized the common occurrence of asymmetry 1 hybrid male sterility directly. We used Drosophila mojavensis and D. arizonae, a recently diverged species pair  and can give insight into how HMS evolves. In a novel design, we have utilized the within-population variation for production of F1 HMS to determine the genomic regions and epistatic interactions that shape the architecture of postzygotic isolation within the F1 hybrid males. Below we address the following questions: What is the nature and number of polymorphic loci within D. mojavensis contributing to the HMS phenotype? And what can that tell us, in conjunction with other information about this species pair, about the evolution of HMS?In the present study, we circumvented the limitations of the hybrid phenotype by exploiting within-species polymorphism for between-species postzygotic isolation to determine the genetic architecture of segregating variation for FD. mojavensis lines were collected from Santa Catalina Island (CI) in April 2001. The Low (CI-10) line exhibiting low levels of HMS was inbred through full-sib mating for six generations and the High (CI-12) line exhibiting high levels of HMS was inbred for eight generations. The lines had minimum inbreeding coefficients of 0.734 and 0.826 respectively D. arizonae males from an inbred line (minimum inbreeding coefficient of 0.859) collected at Peralta Canyon Trail Head, east of Phoenix, Arizona (AZ), in April 1997. All lines show normal male fertility when the males are genetically pure D. mojavensis or D. arizonaeD. mojavensis genome sequencing project, had been confirmed by sequencing five marker loci in 10 females  and an additional seven loci in 10 individuals by Agencourt Biosciences, and none showed sequence variation. The two D. mojavensis QTL lines were homokaryotypic, lacking inversion polymorphism. The lines used showed extreme contrasting HMS (sperm motility) phenotypes in heterospecific crosses  BLASTing D. melanogaster genome sequence to identify which Muller element and thereby to what chromosome in D. mojavensis it belonged; 2) linkage mapping to allozyme markers assigned to chromosome by Zouros We designed fluorescently-tagged PCR primers for flanking sequences of microsatellite loci identified by Ross 2 line cross  for 48 hours. After 48 hours, the females were placed individually into vials, allowed to oviposit for four days, then removed and frozen for later genotyping. Virgin hybrid males were collected from those vials producing viable adults. Hybrid males were allowed to reach sexual maturity at 9–12 days post-eclosion before phenotyping. Use of the F2 mothers' genotypes allowed for a largest possible sample of recombinant genotypes (given limited resources for genotyping) while the use of the sons' phenotypes allowed for a good estimate of their mother's average phenotype, maximizing the quality and quantity of the data. A reciprocal design was also performed; instead of using Low females crossed to High males at the first step (F2 cross) High females were crossed to Low males (RF2 cross). A large number of attempted matings between F2 and RF2 females and D. arizonae males failed to produce offspring as was expected considering the significant prezygotic isolation between the species. Seven percent of the F2 (224 of 3214) and the 12% of the RF2 (278 of 2339) mothers produced larvae after being mated to D. arizonae males.The crossing design used was an innovative modification of a standard Fne cross . Five vihttp://flyfood.arl.arizona.edu/opuntia.php3#s2.1) while virgins were maintained on standard cornmeal food . Cultures were maintained at 24+/−1 degree Celsius and under a strict 12h: 12h light: dark cycle.Mating and/or oviposition took place on standard opuntia-banana food . Reproductive organs were transferred to a fresh slide with an 11 microliter droplet of sperm buffer, stretched with forceps to be linear, and then placed under a coverslip. Pressing the coverslip down on the sample forced all sperm out of the seminal vesicle, allowing motility of the sperm to be observed under dark-field microscopy. The entire area where potentially motile sperm could be found was visually scanned and each seminal vesicle assigned an integer score from 0 to 6 on a sperm motility scale.Male Drosophila sperm are extremely long, forming a tangled wiggling knot, in which the movement of any individual sperm cannot be tracked. The zero-to-six scale is a reasonable means to approximate the amount of motility exhibited by Drosophila sperm, and is a refinement of the presence/absence of motility scheme used in many previous studies of sperm motility in Drosophila2 = 0.79, p<0.0001), validating this method for estimating Drosophila sperm motility suggesting its utility for future studies. Sperm motility is one phenotypic component of sterility. We have shown in previous work that motile sperm are required though not always sufficient for males to be able to reproduce in this system due to additional genetic polymorphism for other phenotypic components of sterility Unlike mammalian systems that have short sperm, 2 or RF2 females that successfully produced hybrid male offspring were genotyped for the 25 microsatellite markers described above. Maximum likelihood marker  uses cofactors from other regions to control for genome wide influences when regression analysis at any given genomic location is being tested QTL mapping was conducted using Windows QTL Cartographer 2.5 sons, sons of each mother were analyzed with their mother's genotype and their individual motility phenotype to account for the within mother variation in her sons' motility scores. Analyses were also conducted on the second dataset using the mother's Best Linear Unbiased Prediction (BLUP) sons data set is better for detecting genetic loci experiencing non-additive effects.Two datasets were used in mapping. In the one called 2 vs. RF2), M1 is the effect of genotype at marker 1 and M2 is the effect of genotype at marker 2. All 300 possible two-way interactions were tested and significance of the interaction term (M1*M2) was assessed by Bonferroni correction and False Discovery Rate (FDR). All calculations were implemented using the SAS Mixed procedure .We assessed epistatic interactions using methods modified from Moehring and Mackay 2 vs. RF2) in the multiple regression model of the form:1 is the genotype at the marker associated with one of the main effect QTL.Maternal effects were identified as a significant cross by main effect interaction. Hybrid male phenotype (n = 304) was regressed against the interaction between marker genotypes as the four main effect loci and the direction of the cross  intercross QTL mapping design , of the species D. mojavensis, were crossed to male D. arizonae for QTL analysis. Sperm motility was scored, from 0 to 6, in the interspecific hybrid males. 