Treatment-related fertility impairment in long-term female childhood, adolescent and young adult cancer survivors: investigating dose-effect relationships in a European case-control study (PanCareLIFE).

STUDY QUESTION: Which chemotherapeutic agents and body site-specific radiation fields are dose-dependently associated with an increased risk of fertility impairment in long-term female childhood, adolescent and young adulthood (CAYA) cancer survivors? SUMMARY ANSWER: Busulfan, lower abdominal radiotherapy (RT) and total body irradiation (TBI) seem to be associated with fertility impairment at any dose, whereas gonadotoxicity of melphalan and procarbazine is suggested at medium/high (>140 mg/m2) or high dose (>5600 mg/m2) therapy, respectively. WHAT IS KNOWN ALREADY: Several treatment-related fertility deficits, as assessed by both self-reported outcomes and hormonal markers are known to occur following treatment of CAYA cancer. However, knowledge regarding precise dose-related estimates of these treatment-related risks are scarce. STUDY DESIGN, SIZE, DURATION: The current case-control study was nested within the PanCareLIFE cohort study. In total, 1332 CAYA survivors from 8 countries, 9 institutions and 11 cohorts, participated in and contributed data to the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants were female 5-year CAYA cancer survivors. In total, 450 cases (fertility impaired survivors) and 882 matched controls (not fertility impaired survivors) were included. Fertility impairment was defined using both questionnaire data (primary or secondary amenorrhea; use of artificial reproductive techniques; unfulfilled wish to conceive) and hormonal data (FSH and anti-Müllerian hormone (AMH)). Multivariable logistic regression models were used to investigate the effect of (i) alkylating agent exposure, and (ii) dose categories for individual chemotherapeutic agents and for RT-exposed body sites. MAIN RESULTS AND THE ROLE OF CHANCE: A positive dose-effect relationship between cyclophosphamide equivalent dose (CED) score and fertility impairment was found, with survivors with a CED score > 7121 mg/m2 being at a significantly increased risk of fertility impairment (odds ratio (95% CI) = 2.6 (1.9-3.6) P < 0.001). Moreover, cumulative dose variables of the following treatments were significantly associated with fertility impairment: busulfan, carmustine, cyclophosphamide, melphalan, procarbazine, lower abdominal RT and TBI. Busulfan, lower abdominal RT and TBI seem to be associated with fertility impairment at any dose, whereas gonadotoxicity of melphalan and procarbazine is suggested at medium/high (>140 mg/m2) or high dose (>5600 mg/m2) therapy, respectively. LIMITATIONS, REASONS FOR CAUTION: Our study may have been subject to selection bias since data from about half of the original base cohorts were available for the current study. This could impact the generalizability of our study results. WIDER IMPLICATIONS OF THE FINDINGS: We identified survivors at high risk for fertility impairment and, consequently, for a reduced or even absent reproductive life span. Both girls and young women who are about to start anti-cancer treatment, as well as adult female survivors, should be counselled about future parenthood and referred to a reproductive specialist for fertility preservation, if desired. STUDY FUNDING/COMPETING INTEREST(S): This study has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 602030. There are no competing interests. TRIAL REGISTRATION NUMBER: n/a.

Maintenance of cardiorespiratory fitness, body composition, and a physically active lifestyle after structured exercise interventions in individuals with overweight and obesity: A mixed-method follow-up study.

