Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice The ability to genetically manipulate mice has led to rapid progress in our understanding of the roles of different gene products in human disease . Transgenic mice have often been created in the FVB/NJ -LRB- FVB -RRB- strain due to its high fecundity , while gene-targeted mice have been developed in the 129/SvJ-C 57Bl/6J strains due to the capacity of 129/SvJ embryonic stem cells to facilitate germline transmission . Gene-targeted mice are commonly backcrossed into the C57Bl/6J -LRB- B6 -RRB- background for comparison with existing data . Genetic modifiers have been shown to modulate mammary tumor latency in mouse models of breast cancer and it is commonly known that the FVB strain is susceptible to mammary tumors while the B6 strain is more resistant . Since gene-targeted mice in the B6 background are frequently bred into the polyomavirus middle T -LRB- PyMT -RRB- mouse model of breast cancer in the FVB strain , we have sought to understand the impact of the different genetic backgrounds on the resulting phenotype . We bred mice deficient in the inducible nitric oxide synthase -LRB- iNOS -RRB- until they were congenic in the PyMT model in the FVB and B6 strains . Our results reveal that the large difference in mean tumor latencies in the two backgrounds of 53 and 92 days respectively affect the ability to discern smaller differences in latency due to the Nos2 genetic mutation . Furthermore , the longer latency in the B6 strain enables a more detailed analysis of tumor formation indicating that individual tumor development is not stoichastic , but is initiated in the # 1 glands and proceeds in early and late phases . NO production affects tumors that develop early suggesting an association of iNOS-induced NO with a more aggressive tumor phenotype , consistent with human clinical data positively correlating iNOS expression with breast cancer progression . An examination of lung metastases , which are significantly reduced in PyMT/iNOS − / − mice compared with PyMT/iNOS + / + mice only in the B6 background , is concordant with these findings . Our data suggest that PyMT in the B6 background provides a useful model for the study of inflammation-induced breast cancer . Introduction Transgenic mouse models that develop spontaneous mammary adenocarcinomas have proven valuable in revealing molecular mechanisms underlying tumorigenesis and metastasis -LRB- Cardiff 2003 ; Fargiano et al. 2003 -RRB- . Models target specific pathways depending on the transgene being expressed under the control of the mouse mammary tumor virus long terminal repeat -LRB- MMTV-LTR -RRB- or whey acid protein -LRB- WAP -RRB- mammary gland promoters and thereby replicate genetic defects in subsets of human tumors -LRB- Cardiff and Wellings 1999 ; Rosner et al. 2002 -RRB- . Molecules from pathways known to be important in the pathogenesis of human breast cancer such as the Wnt , Ras and ErbB2/Neu pathways have been utilized , however few spontaneous mammary tumor model systems develop metastasis , a crucial aspect of cancer progression . As demonstrated by gene expression analysis -LRB- Ma et al. 2003 -RRB- , the polyomavirus middle T -LRB- PyMT -RRB- model in the FVB/N -LRB- FVB -RRB- background activates the same signaling pathways as erbB2 , an oncogene amplified or overexpressed in approximately 30 % of human breast cancers . The model has been particularly useful for genetic studies due to its short tumor latency and high incidence of pulmonary metastasis -LRB- Guy et al. 1992 -RRB- . These features have enabled global expression profiling of PyMT tumors from different genetic backgrounds to identify signatures of tumor virulence -LRB- Qiu et al. 2004 -RRB- that are consistent with a gene set predictive of metastasis in human tumors -LRB- Ramaswamy et al. 2003 -RRB- . Hence , the model has been used effectively to dissect early and late stages of mammary tumor development -LRB- Guy et al. 1994 ; Lin et al. 2001 ; Webster et al. 1998 ; Williams et al. 2004 -RRB- . In women with breast cancers involving this pathway , the course of the disease varies widely . One powerful means to understand that variability is to identify useful animal models . The PyMT breast cancer model can be used in genetic crossing experiments to identify loci having modifier effects on either tumorigenesis or metastasis -LRB- Le Voyer et al. 2000 -RRB- . An understanding of the genetic polymorphisms underlying these effects is important in the study of human cancer as genetic variability affects host-tumor interactions in numerous ways and is an important factor in the response to therapy . Widely varied tumor responses to polyoma virus infection among different inbred strains of mice have been well documented -LRB- Freund et al. 1992 ; Gross 1983 -RRB- . Much of this difference can be attributed to the major histocompatibility complex -LRB- MHC -RRB- which results in immunologically mediated resistance associated with an H-2b haplotype such as in C57Bl strains -LRB- Law et al. 1967 ; Ting and Law 1965 -RRB- . Other inbred strains , notably those with the H-2d and H-2k haplotypes have moderate and weak tumor resistance respectively -LRB- Freund et al. 1992 -RRB- . The FVB strain has an H-2q haplotype , which may be an additional MHC locus that confers susceptibility to polyoma virus induced tumorigenesis . Alternatively , non-MHC genes can also result in a dominant pattern of tumor susceptibility as in the case of the C3H/BiDa strain -LRB- Freund et al. 1992 -RRB- . Strain variations are also relevant to the study of genetic mutations in mouse models of disease . An analysis of targeted disruptions on mammary tumorigenesis in the PyMT model requires breeding of knockout models usually developed in the 129/SvJ-C 57Bl/6J background into the FVB background . It is commonly recognized that the FVB strain is susceptible while the C57Bl/6J -LRB- B6 -RRB- strain is more resistant to PyMT mammary tumorigenesis and F1 crosses of these strains increase tumor latency by approximately 6 days -LRB- Lifsted et al. 1998 -RRB- . However , it is not clear what effects continued backcrossing has on the analysis of targeted mutations in the PyMT model . Previously we reported that inducible nitric oxide synthase -LRB- iNOS -RRB- deficiency in the PyMT model in the B6 background resulted in an increase in tumor latency that was not observed in the FVB background -LRB- Ellies et al. 2003 -RRB- . Since the issue of strain background is an important one for analysis of mouse models we have made iNOS knockout mice congenic in the FVB and B6 backgrounds and carried out a comparison of tumorigenesis and metastasis in both strains . Materials and methods Animals PyMT -LRB- FVB/N-Tg -LRB- MMTV-PyVT -RRB- 634Mul/J -RRB- mice -LRB- Guy et al. 1992 -RRB- were obtained from W. Muller -LRB- McMaster University , Ontario , Canada -RRB- . We acquired PyMT mice that had been backcrossed 5 generations into the B6 background from A. Varki -LRB- UCSD , La Jolla , CA -RRB- . Control -LRB- C57Bl/6J -RRB- and iNOS deficient mice -LRB- B6.129P2-Nos2tm1Lau / J ; Laubach et al. 1995 -RRB- were purchased from The Jackson Laboratory , Bar Harbor , ME . The PyMT B6 mice were further backcrossed until congenic in the B6 background and then bred with iNOS − / − mice . iNOS − / − mice were bred into the FVB background until congenic -LRB- > 10 generations -RRB- and then crossed with PyMT FVB mice . Female mice heterozygous for the PyMT transgene and homozygous for the wild type or mutated iNOS gene were used in these studies . For clarity and brevity , we have designated FVB/N-Tg -LRB- MMTV-PyVT -RRB- 634Mul/JxB6 .129 P2-Nos2tm1Lau / J + / + mice PyMT/iNOS + / + and FVB/N-Tg -LRB- MMTV-PyVT -RRB- 634Mul/JxB6 .129 P2-Nos2tm1Lau / J − / − mice PyMT/iNOS − / − . FVB mice were palpated twice weekly from 4 weeks of age and B6 mice once weekly from 8 weeks of age to monitor mammary tumor development . Tumors were measured in 2 dimensions using calipers and tumor volume estimated using the standard calculation for a sphere 4/3 × 3.14 × a × b2 where a is the smaller diameter and b is the larger diameter . After euthanizing the mice , mammary tumors were dissected and weighed and the total tumor burden calculated -LRB- tumor weight/body weight -RRB- . All studies