304 F2 mothers were genotyped at 25 microsatellite markers across the genome and used to generate the linkage map. Phenotypes were mapped relative to genotype in two ways. One was to use each son's motility score, hereafter referred to as sons, as a sample of the mother's phenotype and the other was to map a mother's Best Linear Unbiased Prediction (BLUP) for her sons' motility scores. Sons better captures non-additive effects, while BLUP highlights the additive effects in the architecture.Female F2 and RF2 mapping populations did not match more closely, and second, no main effects were detected on the X-chromosome. Main effect QTL were detected, however, on the 2nd, 3rd, and 5th chromosomes  and across all population samples . The RF2 population sample did not reveal any other significant QTL.There were two striking outcomes of the main effects analysis; first that the Fomosomes . Note thnd chromosome. Analysis of the sons dataset in the F2 and combined populations found two and one QTL on the 2nd chromosome respectively (sons QTL at 58 cM has a large non-additive effect of 2.23 motility units and estimated effect size is 55.3%. The two QTL found in the F2 population sons dataset (at 32 and 66 cM) also had large (and opposite) non-additive effects of −2.17 and 2.16 motility units, respectively. The RF2sons dataset had an elevated likelihood ratio on the 2nd chromosome but it did not reach significance. The BLUP datasets exposed a significant QTL on 2nd chromosome for the combined population (60 cM) and the RF2 population (50 cM) with a non-additive effect of 0.92 to 1.10 motility units, with estimated effect size of 44.3 to 59.3%. The lack of a significant 2nd chromosome QTL in the F2 BLUP dataset can be explained by the apparent equal and opposite non-additive effects of the two QTL found in the sons dataset.QTL were also identified on the 2ectively . The com2 population sons dataset. It was at 54.5 cM with a relatively large additive effect of −0.80 and moderate non-additive effect of −0.43 motility units with an estimated effect size of only 9%.Chromosome five also contained a QTL, but only in the Fsons datasets on families limited to three randomly selected sons. All QTLs replicated with controlled family size (data not shown) so we are confident that the mapping results are not due to biases in family size. We also looked for any biases introduced by segregation bias due to prezygotic isolation between the species or hybrid male over dominance in viability. We found that 15 of the 25 markers were not in the expected 1:2:1 (autosomes) or 1:1 (x-chromosome) ratios having excess heterozygotes, but there was no association between violation of these ratios and the location of the QTL . Thus, there is evidence of genetic variation for prezygotic isolation with a possible heterozygote advantage that may be worth mapping in future studies, but that variation did not introduce bias into the mapping for HMS. The linkage map, though, is likely only accurate for the population that was genotyped and cannot be applied to the F2-females that failed to mother hybrid sons, because two or more segregation distortion loci within a given marker interval can bias estimates of recombination rate We tested for biases due to variation in family size by rerunning all analyses of both the BLUP and 2 and RF2 populations. Specifically, four main effect QTL were identified in the F2 population; two had largely additive effects on the 3rd and 5th chromosomes and two had largely non-additive effects on the 2nd chromosome. In the RF2 population, however, only a single, largely additive QTL was found on the 3rd chromosome. The combined population data set shows one 2nd chromosome QTL and the 3rd chromosome QTL. These unexpected results motivated further analyses of the underlying architectural complexity of HMS.The lack of a segregating main effect on the X-chromosome was surprising, as this chromosome has been generally shown to play a major role in the evolution of hybrid male sterility 2 and RF2 populations. First, we may have failed to detect QTL due to simple sampling variance between the F2 and RF2 populations. Second, the different compositions of the X-chromosomes in the reciprocal populations could have lead to an X-by-autosome interaction. Finally, an interaction between the nature of the cross (e.g. cytoplasm) and the QTLs could account for (or underlie) the observed differences between reciprocal crosses. We address each of these below.There are several possible explanations for the discrepancy between FSampling Variance: To test whether the inevitable force of sampling variance was driving the differences observed between the F2 and RF2 populations, we subsampled from the datasets to test for consistency of the signal. From each population 100 datasets were created by randomly selecting 80% of the data points from the original dataset. Complete mapping analyses, including permutations for significance thresholds, were then performed on the subsampled datasets. Despite the reduced power of the smaller datasets, the QTLs found on the full dataset remained detectable in between 27% and 70% of the subsampled datasets (e.g. see left portion of chromosome 5 in the F2 dataset). Subsampling indicates that QTL identified in the complete dataset remain detectable in a large portion of the subsets despite variation in sampling and spurious QTL (those not detected in the complete dataset but in the subsets) rarely arise due to sampling variance. The consistency between our subsampled datasets and our complete dataset suggests that our findings are fairly robust to variation in sampling and that the differences between the F2 and RF2 are likely biologically real, though some of the differences could still be due in part to sampling variance.datasets , while sInheritance of the X-chromosome: If an important X-linked genetic difference between the parental lines interacts with the autosomes, the cross direction will affect the frequency of that factor in the mapping population. This, in turn, would influence the detection and nature of autosomal QTL that interact with the X. The X-linked factor could primarily influence the penetrance or expression of the genetic variation at the autosomal factor and thus have a significant epistatic effect without having a main effect. To test for X-by-autosome interactions, as well as for other epistasis, we looked for significant pairwise interactions between all markers across the combined dataset (n = 304). We found seven interactions with a FDR significance of 0.05 or less, two of which were between the X-chromosome and the autosomes  resides in or near a main effect QTL. Two additional markers (a1_2_1 on chromosome 3 and m1_10_11 on chromosome 4) did not appear in the list of significant marker by marker interactions, but exhibited significant deviations from the additive expectation after a Bonferroni correction, indicating that there are at least two other unidentified epistatic interactions. Taken together, not only does the X-chromosome appear to play an epistatic role in shaping the genetic architecture of HMS in this system, but so does within-species epistasis throughout the rest of the genome.utosomes . EpistatMaternal effects: Cytoplasmic factors may also underlie interactions between the direction of the cross and the QTLs. We tested for marker by cross interactions for the four markers showing main effects and found a significant interaction effect of marker dmoj2210 on the 2nd chromosome with cross (p = 0.0142), after controlling for the main effect of the 3rd chromosome . Our present QTL study reveals that HMS, even at the earliest stages of speciation, can be surprisingly complex. We found within-population variation not only for several QTL of main effect, but also for significant within-species epistatic interactions. Similar complexity has been seen in other systems, such as with the Lhr-Hmr gene pair, where the incompatibility requires an epistatic interaction with the hybrid genetic background to be expressed TriboliumThe first question we proposed to answer in this study was: what is the basic architecture of HMS in the early stages of its evolution? Is HMS caused by only a few factors of large effect or does it have a more complex basis, involving multiple loci and epistatic interactions? An earlier study In addition to the unexpected complexity of HMS in this incipient species pair, we were surprised to