Endometrioid adenocarcinoma of the uterus and ovarian endometrioid carcinoma share many morphological and molecular features. Differentiation between simultaneous primary carcinomas and ovarian metastases of an endometrial cancer may be very challenging but is essential for prognostic and therapeutic considerations.In the present case study of a 33 year-old patient we used targeted amplicon next-generation re-sequencing for clarifying the origin of synchronous endometrioid cancer of the corpus uteri and the left ovary. The patient developed a metachronous lung metastasis of an endometrioid adenocarcinoma four years after hyster- and adnexectomy, vaginal brachytherapy and treatment with the synthetic steroid tibolone. Removal of the metastasis and megestrol treatment for seven years led to a complete remission.ARID1A, CTNNB1, PIK3CA and PTEN were identified and confirmed by Sanger sequencing. Primary endometrial as well as ovarian cancer showed an identical mutational profile, suggesting the presence of an ovarian metastasis of the endometrial cancer, rather than a simultaneous endometrial and ovarian cancer. The metachronous lung metastasis showed a different mutational profile compared to the primary cancer. Immunohistochemical staining of the corresponding proteins suggested that the tumour development was driven by alterations in the protein function rather than by changes of the protein abundance in the cell.A total of 409 genes from the Ampliseq Comprehensive Cancer Panel  were analysed by next generation sequencing and mutations in 10 genes, including Our results have demonstrated next generation sequencing as a valuable tool in the differentiation of synchronous primary tumours and metastases, which has an important impact on the clinical decision making process. Similar to breast cancer, targeted therapies based on mutational tumour profiling will become increasingly important in endometrial and ovarian cancer. In summary, our results support the usage of next generation sequencing as a supplementary diagnostic tool, assisting in personalized precision medicine.The online version of this article (doi:10.1186/s12885-017-3054-6) contains supplementary material, which is available to authorized users. Endometrioid adenocarcinoma of the uterus and ovarian endometrioid carcinoma share many morphological and molecular features , 2. CoinA novel powerful tool to identify common genetic alterations specific for a cancer entity and to provide information on the tumour clonal evolution is next-generation sequencing (NGS) . To dateIn the present case report we investigated the mutation pattern in a 33-year-old female patient diagnosed with synchronous endometrioid adenocarcinoma of the corpus uteri and the left ovary. The patient developed a lung metastasis of an endometrioid type adenocarcinoma four years after laparoscopic hysterectomy, vaginal brachytherapy and tibolon therapy. Based on histological findings, it is difficult to decide whether the gynaecological carcinomas are two independent primary carcinomas occurring in parallel. Furthermore, it is almost impossible to recognize the origin of the lung mass. Thus, we employed targeted NGS of 409 tumour-related genes  in order to identify the relation between the two carcinomas and the source of the distant metastasis. Moreover, we were interested in the genetic fingerprint of the metastasis to clarify the recurrence-free survival of the patient after its removal and megestrol treatment for seven years. Finally, we looked for possible germline mutations from blood samples of the patient that could explain the early onset of the disease.A 33-year-old woman, who has undergone a diagnostic curettage due to hypermenorrhoea and menorrhagia, was diagnosed with endometrial endometrioid adenocarcinoma of moderate differentiation and with squamous components. Subsequently, a laparoscopic hysterectomy with pelvic washing, bilateral adnexectomy and pelvic lymphadenectomy was performed. In the hysterectomy, well-differentiated residual cancer was found with infiltration less than one half of the myometrium, consistent with stage pT1a.n = 20) were free of tumour. In summary, the former pathological TNM classification for the corpus uteri was pT1a pN0 (0/20) G2 (FIGO stage IA) and for the ovary pT1a pN0 G1 (FIGO stage IA). Vaginal brachytherapy of 4x5 Gy was administered because of a G2 tumour and the quite young age of the patient. After treatment with tibolon (2.5 mg/day) for 4 years, the patient presented with chest pain in the right upper thorax. Through CT-scan and the following thoracoscopic resection of the right upper lobe of the lung, a metastatic endometrioid adenocarcinoma was removed. Treatment with megestrol (160 mg/day) was started. In addition to the clinical check-up, a CT-scan was performed each year. The latest PET–CT scan after 7 years being treated with megestrol acetate showed no evidence of tumour recurrence. Informed consent was obtained and the study has been approved by the local ethics committee (KEK-ZH-No. 2010–0358).The tumour tissue showed high expression of oestrogen and progesterone receptors , progesterone receptor , mismatch repair proteins as MSH2 , MSH6 (BD Biosciences 1:500), MLH1 , PMS2 PharMingen BD 1:300), PTEN , beta-Catenin , ARID1A/BAF250a , and γH2AX .After antigen retrieval, the slides were incubated with the primary antibodies. After incubation for 1 h at room temperature, the staining of ER, PR, beta-Catenin, and γH2AX was further conducted with the Ventana Benchmark automated system  using Ventana reagents such as UltraView HRP , UltraMap™ DAB detection kit for γH2AX, and Optiview (beta-Catenin). The antibodies against the mismatch repair proteins and ARID1A were incubated for 30 min and the staining procedure was carried out with the automated Leica BOND system using the Bond Polymer Refine Detection Kit (Leica Biosystems).Histological slides from FFPE tissue specimens were reviewed for tumour content and the tumour area was marked by a trained gynaecopathologist for tissue punching Fig. . DNA froTargeted NGS of 409 cancer related genes was performed with 40 ng DNA using the Ion AmpliSeq Comprehensive Cancer Panel (Thermo Fisher Scientific) according to the manufacturer’s protocol. Alignment, variant calling and filtering were performed with Ion Reporter 4.4 (Thermo Fisher Scientific). The following filter chain was used: “Location in utr_3, splicesite_3, exonic, splicesite_5, utr_5” in, “variant effect in stoploss, nonsense, missense, frameshiftInsertion notframeshiftInsertion, nonframeshiftBlockSubstitution, frameshiftDeletion, nonframeshiftDeletion, frameshiftBlockSubstitution” in, “USCS Common SNPs” not in, allele ratio between 0.05 and 1.0 in, allele read-count between 100 and 100 000 in, variant type “INDEL, LONGDEL, SNV, MNV” in. Variants selected after visual inspection of the reads in IGV that displayed more than 15% variant frequency were verified with Sanger sequencing  ovarian cancer as well as the lung metastasis revealed the same tumour morphology, showing an endometrioid adenocarcinoma with low grade features and squamous components Fig. . SquamouIn summary, the endometrial and ovarian neoplasms showed the very same histomorphology and differentiation, and no precursor lesions were found, neither in the ovary, nor in the uterus. The primary site of cancer origin, however, could not be clarified with this observation. To further address this key question we first compared the size of the lesions. Since the endometrial carcinoma was larger than the ovarian tumour, the uterus is in favour of the primary origin. However, a common or multifocal origin can also be hypothesized.To gain insight into the tumour biology we used targeted deep re-sequencing. DNA was extracted from the tumour area and the correct excision of the FFPE punches was confirmed with HE staining  and PTPRT , and one-base indels in RAF1 (p.Met350fs) and ARID1A (p.Arg1053fs) were shared by the endometrial and ovarian carcinomas but not found in the lung metastasis. On the other hand, the lung metastasis carried mutations in ESR1 (p.Tyr537Cys) and PTEN (p.Asp92Tyr) that were not detected in the endometrial and ovarian carcinomas. USP9X (p.Met1478Leu) was mutated only in the ovarian adenocarcinoma. In contrast, PIK3CG (p.Ser931Cys) was altered only in the endometrial adenocarcinoma.Interestingly, SNVs in The general distribution of the detected mutations showed that most of them (8/12) are shared by the endometrial and ovarian tumour. Only a single private mutation in the ovarian and endometrial lesion was found Fig. . Three oThe immunohistochemical profile was consistent in all three cancer manifestations. The hormone receptor expression was strong in almost 100% of tumour nuclei. The expression of four mismatch repair proteins  was visible in normal and tumour tissue, suggestive of microsatellite stable (MSS) carcinomas Fig. . ExpressIn the present study, we investigated the molecular profile of a simultaneous low-grade endometrioid endometrial and ovarian carcinoma as well as of a lung metastasis in a young female patient. From the histology alone, the primary origin is sometimes difficult to determine. The identification of precursor lesions, the pattern of spread and tumour size are valuable parameters for this differentiation. Immunohistochemical analysis of hormone receptors, PTEN and beta-Catenin may show overlapping staining patterns and is therefore not always helpful. The simultaneous occurrence of an endometrial and ovarian carcinoma in women below 50 years of age is a common combination , howeverARID1A, CTNNB1, PIK3CA, and PTEN which are frequently affected in these tumour entities [In our molecular analysis, we detected and verified mutations in genes such as entities , 8. HoweWe further showed that some mutations were lost during the metastasis formation, either due to the treatment or because of redundancy with other alterations affecting the same pathway. Last but not least, we identified enrichment of mutations causing upregulation of PI3K signalling that could explain the prolonged complete remission under megestrol acetate.PTEN is more frequently mutated in low-grade endometrial endometrioid carcinomas (67%) compared to low-grade ovarian endometrioid carcinomas (17%). In contrast, CTNNB1 mutations are significantly different in low-grade ovarian endometrioid carcinomas (53%) compared to 28% of the low-grade endometrial endometrioid carcinomas [Somatic mutations were identified with targeted PCR-based NGS and verified with Sanger sequencing. The fact that 67% (8/12) of the detected alterations were shared by the ovarian and endometrial tumour with similar variant frequencies suggests a common origin of the two lesions. rcinomas . In the PTEN gene showed no inconsistencies. Therefore, larger