The combined group showed continuous improvement during the follow-up period (p < 0.05). Quality of life was significantly improved (p < 0.05), and treatment credibility was maintained in the BVA and combined groups. No serious adverse events were reported. Combined treatment of BVA and NSAIDs are feasible for the treatment of NCNP, showing high persistence of the effect, credibility, and safety. Additional trials with longer follow-up are needed to confirm this effect.This study aimed to investigate the feasibility of a combined treatment of bee venom acupuncture (BVA) and non-steroidal anti-inflammatory drugs (NSAIDs) for the treatment of non-specific chronic neck pain (NCNP). Patients with NCNP for ≥3 months were randomly allocated to a BVA, NSAIDs, or combined group (1:1:1), receiving 6 sessions of BVA, loxoprofen (180 mg daily), or a combination, respectively, for 3 weeks. Recruitment, adherence, and completion rates were calculated to assess feasibility. Bothersomeness, pain, disability, quality of life, depressive status, treatment credibility, and adverse events were assessed. In total, 60 participants were enrolled, and 54 completed the trial. Recruitment, adherence, and completion rates were 100%, 95%, and 90%, respectively. Bothersomeness, pain, disability, and depressive symptoms significantly improved in all groups after treatment ( Non-specific chronic neck pain (NCNP) refers to persistent pain or severe discomfort in the neck for at least 3 months, without pathological and neurological findings and traumatic causes ,2. The lConventional treatments for NCNP include pharmacotherapy, physical therapy, and surgical interventions. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used for the treatment of NCNP due to anti-inflammatory and analgesic effects . HoweverPharmacopuncture is an acupuncture technique that injects herbal medicine into acupoints, inducing the effects of both herbal medicine and acupuncture. Bee venom acupuncture (BVA) is a representative pharmacopuncture that uses diluted bee venom, exerting both mechanical function from acupoint stimulation and pharmacological action from the bioactive compounds isolated from bee venom. Bee venom induces allergic immune responses such as increased CD16 surface expression . HoweverAs the importance of multimodal approach in pain management is being emphasized ,21, combA total of 62 participants were screened for eligibility, of which 60 were randomly assigned to the BVA (n = 20), NSAIDs (n = 20), and combined treatment (n = 20) groups . Of these, 54 completed the study, and 6 dropped out . Two participants (1 in the BVA group and 1 in the combined group) withdrew their consent to participate in the trial because of personal reasons not related to the BVA and four participants (3 in the NSAIDs group and 1 in the combined group) violated the study protocol by taking prohibited medications, including steroid injection and acupuncture  File S1File S1.The BVA adherence rates in the BVA and combined groups were each 100% (20/20) and 90% (18/20), respectively , and the completion rates of the BVA, NSAIDs, and combined groups were 95% (19/20), 85% (17/20), and 90% (18/20), respectively . In the BVA and combined groups, compliance with BVA treatment for the corresponding clinical trial period was 100%. In addition, mean compliance rate with NSAIDs in the NSAIDs and combined groups was 87.10%  82.45, 91.75) and 90.95% , respectively .p > 0.05; Fisher’s exact test  and one-way analysis of variance (ANOVA) ), except age . Post hoc analysis, according to Scheffé’s method, showed that the age of the NSAIDs group was significantly lower than the other groups; therefore, age was used as a covariate for the outcome analysis to minimize potential bias. Additionally, the proportion of women was higher than that of men in the study population. Body mass index was in the normal or overweight range for most patients, and most did not smoke or drink alcohol . The difference between the three groups was significant (p = 0.0317). Post hoc analysis, according to Scheffé’s method, showed that the VAS for bothersomeness significantly improved in the BVA group when compared with the combined group (mean difference (MD) −16.30, 95% CI −31.48, −1.11). At Week 8 follow-up, the BVA group maintained a decreased state of VAS for bothersomeness, and the NSAIDs group slightly increased, while VAS in the combined group continued to decrease after the treatment ended. The difference between the three groups was significant (p = 0.0214). Post hoc analysis revealed that the BVA group was significantly improved when compared with the NSAIDs group . During the clinical trial period, bothersomeness significantly decreased in all groups compared with before treatment, and the repeated measured analysis of variance (RM-ANOVA) results showed that the interaction effect on time and treatment group was significant  for bothersomeness, there was significant reduction of 34.60 , 24.55 , and 22.25  at Week 4 (our primary time point) compared with baseline in each group  and neck disability index (NDI) score  compared with baseline. Interestingly, the VAS for pain intensity and NDI score continued to decrease at Week 8 follow-up in the BVA  and combined groups (p = 0.0002 and p = 0.0008). Post hoc analysis showed that there was a significant difference between BVA and combined groups at Week 4 in VAS for pain intensity  and between BVA and NSAIDs groups in NDI score at Week 8 . In addition, there was a borderline significant difference between NSAIDs and combined group in favor of the combined group in NDI score at Week 8  and at Week 8 in the BVA and combined groups (p = 0.0002 and p = 0.0245), compared with baseline. There was significant difference between groups at Week 8 (p = 0.0182); post hoc analysis showed a significant difference between BVA and NSAIDs groups  and a borderline difference between NSAIDs and combined groups . At Week 4, depressive mood, as measured by the Beck Depression Inventory (BDI), significantly improved in all groups . These significant effects persisted in the BVA (p = 0.0001) and combined groups (p = 0.0041) at Week 8. There was a significant difference between groups at Week 8 (p = 0.0250); post hoc analysis showed a significant difference between the BVA and NSAIDs groups  and a borderline difference between the NSAIDs and combined groups  score significantly increased in the BVA and combined groups compared with before treatment during the trial period, although the difference between groups was not significant. The 36-item Short Form Health Survey (SF-36) scores were significantly improved in the BVA group at Week 4 (p > 0.05). In the NSAIDs group, credibility decreased significantly at Week 4 compared with baseline (p = 0.0308), especially in willingness to recommend to others (p = 0.0421) and rationality of treatment . In the BVA and combined groups, two and one case of mild itching and redness at injection sites was reported in >179 (1.12%) and >170 visits (0.59%), respectively. These cases were judged to be definitely related to BVA treatment. In the NSAIDs group, one case each of skin urticaria, abdominal pain and nausea, and low back pain due to lumbar sprain (3 cases/170 visits [1.76%]) was reported. Only abdominal pain and nausea were judged to be definitely related to NSAIDs, and the severity of all adverse events was mild. All adverse events spontaneously subsided, and no serious adverse events occurred during the clinical trial period. In addition, there were no significant changes in vital signs or in laboratory liver and renal function tests before and after treatment in all groups  in the BVA and combined groups. It was also high for NSAIDs (89.03%) in the NSAIDs and combined groups. The recruitment, adherence, and completion rate were very high  during the clinical period, showing high feasibility. Treatment credibility did not change before and after treatment in the BVA and combined groups. In addition, our results show that 3 weeks of BVA incremental treatment, NSAIDs treatment, or combined treatment significantly improved NCNP bothersomeness. Interestingly, there was no superior effect of combined treatment compared with single treatment; however, bothersomeness continued to decrease at Week 8 follow-up after treatment ended in the combined group alone. This suggests a long-term effect. Pain intensity and disability significantly improved at Week 4 in all groups, and health-related quality of life significantly improved in the BVA and combined groups. In addition, depressive mood significantly improved in all groups at Week 4, and this effect persisted until Week 8 in the BVA and combined groups. Interestingly, the BVA group showed the greatest improvement, and the combined group showed borderline significant differences, compared with the NSAIDs group at Week 8. The compliance rate of NSAIDs was high; therefore, these results could not be interpreted as differences in compliance. In the BVA and combined groups, adverse reactions related to BVA were mild local reactions despite our BVA incremental dose protocol, and no serious adverse reactions occurred.Interestingly, the treatment effect in the BVA and combined groups persisted for 5 weeks in all outcomes, even after treatment had finished. The NSAIDs group showed only a short-term effect during the 3-week treatment period. The primary outcome, VAS for bothersomeness, showed continuous improvement in the combined group alone at Week 8 follow-up, although the difference was not significant compared with the single treatment groups. These results are similar to a previous study showing that the treatment effect of 2 months of BVA and physiotherapy persisted for 1 year compared with normal saline injection plus physiotherapy in patients with adhesive capsulitis of the shoulder . TherefoThe NSAID used in this study was loxoprofen, one of the most frequently prescribed anti-pain drugs in East Asian countries . LoxoproIn our study, BVA alone showed a significant difference compared with the combined group in VAS for bothersomeness and pain intensity at Week 4. This is different from a previous study that reported a combined treatment of morphine and BVA was most effective compared with single treatment . MorphinOur study has the strength of assessing the feasibility of the combined treatment of BVA incremental treatment and NSAIDs for participants with NCNP for the first time, which reflects the real-world practice. However, due to the nature of the research design and interventions, we could not perform blinding of participants and practitioners, which might affect the subjective outcome measures of our study. Nevertheless, we sought to apply a rigorous methodology by minimizing detection bias through blinding of the outcome assessor. Additionally, since there was no placebo group in our study, it is difficult to evaluate the effect of each treatment, and it is uncertain whether symptom relief occurred according to the natural course. In future studies, it will be necessary to evaluate the efficacy of the combined treatment of BVA and NSAIDs compared with appropriate controls.In conclusion, the current study showed high feasibility of combined treatment of BVA and NSAIDs on people with NCNP, showing high recruitment, adherence, and completion rates and high treatment compliance and credibility without serious adverse events. Additional