discover no main-effect QTL segregating on the X-chromosome, though we did find an epistatic effect. While we may have lacked the statistical power to detect X-linked main effects, main effects were detected on other chromosomes indicating that sample size alone is not the full explanation. There may still be fixed X-linked effects that could not be detected by this design, and fixed effects on the X are more likely due its smaller effective population size. Demuth and Wade did demonstrate the role of epistatic variation on the X-chromosome on Haldane's Rule both theoretically D. arizonae mated to male D. mojavensis) to the third, fourth and Y-chromosomes D. arizonae mothers is a function of the same factor or factors as revealed in our study remains unknown.Finally, our data show a striking difference between reciprocal crosses, suggesting a potential role for maternal effects and genome-wide epistatic variation on HMS. Cytoplasmic and epistatic effects have been demonstrated in other systems as well Drosophila species groups, Tribolium beetles Chorthippus parallelus grasshoppers Mus musculusXiphophorus fish Crepis hawksberg weed Gossypium cotton Mimulus monkey flowers et al. The second question we proposed to answer was: what can the genetic architecture of HMS tell us, in conjunction with other information, about its evolution? Substantial within-species natural polymorphism for between species postzygotic isolation has been characterized in many independent taxa thus far, including at least nine OdsH in D. mauritiana, Xmrk-2 in Xiphophorus, Nup96, Hmr, Lhr in the D. melanogaster/D. simulans species pair D. mojavensis and in other systems discussed above, however, we believe it is premature to discount the role of drift and balancing selection in speciation. Empirically, we need to characterize more completely the genetic architecture of incompatibility phenotypes during early stages to find reproductive isolation genes before they are fixed and test those loci for evidence of selection. In the present study we have begun the empirical aspect of this process.A handful of genes causing hybrid incompatibilities have been identified in other species and all but one (a gene transposition Table S1Primers for Microsatellites.(0.06 MB DOC)Click here for additional data file.Figure S1Linkage map for D. mojavensis. Distance between markers given in centiMorgans.(0.30 MB DOC)Click here for additional data file.Figure S2Subsampling effect on QTL detection. Bar graphs of count  of 80%-subsampled datasets at each mapping interval showing a likelihood ratio greater than the significance threshold determined by permutation for each subset. Solid lines indicate likelihood ratio (left x-axis) calculated from the complete dataset. A. F2-sons dataset. B. RF2-sons dataset. Subsampling indicates that QTL identified in the complete dataset remain detectable in a large portion of the subsets and spurious QTL (those not detected in the completely dataset) rarely arise due to sampling variance. Thus, patterns observed in the complete dataset appear robust to sampling variance.(0.16 MB DOC)Click here for additional data file.


We present a comprehensive study of cytogenetic alterations that occur during the progression of clear cell renal cell carcinoma (ccRCC). We used high-density high-throughput Affymetrix 100 K SNP arrays to obtain the whole genome SNP copy number information from 71 pretreatment tissue samples with RCC tumors; of those, 42 samples were of human ccRCC subtype. We analyzed patterns of cytogenetic loss and gain from different RCC subtypes and in particular, different stages and grades of ccRCC tumors, using a novel algorithm that we have designed. Based on patterns of cytogenetic alterations in chromosomal regions with frequent losses and gains, we inferred the involvement of candidate genes from these regions in ccRCC tumorigenesis and development. We then proposed a new model of ccRCC tumorigenesis and progression. Our study serves as a comprehensive overview of cytogenetic alterations in a collection of 572 ccRCC tumors from diversified studies and should facilitate the search for specific genes associated with the disease. Cytogenetic changes underlie most genetic diseases, including cancer. Not only are different patterns of cytogenetic alterations associated with different types of tumors, tumors of the same type at different stages of development also exhibit different cytogenetic patterns. It is commonly accepted that tumors at early stages of development have fewer cytogenetic alterations than tumors at more advanced stages. Early mutations in a few key genes are believed to drive the initial steps of tumorigenesis. Key mutational events are loss-of-function mutations in tumor suppressor genes (TSGs) and/or gain-of-function mutations in oncogenes. The resulting changes in gene function are thought to trigger the process of tumorigenesis and set the stage for the accumulation of more genetic abnormalities as the tumor progresses. Here we have treated gene methylation as a special form of mutation, for ease of description.Mutations are rare. In the two-hit theory of cancer formation , loss ofAdvances in technology now allow for cytogenetic analysis in unprecedented detail. Detailed cytogenetic analysis of a given tumor type at different stages of progression will be necessary for a full understanding of tumor development. Cytogenetic profiling of different tumor subtypes can also shed light on the understanding and diagnosis of cancer by revealing genetic alterations specific for each subtype. In this study, we undertook a comprehensive survey of cytogenetic losses and gains occurring in renal cell carcinoma (RCC) by using the high throughput high density Affymetrix 100 K SNP chips. von Hippel-Lindau (VHL) gene [VHL gene are frequently observed in ccRCC tumors (~90%). Cytogenetic profiling of various RCC subtypes has been studied indirectly through genome-wide gene expression profiling [RCC is a heterogeneous disease consisting of multiple subtypes. The most common RCC subtype is clear cell (ccRCC), accounting for about 70% of all RCC tumors. Other subtypes include papillary , chromophobe CH, ~5%), oncocytoma , and collecting duct carcinomas (very rare) , 3. DespHL) gene . Mutatiorofiling , 5, 6. T%, oncocyrofiling , quantitrofiling , fluoresrofiling , 15 haverofiling  and Berorofiling . Howeverrofiling . To our Here we report a systematic study of cytogenetic alteration profiles in ccRCC tumors at different stages of development and in association with tumor grades. Our study provides a detailed map of the sequence of cytogenetic alterations that occur during the progression of ccRCC. Based on these results, we propose a new model of ccRCC tumor genesis and progression. Our results suggest the involvement of cytogenetic alterations seen in ccRCC, and will aid in the identification of genes relevant to ccRCC.http://www.cancer.org/). VHL mutations were obtained through sequencing of all three exons of the gene. 71 pretreatment tissue samples were collected from multiple cancer centers, including Spectrum Health Hospital in Grand Rapids, MI (31 cases), and the Cooperative Human Tissue Network  over the past few years. There were 42 cases of ccRCC, 6 cases of chromophobe, 8 cases of oncocytoma, and 15 cases of papillary RCC. Clinicopathological information for the 42 ccRCC patients was summarized in In addition to our current study, we also selected 5 large-scale cytogenetic studies in the literature for a combined study. Out of the 5 studies, 3 had tumor stage information, 2 did not. There were 572 ccRCC samples in total with different techniques used