Genomic alterations in ovarian endometriosis and subsequently diagnosed ovarian carcinoma.

STUDY QUESTION: Can the alleged association between ovarian endometriosis and ovarian carcinoma be substantiated by genetic analysis of endometriosis diagnosed prior to the onset of the carcinoma? SUMMARY ANSWER: The data suggest that ovarian carcinoma does not originate from ovarian endometriosis with a cancer-like genetic profile; however, a common precursor is probable. WHAT IS KNOWN ALREADY: Endometriosis has been implicated as a precursor of ovarian carcinoma based on epidemiologic studies and the discovery of common driver mutations in synchronous disease at the time of surgery. Endometrioid ovarian carcinoma and clear cell ovarian carcinoma are the most common endometriosis-associated ovarian carcinomas (EAOCs). STUDY DESIGN, SIZE, DURATION: The pathology biobanks of two university hospitals in Sweden were scrutinized to identify women with surgically removed endometrioma who subsequently developed ovarian carcinoma (1998-2016). Only 45 archival cases with EAOC and previous endometriosis were identified and after a careful pathology review, 25 cases were excluded due to reclassification into non-EAOC (n = 9) or because ovarian endometriosis could not be confirmed (n = 16). Further cases were excluded due to insufficient endometriosis tissue or poor DNA quality in either the endometriosis, carcinoma, or normal tissue (n = 9). Finally 11 cases had satisfactory DNA from all three locations and were eligible for further analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Epithelial cells were collected from formalin-fixed and paraffin-embedded (FFPE) sections by laser capture microdissection (endometrioma n = 11) or macrodissection (carcinoma n = 11) and DNA was extracted. Normal tissue from FFPE sections (n = 5) or blood samples collected at cancer diagnosis (n = 6) were used as the germline controls for each included patient. Whole-exome sequencing was performed (n = 33 samples). Somatic variants (single-nucleotide variants, indels, and copy number alterations) were characterized, and mutational signatures and kataegis were assessed. Microsatellite instability and mismatch repair status were confirmed with PCR and immunohistochemistry, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: The median age for endometriosis surgery was 42 years, and 54 years for the subsequent ovarian carcinoma diagnosis. The median time between the endometriosis and ovarian carcinoma was 10 (7-30) years. The data showed that all paired samples harbored one or more shared somatic mutations. Non-silent mutations in cancer-associated genes were frequent in endometriosis; however, the same mutations were never observed in subsequent carcinomas. The degree of clonal dominance, demonstrated by variant allele frequency, showed a positive correlation with the time to cancer diagnosis (Spearman's rho 0.853, P < 0.001). Mutations in genes associated with immune escape were the most conserved between paired samples, and regions harboring these genes were frequently affected by copy number alterations in both sample types. Mutational burdens and mutation signatures suggested faulty DNA repair mechanisms in all cases. LARGE SCALE DATA: Datasets are available in the supplementary tables. LIMITATIONS, REASONS FOR CAUTION: Even though we located several thousands of surgically removed endometriomas between 1998 and 2016, only 45 paired samples were identified and even fewer, 11 cases, were eligible for sequencing. The observed high level of intra- and inter-heterogeneity in both groups (endometrioma and carcinoma) argues for further studies of the alleged genetic association. WIDER IMPLICATIONS OF THE FINDINGS: The observation of shared somatic mutations in all paired samples supports a common cellular origin for ovarian endometriosis and ovarian carcinoma. However, contradicting previous conclusions, our data suggest that cancer-associated mutations in endometriosis years prior to the carcinoma were not directly associated with the malignant transformation. Rather, a resilient ovarian endometriosis may delay tumorigenesis. Furthermore, the data indicate that genetic alterations affecting the immune response are early and significant events. STUDY FUNDING/COMPETING INTEREST(S): The present work has been funded by the Sjöberg Foundation (2021-01145 to K.S.; 2022-01-11:4 to A.S.), Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement (965552 to K.S.; 40615 to I.H.; 965065 to A.S.), Swedish Cancer Society (21-1848 to K.S.; 21-1684 to I.H.; 22-2080 to A.S.), BioCARE-A Strategic Research Area at Lund University (I.H. and S.W.-F.), Mrs Berta Kamprad's Cancer Foundation (FBKS-2019-28, I.H.), Cancer and Allergy Foundation (10381, I.H.), Region Västra Götaland (A.S.), Sweden's Innovation Agency (2020-04141, A.S.), Swedish Research Council (2021-01008, A.S.), Roche in collaboration with the Swedish Society of Gynecological Oncology (S.W.-F.), Assar Gabrielsson Foundation (FB19-86, C.M.), and the Lena Wäpplings Foundation (C.M.). A.S. declares stock ownership and is also a board member in Tulebovaasta, SiMSen Diagnostics, and Iscaff Pharma. A.S. has also received travel support from EMBL, Precision Medicine Forum, SLAS, and bioMCC. The remaining 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.