full-scale confirmatory randomized controlled trials on the combined treatment should be performed, especially to examine the long-term effects.ClinicalTrials.gov (registration number: NCT01922466) and a detailed study protocol has been published [This was a 3-arm parallel, randomized, assessor-blinded, clinical trial performed at the spine center of Kyung Hee University Hospital at Gangdong (KHUHGD), Seoul, Republic of Korea from July 2013 to July 2014. Participants who voluntarily signed the informed consent after an explanation of the study and met the eligibility criteria were randomly assigned at a 1:1:1 ratio into the 3 weeks of BVA , NSAIDs, or combined treatment groups. This study was approved by the institutional review board of KHUHGD  before the start and was conducted according to Declaration of Helsinki. The protocol of this trial was registered at ublished .To the best of our knowledge, this is the first study to assess the feasibility of a combined treatment of BVA and NSAIDs compared with BVA or NSAIDs treatment alone in patients with NCNP. As a pilot study, a sample size of 20 participants in each group (N = 60), was determined to be adequate for assessing feasibility considering the practical considerations and following discussions with clinical experts and a statistician.Participants were recruited using online  and offline  advertisements. Written informed consent was obtained after a detailed explanation of the study characteristics, including study objectives, procedures, and potential benefits and harms. Eligibility was evaluated according to pre-determined inclusion and exclusion criteria . In addiAn independent statistician, who was not involved in clinical trial procedure and evaluation, generated random sequence numbers. A simple random number generation was conducted using the “RAND” command in Excel 2007 software , with an allocation ratio of 1:1:1 to each group. Sealed opaque envelopes were used for allocation concealment and were kept in double-locked cabinets at hospital. The envelopes were sequentially opened by the clinical research coordinator (CRC) at the baseline visit. Due to the nature of the clinical trial design, the BVA practitioners and participants could not be blinded. Therefore, we blinded the outcome assessors and data analysts to participant allocation by not involving them in the treatment procedures to minimize detection bias. In addition, training was performed that included the prohibition of any conversation about the treatment procedure to maintain the blindness of outcome assessors.A baseline visit was scheduled within 2 weeks of screening visit for participants who met all inclusion and exclusion criteria. In the BVA group, BVA was administered perpendicularly to 12 fixed acupoints  at a depth of 0.5–1.0 cm (subcutaneously) without needle retention, using 1 mL 26-gauge sterile disposable syringes . Participants felt stiffness when injecting the needle and a skin stimulation response. BVA was performed twice a week for 3 weeks , with a weekly dose incremental protocol to minimize adverse events and maximize the treatment effect. This was based on clinical experience and previous studies ,38: 0.2 The NSAIDs group was prescribed loxoprofen , with a dose of oral administration of 1 tablet at a time, 3 times a day, for 3 weeks. The combined treatment group received both BVA and NSAIDs treatment for 3 weeks at the same time, and the treatment contents were the same as in each group.During the clinical period, except for NSAIDs provided in this clinical trial in the NSAIDs or combined group, any concurrent treatment for neck pain was prohibited, including surgical intervention, steroid injection, muscle relaxant, psychotropic drugs, narcotics, physical therapy, acupuncture, and moxibustion. If the participants had been administered analgesics before the trial, they were enrolled after a 15-day washout period to prevent the previously administered analgesics from affecting the clinical trial results. Concomitant use of other drugs that did not affect the interpretation of the results of this clinical trial was allowed under the judgment of the investigator, and all drugs administered during the clinical trial were recorded in the case report form.Demographic characteristics, including sex, age, height, weight, smoking, drinking, and medical history, were collected from all participants at baseline. To examine the feasibility of a confirmatory clinical trial, we calculated the recruitment rate (number of enrolled participants/number of screened participants), completion rate , and BVA adherence rate (number of participants who completed at least 4 of 6 BVA treatments/number of enrolled participants). In addition, compliance rate with BVA and NSAIDs for the corresponding clinical trial period was calculated.The primary outcome was the 100 mm VAS for bothersomeness due to NCNP measured at Week 4 (1 week after 3 weeks of treatment), the primary time point. Participants rated the clinical severity and discomfort on activities of daily life due to NCNP over the past week, from 0 (absence of bothersomeness) to 100 (worst bothersomeness imaginable) ,41 at scThe secondary outcomes were pain intensity, measured by 100 mm VAS; neck-pain related dysfunction, measured by NDI; quality of life, measured by the EQ-5D and SF-36; and depressive status, measured by the BDI. Pain intensity over the past week was evaluated using 0 (absence of pain) to 100 (worst pain imaginable) VAS at screening, baseline, Weeks 2, 3, 4, and 8 . The NDIThe SF-36 evaluates general health status and quality of life using 36 questions; a higher score indicates a better health status . The BDITreatment credibility was assessed using the credibility test, which consists of 4 questions regarding the expectation of NCNP improvement, willingness to recommend to others, rationality of treatment, and effectiveness for alleviating other complaints . Each itThe safety assessment was conducted by monitoring all adverse events that occurred during the clinical trial by medical examination and participants’ self-report, regardless of their relationship with the intervention. In addition, any significant changes before and after intervention were measured via laboratory liver and renal function tests and vital signs. The adverse events that can occur after BVA include skin reactions at the injection sites, such as pruritus, rash, and swelling, and systemic symptoms, such as headache, generalized myalgia, and nausea . If sucht-test was used to analyze the changes in the results before and after treatment within the groups. RM-ANOVA was performed to explore interactions between time and treatment group. Missing values were corrected by applying the last observation carried forward method. Interim analysis was not performed.Based on the principle of intention-to-treat analysis, all statistical analyses were performed using SAS Version 9.4  by a statistician independent of treatment procedure and evaluation. Normality of data distribution was confirmed by Kolmogorov–Smirnov test and Shapiro–Wilk test, and equal variance was confirmed by Levene’s test. For baseline characteristic of participants, one-way ANOVA was performed for continuous variables, and Fisher’s exact test was performed for categorical variables. A two-sided test with a significance level of 0.05 was performed using an analysis of covariance (ANCOVA) with baseline as the covariate and each group as the fixed factor to test the differences between the three groups. Next, post hoc Scheffé adjustment was performed to compare the differences between two groups. A paired


Lifestyle changes were present among some of the participants, 6.1% stopped smoking, 12.1% stopped consuming alcohol, 53.3% sleep later, 9% became more sedentary, and increased their screen (43%) as well as sitting and lying down time (81.6%). Mexicans with Internet access staying at home during COVID-19 confinement perceived positive changes in the quality of their diet, smoking, and alcohol consumption, but negative changes in the level of physical activity and sleep quality. These results emphasise the relevance of encouraging healthy lifestyle behaviours during and after times of crisis to prevent the risk of complications due to infectious and chronic diseases.Perceived changes in diet quality, emotional eating, physical activity, and lifestyle were evaluated in a group of Mexican adults before and during COVID-19 confinement. In this study, 8289 adults answered an online questionnaire between April and May 2020. Data about sociodemographic characteristics, self-reported weight and height, diet quality, emotional eating, physical activity, and lifestyle changes were collected. Before and after confinement, differences by sociodemographic characteristics were assessed with Wilcoxon, Anova, and linear regression analyses. Most participants were women (80%) between 18 and 38 years old (70%), with a low degree of marginalisation (82.8%) and a high educational level (84.2%); 53.1% had a normal weight and 31.4% were overweight. Half (46.8%) of the participants perceived a change in the quality of their diet. The Diet Quality Index (DQI) was higher during confinement (it improved by 3 points) in all groups, regardless of education level, marginalisation level, or place of residence ( Studies on consumer behaviours describe that purchasing habits change in external contexts such as natural disasters or pandemics. Confinement due to COVID-19 caused changes in human behaviour, services, economy, and food systems . InitialPeople living in social isolation or under a crisis situation may be more likely to initiate harmful behaviours such as smoking, excessive alcohol consumption, and overeating as a mechanism of psychological relief ,7. DespiThe results from a telephone survey conducted by the Mexican Institute of Public Health (ENSARS-CoV-2), in which data from 1000 Mexicans were collected between 11 and 30 May 2020, showed participants had less diversity in their diet, reduced their level of physical activity, and increased sedentary behaviours and sleeping hours . FurtherMexico has a high prevalence of overweight and obesity (75.2% in adults), which places its population at high risk of severe COVID-19 . TherefoFor this cross-sectional study, we structured the “Encuesta sobre hábitos de alimentación y estilo de vida alrededor de la pandemia de Coronavirus (COVID-19) en México” (survey on eating habits and lifestyle around the Coronavirus (COVID-19) pandemic in Mexico: ESCAN-COVID19Mx survey) . SelectiThe questionnaire was distributed using a snowball sampling technique through social media  and email. The sample size was estimated at 2401 participants, hypothesising that a proportion of 50% of the population confined at home would present negative changes in their diet quality, with a margin of error of 2%.TheESCAN-COVID19Mx survey has five components, divided as follows: (I) sociodemographic characteristics, (II) diet quality  (III), eI. Sociodemographic characteristics. We collected information about the participants’ gender, age, place of residence, education, marital status, number of children in the household, and self-reported weight and height. The AMAI system 2018  was usedII. Diet quality. The Mini-Survey to Evaluate the Quality of Food Intake (Mini-ECCA) was included to assess diet quality during and before COVID-19 