Objectives: The aim of this mixed-method study was to explore maintenance of physical activity and health effects one year after completion of exercise interventions in transport and leisure-time domains of everyday life. We hypothesised that routinisation of active commuting would lead to better maintenance of physical activity and health effects compared with leisure-time exercise. Study design: Mixed-methods follow-up study. Methods: Individuals with overweight/obesity, who completed a 6-month exercise intervention (active commuting by bike (BIKE), moderate (MOD) or vigorous intensity leisure-time exercise (VIG)), were after one year invited to participate in a follow-up visit which included measurements of cardiorespiratory fitness during an incremental bicycle test and body composition using dual-energy X-ray absorptiometry. Variability in maintenance practices was assessed in a sub-sample of participants who experienced the greatest improvements ('VO2peak improvers') and reductions ('VO2peak reducers'), respectively, in cardiorespiratory fitness. Semi-structured interviews were conducted (15-30 min) and analysed using systematic text condensation to identify barriers and facilitators associated with maintenance of physical activity. Results: Out of the 74 participants completing an exercise intervention, 46 (62%) completed follow-up (BIKE: n = 14; MOD: n = 14; VIG: n = 18). Improvements in VO2peak and reductions in fat mass were maintained in BIKE and VIG. Body weight decreased in BIKE and fat free mass increased in VIG. Changes in VO2peak and anthropometry at follow-up did not differ between BIKE and MOD + VIG. Fat mass decreased and recreational physical activity increased in 'VO2peak improvers'. Findings from the interviews suggested that self-monitoring, collective exercising, and new personal exercise challenges facilitate maintenance of a physically active lifestyle. Conclusion: Completion of a structured exercise intervention consisting of 6 months of active commuting or vigorous intensity leisure-time exercise was associated with long-term maintenance of improvements in VO2peak and body composition, whereas moderate intensity leisure-time exercise was not. In contrast to our hypothesis, active commuting was not associated with better maintenance of physical activity and health effects after the intervention compared with leisure-time exercise.

TCERG1L allelic variation is associated with cisplatin-induced hearing loss in childhood cancer, a PanCareLIFE study.

In children with cancer, the heterogeneity in ototoxicity occurrence after similar treatment suggests a role for genetic susceptibility. Using a genome-wide association study (GWAS) approach, we identified a genetic variant in TCERG1L (rs893507) to be associated with hearing loss in 390 non-cranial irradiated, cisplatin-treated children with cancer. These results were replicated in two independent, similarly treated cohorts (n = 192 and 188, respectively) (combined cohort: P = 5.3 × 10-10, OR 3.11, 95% CI 2.2-4.5). Modulating TCERG1L expression in cultured human cells revealed significantly altered cellular responses to cisplatin-induced cytokine secretion and toxicity. These results contribute to insights into the genetic and pathophysiological basis of cisplatin-induced ototoxicity.

Radiotherapy for childhood cancer and risk for congenital malformations in offspring: a population-based cohort study.

Offspring of childhood cancer survivors may be at risk of genetic disease due to the mutagenic cancer treatments received by their parents. Congenital malformations were evaluated in a population-based cohort study of 1715 offspring of 3963 childhood cancer survivors and 6009 offspring of 5657 survivors' siblings. The Danish Central Population Register, Cancer Registry and Hospital Register were used to identify study subjects and congenital malformations. Gonadal and uterine radiation doses were characterized based on standard radiation-treatment regimens. The prevalence of congenital malformations at birth in offspring of survivors (44 cases, 2.6%) was slightly higher but not statistically different from that of offspring of siblings (140 cases, 2.3%) [prevalence proportion ratio (PPR), 1.1; 95% confidence interval, 0.8-1.5] or of the general population (observed-to-expected ratio, 1.2; 0.9-1.6). Including malformations diagnosed later in life did not change the ratios appreciably. The risk for malformations was slightly higher in the offspring of irradiated parents than in that of non-irradiated parents (PPR 1.2 vs 1.0) but was unrelated to gonadal dose. This study provides evidence that cancer therapy of children does not increase the risk for malformations in their offspring. Continued monitoring of genetic risks among their offspring, however, is warranted.

Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum.

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.

Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids.

The amino-terminal propeptide of carboxypeptidase Y (CPY) is necessary and sufficient for targeting this glycoprotein to the vacuole of Saccharomyces cerevisiae. A 16 amino acid stretch of the propeptide was subjected to region-directed mutagenesis using randomized oligonucleotides. Mutations altering any of four contiguous amino acids, Gln-Arg-Pro-Leu, resulted in secretion of the encoded CPY precursor (proCPY), demonstrating that these residues form the core of the vacuolar targeting signal. Cells that simultaneously synthesize both wild-type and sorting-defective forms of proCPY efficiently sort and deliver only the wild-type molecule to the vacuole. These results indicate that the PRC1 missorting mutations are cis-dominant, implying that the mutant forms of proCPY are secreted as a consequence of failing to interact with the sorting apparatus, rather than a general poisoning of the vacuolar protein targeting system.