to detect cytogenetic alterations. The clinicopathological information was summarized in XbaI or HindIII to digest the genome materials. Digestion of the DNA, PCR, and scanning followed the instructions from Affymetrix (http://www.affymetrix.com/). All data samples passed the quality controls both before application to the chip and after scanning the hybridized chip.We used Affymetrix 100K SNP chips to obtain genomic information. Each 100K SNP chip contains two subchips using either t-test statistics (with a window size of 31 SNPs for the moving windows). The t-test makes use of the copy number's local constant property, which assumes that the losses or gains of SNP copy numbers tend to happen in segments. Neighboring SNPs tend to have the same copy numbers. Thus, by borrowing the information from its neighbors, this copy number calculation will increase its power of uncovering the underlying DNA alterations and decrease noise. This operation is equivalent to smoothing the calculated raw copy numbers. The correction is necessary because DNA samples of tumor tissues prepared for SNP runs were generally not a homogenous collection of tumor cells, but rather a mixed collection of tumor and other cells . Even the tumor cells may have different cytogenetic patterns with differed combinations of numbers of signal for the target over signals for the control . For example, the low-grade and early-stage tumors tend to have a lower proportion of tumor cells than the high-grade and later-stage tumors. We call this phenomenon partial gain or partial loss, as opposed to complete gain or loss in that all cells used for microarray scan are tumor cells. As a result, without any corrections, the final raw copy numbers of the tumor cells would be a weighted mixture distribution of all the components of cells prepared for the scan, usually pulled toward the normal copy number of 2. A solid cutoff to claim a gain or loss will be improper if one takes this into consideration.Raw SNP copy numbers were calculated based on CNAG software version 2 . We usedreb) package in Bioconductor [t-test) was applied to determine if the raw copy numbers in the region were collectively higher or lower than that of normal. The test statistics were then output for each tumor sample and for each cytogenetic region. Disease-specific survival was used and was defined to be the time from first operation date to either death or last known follow-up date. For each cytogenetic region with cross sample interquartile range of the summarized cytogenetic alteration scores  greater than 2.5, a number of survival models were built to associate the patients' survival with the summarized cytogenetic alteration scores. We set the number of models to be 100 for each cytoband. Each model was built on a randomly selected but fixed number of subset of ccRCC patients. The score test was used to calculate the prognostic significance of P-values for the association. The transformed P-values (−log10(p)) were averaged over that from the 100 models as the final significance of association for the cytoband, as well as the regression coefficients. A studentized test was used to test the difference of summarized cytogenetic alterations between two groups of samples.The smoothed copy numbers were then summarized based on cytoband using the regional expression bias . If the box is well above zero, it is interpreted as majority of the samples in the group have gains in their copy numbers (at least 75% again). Thus the boxes contain both the frequency and the intensity of the gain or loss events in the group. It is a natural combination of information for both. The vertical length of the box reflects the range of sample CNA  values: the longer the box, the more variable the range of sample values. Thus, short box lengths reflect a tighter clustering of SNP copy number values than do longer box lengths. The midpoint of each box represents the median CNA value for all samples. An illustration figure was displayed in To display the collective copy number gains or losses for a group of samples, we first calculated for each sample and for each cytoband, the summarization scores of copy numbers of SNPs within the cytoband through reb algorithm. A value larger than 10 was marked a gain for the whole cytoband for the sample, while a value less than −10 was marked a loss for the cytoband. The proportions of losses or gains for each cytoband across all tumors in the study group were calculated. The proportion of loss (gain) of an arm was determined to be the highest loss (gain) proportion among all cytobands within the arm. If the proportion of loss was higher than that of gain, the arm was an overall loss and vice versa . The numbers and proportions of losses or gains for selected chromosome arms calculated this way were then summarized in To determine loss or gain of a specific chromosome arm for a specific sample to produce First, we compared the cytogenetic profiles of the four RCC subtypes: clear cell, papillary, chromophobe, and oncocytoma Figures . UnsuperVHL gene is located on 3p25-26, this indicates that deregulation of VHL pathway is involved for ccRCC development only, but not for the other tumor subtypes. Thus, different RCC subtypes have unique cytogenetic alterations as well as common alterations. As ccRCC accounts for the majority of RCC tumors, we will focus our analysis on this subtype only in the rest of paper. Comparing cytogenetic profiles of four RCC tumor subtypes, we found that −3p, +5q and −8p are unique to ccRCC tumors . Tumor sTumor stage is an important factor in determining tumor progression. We grouped ccRCC tumors and analyzed their cytogenetic alteration profiling according to tumor stages . As was correlation coefficient = −0.25, P =.12). As unbalanced translocations between 3p and 5q were frequently observed [Our data suggest that −3p and +5q are universal events in all stages of ccRCC tumors  and in aobserved , this reobserved , 25. We t-test was used to test the difference of cytogenetic alterations between the two groups of tumors having stages 1a and 1b. A test statistic below −2 or above 2 was used to declare the differences to be significant. The profile of test P-values was plotted in Supplementary Figure S1C. The identified cytogenetic alterations declared to be significant between tumor groups S1a and S1b were 3p (p25–p22), 5q (q34-q35), 4q23, 11q24 and 17q22. Apart from the single cytoband on chromosomes 4, 11, and 17, the most significant differences were recognized as more gains in 5q and more losses in 3p in stage 1b tumors than in stage 1a tumors. Unlike gains in chromosome 7, +5q is seen to be unique to ccRCC tumors , which were likely to be involved in ccRCC progression and development. However, no mutation genes were found in this region so far.The profiles of cytogenetic alterations between stage 1a and stage 1b tumors were remarkably similar  and EGLq21-q24, ), both oq21-q24, ) and SERq21-q24, ). AKT1 gMET  in 7q31, HGF in 7q21, EPO (Human erythropoietin gene) in 7q22, VGF (nerve growth factor inducible gene) in 7q31 and PDGFA  in 7p22 or IGFBP1 (7p13-p12). Amplification of or activating mutations in these genes may play a role in the development of ccRCC. Unlike +5q, +7 was not unique to ccRCC, but that it occurs in all malignant RCCs; a general role for genes in this region can be suggested during RCC development, such as proliferation and vascular invasion , leading to poor patients' survivals .Gain of chromosome 7 was seen to appear in all stages , indicatThese events did not appear in the early stage S1a or S1b tumors, but occurred progressively as tumors advance in stage. For