Expression of inflammation/pain-related genes in the dorsal root ganglion following disc puncture in rats.

PURPOSE: To determine the expression of inflammation- and pain-related genes at days 1 and 3 in the dorsal root ganglion (DRG) of rats with or without disc puncture, using real-time quantitative polymerase chain reaction (RT-qPCR) with the TaqMan low-density array (TLDA). METHODS: 53 female Sprague-Dawley rats were used. The left facet joint between L4 and L5 was removed, and the DRG and intervertebral disc between the vertebrae were exposed. The L4-5 intervertebral disc was punctured using a 0.4-mm diameter injection needle (disc puncture group) or left unpunctured (sham group). After one or 3 days, the 53 DRGs were harvested, frozen, and assessed for expression of inflammation-related genes. Total RNA was isolated from the DRGs. Expression of 119 genes related to inflammation and pain in the DRG after disc puncture were analysed using RT-qPCR with the TLDA. RESULTS: Of the 95 inflammation-related genes, 78 genes were reliably detected. Two genes were significantly up-regulated: cysteinyl leukotriene receptor 1 (CYSLTR1) at day 3 and interleukin 2 receptor gamma (IL2RG) at day 1, and one gene was significantly down-regulated: phospholipase C beta 3 (PLCB3) at day 1. Of the 24 pain-related genes, 18 genes were reliably detected. Two genes were significantly up-regulated: nitric oxide synthase 1 (NOS1) at days 1 and 3 and 5-HT2A receptor (HTR2A) at day 1. CONCLUSION: Disc puncture may elicit changes in the expression of a variety of genes. Gene expression profiling is a useful tool for detecting new potential pharmaceutical targets for spinal pain syndromes.

The myxoid liposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-kappaB target genes by interaction with NFKBIZ.

FUS (also called TLS), EWSR1 and TAF15 (also called TAF2N) are related genes involved in tumor type-specific fusion oncogenes in human malignancies. The FUS-DDIT3 fusion oncogene results from a t(12;16)(q13;p11) chromosome translocation and has a causative role in the initiation of myxoid/round cell liposarcomas (MLS/RCLS). The FUS-DDIT3 protein induces increased expression of the CAAT/enhancer-binding protein (C/EBP) and nuclear factor-kappaB (NF-kappaB)-controlled gene IL8, and the N-terminal FUS part is required for this activation. Chromatin immunoprecipitation analysis showed that FUS-DDIT3 binds the IL8 promoter. Expression studies of the IL8 promoter harboring a C/EBP-NF-kappaB composite site pinpointed the importance of NF-kappaB for IL8 expression in FUS-DDIT3-expressing cells. We therefore probed for possible interaction of FUS-DDIT3 with members of the NF-kappaB family. The nuclear factor NFKBIZ colocalizes with FUS-DDIT3 in nuclear structures, and immunoprecipitation experiments showed that FUS-DDIT3 binds the C-terminal of NFKBIZ. We also report that additional NF-kappaB-controlled genes are upregulated at the mRNA level in FUS-DDIT3-expressing cell lines and they can be induced by NFKBIZ. Taken together, the results indicate that FUS-DDIT3 deregulates some NF-kappaB-controlled genes through interactions with NFKBIZ. Similar mechanisms may be a part of the transformation process in other tumor types carrying FUS, EWSR1 and TAF15 containing fusion oncogenes.