confinement. The Mini-ECCA has been validated for use in healthy Mexican adults . ParticiIII. Emotional eating. We used the Spanish version of the Adult Eating Behaviour Questionnaire (AEBQ-Esp) validated in Mexican adults from the original AEBQ to assess emotional eating . We did IV. Physical activity and sedentary behaviour. This component was comprised of four questions about the participant’s physical activity and sedentary behaviour during and before the contingency period. The questions about physical activity investigated the days per week that the participant performed 30 min or more of intense physical activity ,17. To mV. Lifestyle factors . We were interested in measuring the impact of different lifestyle factors as a composite score that comprised multiple factors ,20. For Information on six lifestyle factors  was used to construct the Lifestyle Quality Index (LSQI). For diet, the quality response values were from 1 to 4, and for physical activity, from 0 to 4. Subsequently, these values were multiplied by 10 to weight the value of each factor equally. Responses to factors such as smoking, alcohol consumption, sedentary behaviour, and sleeping habits were first assigned a value in the range of −1 to 2, considering −1 or 0 as the least recommended or risky behaviour, and a maximum value of 2 to the recommended or non-risky behaviour. The results were divided by the total number of values of each factor, obtaining a rescaled score within each one, obtaining a maximum value of 10 points. Thus, each factor had the same weight, expressing a gradient from the lowest (0 points) to the highest (10 points) value. For the final lifestyle index, the six components were added with a maximum possible value of 60, which could be interpreted that the higher the score, the better the lifestyle quality.p < 0.05. All data analyses and statistical tests were performed using R .Sociodemographic characteristics of the participants were described using indicators of central tendency and dispersion for quantitative variables, while frequencies and percentages were used for qualitative variables. The DQI, PASBI, and LSQI were described with medians and interquartile ranges (IQR 25–75). To investigate whether there were differences in the above-mentioned indices before and during confinement, by sociodemographic characteristics and weight category, Wilcoxon’s signed ranks test and chi square were used, as appropriate. The means for each emotional eating subscale were obtained and linear regressions for both emotional over and under eating to correlate with BMI were calculated. Statistical significance was set at n = 8289) fully completed the ESCAN-Covid19MX survey between 13 April and 16 May 2020. The majority of participants were women (80%), and participants were within an age range of 18 to 38 years (70%), living in areas with very low levels of marginalisation (82.8%) and had higher education levels (84.2%). Respondents , and 13.3% had a BMI ≥ 30 kg/m2.Half of the respondents (53.1%) had a BMI between 18 and 24.9 kg/mp < 0.005).As shown in The sources used by the participants to keep themselves informed about the COVID-19 pandemic were social media (78.8%); leaders and health professionals (73%); and health authorities, government, and health institutions (78.8%). Regarding money spent on food, 38.9% of the participants perceived having a higher food expenditure than usual during confinement, but 47.8% reported having a lower family income. Nearly half of the households (44%) included minors.p < 0.001) for the full sample. The DQI was higher during confinement in all groups, regardless of education level, marginalisation level, or place of residence (p < 0.001). Moreover, there was a positive association between BMI and the DQI (p < 0.001). Nearly half of the participants (46.8%) perceived a change in the quality of their diet. There was an increase in the consumption of homemade foods (28.4%). The median of the DQI improved by three points .Our results showed that 70% of the respondents perceived no changes in their emotional eating during confinement. The results from the AEBQ-Esp Scale showed that the emotional overeating subscale mean was 2.63 (±0.88), whereas the mean for the emotional undereating subscale was 2.51 (±0.97). Linear regressions between emotional overeating and emotional undereating with BMI showed no significant relationships between either subscales and BMI . However, those who reported performing intense physical activity 3-4 times/week prior to confinement decreased from 25 to 18.5% (p < 0.001). Moreover, Physical Activity and Sedentary Behaviour Index (PASBI) Participants who did not engage in any intense physical activity prior to confinement reported engaging in more intense physical activity during quarantine, as the rate of people engaging on intense physical activity increased from 31.2% to 39.9% . Nearly half of the participants (47.4%) were concerned about their health.Some (29.2%) of the participants described having gained body weight, 6.1% stopped smoking, and 12.1% stopped consuming alcoholic beverages. However, 3% smoked more and 10.2% consumed more alcohol. With regard to sleeping habits, 53.3% of the participants reported sleeping later, 27% had interrupted night sleep, and 18% had disturbed sleeping schedules  showed that during the COVID-19 confinement, their participants had a higher consumption of cereals, fresh fruits and vegetables, as well as meat, fish and shellfish, eggs, dairy products, legumes, oils, condiments, coffee and tea. Also, they increased their consumption of sweetened beverages, sweets and snacks. Unlike our study, their report describes a diet diversity index with an average score of 13.5 (0–18) .In France, the results of the Nutri-Net cohort showed a reduction in the consumption of fresh foods and an increase in foods high in sugar at the beginning of the COVID-19 pandemic . On the Recent studies in other countries have also investigated eating behaviours during the COVID-19 pandemic, reaching conflicting results ,26,27,28p < 0.001).The results from our study suggest that confinement negatively impacted on the physical activity of the population. Our results are similar to findings from studies in other countries ,31, whic2 (p = 0.298) and ≥ 30 kg/m2 (p = 0.609); with a basic education level (p = 0.081); medium, low, to very low degree of marginalisation ; and those who lived in the southern region of Mexico (p = 0.096). Studies prior to the COVID-19 pandemic showed that people in social isolation were likely to initiate or increase behaviours such as smoking or alcohol consumption [The LSQI, constructed with information on smoking, alcohol consumption, diet quality, physical activity, and sedentary behaviour, showed a deterioration in lifestyle factors in both young adults and middle age adults during COVID-19 confinement, but not in the elderly. However, it is striking that the change was not significant in participants with BMI < 18 kg/mm2 p = 0.09; with sumption .In Mexico, the prevalence of excessive alcohol consumption during COVID-19 confinement was 40.43%, being higher in men than women , in individuals aged 20 to 29 years, and those who were single and with more education. A higher prevalence was observed in those who had to go out to work daily during confinement and those who worked from home all the time. In women, high risk consumption of alcohol was higher in urban areas compared to rural areas. Both in men and women, the possibility of excessive alcohol consumption increased with a higher level of schooling and decreased with age . This isIn our study, some of the participants reported having changes in their smoking and alcohol habits, both positive and negative; however, the majority these behaviours remained constant as smoking increased by 3%, while alcohol consumption rose to 10.2%. This situation is in contrast with the findings of the ENSARS-CoV-2, where cigarette consumption increased to 21% and alcohol to 12% .2, while it is known that the prevalence of overweight and obesity in Mexico is around 75% [It is possible that the reduction of tobacco and alcohol consumption may have been for health concerns or that participants solely engaged in these behaviours socially. More research is needed to understand the factors that influenced these behaviour changes and whether these changes were permanent. Sleeping habits were affected in more than a quarter of the participants. Our results were similar to those in ENSARS-CoV-2 . It is wound 75% . This coConfinement during the pandemic magnified existing gender inequalities in Mexico. Domestic violence against women is not uncommon and may have been exacerbated due to economic uncertainty, overcrowding at home, and limited access to support networks. In addition, during quarantine, many women were responsible for increased household chores, caring for children, helping with their school activities as all schools closed during the pandemic, and taking care of sick and elderly family members (including family members with COVID) . This maOne possible limitation of ESCAN-COVID19Mx is that the instrument did not collect information on the type of economic activities, household income, or food security of the participants. In addition, we did not inquire on the reasons for making the behaviour changes and we did not reapply the survey at the end of confinement. Therefore, it is not possible to understand the causality of these changes or whether these were maintained. The recruitment strategy through social networks and snowball methodology resulted in a sample that consisted mainly of women, with an age range of 18 to 38 years, with a high level of education, and living in areas with very low levels of marginalisation, with access to the Internet, and therefore it is not representative of the Mexican population . Due to Despite the study’s limitations, the main strengths of our study are that, to our knowledge, this is the first report with a sample size greater than the estimated that obtained responses from all over the country about the changes in diet quality, physical activity, and lifestyle in Mexicans who stayed home during COVID-19 confinement. The results from this study underscore the importance of improving diet quality in people who stay at home or work from home. A possible reason for this could be that most of the people who participated in this survey continued working from home during lockdown, however in this study we did not collect information about the participant’s current working status to confirm this.ESCAN-COVID19Mx was carried out before the new Mexican frontal warning labelling regulation came into place on the 1 October 2020 ; thus, tThe COVID-19 pandemic has emphasised the importance of preventing chronic degenerative diseases, as evidence suggests that people living with these conditions are at higher risk of developing a more severe condition . TherefoThe results from this study showed that during COVID-19 confinement, Mexicans with access to the Internet that were staying at home and who had a low degree of marginalisation and a higher educational level, increased the quality of their diet through consuming more fresh foods prepared at home, yet they also increased the consumption of sugar-sweetened beverages, reduced their physical activity and possibly the general quality of their lifestyle decreased.