deletions on chromosome 10 were unlikely. In addition, germline PTEN mutations could be ruled out.The early onset of the malignant disease in this young woman at age 33 suggests possible germline mutations that accelerated the accumulation of genetic aberrations promoting cancer development. A common genetic defect that leads to the development of endometrial cancer in the context of Lynch syndrome is aberrations in the mismatch repair (MMR) proteins. Interestingly, neither mutations in the MMR genes nor increased double strand breaks could be detected. Another possible cause of the early disease onset is Cowden’s syndrome. Targeted PCR-based re-sequencing, however, is not the ideal method to detect large gene deletions. The uniformity blot (sequencing depth across amplicons) for the ARID1A causes a frame shift at codon 1053 of the protein. Since the percentage of the mutation suggested a heterozygous alteration, the sustained immunohistochemical staining is probably due to wild-type ARID1A expressed in the tumour samples. However, our study does not provide information on the expression status of the mutated protein or possible dominant negative interference with wild-type ARID1A. The mutation was not present in the metastasis or present at a low percentage from a marginal subclone indicating that this mutation was not substantially contributing to the tumour spread.The one base insertion detected in gene CTNNB1 and PIK3CA were detected not only in the endometrial and ovarian tumours, but also in the lung metastasis. Both mutations are reported in COSMIC, providing experimental evidence that both mutations affect the function of the each protein and are pathogenic. The amino acid change p.Thr41Ile caused by the SNV c.122C > T in CTNNB1 increases protein stability of beta-catenin and leads to its relocalisation to the nucleus [PIK3CA results in a mutant PI3K displaying increased substrate phosphorylation [In contrast, the somatic mutations in  nucleus . The SNVrylation . Hyperacrylation .PTEN SNV c.370 T > A results in replacement of the cysteine in the active site against serine (p.C124S) leading to an enzymatically inactive protein. Although, as shown by immunohistochemistry, the protein level was not affected and the mutation was likely to be heterozygous, it has been recently reported that this enzymatically dead mutant inhibits wild-type PTEN in a dominant negative manner [PTEN mutation (c.376G > A) that leads to a threonine replacing the alanine at the adjacent position 126 of the protein, likely to distort the tertiary structure of the active site. None of the PTEN mutations could be detected in the lung metastasis. Instead, another PTEN mutation (p.Asp92Tyr) was detected in the lung metastasis that is also annotated in COSMIC. A possible explanation for the loss of the dominant negative PTEN mutation is the biological redundancy of the PTEN inactivating and PIK3CA activating mutations that both lead to an elevated signalling through the PI3K pathway. Indeed, there is evidence that single clones from the primary tumour are able to colonise distant organs and that the metastases carry rather less mutations than the primary tumour due to natural selection process that sustains only the aberrations indispensable for tumour cell survival and proliferation [Another key protein that is a part of the same signalling cascade and was affected by gene mutations in the described case is the tumour suppressor PTEN. The e manner . Additioerine p.C4S leadinferation , which mPTEN (p.Cys124Ser and p.Ala126Thr) and ARID1A (p.Arg1053fs) which were found in both, the endometrial and the ovarian endometrioid carcinoma, but were not conserved in the distant lung metastasis. Co-occurrence of mutations in ARID1A and PTEN have been shown as potential cancer driving mutations in endometrioid ovarian carcinomas in animal models [PTEN nor the ARID1A mutation has given rise to the distant metastasis. To our knowledge this is the first case demonstrating the lack of an ARID1A mutation in a distant metastasis of an ARID1A mutated primary tumour. This demonstrates that even very frequent and potential cancer driving mutations such as PTEN and ARID1A in endometrioid endometrial and ovarian cancer may not be found in distant metastases, a fact that is essential for potential future targeted therapies [It has been shown very recently by two independent groups that a majority of synchronous endometrial and ovarian endometrioid carcinomas show clear evidence of clonality , 15. Thel models . Interesherapies –19. HoweIn conclusion, our results demonstrate that next generation sequencing is an important tool in the differentiation of simultaneous primary tumours and metastases, which has an important impact for clinical practice. This may not only be valuable information for prognostic considerations, but also may be of increasing importance for future targeted therapies.


Microbial communities in human-impacted soils of ancient settlements have been proposed to be used as ecofacts (bioindicators) of different ancient anthropogenic activities. In this study, bacterial, archaeal and fungal communities inhabiting soil of three archaic layers, excavated at the archaeological site on Monte Iato  and believed to have been created in a chronological order in archaic times in the context of periodic cultic feasts, were investigated in terms of (i) abundance (phospholipid fatty acid (PLFA) analysis and quantitative PCR)), (ii) carbon(C)-source consumption patterns (Biolog-Ecoplates) and (iii) diversity and community composition (Illumina amplicon sequencing). PLFA analyses demonstrated the existence of living bacteria and fungi in the soil samples of all three layers. The upper layer showed increased levels of organic C, which were not concomitant with an increment in the microbial abundance. In taxonomic terms, the results indicated that bacterial, archaeal and fungal communities were highly diverse, although differences in richness or diversity among the three layers were not detected for any of the communities. However, significantly different microbial C-source utilization patterns and structures of bacterial, archaeal and fungal communities in the three layers confirmed that changing features of soil microbial communities reflect different past human activities. In this way, the few existing works studying the microbial communities associated with archaeological samples such as dental calculi2, bones3, different kinds of organic remains4, anthropogenically-impacted soils8 and mud9 have confirmed the efficacy of this new direction of analysis in archaeology that may impact on the formulation of theories and our understanding of past events1.The coming together of archaeology and microbiology promises to revolutionize the analysis and interpretation of archaeological deposits10. It has been suggested that changing features of microbial communities associated with archaeological human-impacted soil layers can be considered as a kind of archive of both environmental and anthropogenic conditions during the period of their formation, adding precise information about the nature of specific activities in the past. This is based, firstly, on the idea that the anthropogenic factor is most likely the crucial one in soil formation within a settlement, and secondly, on the fact that microorganisms are an integrated soil component, highly sensitive to environmental changes and able to effectively respond to changing environmental conditions11. Likewise, soil microbial communities are able to survive long periods of time under adverse environmental conditions through retreating into a dormant state12. Therefore, since microorganisms can specifically react to the input of nutrients in the form of organic substances, human activities changing the intensity and mode of those inputs may be reflected in soil microbial communities in quantitative and qualitative terms11. Former studies have already used microbial communities as bioindicators, termed as ecofacts by Margesin, et al.5, of human activities in the past. Soil fungal communities inhabiting anthropogenically transformed layers of medieval Russian settlements differed from those associated with non-impacted surrounding soils13. Studies at another medieval Russian settlement showed how spatial variations in the presence of keratin-decomposing fungi in sediments of streets, living-floors in and outside houses and from wall-filling revealed differences in the activities and the intensity of anthropogenic impact8. More recently, Peters, et al.11, in a case study from a Late Bronze Age settlement in the North Caucasus (Russia), confirmed that both activities and abundances of soil microbial communities change among the different layers analyzed depending on the intensity and the character of past human activities. Notwithstanding all these works, there is still a lack of surveys considering overall microbial  communities in terms activity, abundance, diversity and composition at archaeological sites, in general, and at anthropogenically transformed sites as a tool to differentiate layers that cannot be perceived by macroscopic analysis, in particular.In the context of the archaeological study of soils at ancient settlements, microbial communities can be used as precise indicators of ancient anthropogenic activities during soil formation in the distant past16. Details regarding the history of this settlement have previously been reported5. The position and the structure of the settlements followed the common pattern of so-called archaeological compounds (hamlet-like structures), which were loosely spread over this settled area. These compounds consisted of several rectangular, round and oval-shaped buildings that were mostly grouped around an open space. Each compound was occupied by an extended family and had its special family-internal cult area, normally comprising of a round building, where sacrificial meals took place within the family or clan. Parts of a protohistoric dwelling of central ritual importance have been excavated in the last years in one of these compounds19. The cultic character is attested by two ritual deposits outside and within the building which have been created in the course of the 6th century BC20.The archaeological settlement on Monte Iato is located in the inland of Sicily , about 30 km south-west of Palermo , and was scattered over the slightly sloping 40 hectare wide plateau of the hill. Its strategic location for defence purposes is typical for Iron Age settlements of the Sicilian hinterland21. Especially the latter are indicative of the formation of these layers of stone packings in the context of sacrificial feasts. A rim fragment found underneath, directly reminiscent of amphora-like pithoi from the Necropolis of