Chloride retention and release in a boreal forest soil: effects of soil water residence time and nitrogen and chloride loads.

The common assumption that chloride (Cl-) is conservative in soils and can be used as a groundwater tracer is currently being questioned, and an increasing number of studies indicate that Cl- can be retained in soils. We performed lysimeter experiments with soil from a coniferous forest in southeast Sweden to determine whether pore water residence time and nitrogen and Cl- loads affected Cl- retention. Over the first 42 days there was a net retention of Cl- with retention rates averaging 3.1 mg CI- m(-2) d(-1) (68% of the added Cl- retained over 42 days). Thereafter, a net release of Cl- at similar rates was observed for the remaining experimental period (85 d). Longer soil water residence time and higher Cl- load gave higher initial retention and subsequent release rates than shorter residence time and lower Cl- load did. Nitrogen load did not affect Cl transformation rates. This study indicates that simultaneous retention and release of Cl- can occur in soils, and that rates may be considerable relative to the load. The retention of Cl- observed was probably due to chlorination of soil organic matter or ion exchange. The cause of the shift between net retention and net release is unclear, but we hypothesize that the presence of O2 or the presence of microbially available organic matter regulates Cl- retention and release rates.

Nutrient intakes of middle-aged men and women in China, Japan, United Kingdom, and United States in the late 1990s: the INTERMAP study.

The purpose of the study was to compare nutrient intakes among Chinese, Japanese, UK, and US INTERMAP samples, and assess possible relationships of dietary patterns to differential patterns of cardiovascular diseases between East Asian and Western countries. Based on a common Protocol and Manuals of Operations, high-quality dietary data were collected by four standardized 24-h dietary recalls and two 24-h urine collections from 17 population samples in China (three samples), Japan (four samples), UK (two samples), and USA (eight samples). There were about 260 men and women aged 40-59 years per sample--total N=4680. Quality of dietary interview and data entry were monitored and enhanced by extensive systematic ongoing quality control procedures at local, country, and international level. Four databases on nutrient composition of foods from the four countries were updated and enhanced (76 nutrients for all four countries) by the Nutrition Coordinating Center, University of Minnesota, in cooperation with Country Nutritionists. The mean body mass index was much higher for Western than East Asian samples. Macronutrient intakes differed markedly across these samples, with Western diet higher in total fat, saturated and trans fatty acids, and Keys dietary lipid score, lower in total carbohydrate and starch, higher in sugars. Based on extensive published data, it is a reasonable inference that this pattern relates to higher average levels of serum total cholesterol and higher mortality from coronary heart disease in Western than East Asian populations. The rural Chinese diet was lower in protein, especially animal protein, in calcium, phosphorus, selenium, and vitamin A. Dietary sodium was higher, potassium lower, hence Na/K ratio was higher in the Asian diet, especially for Chinese samples. This pattern is known to relate to risks of adverse blood pressure level and stroke. At the end of the 20th century, East Asian and Western diets remain significantly different in macro- and micronutrient composition. Both dietary patterns have aspects that can be regarded, respectively, as adverse and protective in relation to the major adult cardiovascular diseases. In both Asian and Western countries, public efforts should be targeted at overcoming adverse aspects and maintaining protective patterns for prevention and control of cardiovascular diseases.

INTERMAP: the dietary data--process and quality control.