followed the NIH guidelines for the care and treatment of experimental laboratory rodents . Mammary gland whole mounts Pairs of inguinal mammary glands were fixed in formalin or Carnoy 's fixative , dehydrated through a graded series of ethanol solutions and defatted in xylene . Following rehydration , the mammary epithelium was stained with carmine stain -LRB- Sigma Chemical , St Louis , MO -RRB- for 30 min . After removing excess stain by washing in water , samples were dehydrated and stored in methyl salicylate -LRB- Sigma Chemical , St Louis , MO -RRB- . Tumor microvascular density Cryosections of OCT embedded tumor samples were cut at 7 μm and stained with anti-CD31 clone 13.3 -LRB- 1:200 , BD Pharmingen , San Diego , CA -RRB- as previously described -LRB- Ellies et al. 2003 -RRB- . Five random fields per tumor were captured on the Spot RT digital camera system -LRB- Diagnostic Instruments , Sterling Heights , MI -RRB- at 200 × magnification and analyzed using the public domain NIH Image program -LRB- http://rsb.info.nih.gov/nih-image/ -RRB- . Microvessel areas were normalized to 1 mm2 of epithelium . Lung metastases Paraffin embedded lungs were serial sectioned and stained with hematoxylin and eosin . Both the numbers and areas of lung metastases were measured by light microscopy using images and software from the Spot RT digital camera system . Lung area per section was measured using a dissecting scope and metastatic tumor burden calculated -LRB- mm2 of lung metastases/cm2 of lung tissue -RRB- for each animal using data from 3 sections at least 100 μm apart . At least 13 PyMT/iNOS + / + or PyMT/iNOS − / − mice were used in this analysis . Statistical analysis The percentage of mice tumor free in each group was analyzed using a Kaplan-Meier survival analysis , and the log-rank statistic was used for comparison of the curves between groups . The t-test was used for comparison of 2 groups and the analysis of variance -LRB- ANOVA -RRB- followed by the Bonferroni post-test was applied to multiple comparisons . The Mann -- Whitney U test was used to compare metastastic burden in the PyMT/iNOS + / + and PyMT/iNOS − / − groups . Results PyMT tumorigenesis is delayed in the C57Bl/6 strain The PyMT model was originally developed in the FVB strain due to its high fecundity . Tumor growth occurs rapidly and metastatic tumors form in the lungs -LRB- Guy et al. 1992 -RRB- . To compare tumorigenesis in the FVB strain with that in the C57Bl/6 strain onto which many genetically modified strains are bred , we made PyMT mice congenic in the B6 background . Examination of tumor latency showed a dramatic difference with a mean latency of 53 days for PyMT tumor development in FVB mice and 92 days in B6 mice , a delay of approximately 6 weeks -LRB- Fig. 1A -RRB- . Whole mounts of mammary glands from the two strains also showed that even at weaning , hyperplasias were more advanced in FVB than B6 PyMT mice -LRB- Fig. 1B -RRB- . Fig. 1PyMT tumorigenesis in the FVB and B6 strains . -LRB- A -RRB- The day at which the first mammary tumor was detected by palpation in each mouse was recorded and a Kaplan-Meier survival plot generated . Log rank analysis indicated a highly significant difference , P < 0.001 between the curves . FVB mice N = 44 , B6 mice N = 46 . -LRB- B -RRB- Whole mounts of mammary glands from 3 week old PyMT/iNOS + / + mice of the FVB -LRB- left panel -RRB- and B6 -LRB- right panel -RRB- strains Effects of iNOS deficiency are more apparent in the C57Bl/6 background To determine how this difference in tumor latency may affect the phenotype observed following crossbreeding with genetically altered mice , we bred in iNOS − / − mice congenic in the two backgrounds . As previously reported , we observed a 3 -- 4 week delay in palpable tumors in B6 PyMT mice lacking iNOS activity -LRB- Ellies et al. 2003 -RRB- . However , this difference was difficult to discern in the FVB background due to the rapid tumor onset -LRB- Fig. 2A -RRB- . Mammary gland whole mounts reflected the latency data with a clear difference in tumor growth with iNOS deficiency being observed in the # 4 glands of B6 PyMT mice -LRB- Fig. 2A -RRB- . Fig. 2Effects of iNOS deficiency on mammary tumor development . -LRB- A -RRB- Whole mounts of PyMT/iNOS + / + and PyMT/iNOS − / − # 4 mammary glands from 11 week FVB mice or 20 week B6 mice . LN = lymph node . -LRB- B -RRB- Kaplan Meier plots of the tumor latency of PyMT/iNOS + / + annd PyMT/iNOS − / − mice of the 2 strains . Data were analyzed by the log rank test . PyMT/iNOS + / + FVB N = 44 , PyMT/iNOS − / − FVB N = 57 , PyMT/iNOS + / + B6 N = 46 , PyMT/iNOS − / − B6 N = 26 PyMT tumor growth kinetics vary in FVB/N and C57Bl/6 strains The pattern of tumor growth differed between strains and may in part account for the greater disparity in tumor development observed in the absence of iNOS in PyMT B6 mice . Once the mean tumor volume reached 1 cm3 , tumor growth proceeded exponentially in the PyMT FVB mice leading to sacrifice within 3 weeks -LRB- Fig. 3A -RRB- . To achieve the same final tumor volume , the PyMT B6 mice required approximately 7 weeks and growth followed a more linear pattern , suggesting more variable rates of growth of individual tumors . Indeed , when we analyzed the tumor latency of all tumors , we were able to define two distinct phases of tumor growth in the B6 strain : early , from 12 -- 17 weeks and late , from 19 -- 23 weeks -LRB- Fig. 3B -RRB- . The major effect of iNOS deficiency appears to be a delay in the early phase of tumor development . Fig. 3Mammary tumor growth kinetics . -LRB- A -RRB- Mean tumor volume per mouse . FVB N = 32 , B6 N = 44 . Data are means ± SEM . -LRB- B -RRB- Tumor latency for individual B6 PyMT/iNOS + / + and PyMT/iNOS − / − tumors . Data were derived from examining 460 B6 PyMT/iNOS + / + and 260 PyMT/iNOS − / − tumors Deficiency in iNOS-modulated tumor growth is site specific In concordance with increased tumor latency and decreased tumor growth -LRB- Ellies et al. 2003 -RRB- tumor burden was significantly lower in PyMT/iNOS − / − mice -LRB- Fig. 4A -RRB- . An examination of tumor burden in individual mammary glands revealed a reduction in all PyMT/iNOS − / − glands compared to controls , although the numbers were only significant in the # 3 and # 4 mammary glands -LRB- Fig. 4A -RRB- . It is interesting to note that these are the glands with the largest fat pads and may indicate a role for the stroma in the iNOS effect . To assess a role for tumor vascularization in this effect we examined tumor microvascular density -LRB- MVD -RRB- in # 1and # 2 -- 5 tumors of both genotypes . Although there was a slight reduction in the mean MVD in # 1 versus # 2 -- 5 tumors in the PyMT/iNOS + / + and PyMT/iNOS − / − genotypes , it was not statistically significant , suggesting this is not the mechanism for the difference observed in tumor growth at different locations -LRB- Table 1 -RRB- . Fig. 4Distribution of B6 mammary gland tumor burden . -LRB- A -RRB- Total tumor burden was significantly lower in PyMT/iNOS − / − mice , *** P < 0.001 . -LRB- B -RRB- Examination of individual tumors indicated that a significant difference in genotypes was only found in the # 3 and # 4 mammary glands , although in each gland the mean tumor burden was lower in PyMT/iNOS − / − mice . * P < 0.05 ANOVA followed by Bonferroni post tests . PyMT/iNOS + / + B6 N = 24 -LRB- black bars -RRB- , PyMT/iNOS − / − B6 N = 25 -LRB- grey bars -RRB- Table 1Quantitative analysis of tumor microvascular densityGenotypeLocationNMVD -LRB- μm2/mm2 -RRB- PyMT/iNOS + / + # 19216 ± 20PyMT/iNOS + / + # 2 -- 59176 ± 12PyMT/iNOS − / − # 16201 ± 11PyMT/iNOS − / − # 2 -- 511189 ± 11Data are means ± SEM This site-specific effect prompted us to examine tumor latency in individual glands . We had observed during palpations that the # 1 mammary gland was usually the first to develop tumors , however we were surprised to find a clear difference between the latency of the # 1 tumors compared with all other tumors -LRB- Fig. 5 -RRB- . This difference was more noticeable in the absence of iNOS . The greater difference in mean latency in the # 4 glands compared with the # 1 glands was consistent with the tumor burden results being more significant in the # 4 glands . Fig. 5Tumor latency of individual B6 mammary glands . Tumor latency was lower in the # 1 mammary gland in PyMT/iNOS + / + mice compared with all other mammary glands -LRB- upper panel -RRB- . A similar result was observed in PyMT/iNOS + / + mice , with an increase in disparity between the latency of the # 1 gland and all other glands -LRB- middle panel -RRB- . When the # 1 and # 4 latencies for both genotypes were overlayed , a greater difference in mean tumor latency was observed between the # 4 glands when compared with the # 1 glands -LRB- lower panel -RRB- iNOS effects on tumor metastasis The PyMT model has been useful for the study of tumor metastasis due to the high penetrance of this clinically important characteristic of tumor progression . However , we found that in the FVB strain , tumors were so aggressive that no significant difference could be observed in the metastatic burden occurring in PyMT/iNOS + / + and PyMT/iNOS − / − mice Fig 6 . Although the incidence of tumor metastasis was less in the B6 strain , a statistically significant difference between the genotypes was found . We have maintained PyMT/iNOS − / − mice for up to 32 weeks and found no increase in metastatic burden -LRB- data not shown -RRB- , suggesting that the decrease in metastasis is not due simply to the delayed tumor latency in these mice . Fig. 6Tumor metastasis . -LRB- A -RRB- HE stained paraffin embedded sections of PyMT/iNOS + / + -LRB- left panel -RRB- and PyMT/iNOS − / − -LRB- right panel -RRB- lungs . -LRB- B -RRB- Comparison of metastatic tumor burden in FVB and B6 mice * P < 0.05 Mann-Whitney U test . PyMT/iNOS + / + FVB N = 14 , PyMT/iNOS − / − FVB N = 13 , PyMT/iNOS + / + B6 N = 17 , PyMT/iNOS − / − B6 N = 21 Discussion Mouse models of mammary tumorigenesis have provided important insights into the molecular basis of human breast cancer and are essential for gathering preclinical data regarding therapeutic drug efficacy . The PyMT model in the FVB strain has a mean tumor latency of 53 days and has been used extensively in studies of mammary tumorigenesis -LRB- Cardiff 2003 ; Muller and Neville 2001 -RRB- . The model is frequently crossed with gene-targeted strains that have been generated in the B6/129 mixed background or backcrossed onto the B6 background . We had highly variable results examining iNOS effects in a mixed B6/FVB PyMT background and sought to characterize the phenotype of this mutation in congenic FVB and B6 strains . Our study demonstrates that the genetic background of the PyMT model can affect the ability to discern differences in phenotype due to genetic alterations that require time to influence tumor progression . Differences observed in the B6 background were obscured in the FVB background due to the signficantly shorter tumor latency and more rapid tumor growth in this strain . Further analysis of mammary tumorigenesis in the B6 background indicated that tumor formation occurred in 2 phases : early , from 12 -- 17 weeks and late , from 19 -- 23 weeks . It is the early phase of tumor formation that is most affected in the absence of iNOS , consistent with recent data linking iNOS expression with transcriptional upregulation of signal transducers and activators of transcription 3 -LRB- STAT3 -RRB- by nuclear epidermal growth factor receptor -LRB- EGFR -RRB- -LRB- Lo et al. 2005 -RRB- . EGFR/ErbB1 , a member of the ErbB family of receptor tyrosine kinases that signal via the same pathways as PyMT -LRB- Webster et al. 1998 -RRB- , is also found in the nucleus of highly proliferative cells where is functions as a transcription factor -LRB- Marti et al. 1991 ; Wells and Marti 2002 -RRB- . Structural and functional characterization of the iNOS promoter has identified binding elements for both EGFR and STAT3 -LRB- Lo et al. 2005 -RRB- . Immunohistochemical studies of breast carcinomas show that nuclear EGFR positively correlates with iNOS and survival analysis demonstrates that high levels of iNOS are a prognostic indicator of poor survival -LRB- Lo et al. 2005 -RRB- . Thus the B6 PyMT mice in the presence or absence of iNOS deficiency are likely to provide an important model for further analysis of these novel molecular interactions . Mammary tumor latency for each of the ten murine mammary glands has generally been assumed to be stoichiometric . However our data demonstrate that the # 1 mammary glands have tumor initiating properties that are distinct from the other 4 pairs of mammary glands . The # 1 mammary glands have the smallest fat pads and are located in the head and neck region , which is highly vascular , so we hypothesized that an increase in vascular density in the # 1 tumors could be responsible for this difference . An examination of the microvacular density -LRB- MVD -RRB- of # 1 tumors versus all others showed a trend in which # 1 tumors had a higher mean MVD than # 2 -- 5 tumors as determined by quantitative analysis of CD31 staining . However , these values were not statistically significant , suggesting a different mechanism is responsible for the observed site-specific differences in tumor burden and tumor latency . Other factors in the stromal microevironment may also play a role in mediating this effect . The release of epidermal growth factor -LRB- EGF -RRB- from the submandibular salivary gland has been shown to promote mouse mammary tumorigenesis in virus mediated and chemical carcinogen induced models -LRB- Kurachi et al. 1985 ; Molinolo et al. 1998 -RRB- . The proximity of the # 1 glands to the submandibular gland make EGF induced proliferation a plausible explanation , however further investigations will be required to determine whether the release of this growth factor primarily affects the # 1 glands . Another possible explanation for this intriguing difference is that the # 1 mammary gland is developmentally distinct from the other mammary glands . The # 1 gland is not the first to develop since placode 3 develops first at E11 .0 -- 11.5 on the ectodermal streak between the forelimb and hindlimb -LRB- Eblaghie et al. 2004 ; Mailleux et al. 2002 -RRB- . Placodes 1 and 5 arise from the axillary and inguinal streaks respectively and placodes 4 and 2 form where the mammary line abuts these streaks -LRB- Veltmaat et al. 2004 -RRB- . Recent studies indicate that signaling molecules essential for mammogenesis are placode dependent . Placodes 2 and 3 do not form in the absence of Lef1 mediated canonical Wnt signaling -LRB- Boras-Granic et al. 2006 -RRB- and placodes 1 , 2 , 3 and 5 require Fgf10 signaling from somites underlying the ectoderm -LRB- Mailleux et al. 2002 ; Veltmaat et al. 2006 -RRB- . It is clear there are developmental differences between mammary glands and it is possible that early developmental factors result in an increased propensity for malignant transformation in the # 1 mammary glands , though the identity of these factors remains to be determined . Since metastasis is an aspect of tumor progression that is critical to patient survival , we examined the effect of iNOS on tumor metastasis in both the FVB and B6 backgrounds . No significant difference in metastatic burden was found in PyMT/iNOS − / − mice in the FVB background , however the tumor burden was reduced in lungs of PyMT/iNOS − / − mice in the B6 background compared with controls . Furthermore , the reduction in metastatic burden was not due simply to the delayed tumor latency as we maintained PyMT/iNOS − / − mice longer to compensate for the difference and found that this did not alter our previous findings -LRB- Ellies et al. 2003 -RRB- . Human clinical studies have associated increased iNOS expression with poor prognosis in breast cancer patients -LRB- Loibl et al. 2005 -RRB- , and the use of non-steroidal anti-inflammatory drugs -LRB- NSAIDs -RRB- with a 22 -- 39 % reduction in the risk of breast cancer -LRB- Harris et al. 2005 ; Khuder and Mutgi 2001 -RRB- . To increase our understanding of the molecular pathways involved in these effects , it is essential to have animal models that recapitulate the clinical findings . We have shown that in the PyMT B6 background loss of iNOS , an important inflammatory mediator , results in increased tumor latency and decreased tumor metastasis that is not due simply to the difference in latency . This model will be useful in determining molecular pathways underlying the role of iNOS in breast cancer .