Functional differences in yeast protein disulfide isomerases.

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

Active-site residues of procarboxypeptidase Y are accessible to chemical modification.

The accessibility of the active-site cleft of procarboxypeptidase Y from Saccharomyces cerevisiae has been studied by chemical modifications of two specific amino-acid residues. Previous studies have shown that these residues, Cys-341 and Met-398 in the mature enzyme, are located in the S1 and S'1 substrate binding sites, respectively, of carboxypeptidase Y. We have found that these residues also in proCPY are accessible to modification with fairly bulky reagents and in the case of Met-398 the rate of modification is even faster than in carboxypeptidase Y. While the catalytic serine in the mature enzyme reacts with diisopropylfluorophosphate, this is not the case for procarboxypeptidase Y.

Decreasing late mortality among five-year survivors of cancer in childhood and adolescence: a population-based study in the Nordic countries.

PURPOSE: To assess the risk of death in patients who survive more than 5 years after diagnosis of childhood cancer and to evaluate causes of death in fatal cases. PATIENTS AND METHODS: This was a population-based study in the five Nordic countries (Denmark, Finland, Iceland, Norway, and Sweden) using data of the nationwide cancer registries and the cause-of-death registries. The study cohort included 13,711 patients who were diagnosed with cancer before the age of 20 years between 1960 and 1989 and who survived at least 5 years from diagnosis. By December 31, 1995, 1,422 patients had died, and death certificates were assessed in 1,402. Standardized mortality ratios (SMRs) for validated causes of death were calculated based on 156,046 patient-years at risk. RESULTS: The overall SMR was 10.8 (95% confidence interval [CI], 10.3 to 11.5), mainly due to high excess mortality from the primary cancer. SMR for second cancer was 4.9 (95% CI, 3.9 to 5.9) and was 3.1 (95% CI, 2.8 to 3.5) for noncancer death. The pattern of causes of death varied markedly between different groups of primary cancer diagnoses and was highly dependent on time passed since diagnosis. Overall late mortality was significantly lower in patients treated during the most recent period of time, 1980 to 1989, compared with those treated from 1960 to 1979 (hazard ratio, 0.61; 95% CI, 0.54 to 0.70), and there was no increase in rates of death due to cancer treatment. CONCLUSION: Long-term survivors of childhood cancer had an increased mortality rate, mainly dying from primary cancers. However, modern treatments have reduced late cancer mortality without increasing the rate of therapy-related deaths.

Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo.

Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast.

Connective tissue disease and other rheumatic conditions following cosmetic breast implantation in Denmark.

OBJECTIVE: To examine the occurrence of connective tissue diseases (CTDs) as well as ill-defined and other rheumatic conditions among Danish women with cosmetic silicone breast implants. PATIENTS AND METHODS: A total of 2761 women with breast implants and 8807 control subjects were identified from plastic surgery private clinics and from public hospital plastic surgery departments. Women operated on at plastic surgery private clinics were identified through the files of each clinic, while women operated on at public hospitals were identified using the nationwide Danish National Registry of Patients. The control group consisted of women who underwent cosmetic surgery other than breast implantation or who only had a consultation. All women were followed up from January 1, 1977, through December 31, 1996, through the Danish National Registry of Patients for the occurrence of CTD as well as ill-defined and other rheumatic conditions. For the study period January 1, 1977, through December 31, 1994, the Danish National Registry of Patients contains information on hospitalization only, whereas data on outpatient visits are included from 1995 on, thus improving the sensitivity of the data. The implant and control groups were compared with the Danish population rates for CTD and ill-defined and other rheumatic conditions, and a direct comparison between the implant and control groups was also performed. RESULTS: When compared with rates from the general population, no excess of definite CTD was observed in the implant cohorts. For ill-defined and other rheumatic conditions, statistically significant excesses of unspecified rheumatism were observed in both the implant and control cohorts when compared with national rates. A direct comparison between the implant and control cohorts found no material differences between the groups. CONCLUSIONS: The findings of this study support previous investigations and independent review panel conclusions that an association between silicone breast implants and definite CTDs is unlikely. The observation of an excess of unspecified rheumatism among women with implants and among control women suggests that women undergoing cosmetic plastic surgery have hospitalization rates for this condition in excess of those from the general population.