Himera, shows that the three layers cannot be dated before 575–50 BC22. The layers of stone packings were clearly separated by thin layers of alluvial material. These alluvial separation layers within the otherwise homogenous material demonstrate that the different layers of stone packings were lying open for some time and therefore proof their formation in the context of periodically celebrated sacrificial feasts, which has not yet been documented before; maybe the packings were meant to compress the clayey ground, so that feast participants from afar would be able to erect mobile dwellings during the second and third quarters of the 6th century BC21.22 m to the east of this structure, three clearly separated layers of stone packings, laying on top of each other and composed of stones and soil, were discovered in September 2015. Between the stones of the individual packings, numerous incised sherds and fragments of matt-painted pottery were found. Additionally, noteworthy amounts of bone fragments with hack and slaughter marks came to light5.The objectives of this study were: (i) to characterize soil microbial  communities in the three layers of stone packings in terms of abundance, physiological profiles, diversity and taxonomic composition, and (ii) to evaluate whether characteristics of soil microbial communities in the three layers, formed chronologically on top of each other, are significantly different. We hypothesize (i) that bacterial, archaeal and fungal communities present in the studied occupation layers are characterized by a wide diversity and (ii) that the structure of the microbial communities differs among the layers as a consequence of different cultic events. Thus, changing microbial features can serve as ecofacts in these human-impacted layers created in the context of cultic events on Archaic Monte Iato and give evidence of ancient human behavior and ritual praxis. This work complements our previous study on the characterization of soil bacterial and fungal communities at another location of the same archaeological settlement differently impacted by ancient human activitiesThe studied soil samples were characterized by close to neutral pH values and low levels of nutrients Table . Soil sagtlA, as a measurement of potential aerobic bacterial respiration, was detected in all three layers and its abundance was significantly highest in the upper layer A14  did not significantly differ among the soil samples in the three layers, although Shannon index and evenness were higher in the intermediate layer A16 respect to the upper layer A14 Table . Global Proteobacteria (the number of classified sequences in this phylum ranged from 18.0 to 35.1% in the 18 libraries), Actinobacteria (16.3–27.8%), Acidobacteria (9.5–17.4%), Firmicutes (3.0–10.7%) and Chloroflexi (4.2–5.2%); these five phyla accounted for more than 63% of total sequences . These sequences were classified into 27 different phyla, being the predominant ones: ces Fig. . GammaprThaumarchaeota  and Euryarchaeota (4.5–14.1%) accounted for more than 94% of the total number of sequences across all the analyzed libraries , Basidiomycota (2.8–34.2%) and Chytridiomycota (0.2–18.2%) were the predominant phyla, which accounted for more than 88% of the total number of sequences  and Gammaproteobacteria dominated in the intermediate layer A16; and Betaproteobacteria and Anaerolineae (with no differences respect to A16) were found to a higher extent in the lower layer A18 . The differences in soil fungal communities among the layers were also evidenced at taxonomic level; Sordariomycetes class was significantly more abundant in the intermediate layer A16, while Leotiomycetes predominated in the upper layer A14 (Table Tetracladium (OTU5), unidentified Ascomycota (OTU17 and OTU49) and Ramicandelaber predominated in the upper layer A14, other OTUs belonging to Ilyonectria (OTU1) and Davidiella (OTU43) genera were found to a higher extent in the intermediate layer A16, and OTUs classified as Ilyonectria (OTU3) or unidentified Russulales (OTU33) were significantly more abundant in the lower layer A18  in the main room of an indigenous building and above the fireplace in the annex of the same building, from the same archaeological area (Monte Iato settlement) as studied here, were characterized in terms of abundance and taxonomic composition5. Since, on one hand, archaeological investigations proved clearly that the ancient human activities responsible for soil formation at the locations studied in both works differed completely20, and, on the other hand, environmental conditions at the studied sites were shown to be similar ), the differences in the features of soil microbial communities inhabiting locations from both surveys are thus mainly driven by the impact of the different past human activities. In this way, samples analyzed in our earlier survey were used as reference for the samples investigated in this study to discuss the results obtained for bacterial and fungal communities, as shown below.Changing features of microbial communities in human impacted soils of ancient settlements can be used as a tool to evidence different anthropogenic activities in the distant past23. Unfortunately, there are no previous studies quantifying the total abundance of microbial communities at archaeological sites in order to do comparisons in absolute terms. The upper layer A14 showed significantly increased levels of organic C, which were not concomitant with an increment in living bacterial or fungal biomass, probably because this C increase is insufficient to sustain higher microbial biomass. Also Peters, et al.11, studying microbial communities in different anthropogenically-impacted soils of a Late Bronze Age settlement located in the North Caucasus (Russia), did not find a positive correlation between higher organic C contents and increased levels of microbial biomass  staining). Yet, we observed significantly increased copy numbers of gtlA gene in soil samples of the layer A14 respect to soil samples of the other two layers, which could indicate higher bacterial respiration rates in this layer due to the presence of a higher amount of easily decomposable C sources. CLPP assays showed that microbial C-consumption patterns differed among soil samples of the three layers; easily decomposable carbohydrates such as pyruvic acid methyl ester and D-xylose were consumed to a higher extent in the upper layer A14, while more recalcitrant C compounds such as Tween 80 and Tween 40 were preferably consumed in deeper layers A16 and A18. We thus see the results of CLPP analysis in connection with the potential higher bacterial respiration detected in upper layer A14. Interestingly, also Peters, et al.11, in the aforementioned study, found higher microbial respiration rates in soils with higher organic C content.Soil samples of the three studied layers were characterized by low C and N contents. These low nutrient contents, along with other environmental factors presumably present in buried layers, such as low moisture, scarce oxygen availability and poor gaseous exchange, were probably restricting microbial life. However, our analyses demonstrated, in all soil samples of the three layers, the presence of both total  and living biomass 21. The specific human activities as well as the characteristics of the remains left by humans  likely differed among the three layers, which presumably determined the heterogeneity of the original physicochemical and environmental conditions  existing in the three layers. These conditions thus converted these three layers into a suitable system to assess whether microbial communities can act as ecofacts (bioindicators) of human activities in the distant past. In concordance with our hypothesis, we found significant differences in the structure of the soil microbial communities among the three layers; i.e., each layer presented a specific bacterial, archaeal and fungal community structure. Previous studies have argued that present microbial communities existing in an archaeological human-impacted soil are the same as those originally inhabiting the soil11. However, although soil bacterial and archaeal community structures differed between the intermediate and lower layers A16 and A18, the higher similarity among them in comparison with the upper layer A14 may be the result of a similar adaptation to changing environmental conditions over the time. Since we analyzed soil microbial communities using DNA-based methods, both living and non-living  microorganisms were targeted23, capturing thus the soil microbial dynamics in the three layers over the time.The impact of human activities on soil samples of the three layers studied here seems clear since they were created in a chronological order on top of each other in order to produce a compact surface for the erection of ephemeral buildings or tents in the context of periodic cultic feastset al.6 also found an unexpectedly high Illumina-based bacterial diversity in buried soil samples at the No. 1 Wangshanqiao Chu tomb in China. The community structure of the bacterial community in the studied layers was significantly different  from that reported in our previous study on soils  and above the fireplace of a residential building (A2 and A3) of the aforementioned protohistorical dwelling20) at the same archaeological area on Monte Iato as studied here5. Pairwise PERMANOVA and ANOSIM also showed that structure of bacterial communities significantly differed between each pair of archaeological locations from both studies  environments and pH conditions27. Previous studies have confirmed the presence of Pseudomonas in archaeological remains. In a culture-dependent study of the mud from a Roman thermae, strains classified as Pseudomonas were reported9. Xu, et al.6 suggested that Pseudomonas, among other bacterial genera, could play a key role in degrading organic culture heritage in burial conditions.The three studied layers harboured a huge microbial diversity in taxonomic terms. In the case of bacterial community, 27 different phyla (using a 50% confidence threshold for taxonomic affiliation of sequences) were shown to inhabit soils of these layers. Xu, es Table . At taxody Table . This fiThaumarchaeota and Euryarchaeota phyla, which have been shown to dominate soil habitats28. OTUs affiliated to Nitrososphaera genus (Thaumarchaeota) were the most abundant and contributed the most to dissimilarity of community structures among the three layers. The variations in the OTUs belonging to Nitrososphaera are probably a consequence of qualitative and quantitative changes in ammonia and carbon sources among the three layers over time29.In comparison with bacterial community, soil archaeal community showed a much more limited diversity in the studied layers