The aim of this report is to describe INTERMAP standardized procedures for assessing dietary intake of 4680 individuals from 17 population samples in China, Japan, UK and USA: Based on a common Protocol and Manuals of Operations, standardized collection by centrally trained certified staff of four 24 h dietary recalls, two timed 24-h urines, two 7-day histories of daily alcohol intake per participant; tape recording of all dietary interviews, and use of multiple methods for ongoing quality control of dietary data collection and processing (local, national, and international); one central laboratory for urine analyses; review, update, expansion of available databases for four countries to produce comparable data on 76 nutrients for all reported foods; use of these databases at international coordinating centres to compute nutrient composition. Chinese participants reported 2257 foods; Japanese, 2931; and UK, 3963. In US, use was made of 17,000 food items in the online automated Nutrition Data System. Average time/recall ranged from 22 min for China to 31 min for UK. Among indicators of dietary data quality, coding error rates (from recoding 10% random samples of recalls) were 2.3% for China, 1.4% for Japan, and UK; an analogous US procedure (re-entry of recalls into computer from tape recordings) also yielded low discrepancy rates. Average scores on assessment of taped dietary interviews were high, 40.4 (Japan) to 45.3 (China) (highest possible score: 48); correlations between urinary and dietary nutrient values--similar for men and women--were, for all 4680 participants, 0.51 for total protein, range across countries 0.40-0.52; 0.55 for potassium, range 0.30-0.58; 0.42 for sodium, range 0.33-0.46. The updated dietary databases are valuable international resources. Dietary quality control procedures yielded data generally indicative of high quality performance in the four countries. These procedures were time consuming. Ongoing recoding of random samples of recalls is deemed essential. Use of tape recorded dietary interviews contributed to quality control, despite feasibility problems, deemed remediable by protocol modification. For quality assessment, use of correlation data on dietary and urinary nutrient values yielded meaningful findings, including evidence of special difficulties in assessing sodium intake by dietary methods.

Higher blood pressure in middle-aged American adults with less education-role of multiple dietary factors: the INTERMAP study.

Extensive evidence exists that an inverse relation between education and blood pressure prevails in many adult populations, but little research has been carried out on reasons for this finding. A prior goal of the INTERMAP Study was to investigate this phenomenon further, and to assess the role of dietary factors in accounting for it. Of the 4680 men and women aged 40-59 years, from 17 diverse population samples in Japan, People's Republic of China, UK, and USA, a strong significant inverse education-BP relation was manifest particularly for the 2195 USA participants, independent of ethnicity. With participants stratified by years of education, and assessment of 100+ dietary variables from four 24-h dietary recalls and two 24-h urine collections/person, graded relationships were found between education and intake of many macro- and micronutrients, electrolytes, fibre, and body mass index (BMI). In multiple linear regression analyses with systolic BP (SBP) and diastolic BP (DBP) of individuals the dependent variables (controlled for ethnicity, other possible nondietary confounders), BMI markedly reduced size of education-BP relations, more so for women than for men. Several nutrients considered singly further decreased size of this association by > or =10%: urinary 24-h Na and K excretion, Keys dietary lipid score, vegetable protein, fibre, vitamins C and B6, thiamin, riboflavin, folate, calcium, magnesium, and iron. Combinations of these dietary variables and BMI attenuated the education-SBP inverse coefficient by 54-58%, and the education-DBP inverse coefficient by 59-67%, with over half these effects attributable to specific nutrients (independent of BMI). As a result, the inverse education-BP coefficients ceased to be statistically significant. Multiple specific dietary factors together with body mass largely account for the more adverse BP levels of less educated than more educated Americans. Special efforts to improve eating patterns of less educated strata can contribute importantly to overcoming this and related health disparities in the population.

The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses.