Increased risk of antidepressant use in childhood cancer survivors: a Danish population-based cohort study.

AIM: Childhood cancer survivors are at risk of both somatic and mental late effects, but large population-based studies of depression are lacking. METHODS: Risk of antidepressant use was evaluated in a population-based cohort of 5452 Danish children treated for cancer in 1975-2009 by linkage to the National Prescription Drug Database, which worldwide is the oldest nationwide registry of prescription medication. Hazard ratios (HRs) for antidepressant use were estimated in a Cox proportional hazards model stratified on sex, with population comparisons as referents. RESULTS: Overall, childhood cancer survivors were at increased risk of having antidepressants prescribed (HR, 1.4; 95% confidence interval (CI), 1.3-1.5). The excess absolute risk of antidepressant use was 2.5 per 1000 person-years (95% CI, 1.7-3.3), equivalent to an excess of 2.5 survivors for every 100 survivors followed for 10years. Increased HRs of 30-50% were seen for survivors of cancers of all main groups (haematological malignancies, central nervous system (CNS) and solid tumors); the highest risk was among children treated with haematopoietic stem cell transplantation (HR, 1.9; 95% CI, 1.2-3.1). Our data suggested that the risk was most pronounced for children treated in the most recent calendar periods (test for interaction between cancer and calendar periods: P<0.001), especially for survivors of haematological cancers (P=0.007). Interaction analysis of the effect of parental socioeconomic position and psychiatric disease on the association between childhood cancer and antidepressant use indicated no modifying effect. CONCLUSION: Childhood cancer survivors should be followed-up for depression. Our results indicate an increasing need for follow-up especially in survivors treated by more recent, intensive anticancer treatment.

Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases.

Barley LTP1 belongs to a large family of plant proteins termed non-specific lipid transfer proteins. The in vivo function of these proteins is unknown, but it has been suggested that they are involved in responses towards stresses such as pathogens, drought, heat, cold and salt. Also, the proteins have been suggested as transporters of monomers for cutin synthesis. We have analysed the stability of LTP1 towards denaturant, heat and proteases and found it to be a highly stable protein, which apparently does not denature at temperatures up to 100 degrees C. This high stability may be important for the biological function of LTP1.

Neurologic disease among women with breast implants.

OBJECTIVE: To investigate the risk of neurologic disease among women with silicone breast implants. BACKGROUND: Since 1992, several case series reported an association between silicone breast implants and neurologic diseases. METHODS: Between 1977 and 1992, 1,135 women received cosmetic silicone breast implants, and 7,071 women had breast reduction surgery, as identified by the Danish National Register of Patients (NRP). NRP files provided information on numbers and types of subsequent neurologic disorders at hospital discharge, which were compared with expected numbers, calculated on the basis of national hospital discharge rates. RESULTS: In the two study cohorts, hospital discharge rates for neurologic diseases were raised by some 60% to 70% compared with Danish women in general. Among women with silicone breast implants, 13 subsequently developed a neurologic disorder compared with 7.7 expected; whereas in the comparison group, 63 observed versus 39.1 expected disorders were recorded. These results indicate that relative to the comparison cohort, women with implants had no excessive levels of definite neurologic disease. Furthermore, medical record reviews revealed that the majority of women with implants discharged with a neurologic diagnosis had either symptoms before implant surgery or neurologic symptoms secondary to degenerative diseases. CONCLUSIONS: Our findings do not support the hypothesis of silicone-induced neurologic disease. The reasons for the elevated rates of neurologic disease in both the exposed and comparison cohorts remain unclear, but may reflect selection processes associated with these women seeking medical care more often than the general population.

Effect of photofrin II and light energy on retinoblastoma-like cells in vitro. Dose-response relationships, effect of light dose rate and recovery ratio.