Disorders of metabolic obesity with normal body weight (MONW) are widely recognized risk factors for the development of cardiovascular diseases and type 2 diabetes. Despite this, MONW is not diagnosed in clinical practice. There is no consensus on the definition of MONW, and measuring the degree of insulin resistance or obesity among apparently healthy, non-obese patients is not widely applicable. The awareness of the relationship between metabolic disorders such as MONW and a higher risk of mortality from cardiovascular causes and other related diseases prompts the need for action to be taken aimed at creating appropriate diagnostic models that will allow for the effective detection of those with metabolic abnormalities among people with normal body weight. Such actions are decisive in the prevention and treatment of diseases. Therefore, the purpose of this article is to review the MONW diagnostic criteria used over the years. Modern human lifestyle is not conducive to maintaining health. Sedentary work, low physical activity, improper diet, irregular meals and snacking between them, as well as overeating in the evening, promote obesity . AccordiObesity is usually caused by supplying the body with too many nutrients in relation to the amount needed. This excess is stored in the body as triglycerides, commonly known as fat, and the adipocytes where triglycerides are stored, are known as fat cells. Increased fat mass can manifest itself by increasing the size of the adipocyte cells (hypertrophy) and proliferation (hyperplasia). When adipocytes cannot uptake excess triglycerides it leads to adipogenesis, creating extra space for large amounts of fat to be stored .Excessive body fat is conducive to the development of many diseases, including: metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), hypertension, ischemic heart disease, atherosclerosis, hyperlipidemia, non-alcoholic fatty liver, as well as complications related to the osteoarticular, musculoskeletal and respiratory systems. Moreover, obesity is one of the risk factors for breast, uterine, esophageal and kidney cancer ,6,7.Obesity is a heterogeneous disorder. People with obesity are characterized by inter-individual variability in terms of the distribution of adipose tissue, metabolic profile and the degree of cardiovascular and metabolic risk. Abdominal fat storage is much more conducive to the development of T2DM and coronary diseases than peripheral or gluteal–femoral obesity. Significant anatomical, cellular, molecular, physiological, clinical and prognostic differences are also observed between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) . AlthougVisceral fat became the subject of interest in the 1980s when Ruderman et al.  describeBased on the research carried out so far in the MONW group , it can be concluded that the excessive accumulation of fat, mainly visceral, adversely affects the lipid profile ,14,15, bThe central parts of the complex and still insufficiently recognized pathogenesis of MONW are the increased amount of visceral and subcutaneous fat in the abdominal area, insulin resistance and hyperinsulinemia, which are recognized as key disorders in MONW ,19. The Hypertrophic adipocytes are also a source of pro-inflammatory cytokines that enhance insulin resistance both in the fat cells themselves and in other tissues. Activated by inflammatory mediators , nuclear factor kappa B (NF-kB) and c-Jun N-terminal kinase (JNK) pathways are the link between chronic inflammation and insulin resistance . ObesityThere is evidence from experimental and clinical studies for a causal relationship between the amount of body fat and insulin resistance and the development and maintenance of elevated blood pressure. The increase in the prevalence of arterial hypertension especially concerns visceral obesity . The etiThe excess of energy substrates flowing into the cell in the form of free fatty acids and glucose causes the formation of an increased amount of acetyl-CoA and, thus, NADP in the mitochondria and, as a result, an increase in the biosynthesis of reactive oxygen species (ROS) and the development of oxidative stress .Therefore, it seems that the results of research on the pathogenesis of MONW to date are not unequivocal. The dominant causes are insulin resistance and abdominal obesity. It is believed that the cause of the changes is the increased mass of adipose tissue and its pro-inflammatory activity. Adipose tissue is an active endocrine and paracrine endocrine organ, and the secreted pro-inflammatory substances (adipokines) are an important link between excess body weight, insulin resistance, atherosclerosis and type 2 diabetes. In addition, there is oxidative stress. The effects of abdominal obesity and insulin resistance are summarized in p = 0.005) associated with the MONW phenotype in 170 men and women of Iranian origin. Research in the Korean population has shown that there is an association between the prevalence of MONW and moderate alcohol consumption, and a small amount of time for moderate-intensity physical activity [It is known that the occurrence of MONW is influenced by both environmental factors—lack of physical activity, unhealthy diet, smoking, alcohol consumption—and genetic factors. While comparing eating habits, controlled studies found that women with MONW consumed more saturated fat and less fiber than metabolically healthy women . The effactivity . Smokingactivity . It is cactivity  showed tactivity  found liA literature review was conducted through the steps of Carnwell and Daly , which wIn this review, relevant articles were searched from electronic databases, including PubMed, ScienceDirect, Scopus and Google Scholar. The search process used the Boolean operator AND/OR on the combination of the following keywords: metabolically obese normal weight, metabolically abnormal but normal weight, metabolically unhealthy normal weight, normal weight obese, metabolically unhealthy non-obese, normal weight metabolically unhealthy, MONW, diagnostics.Articles were included for review if they met inclusion criteria: (1) published in English within the publication date from 2002 to November 2021, (2) article types include original research and review articles, (3) fully accessed articles, of which a copy could be obtained by the authors. Articles were excluded if the main results were not in line with the purpose of this literature review.The author of the first MONW diagnostic criteria is Ruderman et al. , who in This system had its drawbacks, requiring the performance of biochemical tests not routinely performed in healthy people (including uric acid concentration). For this reason, the search for much simpler and more accessible diagnostic criteria was started.2. Values from 25.0 to 29.9 kg/m2 are synonymous with overweight, while obesity is found in people with a BMI over 30 kg/m2 [2, respectively, for overweight and obesity), but not for Creole women or for Indian men and women, whose BMI cut-offs are 3–4 units lower [2 in Lebanon [2 for males and ≥24.5 kg/m2 for females for Ethiopian adults [2 for white, black and Hispanic women, respectively [2 for Brazilian women and ≥29.9 kg/m2 for Brazilian men [Anthropometric measurements are one of the simplest methods by which obesity can be identified. The most commonly used and recommended by WHO and the International Obesity Task Force (IOTF) is BMI . The BMIts lower . Moreove Lebanon , ≥22.2 kn adults , ≥25.5, ectively , or ≥24.lian men . Li et alian men .The best anthropometric indicator of abdominal fat is the waist circumference (WC). Measurement is performed with a non-stretchable tape, at the midpoint between the lower rib and iliac crest . In addiAlthough all anthropometric measurements are simple and convenient methods, their accuracy in assessing obesity has been questioned because they are unable to distinguish lean mass from fat mass .The assessment of the fat depot is possible after measuring the body composition. This test allows for precise and accurate measurement of individual body components including muscle mass, lean mass and, most importantly, the percentage of adipose tissue , the knowledge of which, together with the BMI value, can be used as a screening tool ,54.Body composition can be assessed by various methods: computed tomography (CT), magnetic resonance imaging (MRI), dual-energy X-ray absorptiometry (DXA), electrical bioimpedance (BIA), hydrostatic plethysmography, isotope dilution techniques, skinfold method and air displacement plethysmography . The mosPeople with MONW are characterized by an increased content of adipose tissue—in particular, its visceral deposit . Excessi2) in terms of body composition. Using CT, they determined the area of adipose tissue. A deposit of more than 100 cm2 was tantamount to the diagnosis of MONW. The authors showed a significant correlation between visceral fat areas with serum triglycerides levels  and fasting serum levels of insulin  in subjects with MONW. The applied method of body composition analysis is characterized by high quantitative and qualitative accuracy, image resolution, and precision and sensitivity; unfortunately it is cost-consuming, requires trained operators and exposes the patient to ionizing radiation, which is a great difficulty in routine diagnostics [Katsuki et al.  initiategnostics ,62.2). Their cut-off point for the PBF was 30%, which translated into the diagnosis of MONW in 28 out of 74 women (38%). Compared to CT, DXA is cheaper and, above