The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alga Chlamydomonas reinhardtii. This opsin-related protein (COP) binds retinal and was thought to be the photoreceptor controlling photomovement responses via a set of photoreceptor currents. Unfortunately, opsin-deficient mutants are not available and targeted disruption of non-selectable nuclear genes is not yet possible in any green alga. Here we show that intron-containing gene fragments directly linked to their intron-less antisense counterpart provide efficient post-transcriptional gene silencing (PTGS) in C. reinhardtii, thus allowing an efficient reduction of a specific gene product in a green alga. In opsin-deprived transformants, flash-induced photoreceptor currents (PC) are left unchanged. Moreover, photophobic responses as studied by motion analysis and phototaxis tested in a light-scattering assay were indistinguishable from the responses of untransformed wild-type cells. We conclude that phototaxis and photophobic responses in C. reinhardtii are triggered by an as yet unidentified rhodopsin species.

A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control.

Phospholipase C and two inositol polyphosphate (IP) kinases constitute a signaling pathway that regulates nuclear messenger RNA export through production of inositol hexakisphosphate (IP6). The inositol 1,4,5-trisphosphate kinase of this pathway in Saccharomyces cerevisiae, designated Ipk2, was found to be identical to Arg82, a regulator of the transcriptional complex ArgR-Mcm1. Synthesis of inositol 1,4,5,6-tetrakisphosphate, but not IP6, was required for gene regulation through ArgR-Mcm1. Thus, the phospholipase C pathway produces multiple IP messengers that modulate distinct nuclear processes. The results reveal a direct mechanism by which activation of IP signaling may control gene expression.

Conserved machinery of the bacterial flagellar motor.

Novel periplasmic and cytoplasmic structural modules of the bases of bacterial flagella have been observed in situ and isolated using new biochemical protocols. Flagellar rotation may depend upon interactions of these modules with the intramembrane particle rings, a ubiquitous feature of flagellar bases necessary for torque generation. The outer membrane-associated basal disk of the Wolinella succinogenes polar flagellum has architecture well suited for interaction with the ring particles. However, antibody against the main W. succinogenes basal disk protein did not cross-react with flagella-enriched fractions from Salmonella typhimurium and Bacillus firmus; nor have such structures been observed in these species thus far. Antibodies against two S. typhimurium proteins, FliG and FliM, known to be involved in motor function and part of the cytoplasmic module in this species cross-reacted with flagella-enriched fractions from both W. succinogenes and B. firmus. In addition, flagellar cytoplasmic structure could be isolated from B. firmus. The basal disk may anchor the flagellar motor to the cell wall in some polar bacteria, but this does not seem to be a unique strategy. In contrast, the data indicate that the cytoplasmic module is conserved.

The Dietary Intervention Study in Children (DISC): dietary assessment methods for 8- to 10-year-olds.

OBJECTIVES: The dietary assessment methods used in the Dietary Intervention Study in Children (DISC) are described and the rationale, validity, and/or general usefulness of each are discussed. DESIGN: DISC is the first multicenter, randomized, clinical trial to study the feasibility and long-term efficacy, safety, and acceptability of a fat-moderately diet in 8- to 10-year-old prepubescent children with moderately elevated plasma low-density lipoprotein cholesterol (LDL-C) levels. Final data collection for the original study (DISC I) occurred December 1, 1993; continued intervention and follow-up (DISC II) will extend beyond 1997. SETTING: Six clinical centers across the country participate in DISC. SUBJECTS: Preadolescent boys and girls with fasting LDL-C levels between the 80th and 98th age-specific and sex-specific percentiles established by the Lipid Research Clinics were eligible for the study. The feasibility phase included 140 children who were then enveloped into the full-scale trial. Baseline dietary data for 652 randomized children in the full-scale trial and 6-month results for the feasibility cohort are reported. INTERVENTIONS: Dietary assessment involved several elements: (a) determining eligibility based on consumption of more than 30% of energy from total fat, (b) monitoring adherence to and adequacy of the intervention diet, (c) evaluating acceptability of the diet in the intervention group, and (d) determining appropriate foods for the intervention diet. Methods are described for each purpose. MAIN OUTCOME MEASURES: LDL-C differences between the two groups and differences in total and saturated fat intakes as calculated from three 24-hour recalls were the primary outcome measures. Six-month dietary differences in the feasibility group are reported. STATISTICAL METHODS: Baseline group means and 6-month differences in dietary intake are reported for the full-scale trial and feasibility study, respectively. RESULTS: Baseline mean intake from three dietary recalls for the intervention (n = 328) and control (n = 324) groups, respectively, were as follows: energy = 1,759 kcal and 1,728 kcal; total energy from fat = 33.3% and 34.0%; total energy from saturated fat = 12.5% and 12.7%; and total dietary cholesterol = 209 mg and 195 mg. After 6 months of intervention, percentage of energy from total fat and saturated fat was reduced by 5.1% (P = .004) and 2.9% (P < .001), respectively, in this feasibility subset (n = 73) of the intervention group. Essentially no change in these parameters occurred in the control group (n = 67), which demonstrates a measurable difference in reporting between groups. APPLICATIONS/CONCLUSIONS: Results illustrate the feasibility of implementing a variety of dietary assessment methods among preadolescent children without relying primarily on parental reports.