A clonogenic survival assay on retinoblastoma-like cells (EXP-5 cells) was applied in vitro to assess the effect of photodynamic therapy at relative low concentrations of photofrin II and high doses of light energy. The cellular concentration of photofrin II was estimated by spectrofluorimetry. The cellular content of photofrin II increased with the concentration in the medium (up to 3 micrograms/ml) and the incubation time (up to 24 h) in cells first incubated in photofrin II for 24 h and then in photofrin-II-free medium for 24 h. There was an obvious dose-response relationship between cell damage and photodynamic therapy both for the cellular photofrin II content and the total amount of light energy delivered. The dose rate of light energy had no influence on the cell damage. The cells had a recovery factor of only 1.34, suggesting a low repair of sublethal damage. An approximately linear isoeffect curve at the 10% survival level was described as a function of photofrin II concentration and light energy, suggesting a predictable reversible relationship between them. The results suggest that retinoblastoma cells are very sensitive to photodynamic therapy.

Radionuclides deposition over Antarctica.

A detailed and comprehensive map of the distribution patterns for both natural and artificial radionuclides over Antarctica has been established. This work integrates the results of several decades of international programs focusing on the analysis of natural and artificial radionuclides in snow and ice cores from this polar region. The mean value (37+/-20 Bq m(-2)) of (241)Pu total deposition over 28 stations is determined from the gamma emissions of its daughter (241)Am, presenting a long half-life (432.7 yrs). Detailed profiles and distributions of (241)Pu in ice cores make it possible to clearly distinguish between the atmospheric thermonuclear tests of the fifties and sixties. Strong relationships are also found between radionuclide data ((137)Cs with respect to (241)Pu and (210)Pb with respect to (137)Cs), make it possible to estimate the total deposition or natural fluxes of these radionuclides. Total deposition of (137)Cs over Antarctica is estimated at 760 TBq, based on results from the 90-180 degrees East sector. Given the irregular distribution of sampling sites, more ice cores and snow samples must be analyzed in other sectors of Antarctica to check the validity of this figure.

[Cancer and prevention]. / Kraeft og forebyggelse i Danmark.

INTRODUCTION: The aim of the study was to estimate the preventable potential of various types of cancer in Denmark on the basis of present knowledge. MATERIAL AND METHODS: The well-documented factors in lifestyle and environment causing cancer in Denmark were identified from the IARC Monograph series. The population attributable risk per cent (PAR%) and the annual number of preventable cancers were calculated for each aetiology and cancer type around the year 2000. RESULTS: A large proportion of the cancers occurring in the lungs, larynx, upper digestive tract, skin, lower urinary tract, and the uterine cervix is potentially avoidable, whereas only a small proportion of breast and colorectal cancers is preventable on the given knowledge. The main causative factors include active and passive smoking, alcohol intake, exposure to asbestos and other occupational carcinogens, solar and ionising radiation, obesity, human papillomavirus infection in the female genital tract, and infection with Helicobacter pylori. More than 5000 cancers in men and almost 3500 in women annually in Denmark could have been avoided by eliminating exposure to these known carcinogens. This is equivalent to 39% and 23% of all cancers occurring respectively in men and women, around the year 2000. Smoking habits account for more than half of these avoidable cases. DISCUSSION: The incidence of cancer could be greatly reduced through primary prevention, especially of tobacco smoking, which is the major single factor. A large proportion of the cancers occurring in the lungs, larynx, upper digestive tract, skin, lower urinary tract, and the uterine cervix are potentially avoidable. More research in the field of aetiological factors causing female breast cancer and colorectal cancer is much needed in order to be able to prevent these types of cancer.

Multiple-type human papillomavirus infection in younger uncircumcised men.

A cohort of 388 young men enrolled for military service in the Danish army was established and the participants underwent a clinical examination with human papillomavirus (HPV) testing. In addition, a questionnaire containing questions regarding sociodemographic variables, sexual habits and lifestyle factors was completed. The prevalence of HPV was 33.4% in this cohort of uncircumcised men aged 18-29 years. Multiple HPV types were prevalent with one-third of the HPV-positive men being positive for more than one HPV type. Number of recent sexual partners and infrequent condom use were strong risk factors, particularly in men having multiple HPV types. Our findings re-emphasize the importance of sexual transmission and also point to a role of factors that may be related to individual susceptibility as genital warts, alcohol intake and, to a lesser extent, smoking were strongly associated with having multiple HPV types.