all, exposes the patient to a much lower radiation dose. Radiation in DXA is on average 0.1–4.9 µSv compared to 2000–16,000 in CT [De Lorenzo et al.  used the00 in CT .Kim et al.  assumed Among the presented methods of body composition analysis, DXA is considered the “gold standard”. Studies have shown strong correlations between body composition parameters obtained by DXA and those obtained by CT ,73,74,75In a recent study, Correa et al.  defined 2) aged 35–65 years. MONW diagnostics were based on two models. In model A, the PBF cut-off point was >25% for males and > 30% for females. In model B, the cut-off points depended on age:20–39 years—>19% for men and >32% for women40–59 years—>21% for men and >33% for women60–79 years—>24% for men and >35% for women.Interesting research was performed by Tayefi et al. . They exIn model A, MONW was diagnosed in 38.5% of respondents, and according to model B—in 46.2%. Increasing the amount of adipose tissue with age is a physiological phenomenon, therefore, the adoption of different cut-off points depending on age seems to be the appropriate direction in the correct diagnosis of MONW.Amato et al. , in 2010The authors demonstrated a strong positive correlation between VAI and peripheral glucose consumption during the hyperinsulinemic euglycemia clamp  and the 2), they determined tissue sensitivity to insulin using a metabolic clamp. The glucose consumption of 8 mg/min/kg lean body mass was adopted as the cut-off value for the diagnosis of IO. On this basis, MONW was identified in 13 out of 71 women (18%). The metabolic clamp, although considered the “gold standard”, is technically quite difficult, time-consuming and costly.One of the hallmarks of MONW is the decreased tissue sensitivity to insulin. The first to define MONW as the presence of insulin resistance (IO) was Dvorak et al. . Among pFor the purpose of assessing insulin resistance, HOMA-IR was developed, which is based on measurements of fasting glucose and insulin levels. It is calculated from the formula HOMA−IR=2). After determining the concentration of fasting insulin and fasting glucose, they calculated the measurement of the HOMA-IR index with the cut-off value of 1.69. MONW was identified in 12 women (12.5%). Moreover, the authors concluded that among people with normal body weight, a significant decrease in insulin sensitivity of tissues was observed at lower values.Conus et al.  examined2) with HOMA-IR value within the highest quartile. The prevalence of MONW was 14.2% for men and 12.9% for women.HOMA-IR has also been used by Lee at al. in 2011 . This laMuch interest in the diagnosis of metabolic disorders is focused on the triglycerides–glucose index (TyG), which is the product of fasting blood glucose and triglycerides . It is cIts usefulness as an indicator of insulin resistance has been confirmed in the Asian ,91, LatiTypically, in the identification of MONW, biochemical tests are carried out, mainly involving the measurement of glucose, insulin and triglyceride levels. Nevertheless, there is a trend in searching for new biochemical parameters that may indicate the above-mentioned phenotype of obesity.-BP ≥ 130/85 mmHg or use of antihypertensive medication,-TG ≥ 150 mg/dL or use of lipid-lowering medication,-FG ≥ 100 mg/dL or use of an oral hypoglycemic agent or insulin-HDL cholesterol level <40 mg/dL in men and <50 mg/dL in women or use of medication for reduced HDL cholesterol level-HOMA-IR greater than the 90th percentile in the nondiabetic general Korean population in KNHANES.The concentration of ferritin in the blood serum is a promising indicator. Apart from serving as a biomarker for the assessment of anemia, this protein is associated with insulin function and systemic inflammation. Serum ferritin concentration is elevated in the inflammatory environment and is associated with cardiometabolic disease, which is reflected in insulin resistance . The incIt was concluded that serum ferritin could be a clinical marker reflecting the risk and prevalence of MONW in adult Koreans with a normal BMI. The authors suggested the following serum ferritin cut-off values for the prediction of MONW: 46.87 ng/mL for women and 127.03 ng/mL for men. There are no identical studies for the European population in the literature.In recent years, there has been a tendency to develop indicators using anthropometric measurements and biochemical tests. Kahn  proposedAnother disadvantage in using the index is the fact that it is below zero in women with WC < 58 cm and in men < 65 cm. LAP was intended by the authors to reflect both anatomical and physiological changes resulting from excessive lipid accumulation.Studies found that LAP correlated better with variables of lipid risk, uric acid levels and heart rate compared to BMI. Another study by this author  showed tLipid accumulation product became the foundation for the creation of another marker. Wakabayashi et al., in 2015 , proposep < 0.001). This study assessed the usefulness of CMI in identifying patients with MONW and comparing it with traditional obesity rates. CMI was shown to be positively and independently associated with the presence of the MONW phenotype. Moreover, it was confirmed that CMI is a better screening tool for MONW patients compared to BMI and WC. Li [The TG/HDL-C ratio used reflects small, dense, more atherogenic LDL particles, making it a better predictor of coronary artery disease than classical atherogenic indicators, including the LDL/HDL ratio. In their work, the authors emphasize that CMI may be a useful marker for distinguishing diabetes and suggest greater validity of using this indicator for predicting cardiovascular risk compared to WC or LAP . Researcd WC. Li  showed tSome scientists use diagnostic criteria for classic metabolic syndrome (MetS) in their research on MONW, according to the criteria of the National Cholesterol Education Program Adult Treatment Panel III (NCEPATP III) or proposed by the International Diabetes Federation (IDF). The coexistence of at least three of the five disorders mentioned is synonymous with the occurrence of MetS .2), diagnosed MONW in 12.67% of Asians [In the case of the criteria proposed by the IDF, the percentage of people diagnosed with MONW ranges from 9.46% in the Serbian population  and 12% f Asians . This di-BP ≥ 130/85 mmHg or antihypertensive medication use-Fasting TG level ≥ 150 mg/dL-HDL-C level M: <40 mg/dL, W: <50 mg/dL or lipid-lowering medication use-FG level ≥ 100 mg/dL or antidiabetic medication use-HOMA-IR > 5.13 .Wildman et al., based on the MetS criteria, proposed his own set of criteria :20–34 years—10.3%35–49 years—16.9%50–64 years—41.7%65–79 years—54.7%>80 years—56.2%.The prevalence of MONW depending on age was as follows:2. Based on these criteria, MONW (or as the authors define it—MUNO) was diagnosed in 4762 people (34.4%), of which 2055 (35.4%) were women and 2707 (32.5%) were men. Such a high result is probably the result of adopting such a high cut-off point for BMI.Rotar et al. , in turn-waist circumference > 90 cm for men and >85 cm for women;-TG ≥ 1.70 mmol/L or specific treatment for lipid abnormality;-HDL-C < 1.04 mmol/L or specific treatment for lipid abnormality;-blood pressure ≥ 130/85 mmHg or known treatment for hypertension;-FG ≥ 6.1 mmol/L and/or 2 h plasma glucose ≥ 7.8 mmol/L and/or diabetes mellitus having been diagnosed and currently receiving therapy.Luo et al. , in a stThe cut-off points for PBF were, respectively, <25% for men and <35% for women. According to this criteria, MONW was diagnosed in 90 out of 1186 men (7.6%) and in 95 out of 1578 women (6.0%).The cases of metabolic disorders in people with normal body weight, described in the 1980s by Ruderman , gave ri2 [2 [2 [The key phrase “correct body weight” refers to BMI, which, according to WHO and IOTF recommendations, should be in the range of 18.5–24.9 kg/m2 . Neverth2 [2  or ≤27 k2 [2 [2 .Diagnostics of MONW should begin with anthropometric tests—in particular, waist circumference and body composition testing. The key is to determine the percentage of adipose tissue and its distribution in the body. Particular attention should be paid to visceral fat, which is significantly more metabolically and hormonally active compared to its subcutaneous counterpart. When analyzing the fat deposit, it is reasonable to take into account the adjustment for the age and gender of the test person. There are many methods of body composition analysis, from computed tomography, through dual-energy X-ray absorptiometry, to electrical bioimpedance. The “gold standard” is DXA, which is more precise than BIA and has a lower radiation dose than CT.People with MONW, in addition to increased fatty tissue deposit, are also accompanied by metabolic disorders, including, in particular, carbohydrate metabolism and insulin resistance. Currently, there is a trend in the diagnosis of MONW using the diagnostic criteria of the classical metabolic syndrome. Nevertheless, scientists are looking for further disorders that may indicate abnormalities . There In summary, MONW is undoubtedly a growing problem that should be the focus of further research. Due to the fact that it is a disease that does not show phenotypic signs, screening tests should be carried out, mainly including body composition analysis among young, theoretically healthy people. This will allow for early detection of MONW and appropriate reactions before the occurrence of undesirable consequences—including atherosclerosis or coronary artery disease.