O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O B B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B B O

fvb/nj
c57bl/6j
polyomavirus middle t antigen


Comparison of three perioperative fluid regimes for laparoscopic donor nephrectomy Background Pneumoperitoneum -LRB- PP -RRB- , as used for laparoscopic procedures , impairs stroke volume , renal blood flow , glomerular filtration rate and urine output . This study investigated whether perioperative fluid management can abolish these negative effects of PP on hemodynamics . Laparoscopic donor nephrectomy -LRB- LDN -RRB- has become the method of choice to procure kidneys from living donors , mainly because of the reduced procedure-related morbidity and faster convalescence period -LSB- 1 -- 3 -RSB- . Despite the benefits to the donor , there are concerns over the transient deterioration of renal function in the recipient of the kidney procured by the laparoscopic technique , compared with open donor nephrectomy -LRB- ODN -RRB- -LSB- 2 , 4 -- 6 -RSB- . The exact mechanism of delayed graft function after LDN is not fully understood . Pneumoperitoneum -LRB- PP -RRB- elevates intra-abdominal pressure -LRB- IAP -RRB- , causing a decrease in renal blood flow -LRB- RBF -RRB- and glomerular filtration rate -LRB- GFR -RRB- resulting in oliguria -LSB- 7 -- 10 -RSB- . In an animal model , London et al. have shown that PP resulted in a decrease in RBF during normal saline infusion , whereas RBF did not decrease if volume expansion was given -LSB- 11 -RSB- . From these results vigorous hydration up to 2 l/h of crystalloids during LDN in patients has nowadays been advocated -LSB- 1 , 12 -- 15 -RSB- . In 52 patients , Bergman et al. -LSB- 16 -RSB- found , however , no difference in graft function after LDN between aggressive -LRB- > 10ml/kg/h -RRB- and conservative -LRB- < 10 ml/kg/h -RRB- intraoperative fluid management . Volume loading after establishment of PP is perhaps too late to counterbalance the collapsed venal system . Biancofiore et al. -LSB- 17 -RSB- studied the effect of volume loading on graft function with a crystalloid infusion starting the night before surgery . Early graft function did not differ between ODN and LDN , although the serum creatinine declined earlier , but not significantly , in those receiving kidneys from ODN procedure . Fasting before operation and induction of anesthesia leads to relative hypovolemia and the goal is therefore to compensate this before PP is started . In this study , we compared three different fluid regimes in LDN patients , in which the effect of pre-hydration together with a bolus of colloids given just before induction of anesthesia and a second one just before inflation of PP on hemodynamics was of special interest . Methods Patients undergoing LDN from June 2001 to November 2001 -LRB- N = 21 -RRB- were included in the study . The anesthetic procedure was performed according to a strict protocol for medication , ventilation and fluid regimen . In our hospital the donor patients are admitted the day before the operation , they are fasted during the night from 00:00 and operated on at 08.00 the next morning . Patients were randomized the day before operation by sealed envelopes by the responsible anesthetist to three different fluid regimens -LRB- Table 1 -RRB- : in group 1 fluid administration was started at 22:00 the day before operation with 3 ml/ideal body weight -LRB- IBW -RRB- / h Ringers lactate -LRB- RL -RRB- until operation . Before induction of anesthesia , the patients received 6 ml/IBW of colloid -LRB- 6 % HES 130/0 .4 -RRB- ; thereafter 13 ml/IBW/h RL was started until nephrectomy , before installation of PP another bolus of 6 ml/IBW colloid was given . Group 2 received overnight infusion in the same way as in group 1 with a bolus of 6 ml/IBW colloid just before induction . During operation , an infusion was started with 13 ml/IBW/h RL and 2 ml/IBW/h of colloid was given for three hours . Group 3 was fasted from 00:00 on the day of operation and received only an infusion during operation with 13 ml/IBW/h RL and 4 ml/IBW/h of colloid for three hours . After nephrectomy the infusion protocol was adjusted , so that exactly six hours after start of operation all the patients had received in total 9 ml/IBW/h RL . Patients were fitted with anti-thrombosis stockings . Table 1 . Infusion protocols used in the three groupsGroupnml RL/IBW/h pre-hydrationaml RL/IBW/h preoperativebml HES/IBW before inductioncml HES/IBW before PPdml HES/IBW/h after PPeml HES / IBW total1731366122721363 × 21237163 × 412IBW = ideal body weight n = number of patientsa Prehydration from 22:00 the day before laparoscopic donor nephrectomy , until operationb The amount of Ringer 's lactate given during operation , until nephrectomyc Amount of 6 % HES 130/0 .4 given before induction of anesthesiad Amount of 6 % HES 130/0 .4 given before installation of pneumoperitoneume Amount of 6 % HES 130/0 .4 given per hour after installation of pneumoperitoneumHES = 6 % HES 130/0 .4 Induction of anesthesia was performed with propofol -LRB- 2 mg/kg -RRB- after a bolus of sufentanil -LRB- 0.3 μg/kg -RRB- . Muscle relaxation was achieved with rocuronium -LRB- 0.8 mg/kg -RRB- and monitored by train-of-four -LRB- TOF -RRB- guard , a bolus of rocuronium -LRB- 0.3 mg/kg -RRB- was given for three or more twitches . Anesthesia was maintained with propofol by continuous infusion -LRB- 4 -- 11 mg/kg/h -RRB- , aiming at a bispectral index between 45 and 55 -LRB- BIS monitor ; Aspect Medical Systems , Newton , MA , USA -RRB- , and analgesia was achieved by continuous infusion of sufentanil -LRB- 0.4 μg/kg/h -RRB- until nephrectomy . One hour after the start of operation 20 mg mannitol was given intravenously . After intubation all patients were ventilated in a pressure-controlled mode using a closed-loop ventilator -LRB- Physioflex ® , Dräger , Lübeck , Germany -RRB- with the following initial settings : FiO2 of 0.4 , positive end-expiratory pressure -LRB- PEEP -RRB- of 7 cm H2O and peak inspiratory pressure -LRB- PIP -RRB- of 22 cm H2O . Ventilation frequency was adjusted to keep PetCO2 between 4 and 5.5 kPa . After induction of anesthesia and before positioning of the patient , an esophageal Doppler probe -LRB- HemoSonicTM 100 , Arrow International Inc. , Reading , PA , USA -RRB- was positioned for measuring stroke volume -LRB- SV -RRB- and left ventricular ejection time , corrected for heart rate -LRB- LVETc -RRB- -LSB- 18 -- 20 -RSB- . After positioning the patient in full lateral nephrectomy position , PP was installed with an IAP of 12 mmHg , which was constantly maintained at this level . All operations were done by the same team of anesthesiologists and surgeons . The surgical techniques have been described in detail elsewhere -LSB- 21 -RSB- . Mean arterial pressure -LRB- MAP -RRB- and SV -LRB- available after induction of anesthesia -RRB- were monitored noninvasively every five minutes . Urine output was measured from 22:00 the day before until the introduction of PP -LRB- T0 -RRB- , and was then measured every hour up to six hours thereafter -LRB- T1 -- 6 -RRB- . Blood samples of the donors were collected to determine creatinine levels the day before operation , after induction of anesthesia , six hours after installation of PP , two days , one month , and one year after operation . Creatinine clearance -LRB- CrCl -RRB- was determined using the Cockcroft-Gault formula -LSB- 22 -RSB- . Statistical analysis Data analysis was performed using SPSS for Windows -LRB- version 14.0 , SPSS Inc. , Chicago , USA -RRB- . Data are presented as means with standard deviation -LRB- SD -RRB- . Differences between the groups were analyzed using the independent t-test , depending on Levene 's test , pooled or unpooled . Repeated measures with a general linear model from SPSS were used to assess significance for CrCl . A -LRB- two-sided -RRB- p-value < 0.05 was considered statistically significant . Results Baseline characteristics are shown in Table 2 and were comparable between the three groups . After induction of anesthesia , SV was significantly higher in both pre-hydrated groups compared to the control group -LRB- Fig. 1 -RRB- . After repositioning from supine to lateral , SV decreased significantly in the control group but not in groups 1 and 2 -LRB- Fig. 1 -RRB- . After installation of PP , SV remained stable in group 1 but not in groups 2 and 3 -LRB- Fig. 1 -RRB- . Table 2 . Demographic data on the three groups , mean -LRB- SD -RRB- Group 1Group 2Group 3Age -LRB- yrs -RRB- 56 -LRB- 11 -RRB- 53 -LRB- 9 -RRB- 55 -LRB- 12 -RRB- Weight -LRB- kg -RRB- 72 -LRB- 9 -RRB- 82 -LRB- 8 -RRB- 74 -LRB- 7 -RRB- IBW -LRB- kg -RRB- 70 -LRB- 8 -RRB- 78 -LRB- 8 -RRB- 71 -LRB- 6 -RRB- Male/female 2/54/33 / 4Operation time -LRB- min -RRB- 237 -LRB- 37 -RRB- 251 -LRB- 46 -RRB- 226 -LRB- 31 -RRB- IBW = ideal body weightFig . 