example, −13 and −18 occurred only when tumor stages exceeded 2, and they peaked at the latest stage S3b4, . These eNext, we grouped and analyzed cytogenetic alterations in ccRCC by tumor grade . The patP-values for each cytoband were plotted in Supplementary Figure S2A. Cytobands from chromosomes 1, 2, 4, 6, 7, 8, and 13 were identified to be associated with patients' survivals. Specific cytogenetic bands associated with patient survival were summarized in Supplementary Table 1. Among these regions, −4p/4 and −13 were seen to be most significantly associated with poor survivals. This was confirmed in previous study [Next, we examined cytogenetic alterations in ccRCC tumors in association with patient survivals. The averaged significant us study . +1q wasAssociations between cytogenetic alterations in ccRCC and other clinicopathological features, including VHL mutation status, gender, tumor size, sarcomatoid elements, gross tumor necrosis, renal vein and vascular invasions were examined too; the significances of tests were summarized in Supplementary Table 2 and displayed in Supplementary Figure S2. Of the different profiles associated with different clinical or pathological factors, the profiles for tumor grade , for tumor stage  and for tumor size  are comparably similar, while the profiles for vein invasion  and for vascular invasion  are remarkably similar.These similarities reflect the closeness of being clinicopathological factors. We note that, interestingly, VHL mutation status is not associated with any specific cytogenetic changes and that +1q is associated with sarcomatoid differentiation, tumor vein and vascular invasions as well as poor patient survivals, indicating that some genes in this gene rich area play critical roles in tumor progression or metastasis. The cytogenetic profiles between male and female patients were remarkably similar too , except that male patients were more likely to have amplified chromosome 7 and 1q than their female counterparts, and that female patients were more likely to exhibit 8p loss. Further studies were needed to verify if these differences were gender related.We have studied the whole genome cytogenetic alterations in RCC tumors using high-density high-throughput Affymetrix SNP arrays. We confirmed that different subtypes of RCC had distinct cytogenetic profiles, reflecting the heterogeneity of RCC. We also showed that specific cytogenetic alterations in ccRCC are associated with specific clinicopathological features. Finally, by examining cytogenetic profiles from ccRCC tumors at different stages of progression, we will be able to construct a detailed map of the sequential cytogenetic changes that occur during ccRCC progression below. Based on results in VHL gene mutation for deactivation of the VHL functional pathway. Deactivation of VHL pathway alone is not sufficient to cause the ccRCC phenotype [First of all, based on our experience and mouse model study as well as published papers , we posthenotype , 50.Combining all the information, we have collected, we propose a new model of ccRCC tumorigenesis and progression. This model is illustrated in Supplementary Figure S1 is the summary plot when cytogenetic alteration profiles between Stage 1a and Stage 1b tumors were compared and Figure S2 is the plot when copy number alteration profiles separated in two groups by various clinical features were compared.Supplementary Table 1 is the list of identified cytogenetic bands in which patients' cytogenetic scores were significantly associated with patients'survivals when fitting survival models and Supplementary Table 2 is the list of identified cytogenetic bands in which a formal test was significant whencomparing copy number alterations between two groups of patients based on various clinical features.Click here for additional data file.Click here for additional data file.


Metabolic syndrome (MS) may comprise several clinical conditions such as obesity, diabetes and inflammatory disorders, which are characterized by metabolic imbalances. The study of glucose transport and regulation by insulin in lymphocytes is important, since the way they increase inflammation and susceptibility to infections are common in MS. We studied glucose internalization in isolated thymocytes and splenocytes, its regulation by insulin, and the role of three glucose transporters (Gluts) in control and in MS rats. Control glucose internalization and insulin responses were lower in splenocytes than in thymocytes. Control and insulin-induced glucose internalization in thymocytes declined with age, while transport by splenocyte continued to respond to insulin. Control thymocyte glucose internalization was blocked by antibodies against Glut 1 and 4, while the insulin response also was blocked by an anti-Glut 3 antibody. On four month old control and insulin-induced response, splenocyte transport was only blocked by Glut 1 and 4 antibodies. At six months splenocyte glucose internalization depended on Glut 1 and was less sensitive to the effects of an anti-Glut 4 antibody. In MS splenocytes the capacity of anti-Glut 1 antibodies to inhibit control and insulin-dependent glucose transport was less significant, and we found that in MS rats, glucose internalization was dependent on Glut 3 and Glut 4. In summary, the altered metabolic state present in MS rats shows signs of modulation of glucose internalization by the Glut1, Glut 3 and Glut 4 transporters, compared with its own age control. Metabolic Syndrome (MS) should be considered as a cluster of mostly modifiable risk factors triggering a proinflammatory state, that provide a higher risk of the development of diabetes and cardiovascular diseases. Specific cardiovascular disease risk factors might include obesity, type 2 diabetes, hyperlipidemia, insulin resistance and hypertension. Some other alterations, such as a pro-coagulant state and pro-inflammatory signs, can be included . The chaThe immune system is crucial for the defense against organisms that cause infections and against toxic products; a single defect in any of its components can cause a breakdown in this defense system and lead to serious or fatal diseases. The consequences can be systemic infections, cancer, autoimmune disorders or metabolic impairments. In diabetic patients the incorrect management of sepsis, due to an inappropriate immune response, can cause excess inflammation, which can also decrease longevity .Lymphocytes as part of the adaptive immune response are critical for normal immune functions . These cGlucose transport into the cell, its regulation and the mechanism of action of the hormones involved, varies among different tissues and organs as well as during development ,14. GlucGlucose is transferred across the plasma membrane by facilitated diffusion along a concentration gradient involving transport proteins called glucose transporters (Gluts), which have been characterized according to their transport properties and to the requirements of the tissue in which they are expressed . There aRegulatory factors, such as insulin level and hypoxia -23, enhaet al. [In this study, we used a rat model of MS developed in our institution by Baños et al. . The chaet al. -30.Due to the pro-inflammatory state and metabolic disorders present in the MS, the aim of this study was to study the glucose transport at two different stages of maturation of the lymphocytes in these animals and the participation of three known glucose transporters in these types of cells. Our interest increased due to the inflammatory state and the non-diabetic condition of this model. We studied the modulation of glucose transference in