Tumors of colon and rectum other than adenocarcinomas represent a neglected issue from clinical and laboratory points of view because of their rarity. In this review, we summarize and describe the rare histologic entities occurring in colon and rectum. Clinical and pathologic characteristics, prognostic behavior, treatments, and altered genes are reported to provide readers with a paramount and comparative perspective. In relation to this, we propose that improvements in registries and multidisciplinary research are warranted to ameliorate their management.The most frequent form of colorectal cancer is represented by adenocarcinoma being about 98% of tumor histological types. However, other rare histotypes can be found in colon and rectum . Altogether, these forms account for less than 2% of colorectal tumors. There are no specific diagnostic or therapeutic recommended approaches and most of the information available from literature derives from small and retrospective clinical series. In the present study, we provide a paramount and updated view on clinical and biologic characteristics of rare colorectal tumors. Colorectal cancer (CRC) is the third leading cause of cancer-specific death. It has been estimated that, in 2018, there were 1.8 million new cases of CRC in Western countries. The most frequent form of CRC is represented by  adenocarcinoma with three grades of differentiation: well, moderate, poor , PTEN , CDKN2A (p.W110 *), FBXW7 (p.R465H), and AKT1 (p.R23Q) were found in rectal well-differentiated NETs but not in appendiceal ones. No repetitive mutations were described in appendiceal well-differentiated NETs. Interestingly, PTEN (p.G129R), EGFR (p.E709K), and KIT (p.V555I) were shared between rectal and appendiceal NETs [There are very limited data about key-driver mutations in colorectal NETs. Interestingly, in a study reporting targeted pyrosequencing of e tumors . Mutatioeal NETs . FurtherThere are no specific registries for these exceptional forms of colorectal tumors and it has been estimated that less than 80 new cases/year occur in the United States  . Most ofAngiosarcoma accounts for less than 1% of all colorectal sarcomas and it is extremely rare . Only a TP53 and BRAF (p.V600E) can be altered in GCC. Furthermore, CTNNA1, NOTCH1, NUMA1, TGFBR2, USP9X, and ERBB2 mutations have been described. Alterations in ‘typical’ adenocarcinoma key-driver genes—including APC, KRAS, NRAS, PIK3CA, PTEN, or SMAD4 —have not been detected so far [Goblet cell adenocarcinoma (GCC) is a subtype of appendiceal cancer. It is a tumor composed by neuroendocrine and epithelial cancerous cells with positive staining for both glandular and neuroendocrine differentiation markers . The incidence is about 1 per 2 million individuals/year and it affects mainly patients 50–60 years old . The cliMLH-1 and CDX2 and strong expression of calretinin [IDO-1, WARS, GBP1, GBP4, GBP5, PD-1, and PD-L1. Mutations have been described in ARID1A (p.E896*) determining loss of protein function and expression, and BRAF (p.V600E) genes [Medullary carcinoma of the colon is an extremely rare tumor representing 0.05% of all colon cancers . Clinicalretinin . StudiesE) genes .BRAF and NRAS mutations are less frequent compared to cutaneous melanomas. By contrast, activating KIT mutations are reported in more than 30% of cases [BRCA1, HRAS, MLH1, NF1, PDGFRA, and SF3B1 [BRCA1 and MLH-1) are associated with genome instability and can be predictive of response to immunotherapy; others  are under intensive investigation for their potential ‘actionability’.Melanoma can arise in extra-cutaneous tissues including uvea, leptomeninges, and mucous membranes. About 1% of melanomas can present in the gastrointestinal tract. The most frequent localization is in the anorectal region (more than 50% of cases), while the remaining large intestine is involved in less than 1% of cases . About 6of cases . In thesof cases . Other mnd SF3B1 . Some ofColonic solitary plasmacytoma is extremely rare  with very few descriptions available in literature . GeneralK- and N-RAS, PIK3CA, APC, and TP53 mutations are lower compared to adenocarcinoma suggesting that alternate pathways may be involved in SRCC tumorigenesis [Signet ring cell carcinoma (SRCC) represents about 1% of CRCs. It has an intracytoplasmic mucinous component that pushes the nuclei to the periphery of cell, conferring it the typical appearance of a “signet ring”. SRCC of the colon frequently expresses MUC2, CDX2 (nuclear staining), and MUC5AC . The meaigenesis .TP53 (p.Q331 *) and no alterations of KRAS [Primary small cell carcinoma or small cell undifferentiated carcinoma or small cell neuroendocrine carcinoma of the colon is an aggressive tumor, showing early locoregional and distant metastases. It is a subtype of neuroendocrine carcinomas. It is treated separately from other neuroendocrine tumors due to its exceptional incidence. In fact, these tumors account for about 0.2% of all CRCs  ,84. An a of KRAS .Squamous cell carcinoma arising in colon and rectum is a very rare tumor. The incidence is estimated to be less than 1 on 100,000 patients affected by a colorectal tumor . The etiWe have provided a descriptive overview of rare forms of CRCs. However, some issues deserve to be raised and discussed.First, specific staging systems for these forms of CRCs are currently lacking due to their rarity. Thus, the AJCC (American Joint Committee on Cancer) TNM staging system working for the classical adenocarcinoma is applied. However, it does not take into account some biological characteristics  that could have a major role in some forms. This suggests the need for including in the staging systems molecular or biological data with both pathogenic and therapeutic significance. Moreover, the prognostic value of some characteristics overcome that of the volume of masses  such as necrosis in GISTs, density or anarchy of vascular channels in angiosarcomas, squamous components in mixed forms, etc.Second, the improvement of the understanding in biology and pathogenesis of these forms is required and it could inform about the clinical significance of specific mutations (unique for some of them) in publicly available archives . These tools provide practical information for interpreting the clinical and diagnostic significance of some variants and may also help researchers to drive and focus their scientific efforts.In our opinion, improvement of diagnostic tools, including molecular assessment with NGS, will allow better definition of the histologic entities of CRCs. Consequently, it is argued that, in this case, we will be able to increase the diagnosis of ‘rare’ forms of CRCs. This is a phenomenon recently described for NETs and thyroid cancer: in synthesis, the amelioration of detection tools (more sensitive imaging and laboratory methods) optimizes the diagnosis inducing an incidence increase ,94. A siFurthermore, CRC cases are classified according to standardized codes from the International Classification of Diseases (ICD). The code for oncology integrating topographical, morphological and biologic information for CRC is ICDO3-M 8140.3. The question has an apparent formal or bureaucratic significance, but it deserves appropriate discussion. In fact, although specific morphological codes exist for rare entities, they are often not documented in the pathology reports. In other words, assignment of generic morphological codes for CRC accounts for an underestimation of rare CRC frequency into National Tumor Registries as well as of related financial needs. Therefore, awareness of pathologists about the importance to register by using precise codes should be improved.The present study has at least two limits. First, the paucity of specific data on genetic alterations do not give the chance to draw any conclusion on the involvement of the genes in the biology and pathogenesis of the tumors. Second, the source of data is limited by the intrinsic rarity of these tumors as stated in Methods. Thus, the role of many clinic-pathological and prognostic characteristics deserves confirmation in larger series.The lack of specific molecular or biologic signatures can make differentiation between different histotypes of this group of tumors difficult and lead to misinterpretations based only on a ‘morphologic’ point of view. In this context, the emergence of NGS techniques will open a new scenario based upon the complete exome sequencing of these rare tumors to find somatic mutations that could have diagnostics and therapeutic consequences. In fact, day by day, the therapeutic weaponry available for the treatment of tumors is increasing giving the opportunity to include the patients in ‘basket’ trials for the evaluation of the efficacy of these agents in these orphan-drug diseases. Hopefully, implementation of specific registries to collect, systematize, and connect clinical, therapeutic, and genetic information beyond the difficult and utopian design of prospective clinical trials is warranted.