1 . Stroke volume changes during laparoscopic donor nephrectomy , comparing three different fluid regimens : # symbol p < 0.05 for groups 1 and 2 versus control group 3 ; o symbol p < 0.05 for group 1 versus group 2 ; × symbol p < 0.05 versus supine position . Data are mean ± standard deviation . After induction , LVETc was higher in group 1 compared to the control group during the whole procedure and remained stable -LRB- Table 3 -RRB- . In all groups MAP decreased after induction of anesthesia ; in the control group MAP decreased significantly more compared to group 1 -LRB- p = 0.03 -RRB- . HR was comparable between the three groups -LRB- Table 3 -RRB- . Table 3 . Data on hemodynamic parameters : heart rate -LRB- HR -RRB- , mean arterial pressure -LRB- MAP -RRB- and left ventricular ejection time -LRB- LVETc -RRB- , mean -LRB- SD -RRB- . Columns correspond to seven times : before anesthesia -LRB- preop -RRB- ; after induction of anesthesia , supine position -LRB- supine -RRB- ; full lateral position -LRB- lateral -RRB- ; and mean measurement for the first , second , third and fourth 30 minutes after installation of pneumoperitoneum -LRB- PP30 , PP60 , PP90 and PP120 , respectively -RRB- PreopSupineLateralPP30PP60PP90PP120HR Group 165 -LRB- 6 -RRB- 65 -LRB- 13 -RRB- 63 -LRB- 20 -RRB- 60 -LRB- 9 -RRB- 61 -LRB- 8 -RRB- 60 -LRB- 10 -RRB- 61 -LRB- 9 -RRB- Group 263 -LRB- 11 -RRB- 60 -LRB- 9 -RRB- 61 -LRB- 14 -RRB- 62 -LRB- 11 -RRB- 61 -LRB- 9 -RRB- 63 -LRB- 6 -RRB- 63 -LRB- 8 -RRB- Group 3 -LRB- control -RRB- 73 -LRB- 9 -RRB- 69 -LRB- 14 -RRB- 61 -LRB- 6 -RRB- 65 -LRB- 10 -RRB- 63 -LRB- 6 -RRB- 60 -LRB- 7 -RRB- 61 -LRB- 6 -RRB- MAP Group 1102 -LRB- 11 -RRB- 85 -LRB- 13 -RRB- * # 81 -LRB- 19 -RRB- * 93 -LRB- 14 -RRB- 96 -LRB- 15 -RRB- 94 -LRB- 17 -RRB- 98 -LRB- 20 -RRB- Group 2102 -LRB- 14 -RRB- 73 -LRB- 10 -RRB- * 81 -LRB- 15 -RRB- * 107 -LRB- 10 -RRB- 107 -LRB- 18 -RRB- 104 -LRB- 8 -RRB- 102 -LRB- 12 -RRB- Group 3 -LRB- control -RRB- 105 -LRB- 12 -RRB- 70 -LRB- 11 -RRB- * 79 -LRB- 7 -RRB- * 103 -LRB- 9 -RRB- 98 -LRB- 11 -RRB- 95 -LRB- 12 -RRB- 95 -LRB- 15 -RRB- LVETc Group 1336 -LRB- 19 -RRB- # 339 -LRB- 20 -RRB- # 355 -LRB- 26 -RRB- # 345 -LRB- 31 -RRB- 335 -LRB- 30 -RRB- 336 -LRB- 33 -RRB- Group 2313 -LRB- 29 -RRB- 308 -LRB- 39 -RRB- 295 -LRB- 58 -RRB- ° 311 -LRB- 20 -RRB- ° 308 -LRB- 27 -RRB- 300 -LRB- 28 -RRB- Group 3 -LRB- control -RRB- 294 -LRB- 26 -RRB- 284 -LRB- 31 -RRB- 292 -LRB- 26 -RRB- 307 -LRB- 18 -RRB- 309 -LRB- 18 -RRB- 311 -LRB- 25 -RRB- * p < 0.05 versus preoperative values # p < 0.05 groups 1 and 2 versus control group ° p < 0.05 group 1 versus group 2 Urine output , measured from the start of operation until the moment of kidney extraction , was 1.9 ml/kg/h -LRB- range 1.2 -- 3.2 -RRB- for group 1 , 1.4 ml/kg/h -LRB- range 0.8 -- 2.3 -RRB- for group 2 , and 1.1 ml/kg/h -LRB- range 0.6 -- 1.6 -RRB- for group 3 . In controls , the urine production was significantly lower compared to group 1 -LRB- p = 0.01 -RRB- . CrCl decreased in the control group directly after PP , but not in the other groups -LRB- Table 4 -RRB- . From two days postoperative , CrCl was comparable between the three study groups -LRB- Table 4 -RRB- . Table 4 . Creatinine clearance -LSB- mean -LRB- SD -RRB- -RSB- in the three groups at six measurement points : one day before operation ; just after induction of anesthesia ; at 14:30 , six hours after installation of pneumoperitoneum T6 ; two days after operation ; one month after operation ; and one year after operationGroup 1Group 2Group 3 -LRB- control -RRB- CrCl preop -LRB- ml/min -RRB- 89 -LRB- 19 -RRB- 101 -LRB- 25 -RRB- 102 -LRB- 34 -RRB- CrCl after induction -LRB- ml/min -RRB- 104 -LRB- 18 -RRB- 109 -LRB- 20 -RRB- 96 -LRB- 31 -RRB- CrCl after the operation -LRB- ml/min -RRB- 87 -LRB- 17 -RRB- 94 -LRB- 14 -RRB- 73 -LRB- 23 -RRB- ªCrCl D2 -LRB- ml/min -RRB- 63 -LRB- 10 -RRB- * 65 -LRB- 11 -RRB- * 64 -LRB- 19 -RRB- * CrCl after 1 month -LRB- ml/min -RRB- 63 -LRB- 12 -RRB- * 67 -LRB- 11 -RRB- * 63 -LRB- 24 -RRB- * CrCl after 1 year -LRB- ml/min -RRB- 71 -LRB- 13 -RRB- 71 -LRB- 13 -RRB- * 66 -LRB- 25 -RRB- ** p < 0.05 versus preoperative valuesª p < 0.05 difference between CrCl preop and CrCl after operation , control group versus groups 1 and 2 Discussion This study showed that during LDN preoperative hydration together with a bolus of colloid given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output compared to a fluid regimen with only an intraoperative aggressive fluid infusion . The second group , which received no bolus of colloid before PP in contrast to group 1 , showed a significant reduction in SV after installation of PP . In the control group , LVETc and urine output at the moment of kidney extraction showed significantly lower values compared to both pre-hydration groups . CrCl values six hours after the start of the operation was significantly reduced in the control group compared to preoperative values but not in the two pre-hydrated groups ; this difference was reduced two days postoperatively . Clinical studies yield conflicting data concerning the effect of LDN on recipient graft function compared to ODN . The largest study to date compared more than 5,000 kidney transplants from a database and found that LDN was associated with slower early graft function compared to ODN . However , renal function and graft survival at one year was similar between both groups . This was confirmed by retrospective analysis of 120 LDN and 100 ODN in our own institution in which serum creatinine in the recipients was significantly higher in the LDN group only in the first week after transplantation -LSB- 4 -RSB- . One very important discriminating factor between ODN and LDN is the pneumoperitoneum . From experimental studies it has become clear that PP decreases RBF and that the magnitude of this decrease is affected by the IAP used , the volume status , and positioning . To counterbalance the increased IAP , vigorous intravenous hydration during LDN is nowadays recommended in an attempt to optimize preload and promote diuresis , but randomized clinical data are missing . In a porcine model , Demyttenaere et al. -LSB- 23 -RSB- showed that the decrease in SV and renal cortical perfusion could be prevented by a simple hydration of 15 ml/kg/h saline combined with a bolus 20 ml/kg saline , in accordance with the findings of London et al. -LSB- 11 -RSB- . This was also seen in the present study in which pre-hydration with a normal infusion with crystalloids during operation combined with a bolus of colloids just before PP did not decrease SV but improved diuresis . Besides PP , the kidney lateral decubitus position , which is an anti-Trendelenburg position , contributes to hemodynamic alterations by decreasing preload through the effect of gravity on venous return -LSB- 24 -RSB- . Yokoyama et al. -LSB- 25 -RSB- found no significant change in hemodynamic values after postural change of their patients from supine to lateral but a significant reduction in SV after postural change to kidney position ; these patients received a fluid regime of 20 ml/kg/h of crystalloids . This was confirmed by our study in which the control group showed a significant reduction in SV after postural change from supine to kidney position whereas there was no reduction in the two pre-hydrated groups , which received a bolus of colloid just before induction -LRB- Fig. 1 -RRB- . After pre-hydration with crystalloids we infused colloids to achieve optimal plasma expansion just before installation of PP -LSB- 26 -RSB- . In our hospital we use 6 % HES 130/0 .4 for fluid expansion , because the rate for anaphylactic reactions is considerably lower than for gelatin products -LSB- 27 -RSB- . However , there are concerns that infusion of certain HES types may influence kidney function -LSB- 28 -RSB- . As long as adequate hydration using sufficient amounts of crystalloids are used , the latest generation of HES products -LRB- 6 % HES 130/0 .4 -RRB- do not increase