thymocytes and splenocytes mediated by three different glucose transporters , using antibodies to block glucose internalization, to possibly define the participation of the glucose transporters in the MS.Rattus norvegicus) were studied; young 4 month old rats (150-200 g) and medium age six month old rats (400-500 g). Control animals: 4 month old animals were used to measure the thymus activity, which declines with age [ad libitum. At the end of treatment period, the rats were killed by decapitation to obtain the blood, spleens and thymuses for experiments. Decapitation was chosen to avoid the interference by any anesthetic and to allow blood draining. Serum was obtained by blood centrifugation.Two control groups of male Wistar rats  up to six months of age, with normal rodent diet and a solution of 30% sucrose as the only liquid source. At the end of the treatment these animals were medium age adults (400-500 g), which were killed by the same procedure as the other groups. To determine the MS, their weight, blood glucose and triglyceride concentration, blood pressure and insulin concentration were measured. These variables were also determined to the control animals.Systolic arterial blood pressure was measured in conscious animals using the tail-cuff method; the cuff was connected to a pneumatic pulse transducer  and a programmed electro sphygmomanometer. The mean was calculated. The recordings were taken of six independent determinations by means of a Grass polygraph .After overnight fasting (12 hours), the animals were killed by decapitation and blood was collected. It was spun and the serum was separated by centrifugation at 15,000 rpm during 15 min at room temperature and stored at -70°C until needed.Serum insulin was determined using a commercial radioimmunoassay (RIA) kit specific for rat ; the sensitivity was of 0.1 ng/mL and intra- and inter-assay coefficients of variation were 5 and 10%, respectively.R Plus .Glucose concentration was assayed using an enzymatic Kit SERA-PAKTriglycerides (TGs) were determined by commercially available procedures .Cholesterol was determined by commercial enzymatic procedure SPINREACT cholesterol -LQ (Spinreact S. A. Gerona Spain).HOMA was used as an index to measure the degree of insulin resistance and was calculated by the formula: (insulin (μU/ml) × glucose (in mmol/L)/22.5) -34.2-5%CO2, pO2 160 and pCO2 21.3 mmHg. The organs were extracted from the control and MS rats. Spleens and thymuses were cut separately into small pieces (2 mm2) and filtered through fine gauze and then centrifuged 3 min at 1000 rpm. In the spleen, erythrocytes were lysed by adding 3 ml of a lysis solution to the pellet; (1 part of Tris (0.17 M) and 9 parts of NH4Cl (0.16 M)). The cells were gently agitated in this buffer for 1 min and centrifuged again. They were washed twice with Tyrode to remove the lysis buffer. Thymus cells were filtered and washed once and resuspended in Tyrode. Macrophages from both cell lines were removed by incubating the cell at 37°C for 30 min in plastic Petri dishes. The cells were then washed twice in fresh Tyrode solution by centrifuging 3 min at 1000 rpm and resuspending the pellet. Cellular viability was determined by the percentage of cells that excluded 0.3% Trypan Blue (Gibco-BRL) [Immediately after blood draining, spleens and thymuses were dissected and placed in cold Tyrode solution. All solutions were bubbled with 95%Obco-BRL)  in Tyrod6 cells (approximately 100 μl of the pellet) were placed in Eppendorff tubes, centrifuged and the supernatant substituted by 100 μl of one of the following different solutions added to each tube: 1) Tyrode solution (Tyr), 2) Tyrode solution with 50 mU/L insulin (Insulin) (Eli Lilly), approximately the postprandial dose reached in plasma, 3) Tyrode solution with 1 μM trifluoperazine (TPZ) , a calcium-calmodulin complex blocker [4) Tyrode containing commercial anti-Glut1 antibodies (G1), directed against the extracellular domain (1:1000), , raised in goat, 5) Tyrode containing commercial anti-Glut 3 antibodies (G3), directed against the extracellular domain , , when used in thymocytes and  when used in splenocytes, 6) Tyrode containing commercial anti-Glut 4 antibodies (G4), directed against the extracellular domain , , raised in goat, 7) Tyrode solution plus an Affinipure goat anti-rabbit serum(serum), at the same concentration as the antibodies used to block the glucose transporters (negative control) , 8) Tyrode solution with 0.1 μM cytochalasin B (Cyto) , a glucose transporter blocker (positive control) [Cells were centrifuged again and aliquots of 5 × 10 blocker ,37; 4) Tcontrol) . All dosAfter the incubation time, 2 μl of the supernatants were added to 200 μl of a glucose assay,  to measure the glucose concentration by the glucose oxidase reaction , in a BeAll animals were lawfully acquired and the manipulation of all animals in the research was carried out in accordance with "The European Commmission Enviroment" (Declaration of Helsinki): EC Directive 86/609/EEC for animal experiments . It alsoData were reported as micrograms of glucose subtracted from the incubation media per gram of fresh tissue per hour. Means and standard errors of at least 20 experiments were calculated. ANOVA was used to evaluate the differences on glucose internalization produced by the cell populations. The Mann Whitney test was used to determine which differences were statistically significant considering p < 0.05 as significant. Student's t test was used to compare control and insulin responses.After six months of drinking a high sucrose solution the animals develop many of the characteristics of the MS, showing similar symptoms to the fructose-fed model developed by Reaven . The aniGlucose internalization was significantly larger in immature thymocytes and they incorporated 3.31 ± 0.23 glucose μg/fresh tissue g/h. There were differences between 4 and 6 months of age in control thymocytes, the 4 months old cells being higher glucose consumers. At the stage of 4 months the value of glucose transport in thymocytes was 51%; meanwhile at the age of 6 months thymocytes decreased their glucose transport by 15% Figure .Insulin significantly increased glucose transport in thymocytes at the age of 4 and 6 months old, but glucose transport diminished with time; the insulin-induced response at 6 months old was reduced to 66.4%.Basal glucose internalization in thymocytes from MS rats was the same as in 4 and 6 month old controls, but they stopped responding to insulin Figure .In cells isolated from the spleen the glucose internalization was not as high as in thymocytes. However this insulin effect was more pronounced (19%) in immature thymocytes than in splenocytes; these differences decreased at the age of 6 months. Splenocytes incorporated 2.29 ± 0.12 glucose μg/fresh tissue g/h. and did not change their transport through time. The insulin effect in these cells was present at both ages Figure .MS splenocytes decreased their glucose transport, but they had a greater insulin-induced effect compared with its own age control Figure .Insulin effect on both cell populations was reduced by a dose of 1 μM of trifluoperazine, which is a calcium-calmodulin inhibitor. Therefore calcium-calmodulin could be one of the possible pathways of the insulin effect. Trifluoperazine had no effect on control glucose transport Figure .At the age of 4 months, the participation of glucose transport molecules in both basal and insulin-induced glucose internalization was studied by incubating thymocytes (immature cells) in the presence of commercial anti-Glut1, anti-Glut 3 and anti-Glut4 antibodies. Antibodies against the Glut1 