as it was basically comprised of p = 0.0001; ANOSIM, r = 0.496, p = 0.0001; Table 5. These differences in community structure were evidenced at taxonomic level, while Dothideomycetes and Pezizomycetes classes were significantly more abundant in the soils of the protohistorical dwelling, Sordariomycetes and Agaricomycetes were significantly more dominant in the soils of the present study  in soil samples of the three layers is seen as an evidence of the accumulation of vegetal material  as a consequence of the ancient anthropogenic activity since species of this genus have been associated with root and decay of woody and herbaceous plants32.Soil fungal community inhabiting the studied layers was also highly diverse and its structure significantly differed  in the soil samples of all three studied layers. Soil bacterial, archaeal and fungal communities were characterized by a wide diversity, which confirms our first hypothesis. We also demonstrated that soil microbial communities of the three layers differed significantly (in agreement with our second hypothesis) in terms of C-source consumption patterns, community structure and taxonomic composition and relate this fact to the different past human activities that resulted in heterogeneous conditions for soil microbial communities in each of the layers. The high degree of similarity between microbial characteristics  in the intermediate and lower layer and the dissimilarity of these two layers with the upper layer might be a consequence of the adaptation of microbial communities to the prevailing physicochemical and environmental conditions in each layer. Soil bacterial and fungal communities studied here significantly differed from those in other human-impacted soils of the same settlement, which demonstrates the high variability of microbial communities within an ancient settlement. Altogether, our data evidence that changing characteristics of soil microbial communities reflect different ancient human activities. In the future, further works studying the features of soil microbial communities from a wider range of archaeological locations are needed to identify key microbial groups as indicators of specific past anthropogenic activities and to generate more general conclusions regarding the use of the characterization of soil microbial communities as a complementary tool of the traditional archaeological methods.The three clearly separated layers described in the introduction were discovered during a field campaign in September 2015. An area of 1.5 × 1.5 m, with a depth of 50 cm, was excavated. The superimposed layers of stone packings were clearly separated by thin layers (a few cm) of alluvial material The upper layer (A14) had a depth of 27 cm while the intermediate A16) and lower layers (A18) measured 10 cm each , three sampling points in the vertical mid of each layer were chosen, which were separated by ca. 40 cm from each other. From each of these sampling points, several sub-samples were collected with sterile spatulas, trying to get as much soil as possible and avoiding to sample stones or remains, merged and placed in sterile vials. This way, we obtained 3 soil samples  per layer. Immediately after sampling, the soil samples were stored in cooled boxes, shipped to the laboratory in Innsbruck (Austria) and stored at 4 °C for physicochemical characterization and measurement of microbial activities or at −80 °C prior to DNA-based and PLFA analyses.Unfortunately, there is no information available in the literature on soil microbial communities at places in the vicinity of the studied archaeological site. Therefore, we could not include a pristine reference site in our studies.2), dry mass (105 °C for 24 h) and contents of soil organic matter , total organic carbon  and total N . C/N ratio was calculated as TOC/N.Each of the 9 soil samples was characterized regarding pH  during 1 h and soil suspensions were then serially diluted to 10−4. 130 µl of soil solutions were used for each well and Ecoplates were incubated at 20 °C for 21 days. Colour development for each well was obtained in terms of optical density (OD) at 590 nm every 24 h using an automated plate reader. Microbial catabolic activity was calculated as average well colour development (AWCD) as described by Siles, et al.33. The 10-day OD data, normalized by dividing them by their AWCD, were selected to calculate substrate richness, Shannon index, evenness and the relative consumption rate of each substrate.The Biolog EcoPlate system  was used to determine community level physiological profiles (CLPP) for each soil sample in triplicate according to Siles, 34. Briefly, microbial lipids were firstly extracted from 0.5 g of soil sample using a mixture of chloroform:methanol:phosphate buffer  according to Bligh and Dyer35. Phospholipids were then separated by solid-phase extraction cartridges , and the samples were subjected to mild alkaline methanolysis. The free methyl esters of phospholipid fatty acids were analyzed by gas chromatography-mass spectrometry .Phospholipid fatty acid (PLFA) analysis was used as a proxy to quantify living microbial biomass in each soil sample. Extraction and analysis of PLFAs were carried out as previously described36. The sum of all the identified phospholipids was used to estimate the total microbial biomass. Ratios between PLFA markers derived from fungi and bacteria (F/B) were calculated.The PLFA i14:0, i15:0, a15:0, 16:1ω9, 16:1ω7, 16:1ω5, 10Me-16:0, i17:0, a17:0, cy17:0, 17:0, 10Me-17:0, 10Me-18:0 and cy19:0 were addressed as bacterial signature markers. The PLFA 10Me-16:0, 10Me-17:0 and 10Me-18:0 were used as actinobacterial markers, while the fatty acid 18:2ω6,9 was selected as fungal marker. The fatty acids found both in bacteria and fungi, such as 15:0 and 18:1ω7, were excluded from the analysis characterizing the different microbial groups260/A280 and A260/A230. Subsequently, the DNAs were quantified using QuantiFluor™ dsDNA System  and DNA concentration for each sample was standardized to 1 ng μL−1.Total DNA from each of the 9 soil samples was extracted in triplicate from 250 mg of soil fresh mass, using Power Soil™ DNA Isolation Kit . Triplicate DNA extracts from each soil sample were then pooled, and DNAs were purified using DNA Clean & Concentrator Kit  according to manufacturer’s instructions. The quality of DNAs was spectrophotometrically checked by NanoDrop  based on the absorbance ratios AgtlA, as functional marker of bacterial respiration activity) was carried from each of the 9 DNA samples in duplicate with the pairs of primers Eub338/Eub518 for bacteria37, Arch-967F/Arch-1060R for archaea38, FR1/FF390 for fungi39 and CS680F/CS904R for gtlA gene40 using SYBR® Green as detection system . The PCR mixtures and thermal conditions for quantification of ribosomal bacterial, archaeal and fungal genes were as previously described41. In the case of gtlA gene, the amplifications were carried out using the PCR mixtures described by Siles and Margesin41 under the following thermal conditions: 94 °C for 5 min followed by 40 cycles of 94 °C for 20 s, 61 °C for 30 s, and 72 °C for 30 s. In all the cases, after amplification reactions, melting curve and gel electrophoresis analyses were conducted to confirm that the amplified products had the appropriate size. Copy number for each gene was quantified by comparing the cycle at which fluorescence crossed a threshold to a standard curve constructed using a serial dilution of a plasmid containing an appropriate template, as previously described41. Standard curve for gltA gene was generated using Sphingomonas alpina S8-3T (DSM 22537) genomic DNA. The relations A/B  and F/B  were calculated on the basis of the log-transformed copy number values.Quantitative PCR (qPCR) was used to determine total (living and non-living (dead or in a dormant state)) bacterial, archaeal and fungal abundance and to estimate the potential aerobic bacterial respiration in each soil sample. The quantification of bacterial and archaeal 16S rRNA and fungal 18S rRNA genes as well as bacterial citrate synthase gene , quantified with the QuantiFluor™ dsDNA System and pooled in equal proportions. The pooled product was then used to prepare Illumina DNA library following TruSeq DNA library preparation protocol. Paired-end sequencing (2 × 300) was performed on the Illumina MiSeq sequencing platform  at MR DNA .For Illumina amplicon sequencing analyses, two technical replicates of each of the 9 DNA samples were carried out, obtaining a total of 18 libraries for each microbial  community. Diversity and structure of bacterial communities were characterized by amplifying a fragment of 16S rRNA gene (and capturing the hypervariable V1-V3 regions) with the primers 27 F (5′-AGRGTTTGATCMTGGCTCAG-3′) and 519 R (5′-GTNTTACNGCGGCKGCTG-3′)Plus Master Mix Kit  and barcoded forward primers, under the following thermal conditions: initial denaturation at 94 °C for 3 minutes, followed by 28 cycles of denaturation at 94 °C for 30 s, primer annealing at 50 °C for 40 s and extension at 72 °C for 60 s as well as a final elongation step at 72 °C for 5 minutes. The processing of PCR products for paired-end sequencing (2 × 300) on Illumina MiSeq sequencing platform was done as described for bacteria.Archaeal communities were analyzed using the pair of primers arch519F (5′- CAGCMGCCGCGGTAA-3′) and arch806R (5′-GGACTACVSGGGTATCTAAT-3′), which captures the V4 hypervariable region of 16S rRNA gene. DNA samples were amplified in triplicate using HotStarTaq 44 under the PCR and thermal conditions reported by Margesin, et al.5. Secondly, the processing of PCR products for paired-end sequencing (2 × 300) on Illumina MiSeq sequencing platform was conducted as described for bacteria.In the case of fungal communities, internal transcribed spacer (ITS) 1 region was firstly amplified using the ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) primersThe raw bacterial, archaeal and fungal sequences associated with this study were deposited in the GenBank SRA database under BioProject accession number PRJNA393946.41. Next, sequences from each library were processed using USEARCH pipeline and UPARSE-OTU algorithm45. Briefly, reads were separately quality-filtered , trimmed (to 450-bp (base pair), 220-bp and 230-bp for bacterial, archaeal and fungal libraries, respectively), dereplicated and sorted by abundance (removing singleton sequences) prior chimera detection and OTU  determination at 97% sequence identity. Finally, original high-quality sequences were mapped to OTUs at the 97% identity threshold obtaining three OTU tables, one for each microbial community. The taxonomic affiliation of each OTU was obtained using RDP  taxonomic classifier46 against 16S rRNA training set 16 for bacterial and archaeal sequences and UNITE Fungal