the risk for renal dysfunction even when used in large amounts -LSB- 29 , 30 -RSB- . Lang et al. -LSB- 31 -RSB- even demonstrated that 6 % HES 130/0 .4 improved tissue oxygenation during and after major surgical procedures compared with a crystalloid-based volume strategy . In this study , we used the HemoSonicTM , a transoesophageal Doppler ultrasonography -LRB- TOD -RRB- device , to measure blood flow in the descending aorta . Several studies have confirmed good correlation with cardiac output measured by the thermodilution technique -LSB- 18 , 32 -RSB- . It has been shown that the accuracy of the device is somewhat operator-dependent -LSB- 20 -RSB- and therefore the same two people did all the measurements with this device in the present study . Feldman et al. -LSB- 33 -RSB- used LVET to guide their fluid management in LDN patients . In the present study it was shown that LVETc was significantly lower in the control group that did not received pre-hydration and increased over time -LRB- Table 2 -RRB- . It should , however , be taken into account that the blood flow with this device is measured in the descending aorta , which is around 70 % of the total cardiac output . This could influence our measurements if redistribution of flow away from the descending aorta occurs because of elevated IAP and this is more pronounced in hypovolemic patients . Some other limitations of this study should be noted . In only four patients a MAG3 scan was performed , which provides the distribution of the function from the two kidneys of the donor . In the four measured patients the harvested kidney contributed 43 -- 48 % of the total kidney function , these four patients were divided over all three study groups . However because we do not have the data on the other patients , this could have biased our data on postoperative CrCl . Prehydration of the donor patients conform our protocol , started the night before operation , which contradicts fast-track surgery where kidney donor patients are admitted to hospital on the day of surgery . Also these patients can receive adequate pre-hydration , but further research should be done . In this study we focused on intraoperative hemodynamic changes . Our data show that preoperative hydration together with a colloid bolus given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output during LDN , compared to controls that received only an aggressive intraoperative infusion . While under-hydration may contribute to renal dysfunction , perioperative fluid excess can also cause problems , such as pulmonary edema , ileus and increased risk of cardiopulmonary and wound healing complications , which might result in longer hospital stay -LSB- 34 -RSB- . However , there is a need to ensure adequate hydration status during PP without being overaggressive . First , our fluid regime will be tested in a large prospective study in order to prevent the negative effect of PP on early graft function in the recipient , and to study possible side-effects in the donor .

O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

renal (kidneys)
anaesthesia
urbanization


Position paper on the use of mandibular advancement devices in adults with sleep-related breathing disorders Custom-made mandibular advancement devices are an effective treatment option for snoring , upper airway resistance syndrome , and obstructive sleep apnea -LRB- OSA -RRB- . Evidence-based data indicates their efficacy , and international sleep societies recommend oral appliance -LRB- OA -RRB- therapy for patients with sleep-related breathing disorders . The following position paper by the German Society of Dental Sleep Medicine -LRB- DGZS -RRB- is to guide the interdisciplinary team -LRB- sleep physician and sleep disorder dentist -RRB- in detail when to prescribe oral appliances . This position paper supports the responsible use of OA as an effective treatment option for patients with sleep-related breathing disorders . The paper advises of proper indication regarding OSA severity , body mass index -LRB- BMI -RRB- , and dentition . It emphasizes the interdisciplinary approach of oral appliance therapy and suggests treatment under the guidance of dentists trained in dental sleep medicine . Introduction Various options are available in the treatment of obstructive sleep-related breathing disorders -LRB- SRBD -RRB- : Continuous or bi-level positive airway pressure -LRB- nCPAP/biPAP -RRB- Mandibular advancement devices -LRB- MADs -RRB- Surgical proceduresOther procedures The use of oral appliances in treating SRBD was first described in 1982 -LSB- 3 -RSB- . The first findings , which document the therapeutic effect of MADs , were presented in 1984 -LSB- 8 -RSB- . Of the many oral appliances designed for use in the treatment of SRBD , mandibular advancement devices have been the most intensely researched . Progress in the areas of dentistry and appliance technology has lead to fundamental modifications in the design of mandibular advancement devices . The clinical effectiveness of this treatment option has been documented with varying degrees of evidence in numerous studies . The clinical importance of this form of therapy is constantly being reinforced by the gathering of improved data on the validity of this form of treatment . The effectiveness of mandibular advancement devices in the treatment of SRBD has been documented by the Cochrane Collaboration as the highest level of scientific evidence -LSB- 5 -RSB- . The clinical analysis has been reviewed and revised twice since Cochrane 's first evaluation in 2002 -LSB- 6 , 7 -RSB- . The German Society of Dental Sleep Medicine -LRB- DGZS -RRB- takes this opportunity to issue its position with regard to this form of therapy and to make recommendations . The definition of mandibular advancement devices in the treatment of SRBD A system of laboratory fabricated custom-made adjustable splints manufactured for the individual patient and adjusted to fit the upper and lower jaws . These splints position the lower jaw , tongue , and additional structures more anteriorly , and increase the occlusal vertical dimension , resulting in an opening of the pharynx lumen , which reduces resistance in the airway and mechanically keeps the airway open during sleep . Guidelines and indication recommendations from professional sleep-medicine societies As early as 1995 , the American Academy of Sleep Medicine -LRB- AASM -RRB- published recommendations on the indication and application of oral appliances in the treatment of SRBD -LSB- 1 -RSB- . These were updated and stated more precisely in 2005 -LSB- 2 -RSB- . In 2001 , the German Society of Pneumology -LRB- DGP -RRB- in co-operation with the German Sleep Society -LRB- DGSM -RRB- published the `` S2 '' guidelines on the diagnosis and treatment of SRBD . These guidelines envisaged the use of mandibular advancement devices as a treatment option -LSB- 4 -RSB- . A new description of the significance of mandibular advancement devices was formulated by the `` Task Force Apnea of the DGSM '' in the year 2006 -LSB- 9 -RSB- . Based on scientific appraisals and international guidelines , the DGZS recommends the use of mandibular advancement devices in the treatment of SRBD : As an initial treatment for Primary snoringUpper airway resistance syndromeMild to moderate obstructive sleep apnea -LSB- apnea hypopnea index -LRB- AHI -RRB- up to about 25/h -RSB- with minor clinical symptoms with a sufficient number of retaining teeth and a body mass index -LRB- BMI -RRB- of up to 30 kg/m2In cases of SRBD with an approximate AHI of over 25/h , MADs can be used as an alternative after a previous therapeutic trial with nCPAP.Regardless of SRBD severity in patients with CPAP failure and CPAP noncompliance.A special dental , oral , and functional diagnosis is necessary to check the dental requirements for long-term use before prescribing MADs.The procedure should take into account the various interdisciplinary and intradisciplinary treatment flows , particularly the preliminary medical diagnosis and the subsequent medical follow-ups . This service should be delivered by dentists who have specialized training in the field of sleep medicine . The DGZS supports the specialized training of dentists and physicians in the field of dental sleep medicine . Patients and referring physicians can find the addresses of certified practitioners on the DGZS website at http://www.dgzs.de .