and Glut4 molecules significantly decreased basal and insulin-induced response. Anti-Glut 3 antibodies had no effect on the basal transport Figure . The higBasal and insulin-induced glucose transport were significantly decreased when thymocytes were incubated with cytochalasin B. Control goat serum without anti-glucose transporter antibodies did not alter either basal or insulin-induced glucose internalization Figure .Glucose transporter molecule participation in both basal and insulin-induced glucose internalization was also studied in splenocytes (mature cells) in the same way as in thymocytes and at the same age. Control and insulin-induced glucose consumption by splenocytes was blocked by anti-Glut 1 and anti-Glut 4 but not by anti-Glut 3 antibodies Figure .Basal and insulin-induced glucose internalization were significantly decreased when splenocytes were incubated with cytochalasin B. Control goat serum without anti-glucose transporter antibodies did not alter basal or insulin-induced glucose capture Figure .Due to the fact that the stock of thymocytes is built up in early life and the thymus functions decline as age advances, we decided to continue to study splenocytes only. This decision was also made because the glucose transport at 6 months old and in the MS thymocytes declined and there was no response to insulin.Different glucose transporter participation was observed at the two ages in splenocytes control cells. Splenocytes glucose internalization did not change through time, but the way in which cells captured glucose was different. At the age of 4 months, these cells incorporated glucose through Glut 1 and Glut 4 and insulin response promoted the entrance by the same transporters. At 6 months, Glut 1 also facilitated the entrance of glucose, but when the cells were incubated with the antibody against Glut 4  it no longer participated, although a tendency to do so by insulin was observed. In the event, we concluded that the transporters could be modified by age Figure .Then the cells were incubated with a higher concentration of anti Glut 4 antibodies (1:500) and a blockade in both cases was observed (control and insulin). The behavior of Glut 3 did not change with age Figure .Glucose transport in MS animals is lower compared with control animals of the same age, but the response to insulin is enhanced. In this model, it seems that Glut 1 does not mediate basal glucose intake, but when an increase in the antibody concentration was used (1:500), a blockade was observed, as well as in the insulin response. This might suggest that in this condition the splenocytes need more glucose transporter in the membrane. Under control conditions, the participation of Glut 4 in the MS cells is blocked, with the same concentration (1:500) of antibodies as previously used in the 6 month old animals. When the MS splenocytes were stimulated by insulin at the same concentration of antibodies against Glut 4, the blockade was complete. This suggests that the quantity of antibody was sufficient to block the response. An activation of Glut 3 in the basal and insulin-induced transport was now observed in the MS model (Figure Lymphocytes, as part of the adaptive immune system, are critical for normal immune functions . They reIn accordance with the observation that glucose transport changes during development and maturation in many cell types ,14, we fGlucose enters cells by facilitated diffusion  via glucLymphocytes express Glut 1, Glut 3 and Glut 4 molecules -18. In tRegulation of glucose transport is also modified during development ,14. The Basal and insulin-induced glucose internalization by thymocytes declines after some time and this can be due to thymus atrophy. The stock of T lymphocytes is built up in early life, so the function of the thymus is diminished in adults. In elderly adults it consists of fatty tissue. However, the thymus still functions as an endocrine gland that stimulates the immune system and this phenomenon is related to post-receptor defects in the mechanism of insulin-mediated glucose uptake in target tissues . In the Splenocytes formed in the bone marrow are naive and have never been exposed to antigens; they get activated and migrate to lymphoid organs. This route is a very high glucose-consuming process. In splenocytes a movement of the glucose transporter 4 with aging is observed and this can be explained by the fact that this transporter has a developmental expression. It has been reported that this Glut is regulated, in a tissue-specific manner, by a developmental program at early stages in different tissues and this program may coordinate the expression of other proteins of metabolic importance; therefore, it should not be surprising that this change in expression might also happen at later stages of development to adjust to metabolic changes . B cells2+ and calmodulin as the intracellular messenger of the insulin receptor complex, since calmodulin must interact with Ca2+ to induce vesicle fusion to the plasma membrane. It has also been previously reported that an increase in intracellular calcium level leads to increases in cellular proliferation and secretion of intracellular products [In the present paper we studied the participation of the calcium-calmodulin complex in insulin increases in glucose internalization through its inhibition, by using trifluoperazine ,53. It hproducts .MS is associated with abdominal obesity, blood lipid disorders, inflammation, insulin resistance and an increased risk of cardiovascular diseases. Our model concedes with many of the MS characteristics and the obesity that these animals develop may be a risk factor for deteriorating cellular immune function .The fact that the MS cells decrease their glucose transport compared to control cells might be due to the phenomenon called immunosenescence, which covers numerous immune system changes with age . The MS We postulate that, in our model, there are glucose transporter movements due to the metabolic disorders that these animals suffer -29. Fu eWe conclude that in rat, thymocytes consume more glucose than splenocytes and respond better to regulatory factors such as insulin when they are at an active stage. Lymphocytes show three different glucose transporters: Glut1, Glut 3 and Glut4. Thymocytes use Glut 1 and Glut 4 for basal glucose consumption and the three transporters participate in the insulin-induced response. Meanwhile, in splenocytes, glucose transport is made by Glut 1 and Glut 4 in basal and in the MS the glucose transport is diminished and the three glucose transporters are activated. There is a regulation of the transporters, where Glut 1 raises its participation, Glut 3 is activated and Glut 4 diminishes its response in the insulin stimulation. The participation of the calcium-calmodulin complex, as a possible intracellular signaling pathway for insulin, could suggest part of the regulation of this hormone in the incorporation of glucose into the cell and in the glycemic control of these animals. These results could contribute to a better understanding of why in the MS the immune system is constantly activated to respond to persistent infections, through the regulation of glucose transport.The authors declare that they have no competing interests.VG suggested the idea and supervised the work; RC structured and performed the experiments and analyses of the data. Both authors participated in the elaboration of the manuscript and have read the complete manuscript and take responsibility for its content and completeness and understand that if the paper or part of the paper is found to be faulty or fraudulent they share responsibility.