ITS train set 07-04-2014 for fungal sequences with a 50% confidence threshold for the three communities. Next, OTUs classified as Bacteria were removed from archaeal OTU table, and vice versa. In the case of archaeal dataset, also the OTUs that could not be classified as Archaea were discarded. All the remaining bacterial OTUs and all the OTUs from fungal dataset could be classified as Bacteria and Fungi, respectively, and they were thus retained. These three OTU tables were used for downstream analyses.First, raw MiSeq paired-end reads from bacterial and archaeal 16S rRNA and fungal ITS1 amplicons were independently assembled and reoriented using MR DNA pipeline. Subsequently, sequences were demultiplexed and formatted for processing using a Phython script47.For diversity characterization of microbial communities and statistical analyses, the number of sequences per sample was normalized to 16,000, 1,399 and 12,000 for bacterial, archaeal and fungal OTU-tables, respectively. The bacterial, archaeal and fungal communities were characterized in terms of diversity by calculating richness (number of OTUs), Shannon index, Smith-Wilson evenness and the richness estimator indices ACE (abundance-based coverage estimation) and Chao 1 using Mothur v.1.39.3One-way analysis of variance (ANOVA) was applied to determine significant differences (p ≤ 0.05) among the three layers regarding: (i) soil physicochemical properties; (ii) PLFA- and qPCR-based measurements of microbial abundances; (iii) diversity measurements of CLPP dataset as well as bacterial, archaeal and fungal communities; (iv) relative abundance of bacterial, archaeal and fungal taxonomic groups; and (v) relative abundance of the top bacterial, archaeal and fungal OTUs extracted from SIMPER analysis. When ANOVA resulted in significant results, Tukey’s honest significance difference (HSD) post-hoc test was subsequently applied for multiple comparisons of means at a 95% confidence interval. Normality and heteroscedasticity of data were tested by the Shapiro-Wilk and Levene tests, respectively. In case that one of those conditions was not met, the values were log transformed.5. SIMPER (similarity percentage) analysis was applied to assess which C sources and microbial OTUs were primarily responsible for the observed differences using PAST ver. 3.07 software. One heat map for each microbial community, considering the OTUs contributing ≥0.2% to microbial community dissimilarities among the layers, was modeled with the “pheatmap” package in R.Multivariate ordination methods were applied to reduce the multiple dimensions in datasets and visualize patterns. CLPP dataset was subjected to principal component analysis (PCA) since a previous detrended correspondence analysis of the data revealed a gradient length of the first axis <1. In the case of bacterial, archaeal and fungal community structures, non-metric multidimensional scaling (NMDS) analysis based on Bray-Curtis similarities at OTU level was used as ordination method. The significance of the differences found in CLPPs and OTU-based structures of bacterial, archaeal and fungal communities among the layers was assessed through permutational multivariate analysis of variance (PERMANOVA) and analysis of similarity (ANOSIM) with Bray-Curtis similarities after 9,999 permutations using the “vegan” package in R. PERMANOVA and ANOSIM were also used to compare the structure of soil bacterial and fungal communities of the present study with those of our previous survey investigating other soil samples of the same archaeological settlementSupplementary Material


According to recent WHO reports, alcohol remains the number one substance used and abused by adolescents, despite public health efforts to curb its use. Adolescence is a critical period of biological maturation where brain development, particularly the mesocorticolimbic dopamine system, undergoes substantial remodeling. These circuits are implicated in complex decision making, incentive learning and reinforcement during substance use and abuse. An appealing theoretical approach has been to suggest that alcohol alters the normal development of these processes to promote deficits in reinforcement learning and decision making, which together make individuals vulnerable to developing substance use disorders in adulthood. Previously we have used a preclinical model of voluntary alcohol intake in rats to show that use in adolescence promotes risky decision making in adulthood that is mirrored by selective perturbations in dopamine network dynamics. Further, we have demonstrated that incentive learning processes in adulthood are also altered by adolescent alcohol use, again mirrored by changes in cue-evoked dopamine signaling. Indeed, we have proposed that these two processes, risk-based decision making and incentive learning, are fundamentally linked through dysfunction of midbrain circuitry where inputs to the dopamine system are disrupted by adolescent alcohol use. Here, we test the behavioral predictions of this model in rats and present the findings in the context of the prevailing literature with reference to the long-term consequences of early-life substance use on the vulnerability to develop substance use disorders. We utilize an impulsive choice task to assess the selectivity of alcohol’s effect on decision-making profiles and conditioned reinforcement to parse out the effect of incentive value attribution, one mechanism of incentive learning. Finally, we use the differential reinforcement of low rates of responding (DRL) task to examine the degree to which behavioral disinhibition may contribute to an overall decision-making profile. The findings presented here support the proposition that early life alcohol use selectively alters risk-based choice behavior through modulation of incentive learning processes, both of which may be inexorably linked through perturbations in mesolimbic circuitry and may serve as fundamental vulnerabilities to the development of substance use disorders. Adolescence is characterized by risky and impulsive behaviors, often including initial experimentation with drugs of abuse and most commonly, alcohol  tasks, respectively. The purpose of these studies is to further test our hypothesis that incentive based learning and assignment of motivational value through reinforcement learning, not impulsivity, is the underlying psychological mechanism of maladaptive decision making after early life alcohol use. Our results show that adults exposed to alcohol during adolescence exhibit enhanced attribution of value to reward-predictive cues compared to controls. Alternatively, we find no significant difference between alcohol-exposed and control animals on either measure of impulsivity (impulsive choice and action). Therefore, we provide further compelling evidence that adolescent alcohol use promotes disadvantageous decision making by altering a learning-based incentive attribution process and not through a general mechanism of impulsivity. ad libitum except as noted. Rats were weighed and handled daily throughout the course of the experiment. All work in this manuscript was conducted under the guidance and permission of the Institutional Animal Care and Use Committee at the University of Washington and pursuant to federal regulations regarding work with animals.Male Sprague-Dawley rats  were weaned at postnatal day (PND) 21 and aged PND 27 at the start of experiments. All animals were housed individually in polycarbonate tubs on a 12-h light/dark cycle (lights on at 06:00). Animals were housed individually for accurate assessment of alcohol gel consumption. Water and Teklad  rodent chow was availablead libitum water and chow. Alcohol gel intake levels were monitored daily and expressed in g/kg of body weight using individual gel consumption and body weights measured daily. Rats failing to consume gel during the control gel pre-exposure, exhibiting three consecutive days of no consumption, or burying of the gel in bedding once the alcohol gel exposure began were excluded from the study. Experiments began with 3 days of pre-exposure to control gel. Subsequently, adolescent rats were each split into alcohol gel and control gel groups matched by weight and baseline intake. Twenty days of 24 h/day gel exposure followed. Upon completion of the 20-day exposure to ethanol rats were monitored daily for withdrawal symptoms  for the following 20 days. No overt signs of withdrawal were observed.Alcohol was presented to adolescent (PND 30–49) rats in a gel matrix consisting of distilled water, Knox gelatin, polycose (10%) and 190-proof ethanol (10%). Control gels had ethanol replaced with distilled water. Preparation was as previously described . For all tasks, prior to the beginning of training, animals were food restricted to maintain them at ~90 ± 2% free-feeding weight. Free-feeding weight was based on pre-restriction weights. The rewards for instrumental responses and during Pavlovian conditioning were 45-mg sucrose pellets . Rats underwent magazine training before all tasks where 10 sucrose pellets were delivered over 15 min. For delay discounting and DRL, rats were trained on a fixed ratio 1 schedule to a criterion of ≥24 responses in a 30-min session. For delay discounting, once criterion was met, rats were auto-shaped over the course of 5 days  from 0 s to 15 s; day 3 reduced the time to perform trial-initiating nosepoke to 10 s; day 4 increased the ITI from 15 s to 30 s; day 5 increased the ITI from 30 s to 45 s).Following magazine training, rats underwent 7 days of pavlovian training with 25 trials per day. For each training session, a trial consisted of the extension of a lever  into the chamber along with illumination of a cue light above the lever  for 8 s, followed immediately by the delivery of two sucrose pellets  into the food tray and illumination of the tray light. The CS-US presentations occurred on a 60 s variable ITI schedule for each session. Lever presses and cup entries during lever presentation were recorded as measures of pavlovian conditioned approach. Total number of lever presses and food tray head entries during lever presentation were recorded but had no programmable response.After 7 days of pavlovian training, all animals underwent a test for conditioned reinforcement. The chambers were rearranged such that the left and right retractable levers flanking the food tray were replaced with nosepoke ports and the food tray was replaced with a single retractable lever. The conditioned reinforcement session lasted a total of 40 min and the houselight was on for the full duration. During the session, nosepokes into the designated “active” port  resulted in insertion of the illuminated lever for 2 s. Nosepokes into the “inactive” port had no programmable consequences. Total number