B I O O O O O O O O O O O O O O O O O O O O O O O B O B I I I O O B I I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O B I I O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I I O O O O O O O O O O O O O O B O O O O

position paper
oral appliance
dental sleep medicine
obstructive sleep apnea
upper airway resistance syndrome
snoring
dgzs

mandibular advancement device
german society of dental sleep medicine


TNM staging of foregut -LRB- neuro -RRB- endocrine tumors : a consensus proposal including a grading system The need for standards in the management of patients with endocrine tumors of the digestive system prompted the European Neuroendocrine Tumor Society -LRB- ENETS -RRB- to organize a first Consensus Conference , which was held in Frascati -LRB- Rome -RRB- and was based on the recently published ENETS guidelines on the diagnosis and treatment of digestive neuroendocrine tumors -LRB- NET -RRB- . Here , we report the tumor -- node -- metastasis proposal for foregut NETs of the stomach , duodenum , and pancreas that was designed , discussed , and consensually approved at this conference . In addition , we report the proposal for a working formulation for the grading of digestive NETs based on mitotic count and Ki-67 index . This proposal , which needs to be validated , is meant to help clinicians in the stratification , treatment , and follow-up of patients . Background It has been known for a long time , and was finally defined within the World Health Organization -LRB- WHO -RRB- classification of endocrine and digestive tumors , that neuroendocrine tumors -LRB- NET -RRB- arising at different anatomical sites of the digestive system represent tumor entities that differ in their biology -LSB- 5 , 7 , 9 , 31 -RSB- . Several recent publications focused on the application of the `` new '' WHO classification and proved its effectiveness , supporting the concept that the different endocrine tumor types also differ in their clinical behavior -LSB- 3 , 4 , 19 , 20 , 27 , 32 -RSB- . Malignant gastroenteropancreatic NETs may be fatal , though at a significantly slower pace than their exocrine counterparts . A number of retrospective papers and epidemiological data solidly support such statements -LSB- 8 , 9 , 12 -- 14 , 21 , 24 , 25 , 32 -RSB- . This peculiar clinical feature attracted the interest of pathologists very early and was the reason for the special designation of such tumors as `` carcinoid '' by Oberndorfer -LSB- 15 -RSB- . As gastroenteropancreatic NETs are rare -LSB- 9 , 13 , 14 -RSB- , it is tempting to lump them together and equate all digestive `` carcinoids '' with the appendiceal `` carcinoid , '' probably the best known NET with the most benign behavior -LSB- 28 -RSB- . However , in recent years it has become clear that gastroenteropancreatic NETs , especially foregut NETs , are heterogeneous in their morphological and biological features . In the last two decades efforts were therefore made by the WHO to define NET features that discriminate true benign behavior -LRB- low risk -RRB- from low-grade malignant well-differentiated NETs in the different parts of the digestive system . Although the new WHO classification is an important step toward defining the diverse tumor biology of NETs , further efforts are necessary to improve the prognostic assessment of the individual NET . The demand for standards in the stratification and treatment of patients with gastroenteropancreatic NETs prompted the recently established European Neuroendocrine Tumor Society -LRB- ENETS -RRB- to define guidelines -LSB- 22 , 33 -RSB- . Such guidelines underwent scrutiny for consensus in the first of two meetings entitled `` Consensus Conference on the ENETS Guidelines for the Diagnosis and Treatment of Neuroendocrine Gastrointestinal Tumors , Part 1 : Foregut Tumors '' held in Frascati -LRB- Rome , Italy -RRB- from November 2 -- 5 , 2005 . During this meeting the clinical need for a tumor -- node -- metastasis -LRB- TNM -RRB- classification of gastroenteropancreatic NETs was felt . Here we report the TNM staging classification proposal for foregut NETs that was approved at this consensus conference . In addition , we suggest a simple grading system with some pointers that may help to standardize the prognostic assessment of gastroenteropancreatic NETs . Materials and methods Sixty-two experts in the field of digestive endocrine tumors from 20 different countries attended the Consensus Conference . The attendees represented all medical branches involved in managing patients with gastroenteropancreatic NETs . They formed four working groups according to their specific clinical expertise : -LRB- 1 -RRB- pathology and genetics -LRB- 11 participants , all listed as authors and G. Klöppel -RRB- , -LRB- 2 -RRB- surgery -LRB- 10 participants , including the coauthors H. Alhman and M. Falconi -RRB- , -LRB- 3 -RRB- imaging and radiology -LRB- 10 participants -RRB- , -LRB- 4 -RRB- medicine and clinical pathology -LRB- 31 participants , including the coauthors M. Caplin , W.W. de Herder , B. Erikssson , and B. Wiedenmann -RRB- . The Conference was divided sequentially into eight sessions devoted to specific topics on an anatomical basis -LRB- gastric NET sessions 1 -- 2 , duodenal NET , pancreatic NET sessions 1 -- 4 , and poorly differentiated endocrine carcinomas -RRB- . A working booklet with the ENETS guidelines and specific queries had been prepared in advance by the Organizing Committee . The work was organized such that , after a short case presentation in a plenary session , each working group gathered separately to discuss group-specific questions . Once agreement was reached within each group , consensus statements were discussed and approved or rejected by all participants gathered in the plenary session . This procedure was followed for all eight sessions . The TNM staging proposal was made by the Pathology and Genetics working group and amended and approved by the plenary session of the consensus conference . The grading system was discussed and defined by the Pathology and Genetics working group only . Results and discussion The consensus guidelines are reported elsewhere . Here , we report the TNM staging proposal for gastroenteropancreatic NETs of the foregut together with a grading system that may be relevant for the prognostic assessment by the pathologist . The foregut NETs were separated into gastric , duodenal -LRB- including ampulla and proximal jejunum -RRB- , and pancreatic NETs , but were not distinguished according to specific functional activity , main tumor cell type , and specific genetic background . TNM staging proposal -LRB- see Tables 1 , 2 and 3 -RRB- The currently published TNM format was adopted as working template -LSB- 29 -RSB- . Tumor The proposed definition of tumor in situ applies to the stomach only and adheres to the literature -LSB- 30 -RSB- . No definition is given for the duodenum and pancreas because none has been agreed upon in spite of recent working proposals -LSB- 1 , 2 -RSB- . The size limits indicated for T1 are those defined by the WHO for tumors with `` benign behavior '' according to site-specific clinicopathological correlations -LSB- 5 , 7 , 31 -RSB- . Similarly , for T2 of the stomach and duodenum , the sizes are those indicated for tumors of `` uncertain behavior . '' In the pancreas the size limit given for T2 needs to be validated -LSB- 5 -RSB- . Deeply invasive tumors are included under the T3 and T4 definitions , taking into account site-specific features . Nodes N1 indicates the presence of any single or multiple metastases in regional lymph nodes , according to TNM rules . Although the presence of regional lymph-node metastases is , per se , a negative prognostic factor in gastroenteropancreatic NETs -LSB- 11 -RSB- , the prognostic significance of the number of metastatic nodes is not known . In light of this , stage 3B of Tables 1 , 2 and 3 is proposed to mark the N1 status for future validation . Table 1Proposal for a TNM classification and disease staging for gastric endocrine tumorsTNMT -- primary tumor TXPrimary tumor can not be assessed T0No evidence of primary tumor TisIn situ tumor/dysplasia -LRB- < 0.5 mm -RRB- T1Tumor invades lamina propria or submucosa and ≤ 1 cm