Biologic predictors or biomarkers of survival in pulmonary fibrosis with a worse prognosis, more specifically in idiopathic pulmonary fibrosis would help the clinician in deciding whether or not to treat since treatment carries a potential risk for adverse events. These decisions are made easier if accurate and objective measurements of the patients' clinical status can predict the risk of progression to death.A literature review is given on different biomarkers of survival in interstitial lung disease, mainly in IPF, since this disease has the worst prognosis.Serum biomarkers, and markers measured by medical imaging as HRCT, pertechnegas, DTPA en FDG-PET are not ready for clinical use to predict mortality in different forms of ILD. A baseline FVC, a change of FVC of more than 10%, and change in 6MWD are clinically helpful predictors of survival. Interstitial lung diseases (ILD)  are a heThe clinical course of individual patients with ILD is variable and can manifest long periods of stability, a steady gradual decline, and/or periods of acute deterioration . Some foIn the search for an effective therapy, biologic predictors of survival in pulmonary fibrosis with a worse prognosis, more specifically in IPF, have been extensively studied in the last 10 years. These surrogate endpoints for survival are so-called biologic markers or biomarkers. They can be subdivided into those markers measured in serum, those measured by lung function testing and those by imaging techniques. Before discussing the different results of the studies performed on predictors of survival, an introduction about what a biomarker is, is given."A biomarker indicates a change in expression or state of a biologic measurement  at a given time point that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment at a future time point ". Once aUse of such a biomarker as surrogate endpoint to assess a clinical endpoint  has potential disadvantages. Biomarkers can be difficult to validate and require different levels of validation depending on their intended use . If a biIn summary, 4 pitfalls are present during the validation process of a biomarker as a diagnostic test : (1) pre1, DLco and resting PA-a O2 [At the end of the 1990s various serum markers were tested for their use in ILD, more specifically in IPF. Many markers have been studied and those with the most promise are surfactant proteins A and D (SP-A and SP-D), KL-6, and lactate dehydrogenase (LDH). In 2009 serum CC-chemokine ligand 18 (CCL-18) was presented as a potential biomarker in IPF . Why the PA-a O2 . The thi PA-a O2 . It was  PA-a O2 . In concIn 1989, Kohno et al. discovered a compound named KL-6, a mucin-like high-molecular weight glycoprotein, which is expressed on type II alveolar pneumocytes . ConcentSome authors suggest that LDH is a potential marker of disease activity and of response to therapy in different forms of ILD -27. CytoCCL18 is a CC-chemokine produced by human myeloid cells . It is c1/FVC ratio) together with a reduction in DLco. However, in early disease, lung volumes and DLco may be within the normal range [ILD are usually characterised by a restrictive lung function, i.e. a reduction in lung volumes with preserved Tiffeneau index  and a DLco > 43%  at baseline were found to influence rate of survival by a Cox's regression analysis. Changes in VC or DLco over 6 or 12 months were not found to be independent variables of survival.The spinoff of the IFIGENIA trial aimed to quantify the risk for mortality in 155 IPF patients after 4 years of the date of inclusion in the IFIGENIA study . The stuLco and VC were measured over a median period of 76 weeks. Twenty one percent died, of which IPF (89%) was the primary cause of death [Lco was observed, although significant intra-patient variability occurred over time [Lco over a period of 76 weeks [Martinez et al. analysed retrospectively data from the placebo group (168 IPF patients with mild to moderate disease) of a trial evaluating interferon gamma as treatment for IPF . At 12 wFlaherty et al. examined retrospectively 80 patients with IPF and 29 patients with NSIP . They foVarious other investigators have also suggested that a decreased FVC at baseline identifies patients at subsequent risk of mortality -40. In aLco over 6 months of follow-up has limited prognostic value and this measurement was not found to add independent predictive value for mortality in IPF [Lco varies to an even greater extent than FVC and clinically significant changes are believed to be more than 20% [Lco compared with those experiencing a decrease of 20% or more after 1 year of therapy [Flaherty et al. demonstrated that the change in Dy in IPF . Althougy in IPF ,50, the than 20% ,46,47,51 therapy .Lco are useful as surrogate markers for survival in trials searching for therapeutic strategy in IPF? This question is critical since change from baseline to a specified time point of these physiologic variables has been used either or both as primary endpoint in the most recent therapeutic trials in IPF [The question remains whether FVC and Ds in IPF .Lco during 77 weeks treatment with either interferon gamma or placebo [The INSPIRE trial, the largest trial ever, included 1373 IPF patients, and concluded that no difference is seen in survival between those treated with interferon gamma and those with placebo . Consist placebo .Lco are valid biomarkers for survival, because the results of above mentioned trials are confusing. Is survival itself the only valid endpoint to be used in therapeutic trials? IPF is a rare disease, and if investigators decide only to use survival as primary endpoint, trials will be difficult to set up and manage to a successful conclusion. Taking the INSPIRE study as an example, the investigators calculated at the onset of the trial that to have 90% statistical power to detect a treatment effect equivalent to a 50% reduction in 3-years, about 600 patients were needed to achieve the targeted number of deaths within the planned duration of the study (77 weeks). Two interim analyses of this trial increased the number to 1200 patients. The economic and logistic efforts (worldwide participation of 82 centres) to bring this to a successful conclusion are huge. Efforts not every investigator or pharmaceutical company can afford [With the available evidence, it is difficult to conclude that a decrease or a change over time in FVC or Dn afford .Different studies have evaluated the correlation between 6 minute walking distance (6 MWD) or change in walking distance and survival -61. Most99mTc-diethylenetriamine pentacetate (DTPA), and positron emission tomography (PET).Two types of medical imaging are discussed. HRCT and nuclear imaging including imaging that measures the alveolar capillary membrane permeability of the lung by use of radioactive isotopes, pertechnegas and It is nowadays unthinkable not to use HRCT in the diagnosis of ILD. The added value of HRCT depends upon its ability to increase confidence of a specific diagnosis, to alter patient management and, if possible, to influence outcome . ResearcCan HRCT predict response to therapy in IPF? Gay et al. set up a study with 38 biopsy proven IPF patients. The study patients received 1 mg/kg prednisone daily during 3 months. The HRCT before treatment was scored (score from 0 to 5 for each lobe) for ground glass and fibrosis by 4 radiologists independently. They demonstrated that a fibrosis score of 2 or more has a 80% sensitivity and 85% specificity in predicting survival . From thThe ILD are characterised by an acute or chronic inflammation of the interstitium, also called the alveolar capillary membrane . A possi18F-FDG PET imaging may serve as a sensitive tool for the evaluation of disease activity in sarcoidosis, with higher sensitivity and interobserver agreement compared to the classical Gallium scintigraphy [18F-FDG PET as a biomarker for disease activity in other ILD is less clear. In IPF the magnitude of 18F-FDG uptake in the lungs is usually low. As 18F-FDG is thought to assess the inflammatory burden and not the fibrosis, the finding of relatively low SUV in IPF can be regarded as confirmative for the concept that inflammation does not play a major role in the pathogenesis of this disease. No studies are present that correlates rate of 18F-FDG uptake with survival in specific forms of interstitial lung diseases.Positron emission tomography (PET) imaging has recently emerged on the scene of biomarkers of ILD. A number of studies has suggested that tigraphy -77. The Interstitial lung diseases are a diverse and complex collection of parenchymal lung diseases that vary widely in aetiology, histopathology, clinical radiological presentation, and clinical course . There iThe authors declare that they have no competing interests.All authors wrote and revised the manuscript, and approved the final version