of lever presses, nosepokes into the active port, and nosepokes into the inactive port were recorded.Following instrumental training as outlined above, animals were tested on a concurrent instrumental task involving the presentation of two levers. Each daily session consisted of 24 forced trials followed by 24 free-choice trials, with a total of seven testing sessions. At the start of each session, the chamber was in the ITI state, completely dark with no light cues. All trials began with illumination of the house light and a light in the food tray cueing the animal to make a nosepoke into the food tray within 10 s. Failure to nosepoke resulted in trial termination, and the chamber returned to the ITI state. During training, animals were exposed to forced trials wherein a successful nosepoke led to the extension of a single lever, presented pseudo-randomly. These trials served to expose the animal to each option and its associated expected value. During each session, forced choice trials were followed by free-choice trials with the same contingency for each lever. Free-choice trials follow the guidelines described above, but each successful nosepoke resulted in the extension of both levers, and the animal was free to choose between the two levers within 10 s. During both forced- and free-choice trials, one lever was associated with the delivery two sucrose pellets after 1 s and the other lever associated with the delivery of four sucrose pellets after an increasing delay of time . Increasing delay times were tested in separate testing sessions (days) with a 1 s delay on the first day of testing, a 2 s delay on the second day of testing, and so on for all animals. This method of testing the different delay times on separate days was done in order to be kept consistent with our previous studies using the probability discounting task where the different probabilistic deliveries of reward were also tested on separate, subsequent days. Each session assessed the animal’s preference between the two levers. Choice of the high reward lever was recorded during free-choice trials.Following instrumental training as described above, animals were tested on the DRL task as a measure of impulsive action. Each 45 min session began with illumination of the house light and lever presentation. Each animal was assigned an active lever (left or right) counterbalanced between rats, which remained extended throughout the duration of the sessions. For the first 5 days, rats were tested using a 5 s DRL interval, in which a lever press resulted in pellet delivery only if at least 5 s elapsed since the previous press. Each lever press that occurred before the required 5 s wait period resulted in a resetting of the 5 s criteria, during which animals would have to wait another 5 s before pressing the lever to receive a pellet. Following five consecutive days of the 5 s DRL, the schedule was then switched to a 10 s DRL interval for 5 days, then to a 20 s DRL interval for 5 days, and finally to a 30 s DRL interval for 5 days. The total number of lever presses and the total number of reinforcers received were recorded. Percent of effective lever presses was also determined by dividing the total number of pellets received by total number of lever presses.t-test was used to analyze total number of lever presses during the conditioned reinforcement test. Performance on the delay-discounting and DRL tasks were analyzed using repeated measures two-way ANOVA with treatment  and delay  or DRL interval  as the independent variables, and preference for the high reward lever or lever presses, reinforcers and effective lever presses, respectively, as the dependent variables. Bonferroni post hoc analyses were used and were corrected for multiple comparisons. All data are presented as mean ± SEM and threshold for statistical significance was set at p < 0.05.All statistical analyses were conducted using GraphPad Prism 6. Behavioral data for the pavlovian training sessions were binned into five-trial epochs. A response bias score, which is a measure of the relative allocation of behavioral responses between the lever and food tray during pavlovian training, was calculated by subtracting the number of food tray entries from the number of lever presses divided by the sum of both responses: (lever press − food tray entries/(lever presses + food tray entries), resulting in a number ranging from −1  to +1  group = 2.1, NS). Across all three behavioral experiments, a total of 10% of rats did not consume the alcohol gelatin during the 20 exposure days and thus were not run in the behavioral studies. These intake levels are comparable to our previous studies further supporting this method of alcohol intake as a consistent model of moderate/recreational alcohol use  trial bin = 57.2 p < 0.0001; Figure F trial bin × treatment = 2.6, p < 0.001; F treatment = 4.3, p < 0.05; Figure F trial bin = 14.0, p < 0.0001; Figure F trial bin × treatment = 3.9, p < 0.0001; F treatment = 4.6, p < 0.05 Figure F trial bin = 40.6, p < 0.0001; Figure F trial bin × treatment = 2.0, p < 0.001; Figure Consistent with previously published data  port = 116.4, p < 0.0001; Figure F treatment × port = 5.2, p < 0.05; Figure p < 0.05). Further, during the 2-s lever extension period following a poke in the active port, alcohol animals also had significantly more lever presses than control animals  = 2.4, p < 0.05; Figure n = 3) and control (n = 5) rats that were not classified as sign trackers (response bias scores between −0.70 and +0.70) did not differ in total number of active or inactive nosepokes  treatment = 0.2, NS; F treatment × port = 0.02, NS; Figure All animals underwent the test of conditioned reinforcement following pavlovian training. However, given that this task measures the effectiveness of the lever-CS as a reinforcer (defining a strong sign-tracker bias), we were primarily interested in the behavior of sign-tracking animals. Therefore, only the 23 animals designated as sign trackers based on the above mentioned criterion (>+0.70 response bias score) were kept in the analyses. One sign tracker from each treatment group was dropped because the incorrect active port was assigned for the conditioned reinforcement test yielding F delay = 62.1, p < 0.0001; Figure F treatment = 0.02, NS; F delay × treatment = 0.4, NS; Figure The delay-discounting task was used to assess the effect of adolescent alcohol intake on impulsive choice in adulthood. Analysis of choice behavior on the delay-discounting task generated standard discounting curves for choice of the larger but delayed reward option over all conditions, with increasing delay of the large reward resulting in decreased choice of the large reward option for both alcohol-exposed and control animals  DRL × treatment = 0.6, NS; F treatment = 0.01, NS; Figure F DRL × treatment = 0.4, NS; F treatment = 0.02, NS; Figure F DRL × treatment = 0.7, NS; F treatment = 0.01, NS; Figure F treatment = 24.9, p < 0.0001; Figure F treatment = 246.8, p < 0.0001; Figure F treatment = 63.9, p < 0.0001; Figure The DRL task was used to assess the effect of adolescent alcohol intake on impulsive action in adulthood, as measured by the ability of the animals to withhold responding for reinforcers during 5, 10, 20, and 30 s DRL intervals. Alcohol-exposed animals did not differ from controls on total number of lever presses (Alcohol is the most commonly abused substance among adolescents and use during this sensitive developmental period is one of the best predictors of risk for development of alcohol use disorder (Grant et al., Cues in an environment that repeatedly predict reward can attain value through a Pavlovian process referred to as stimulus-reward learning. In the realm of addiction, cues associated with drugs often attain enhanced incentive value and play a critical role in promoting drug craving, seeking and relapse following periods of abstinence (O’Brien et al., These findings further lend support to our hypothesis that maladaptive decision making under risk, a consequence of adolescent alcohol use, is a result of altered incentive learning. However, other underlying constructs such as altered valuation of primary rewards, deficits in reinforcement learning where corrupted value is assigned to specific cues and actions, and/or increased impulsivity, could also influence the risk-seeking phenotype. Thus, in a series of experiments begun in Clark et al., Deficits in impulse control are a key feature of alcohol use disorders, with higher levels of impulsivity predisposing to the development of alcohol problems later in life and increased likelihood of relapse following abstinence (Mitchell et al., Previous studies have explored the relationship between probability discounting, delay discounting, incentive learning, sign-tracking behavior and addiction-prone phenotypes with varying results on how these behaviors cluster (Flagel et al., In addition to delay discounting, the probability-discounting task has also been used as a measure of impulsive choice (Flagel et al., Taken together with our previous work, the findings presented here support our hypothesis that the compromised encoding of Pavlovian cues following alcohol exposure in adolescence may underlie suboptimal decision making strategies in adulthood. Our data additionally support delay- and probability-discounting as dissociable decision-making paradigms, and confirm that impulsivity is not likely an explanation for maladaptive decision making under risk in adulthood seen following adolescent alcohol use. One limitation of the present set of experiments is that only the effect of adolescent alcohol intake, but not adult alcohol intake, on incentive value attribution and impulsivity in adulthood was assessed. Thus, we cannot state that the results are specific to adolescent intake. However, we have previously demonstrated that moderate alcohol intake during adulthood does not alter risky decision making (Schindler et al., LCK performed most of the studies, analyzed the data. LCK and JJC wrote the manuscript. JJC, AGS and LCK conceived the studies and edited the manuscript. RGW performed the delay discounting study. SJW performed analyses for the delay data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The status of lymph node involvement is an important prognostic factor for breast cancer. However, the presence of intratumoral lymphatic vessels in primary tumor lesions and the relationship between lymphatic vessel density (LVD) and lymph node metastasis (LNM) have not been firmly established. Therefore, we performed a meta-analysis study to investigate these issues. According to the pre-established inclusion and exclusion criteria, 13 studies, involving 1029 breast cancer patients, were included in this study. Using immunohistochemical