Effects on serum lipids of adding instant oats to usual American diets.

This study was designed as a test of the serum lipid response and dietary adaptation to recommended daily inclusion of instant oats in an otherwise regular diet. Hypercholesterolemic adults were randomly assigned to a control or intervention group. Participants in the intervention group were given packages of instant oats and requested to eat two servings per day (approximately two ounces dry weight), substituting the oats for other carbohydrate foods in order to maintain baseline calorie intake and keep weight stable. Serum lipids were measured in blood collected by venipuncture at baseline, four weeks, and eight weeks. Baseline mean total cholesterol (TC) levels were 6.56 mmol/L and 6.39 mmol/L for intervention and control groups, respectively. After eight weeks, mean serum total cholesterol of the intervention group was lower by -0.40 mmol/L, and mean net difference in TC between the two groups was 0.32 mmol/L (95% CI: 0.09, 0.54). Low-density lipoprotein-cholesterol was similarly reduced with mean net difference of 0.25 mmol/L (95% CI: 0.02, 0.48) between the two groups. Mean soluble fiber intake increased along with slight self-imposed reductions in mean total fat, saturated fat, and dietary cholesterol intake in the intervention group. Neither group changed mean body weight. Daily inclusion of two ounces of oats appeared to facilitate reduction of serum total cholesterol and LDL-C in these hyperlipidemic individuals.

Dietary assessment in children using electronic methods: telephones and tape recorders.

Selection of dietary assessment methodology is important in dietary intervention studies. Few studies have reported the relative merits of various assessment methods or the feasibility of electronic methods in pediatric populations. Parent-child dyads performed telephone recalls (no. = 32) and tape recorded dietary records (no. = 33). Traditional recalls were first conducted to familiarize parents and children with the telephone recall procedure, to introduce two-dimensional food models, and to instruct on the use of the tape recorder. Parents monitored and documented as unobtrusively as possible the child's intake on the day before the telephone recall and also on the day of the taped record. Children were called at random to reduce bias. Simple correlation coefficients (r) were calculated for nine nutrients and calories for both methods. For telephone recalls, values ranged from a low of r = .64 for saturated and polyunsaturated fat to r = .85 for cholesterol and r = .93 for iron. Tape-recorded data yielded r = .80 or above, except calories with r = .68 (p less than .001 for all values, 1-tail tests). Mean nutrient values were within expected ranges, e.g., 1,800 kcal +/- 500, with saturated fat about 14% of calories per day. Comparisons between parents and childrens reports of food frequencies and portion sizes revealed the best correlations for beverages, bread-cereals-crackers, meat-fish-poultry, and mixed dishes. We conclude that preadolescent children are able to provide dietary intake data using electronic methods in a manner that compares favorably with their parents' written records.