T2Tumor invades muscularis propria or subserosa or > 1 cm T3Tumor penetrates serosa T4Tumor invades adjacent structuresFor any T , add -LRB- m -RRB- for multiple tumorsN -- regional lymph nodes NXRegional lymph nodes can not be assessed N0No regional lymph node metastasis N1Regional lymph node metastasisM -- distant metastasis MXDistant metastasis can not be assessed M0No distant metastases M1aDistant metastasisStage Disease stages Stage 0TisN0M0 Stage IT1N0M0 Stage IIaT2N0M0 IIbT3N0M0 Stage IIIaT4N0M0 IIIbAny TN1M0 Stage IVAny TAny NM1aM1 specific sites defined according to Sobin and Wittekind -LSB- 29 -RSB- Table 2Proposal for a TNM classification and disease staging for endocrine tumors of the duodenum/ampulla/proximal jejunumTNMT -- primary tumor TXPrimary tumor can not be assessed T0No evidence of primary tumor T1Tumor invades lamina propria or submucosa and size ≤ 1 cma T2Tumor invades muscularis propria or size > 1 cm T3Tumor invades pancreas or retroperitoneum T4Tumor invades peritoneum or other organsFor any T , add -LRB- m -RRB- for multiple tumorsN -- regional lymph nodes NXRegional lymph nodes can not be assessed N0No regional lymph node metastasis N1Regional lymph node metastasisM -- distant metastases MXDistant metastasis can not be assessed M0No distant metastases M1bDistant metastasisStage Disease stages Stage IT1N0M0 Stage IIaT2N0M0 IIbT3N0M0 Stage IIIaT4N0M0 IIIbAny TN1M0 Stage IVAny TAny NM1aTumor limited to ampulla of Vater for ampullary gangliocytic paragangliomabM1 specific sites defined according to Sobin and Wittekind -LSB- 29 -RSB- Table 3Proposal for a TNM classification and disease staging for endocrine tumors of the pancreasTNMT -- primary tumor TXPrimary tumor can not be assessed T0No evidence of primary tumor T1Tumor limited to the pancreas and size < 2 cm T2Tumor limited to the pancreas and size 2 -- 4 cm T3Tumor limited to the pancreas and size > 4 cm or invading duodenum or bile duct T4Tumor invading adjacent organs -LRB- stomach , spleen , colon , adrenal gland -RRB- or the wall of large vessels -LRB- celiac axis or superior mesenteric artery -RRB- For any T , add -LRB- m -RRB- for multiple tumorsN -- regional lymph nodes NXRegional lymph node can not be assessed N0No regional lymph node metastasis N1Regional lymph node metastasisM -- distant metastases MXDistant metastasis can not be assessed M0No distant metastases M1aDistant metastasisStage Disease stages Stage IT1N0M0 Stage IIaT2N0M0 IIbT3N0M0 Stage IIIaT4N0M0 IIIbAny TN1M0 Stage IVAny TAny NM1aM1 specific sites defined according to Sobin and Wittekind -LSB- 29 -RSB- Distant metastasis M1 indicates the presence of any single or multiple metastases at any distant anatomical site -LRB- including nonregional nodes -RRB- . Because there is evidence that extrahepatic bone metastases are a particularly ominous sign -LSB- 6 , 20 -RSB- , it is recommended to specify the anatomical site of the metastasis according to the TNM classification rules -LRB- PUL , pulmonary ; HEP , hepatic ; OSS , osseous ; etc. -RRB- -LSB- 29 -RSB- . Staging The proposed staging system lists stage 0 only for the stomach because this is the only anatomical site where Tis is defined . Stage I encompasses the T1 NETs with limited growth . Stage II identifies tumors that are larger in size or more invasive , either T2 or T3 , though always in the absence of metastasis . At stage III the increased malignancy refers either to invasion into surrounding structures -LRB- stage IIIa -RRB- or to the presence of regional node metastasis -LRB- stage IIIb -RRB- . Stage IV always implies the presence of distant metastasis . Grading proposal -LRB- see Table 4 -RRB- Grading It has been widely discussed and generally accepted that no histological grading system effectively predicts the behavior of well-differentiated endocrine tumors . The major obstacle to developing a practically effective grading system is the fact that severe cytological atypia , as , for instance , in pheochromocytomas , has no impact on the clinical behavior and malignancy of such tumors . However , recent studies in well differentiated NETs of the foregut , including the pancreas , and of the midgut have shown the usefulness of a grading system -LSB- 10 , 25 , 32 -RSB- . Thus , well-differentiated endocrine tumors with a more solid appearance and distinct proliferative activity , which also lead to difficulties in the differential diagnosis vs poorly differentiated endocrine carcinomas , seem to have a worse prognosis than NETs without these features -LSB- 16 -- 18 , 22 , 23 -RSB- . It was therefore decided to introduce a grading system that could be of help in distinguishing the well-differentiated NETs into G1 and G2 categories . As a working suggestion , we propose to apply to foregut NETs a grading system modified from that adopted by the WHO for endocrine tumors of the lung , though exclusively referring to the proliferation status . In brief -LRB- see Table 4 -RRB- , three tumor categories are identified : G1 , < 2 mitosis per 2 mm2 -LSB- 10 high power fields -LRB- HPF -RRB- 40 × magnification -RSB- and/or Ki-67 index ≤ 2 % ; G2 , 2 -- 20 mitosis per 2 mm2 and/or Ki-67 index between 3 and 20 % ; G3 with 21 or more mitosis per 2 mm2 and Ki-67 index > 20 % . Table 4Grading proposal for foregut -LRB- neuro -RRB- endocrine tumorsGradeMitotic count -LRB- 10 HPF -RRB- aKi-67 index -LRB- % -RRB- bG1 < 2 ≤ 2G22 -- 203 -- 20G3 > 20 > 20a10 HPF : high power field = 2 mm2 , at least 40 fields -LRB- at 40 × magnification -RRB- evaluated in areas of highest mitotic densitybMIB1 antibody ; % of 2,000 tumor cells in areas of highest nuclear labeling In general , G1 and G2 should refer to well-differentiated NETs displaying diffuse and intense expression of the two general immunohistochemical neuroendocrine markers , chromogranin A and synaptophysin -LSB- 26 -RSB- . Punctate necrosis is , per se , indicative of a more aggressive tumor , pointing to a G2 status , which , however , has to be confirmed by the mitotic count . G3 indicates a poorly differentiated neuroendocrine carcinoma . It has high mitotic counts/Ki -67 index , is often associated with fields of necrosis , and shows significantly reduced chromogranin A expression , while maintaining intense staining for synaptophysin . It is relevant to remind here that the diagnosis of G3 carcinoma is based on a specific histologic pattern according to the current WHO criteria -LSB- 5 , 7 , 31 -RSB- . In addition , the clinical behavior of G3 poorly differentiated neuroendocrine carcinomas of the gastroenteropancreatic tract does not necessarily correspond to that of small cell cancers of the lung or of any other sites . Mitotic count and Ki-67 index We propose that mitoses should be counted on hematoxylin and eosin-stained slides in at least 40 HPFs , where possible . The mitoses should be assessed in areas where they are most frequent after a general slide survey . For Ki-67 assessment , the MIB1 antibody is recommended at the conditions that have been established at the laboratory in question . The Ki-67 index should be assessed in 2,000 tumor cells in areas where the highest nuclear labeling is observed -LRB- often but not exclusively at the tumor periphery -RRB- . Concluding remarks Requests for standardization in the management of patients with gastroenteropancreatic NETs recently resulted in the development of several guidelines , including those proposed by ENETS -LSB- 16 , 17 , 22 , 23 -RSB- . However , it was never attempted to reach consensus on specific practical issues . The TNM staging system we propose here was developed to meet a clinical need , is based on the current WHO classifications of endocrine and digestive tumors , and is the result of a consensus conference held by specialists involved in the management of digestive endocrine tumor patients . Along the same line , the grading system described here attempts to close the gap between the advances of the most recent WHO classifications and the need for a better prognostic assessment of NETs . It is obvious , of course , that all our proposals have to be validated by future clinicopathological work .

B B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

neuroendocrine tumors
pancreas
staging
tnm
grading