staining, intratumoral lymphatic vessels were detected in 40.07% of breast cancer patients (240/599), and peritumoral lymphatics were detected in 77.09% (397/515). All studies demonstrated that peritumoral LVD was higher than intratumoral LVD, with a pooled standard mean difference and 95% confidence interval (95% CI) of 1.75 (1.28 to 2.21). Both intratumoral LVD and peritumoral LVD positively correlated with LNM, with correlation coefficients of 0.14 (95% CI 0.05 to 0.23) and 0.31 (95% CI 0.13 to 0.49), respectively. In summary, our study reports the overall detection rate of intratumoral lymphatics and demonstrates the associations between intratumoral LVD, peritumoral LVD, and LNM in breast cancer. Additionally, controlled studies with a larger number of subjects are needed to establish these relationships. Breast cancer is the most common malignant tumor in females. Although great efforts have been made in the field of early diagnosis and adjuvant therapy, the incidence and overall mortality of breast cancer continues to increase24Due to the lack of specific markers, the detection of lymphatic vessels has been hampered in previous studies. Intratumoral lymphatic vessels were considered to be rare and nonfunctional due to mechanical compression8Some studies have suggested that LVD is associated with an increased risk of LNM1012The flow chart of the article selection process is presented in The details of the included studies are exhibited in Table 110111213141516171819202119P < 0.05, I2 = 95%), all studies indicated that peritumoral LVD values were higher than intratumoral LVD, with a pooled SMD of 1.75 (95% CI 1.28 to 2.21). The random-effects model was used to combine the SMD values because of significant heterogeneity.Among the 13 studies included, seven1011131416171011131416r values. The Fisher’s Z transformation was used to convert r values to Z values. The main outcomes are summarized in 2 = 28%, P = 0.002, 2 = 78%, P = 0.002, r values by inverse Fisher’s Z transformation. Both intratumoral LVD  and peritumoral LVD  were positively correlated with LNM in breast tumors.The studies that include detailed data of intratumoral and peritumoral LVD and the presence of LNM were selected to investigate the interrelationships between them10111213P = 0.017).To evaluate the stability of the results, a sensitivity analysis was performed using the random-effects model. Sensitivity analysis, by repeatedly analyzing the data after removing individual studies in turn, demonstrated that no studies were responsible for the disproportionate influence on the pooled estimate . Begg’s P < 0.05). The positive correlation between peritumoral LVD and LNM was moderately stronger than that of intratumoral LVD and LNM. However, there was substantial evidence of heterogeneity among these studies. By conducting a sensitivity analysis, two studies were identified to be the main source of heterogeneity17The current meta-analysis included 13 observational studies with an overall population of 1029 breast cancer patients. By immunohistochemical staining with D2–40/podoplanin antibodies, 40.07% of the specimens exhibited intratumoral lymphatics, and 77.09% of the specimens showed peritumoral lymphatics. Peritumoral LVD is significantly higher than intratumoral LVD in breast cancer . The result is supported by all included studies. Peritumoral LVD is also reported to be higher than that of normal and benign breast lesions1012It is well known that blood vessel density, an indicator of tumor angiogenesis, is closely associated with the clinicopathological outcomes of breast cancer2110312 Although the current meta-analysis study has some definite strengths, some limitations should be considered. All included studies were observational studies with relatively small sample sizes, and several studies6In conclusion, the study demonstrates the existence of intratumoral lymphatic vessels. Although the overall detection rate of intratumoral lymphatic vessels is lower than that of peritumoral lymphatic vessels, it does not change the fact that they are present and constitute a risk factor for tumor metastasis. Both intratumoral LVD and peritumoral LVD are correlated with the increasing risk of LNM, and peritumoral LVD exhibits a moderately stronger correlation with the increasing risk of LNM than that of intratumoral LVD. It might provide a potential target to prevent lymphangiogenesis and lymphatic metastasis in breast cancer.Two independent observers searched the databases of PubMed and Embase. The databases were searched using the following Medical Subject Heading (MeSH) terms or keywords: “breast cancer OR breast carcinoma OR breast neoplasms” AND “lymphatic vessel density OR lymphatic microvessel density OR LVD OR LMVD OR lymphangiogenesis” with no restrictions. All abstracts that indicated LVD assessment in breast cancer, no matter prospective or retrospective, were chosen for further consideration. The reference lists of all selected papers and abstracts were also screened. If it was necessary, we contacted the authors of the original studies for the required data. The search was ended on April 8, 2016.All studies that met the following criteria were included: (1) patients with breast cancer at any age; (2) a sample size larger than 10 patients; (3) no neoadjuvant chemotherapy or radiotherapy administered before the surgical treatment; and (4) specimens stained with the immunohistochemical method. Studies were excluded if they included the following: (1) review articles, case reports, meeting abstracts, or animal studies; (2) an examination of the total LVD of breast tumor lesions without distinguishing intratumoral LVD and peritumoral LVD; or (3) patients previously diagnosed with other diseases that could lead to LNM. Two independent authors followed the above inclusion and exclusion criteria to review the studies. When two or more articles reported duplicated data, we included the study with the most-recent data, the largest dataset, or the most relevant data. In cases of disputes, a third reviewer assessed the study to obtain a consensus.Two authors independently checked each item mentioned by the publications and discussed the data that was extracted. The information extracted from each study was summarized in a table and included the following items: first author’s name, publication year, country, number of patients (size), age, type of breast cancer involved, antibody and dilution, intratumoral LVD and detection rate, peritumoral LVD and detection rate. Two authors conducted the quality assessment based on the criteria of the Newcastle-Ottawa Quality Assessment scale (NOS)r) were used for the meta-analysis because some variables in the original studies were log-transformed before analysisr values to Z values into a normal distribution2 statistic. A random-effects model or, in the absence of heterogeneity, a fixed-effects model was used to combine the SMDs and Z values with 95% CIs. The pooled r values and 95% CIs were obtained from the inverse Fisher’s Z transformation. Additionally, we conducted a sensitivity analysis by STATA 12.0 software to investigate the influence of a single study on the overall result by omitting each study in turn. Publication bias was detected by Begg’s and Egger’s test. In this study, P < 0.05 was considered statistically significant.Spearman correlation coefficients .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Chromosomal aberrations (CAs) in blood lymphocytes have been shown to be associated with overall cancer risk and aging. However, their relationship to bladder cancer risk remains to be elucidated. In a case-control study of bladder cancer in Egypt, we examined the relationship between the increased frequency of CAs in blood lymphocytes and bladder cancer risk. High frequency of CAs was significantly associated with an increased risk of bladder cancer . The associations were somewhat stronger in squamous cell carcinomas  than in urothelial carcinomas . We also identified chromosome specific CAs for chromosomes 3, 4, 5, 8, 9, 10, 11, 12, 17, 19 that were significantly associated with an increased risk of bladder cancer. We observed particularly strong associations between aberrations of chromosomes 12, 13, 17 and risk of SCC . Conclusion: increased frequency of chromosomal aberrations in blood lymphocytes was significantly associated with bladder cancer risk. Overall and chromosome specific aberrations in blood lymphocytes may be a unique set of biomarkers for risk assessments of SCC and UC. Urinary bladder cancer subtypes include urothelial carcinoma (UC), squamous cell carcinoma (SCC), adenocarcinoma, anaplastic, and other rare forms. The most prevalent subtype is UC, accounting for about 90% of incident cases in developed countries2. The genome of bladder cancer cells, like many other human solid tumors, is characterized by chromosomal instability, including structure and numerical chromosomal aberrations (CAs)4. The formation of structural chromosomal aberration is often associated with carcinogen-induced DNA damage or faulty replication of a damaged DNA template5. The capacity to repair damaged-DNA also plays a critical role in the formation of structural CAs6. It has long been hypothesized that the level of genetic damages assessed in blood cells reflects the level of damage in the somatic cells affected by carcinogenesis.Urinary bladder cancer ranks seventh overall among men worldwide but seventeenth among women7 and has also been shown to be associated with aging and an increased risk of leukemia8. Several epidemiological studies provided consistent evidence that CA frequency in blood lymphocytes was associated with elevated overall cancer risk, independent of other cancer risk factors such as exposure to carcinogens, gender, and age13. The strongest association was found in melanoma and carcinomas arising from lung, stomach, liver, and prostate13. However, the number of bladder cancer cases in these previous studies was very small (N < 60), thus lacking statistical power to draw meaningful conclusions. In the present report, we examined the association between the frequency of CAs in blood lymphocytes and bladder cancer risk in a large case-control study that included sufficient numbers of both UC and SCC patients to examine the two subtypes of cancer separately (533 cases and 560 controls). The large sample size also allowed us to examine the associations between chromosome specific CAs in blood lymphocytes and bladder cancer risk for the first time.The frequency of chromosomal aberrations in peripheral blood lymphocytes has been used for the surveillance of healthy individuals exposed to known carcinogensTable The bladder cancer cases were significantly more likely than the controls to have blood lymphocytes with any CAs  of 3.90  and bladder cancer risk. We observed a significantly higher percentage of bladder cancer patients showing CAs in blood lymphocytes for autosomes 3, 4, 5, 8, 9, 10, 11, 12, 17 and 19  than in controls . Chromosome X aneuploidy is extremely rare in males. Multivariate logistic regression analyses revealed that frequency of chromosome X aneuploidy in blood lymphocytes was not significantly associated with bladder cancer risk in females  and found that loss of chromosomes 8p, 9p and 17p, and gain of chromosomes 1q, 3p, 5p, 6p, 8q, 10p, 11q, 12q, 14q, 17q and 20q were frequently observed . The present study, for the first time, provided evidence that instability in chromosomes 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19 are shared between blood lymphocytes and bladder tumors, suggesting that inherent instabilities in these chromosomes may be a contributing factor to bladder carcinogenesis.One of the exceptional strengths of the present study is that chromosomal aberrations were analyzed by karyotyping of G-banded chromosomes. This approach allows the characterization of CA for each of the 23 pairs of human chromosomes. Large sample size provided sufficient power to evaluate the association between chromosome specific CAs and bladder cancer risk. We found that structural CAs of 11 autosomes, namely chromosomes 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19, were significantly associated with bladder cancer risk. Three  of the 11 chromosomes were significantly associated with risk of both UC and SCC. Like many other solid tumors, structure chromosome aberrations are common in bladder tumor cells23, and is classified as a Group 1 carcinogen24. This parasitic disease, characterized by repetitive infections, causes damage to the bladder and kidneys25. It is thought that bladder tumors occur as a result of chronic inflammation, leading to metaplasia. Previous cytogenetic studies have not found a consistently distinct pattern of chromosome alterations specific for SCC29. Loss of chromosome 17p has been reported in both UC and SCC tumors, and was found more frequently in SCC tumors29. There is one small study that reported frequent loss of chromosome 13q in SCC tumors28. Frequent alteration of chromosome 12 has not been reported in SCC. Our study is the first to report that chromosome 12, 13, and 17 aberrations in blood lymphocytes are strongly associated with the risk of SCC. These strong associations warrant further investigation.Most interestingly, we found that aberrations in chromosomes 12, 13, and 17 in blood lymphocytes is particularly strong risk factors for SCC. SCC of bladder is rare in the developed world, but is frequent in regions with high prevalence of Schistosoma infection (SH). SH is a well-established risk factor for SCC31. Loss of chromosome Y in blood lymphocytes has been reported to be associated with non-hematological cancer mortality12 and colorectal and prostate cancer risk32. However, a recent large cohort study found that mosaic loss of chromosome Y in blood lymphocytes was not consistently associated with overall or specific cancer risk  nor with cancer survival after diagnosis33. Loss of chromosome Y is a frequent occurrence in bladder tumors35. In the present study, we found that mosaic loss of chromosome Y in blood lymphocytes was not associated with bladder cancer risks in males. We observed no significant association between loss of chromosome X and bladder cancer risk among females.Sex chromosome aneuploidy, particularly loss of chromosome X or Y, in blood lymphocytes has been shown to be associated with agingA number of strengths are presented in this study. This is one of the largest cytogenetic evaluations undertaken for bladder cancer, which allowed us to assess chromosome specific CAs and identified a set of chromosome specific CAs for UC and SCC separately. The unique epidemiological study setting allowed for evaluation of several known bladder cancer risk factors, such as tobacco smoking and schistosomiasis.Among the limitations of the current study is the case-control study design. Retrospective case-control study design can not rule out the possibility of reverse causality. However, the blood samples in cases were collected before any treatment was implemented, thus minimizing the effect of treatment on the quality of the blood. The frequency of CAs in blood lymphocytes was not associated with tumor grade and muscle invasiveness  who reported as either (i) urothelial carcinoma (UC), (ii) squamous cell carcinoma (SCC), (iii) adenocarcinoma, or other, including undifferentiated carcinoma. Carcinomas that metastasized to the bladder were excluded. This report includes subjects enrolled between January, 2010 and June, 2014.The study population accrual and eligibility criteria were described previouslyNon-cancer controls were randomly selected from the general population to frequency-match the cumulative group of cases on gender and age . All controls fulfilled the following eligibility criteria: (i) no known diagnosis of any cancer; (ii) between ages 19 and 80; and (iii) self-identified as able to participate in an interview. Using a portable ultrasound machine, the physician accompanying the recruitment team conducted an abdominal ultrasound examination to rule out asymptomatic abdominal mass. The interview and phlebotomy were conducted at the participants’ home. The participation rates were 84% for cases and 97% for controls.After explaining the study and obtaining the consent, trained interviewers administered a structured questionnaire, assessing socio-demographic characteristics including current residence and birth governorate, prior medical history with emphasis on schistosomiasis or other urinary tract infection, cigarette and water pipe smoking status and history, and reproductive history (for women). Histories of exposure to environmental tobacco smoke (ETS) at home and outside the home were also recorded.The study was approved by the Institutional Review Boards of Georgetown University, the 2 collaborating cancer centers in Egypt, and the National Scientific and Research Ethical Committee at the Egyptian Ministry of Health and Population. All participants signed an informed consent form and donated a blood sample. Socioeconomic characteristics and epidemiological and clinical data were collected through a structured, in-person interview and review of medical records. All experiments were performed in accordance with relevant guidelines and regulations.37. Briefly, one ml of fresh whole blood was added to 9 ml of RPMI-1640 medium, supplemented with 15% fetal bovine serum, 1.5% of phytohemagglutinin and 100 unites/ml each of penicillin and streptomycin. The blood lymphocytes were cultured at 37 °C for 4 days (92–96 hours) and on the day of harvesting, colcemid (0.2 μg/ml) was added to the culture and incubated at 37 °C for additional one hour. The cells were then treated in a hypotonic solution (0.06 M KCl) and fixed in the fixative (3 parts of methanol with 1 part of acetic acid). The fixed cells were kept at −20 °C for future assays.Blood was obtained by trained interviewers in heparinized tubes and blood samples from cases were collected before any treatments were given to the patients. Blood lymphocyte cultures were set up within 48 hours after blood draw, following the protocol as previously describedChromosome preparations were dropped onto clean microscopic slides to make metaphase spreads. The slides were counterstained with 300 ng/ml 4′-6-diamidino-2-phenylindole (DAPI) in an anti-fade mounting medium. The slides were analyzed using an epifluorescence microscope equipped with a charge-coupled device camera. Metaphase cells were captured with exposure times of 0.05 seconds using a DAPI filter under 1000 x magnification. Digitized metaphase images were analyzed using the Isis software  with karyotyping capability by DAPI banding . For each study subject, 30 metaphase cells were randomly selected from one or two (if the number of good metaphase cells on one slide were less than 30) slides and karyotyped. Structural CAs were classified according to the International System for Human Cytogenetic Nomenclature, including chromosomal translocations, deletions, insertions, dicentric chromosomes, ring chromosomes, marker chromosomes, and structurally abnormal chromosomes. The number of metaphase cells showing any CA or any chromosome specific CA was recorded for data analysis. X or Y chromosome aneuploidy was recorded as number of blood lymphocytes with loss or gain of sex chromosomes. Laboratory personnel who were responsible for blood culture and karyotyping were blind to the case-control status of the subjects. One cytogenetist (Y.L.Z) performed karyotyping for the whole project. Repeated analysis of computerized images from ~5% of the subjects showed 100% agreement in scoring the CAs.The primary exposure of interest was the frequency of blood lymphocytes with structural CAs or with sex chromosome aneuploidy. Tobacco use was categorized as “never users”, “waterpipe only”, “cigarette only”, and “both waterpipe and cigarette”. Participants who had smoked less than 100 cigarettes in their lifetime and had never smoked a waterpipe were classified as “never users”; those who smoked less than 100 cigarettes in their lifetime but reported smoking waterpipe were classified as “waterpipe only” users; “cigarette only” users were those who had never smoked waterpipe but had smoked at least 100 cigarettes in their lifetime; and “both waterpipe and cigarette” users were those reported smoking at least 100 cigarettes in their lifetime and also used waterpipe. Schistosoma infections (any during the lifetime) were self-reported.P-values were two-sided and considered statistically significant if P < 0.05. All analyses were performed using SAS software, version 9.3 .Student’s t-test and Chi-square test were respectively used to compare continuous variables and categorical variables between cases and controls. We used unconditional logistic regression to assess the risk of bladder cancer with the increased frequency of blood lymphocytes showing CAs. The analyses were stratified by age due to the fact that age has been identified in other studies as an important risk factor associated with cancer risk. All models were adjusted for the matching factors – age and gender. In addition, multivariate models were adjusted for body mass index (BMI), education , and tobacco use . The covariates included in the model were selected based on the matching variables and the bivariate relationship between the covariates and the outcome variable. The penalized likelihood estimates for the odds ratios in the logistic models were reported when data were sparse (where the count in sub-category was 5 or less). Supplementary Information