Endocrine-disrupting chemicals (EDCs)—including butyl benzyl phthalate (BBP), perfluorooctanoic acid (PFOA), and zeranol —can interfere with the endocrine system and produce adverse effects. It remains unclear whether pubertal exposure to low doses of BBP, PFOA, and ZAL has an impact on breast development and tumorigenesis. We exposed female Sprague Dawley rats to BBP, PFOA, or ZAL through gavage for 21 days, starting on day 21, and analyzed their endocrine organs, serum hormones, mammary glands, and transcriptomic profiles of the mammary glands at days 50 and 100. We also conducted a tumorigenesis study for rats treated with PFOA and ZAL using a 7,12-dimethylbenz[a]anthracene (DMBA) model. Our results demonstrated that pubertal exposure to BBP, PFOA, and ZAL affected endocrine organs and serum hormones, and induced phenotypic and transcriptomic changes. The exposure to PFOA + ZAL induced the most phenotypic and transcriptomic changes in the mammary gland. PFOA + ZAL downregulated the expression of genes related to development at day 50, whereas it upregulated genes associated with tumorigenesis at day 100. PFOA + ZAL exposure also decreased rat mammary tumor latency, reduced the overall survival of rats after DMBA challenge, and affected the histopathology of mammary tumors. Therefore, our study suggests that exposure to low doses of EDCs during the pubertal period could induce changes in the endocrine system and mammary gland development in rats. The inhibition of mammary gland development by PFOA + ZAL might increase the risk of developing mammary tumors through activation of signaling pathways associated with tumorigenesis. Cancer is the second leading cause of death worldwide according to the Centers for Disease Control and Prevention. In women, breast cancer is the most commonly diagnosed cancer in 159 countries, and is also the leading cause of cancer death in 110 countries .The breast is a hormone-sensitive organ. Changes in circulating or gland-tissue-localized hormones, receptors, or metabolism can have an impact on mammary tissue. Endocrine-disrupting chemicals (EDCs) are a group of chemicals that can mimic or interfere with the body’s hormones. Epidemiological findings support the notion that exposure to environmental pollutants containing EDCs at critical windows during the life course may alter breast development and play a role in changing the susceptibility to breast tumorigenesis ,4. The pBBP is a plasticizer commonly used in construction materials such as vinyl floor tiles, vinyl foams, pipes, coatings, and plastic and rubber products. BBP can be easily released into the environment, where it can enter food via food packaging materials. BBP has been detected in indoor dust, as well as in dietary fats, oils, and infant formulas, at a concentration up to the median of 135 µg/g . The BBPPFOA is a synthetic organic pollutant; it is extensively used in industrial applications, including food packaging, water-resistant coatings, fire extinguishers, floor wax, nonstick coatings on cookware, and many more. PFOA is extremely persistent in the environment and resistant to degradation; it has been detected in the rivers, house dust, popcorn bags, drinking water, and food, at a wide range of concentrations . PFOA haZAL is a metabolite of mycotoxin zearalenone, and is also a synthetic nonsteroidal estrogen used as a growth promoter in livestock under the brand name Ralgro. The use of ZAL has been banned in the European Union since 1985; however, it can still be used in the USA, Canada, and Australia. The study of ZAL in humans and animal models is not as extensive as that of BBP and PFOA. ZAL has been detected in meat, water, grains, and other plant products in the USA, Europe, and Asia . In adolIn addition, EDCs are usually present in the environment as mixtures of low doses. Exposure to mixtures of EDCs may induce additive or synergistic effects that can cause significant adverse effects even when the exposure to a single EDC at such a low dose is not a concern. Therefore, in this study, we also exposed the animals to combinations of BBP, PFOA, and ZAL at low doses, and compared their effects to those of single exposures. Here, we present our novel findings on the effects of pubertal exposure to BBP, PFOA, and ZAL—alone or in combination—on the endocrine system, rat mammary gland development, and DMBA-induced tumorigenesis. We analyzed the mammary gland transcriptome and revealed the pathways and important genes that might potentially contribute to the changes in mammary gland development and tumorigenesis induced by the exposure. p = 0.04) in the D50 study  and 50 (for D100) down- or upregulated genes with the highest or lowest expression for the PFOA + ZAL group are shown in To identify the biological processes (BPs) and pathways enriched in DEGs, lists of up- and downregulated genes were analyzed using the DAVID tool and the Shiny application . At D50,BPs affected by PFOA + ZAL were very different from those affected by PFOA_L and ZAL_L at D50 A,B. MultWnt4, the upregulation of Adcy2, Igf1, and Mecom at D50, and the upregulation of Hes1, Met, and Mecom1 at D100 by PFOA + ZAL. Hes1, Igf1, and Mecom1 were also upregulated by ZAL_L at D100. Selected DEGs from the pathways of interest in the PFOA + ZAL group were validated by qRT-PCR A. We conCd8a was significantly increased in PFOA + ZAL, and tended to be upregulated in the ZAL_L group at D50  and lower overall survival probability  than control rats challenged with DMBA. There were no differences between control and PFOA_L- or ZAL_L-treated rats  D. Urine Rodents are the most widely used animal models for assessing the risk of exposure to EDCs on mammary gland development and tumorigenesis, because their mammary gland morphology and development process are relatively similar to those of humans. Among rodent models, Sprague Dawley rats are highly sensitive to the DMBA-induced mammary tumorigenesis. In the present study, we exposed Sprague Dawley rats to BBP_L at 0.5 mg/kg/day and BBP_H at 5 mg/kg/day, PFOA_L and ZAL_L at 0.01 mg/kg/day, PFOA_H and ZAL_H at 0.1 mg/kg/day, and the combinations of low doses, for 21 days during the pubertal period. These doses were selected based on the literature published before we started the animal study in 2016, with the intention of mimicking environmental exposure levels. We showed that pubertal exposure to BBP, PFOA, and ZAL induced changes in endocrine organs, serum E2 levels, and mammary gland development in rats. We observed that PFOA + ZAL exposure induced more phenotypic changes in the mammary glands than the other groups, and had the greatest number of DEGs in the mammary gland transcriptome at age D50. We found that BBP_H, ZAL_L, and ZAL_H, as well as all combinations, induced large amounts of DEGs in the mammary glands at D100. We revealed that PFOA + ZAL exposure mainly affected biological processes related to organ development and system development, and signaling pathways associated with mammary gland development and tumorigenesis. We demonstrated that pubertal exposure to PFOA + ZAL increased susceptibility to mammary tumorigenesis when challenged with DMBA. The pubertal period is a stage when the mammary gland undergoes dramatic changes under the influence of hormones, growth factors, and other signals. Although we did not observe changes in serum E2 and P4 levels, or in hormone receptors in mammary epithelial cells, as a result of PFOA + ZAL, the number of TEBs in the PFOA + ZAL group was significantly increased at D100 compared to controls. It has been reported that the estrous cycle has an impact on the ductal histo-architecture and lobuloalveolar structure of rat mammary glands ; we evalFor the analysis of serum hormones and uterine weight, in addition to the analysis based on the exposure groups, we also performed analyses in function of the estrous cycle—independently of exposure—due to the limited number of rats in each phase of the estrous cycle per group/timepoint. For the serum hormones, we observed that the physiological peaks of E2 and P4 were disrupted at D50, while they were recovered at D100 . This suTo investigate the molecular mechanisms underlying the phenotypic changes in the mammary gland induced by the exposures, we analyzed the mammary gland transcriptome. PFOA + ZAL exposure induced over 600 DEGs above twofold change at both D50 and D100, whereas there were only 82 common DEGs between the two timepoints, suggesting that the treatments induced different changes in the short and long term. Due to the limited number of animals used for RNA-Seq analysis (six per group), we were not able to include serum hormones and estrous cycles into our analysis to assess whether these were influencing the gene expression changes in PFOA + ZAL. However, we evaluated whether these parameters were significantly different among the rats used for RNA-Seq, and we did not observe any differences in E2 or P4 levels, nor in the estrous cycle, between PFOA + ZAL and control rats .Krt15, Krt25, Krt26, Krt27, Krt28, Krt31, Krt32, Krt34, Krt35, Krt36, and Krt42—were downregulated after ZAL_L and PFOA + ZAL exposure. Among these genes, Krt15, Krt25, Krt27, Krt28, and Krt31 served as estrogen-responsive genes, and were downregulated by estrogen in an estrogen-specific mammary gland outgrowth model [Krt25, Krt27, Krt31, and Krt32 were downregulated in rat seminiferous tubule culture model by exposure to low-dose bisphenol A [Adcy2—in multiple signaling pathways. It was reported that estradiol upregulates Adcy2 and, subsequently, promotes the production of cAMP in mesenchymal stem cells [Adcy2 encodes a member of the adenylyl cyclase family, which catalyzes the conversion of ATP to cAMP. The upregulation of Adcy2 at D50 by PFOA + ZAL  and mast cells , whereas it was 5.5 times lower than the concentrations found in 48 adult women (0.437 ng/mL) ,40,79,80This is the first study to assess the risk of pubertal exposure to BBP, PFOA, and ZAL at environmental levels on rats’ mammary gland development and susceptibility to carcinogen-induced tumorigenesis. We aimed to study the phenotypic and transcriptomic changes of the mammary gland at two timepoints after chemical exposure—50 and 100 days old. Due to the large number of groups studied (11) at the same time, we were not able to increase the number of rats per group, limiting our capability to perform analyses in function of the phases of the estrous cycle for each exposure group. However, the data presented herein are informative, and might contribute to future studies in this field.In conclusion, we showed that pubertal exposure to low doses of BBP, PFOA, and ZAL that mimic the environmental levels of exposure could exert short- and long-term effects on the mammary gland transcriptome. We found that exposure to the mixture of low-dose PFOA and ZAL inhibited mammary gland development and increased susceptibility to mammary tumorigenesis, which might be mediated by the alteration of estrogen signaling, Wnt signaling, and pathways associated with tumorigenesis. These findings may contribute to the development of molecular targets for intervention, and may also potentially contribute to the implementation of guidelines and regulations for the use of EDCs in industry.In this study, BBP , PFOA , ZAL , and DMBA  were used for treating animals. BBP, PFOA, and ZAL were dissolved in sesame oil  to make 15 mg/mL, 300 µg/mL, and 300 µg/mL stock solutions, respectively. Working solutions were made freshly before the treatment, and were administered to rats via oral gavage at 333 µL per 100 g BW. DMBA was dissolved in sesame oil at a concentration of 15 mg/mL on the day of administration.Sprague Dawley rats purchased from Taconic Biosciences , Inc. were bred in the Laboratory Animal Facility at FCCC. Animal studies were conducted at FCCC using protocol #15–14 approved (28 October 2015) by the Institutional Animal Care and Use Committee (IACUC) of FCCC. Female offspring were weaned until the age of 21 days, and housed in polypropylene cages with water bottles (both of which were polycarbonate/bisphenol A-free) in a temperature-controlled room (23–25 °C) with a 12 h light/dark cycle. An irradiated AIN-93G diet  was used throughout the experiment to decrease the exposure to phytoestrogens. BBP, PFOA, and ZAL were used individually at low and high doses, or combined at low doses. Therefore, there were 10 treatment groups and 1 control group (treated with sesame oil only), with 10 to 12 rats per group. The experiments for the two endpoints (euthanasia at 50 and 100 days old) were designed to study the short- and long-term impacts of the exposure, and were conducted separately due to a large number of animals being studied at a time A. In botFemale Sprague Dawley rats were randomized into 4 groups (36–37 rats/group) and exposed via oral gavage to sesame oil as controls, or to low-dose PFOA (0.01 mg/kg BW) and ZAL (0.01 mg/kg BW)—individually or in combination—at the age of 21 to 42 days old. At the age of 50 days, all rats were challenged with one dose of DMBA at 30 mg/kg body weight via gavage, and the development of mammary tumors was followed for 7 months A. Tumor-Mammary gland whole mounts were prepared see the . The ideSerum was prepared when euthanizing rats at 50 and 100 days old. The Mouse/Rat Estradiol (E2) ELISA kit from ORIGENE  and Progesterone (P4) ELISA kit from Enzo Life Sciences  were used to measure serum E2 and P4 concentrations, following the manufacturer’s instructions. Mammary tissues, organs, and tumors were fixed with 10% neutral buffered formalin at least for 24 h, and then processed and embedded in paraffin blocks. Paraffin sections of 4 µm thickness were used for H&E staining and evaluated by Dr. Jose Russo—a senior pathologist. For the analysis of the necrotic area in the adrenal glands, ImageJ was used to trace the necrotic area and the cortex area, and then the percentage necrotic area was calculated. Raw data were collected blindly by at least two individuals. Tissue sections for mast cell analysis were stained with chloroacetate esterase (CAE)  and analyzed with ImageJ.All H&E-stained mammary tumor slides were reviewed, and areas of interest representing the histopathology of each tumor were circled on the slides by Dr. Russo. The paraffin blocks were retrieved, and the tissues corresponding to the selected areas were used to construct the tissue microarray by using the MTA1-Manual Tissue Arrayer developed by Beecher instruments . In total, 24 tissue microarray blocks were constructed, with 1–8 cores per tumor distributed on different blocks. Paraffin sections with a thickness of 4 µm were used for IHC; the antibodies used are listed in RNA extraction was performed see the . Six RNASequence quality was checked using FastQC  prior to alignment. The raw reads were quality filtered and trimmed to reduce positional sequence bias using the Trim sequence tool . Then the cleaned reads were aligned to the Rattus norvegicus (Rnor_6.0) reference genomes (Ensembl rn6) using the CLC Genomics Workbench. The mapping rate ranged from approximately 98 to 99% for all the samples. p-value FDRp < 0.05 were considered as differentially expressed genes (DEGs).The expression value per gene or transcript was defined by total counts using CLC genomic workbench version 12. A differential expression analysis test (Robinson and Smyth’s exact test) that assumes a negative binomial distribution of the data, and takes into account the over-dispersion caused by biological variability, was used to compare expression levels between each treated group and controls. The genes with absolute fold change (FC) > 1.5 and with a false-discovery-rate-adjusted p < 0.01. Venn diagrams, volcano plots, and heatmaps were generated using R version 4.0.3 , with the packages VennDiagram, ggplot2, and pheatmap.In this report, we focused on the analyses of DEGs affected by low doses of PFOA, ZAL, and PFOA + ZAL. We used the DAVID tool and the Shiny application in R version 3.5.3 to identify the biological processes and Kyoto Encyclopedia of Genes and Genome (KEGG) pathways related to the DEGs. Significant terms of Gene Ontology enrichment were determined using Benjamini–Hochberg correction, with cutoff levels of Genes of interest were validated by qRT-PCR. Probe information is shown in We collaborated with Silent Spring Institute for internal dose measurements in the dosed rats. Rat blood serum and urine samples were collected one day after the EDC treatments were completed, at 43 days. Both serum and urine samples were stored in aliquots at −80 °C prior to analysis. The measurement of the BBP metabolite mBzP in urine was conducted at NSF International in Michigan . Serum Pt-test. For the mammary tumorigenesis study, with 36 animals in each arm, we had 80% power to detect (a) a difference in tumor incidence of 39% between groups, and (b) an inter-arm standardized effect size of 0.67 for continuous outcomes. One-way ANOVA or Kruskal–Wallis one-way analysis of variance on ranks were used to compare the phenotypic changes among the 11 groups whenever applicable. Two-sample t-tests, the Mann–Whitney rank sum test, or Fisher’s exact test were used to compare a specific treatment group with controls. SigmaPlot 12.0 software  was used to performed statistical analyses for the mammary gland development study. Associations between TEBs, CD3, or CD8a and endogenous hormones or the estrous cycle were evaluated using linear regression models through the “lm” function of the package “stats” (version 4.1.2) in R (R version 4.1.2). Prism 8.0  was used to perform survival analysis, hazard ratios, and comparison of the tumor numbers. SigmaPlot 12.0 was used for the analysis of Ki67, ER alpha, and PR. All statistical tests used a two-sided 5% type I error; p-values ≤ 0.05 were considered statistically significant. The number of animals was calculated before we conducted the animal study, based on the data from our previous experiments. In brief, for the mammary gland development study, with data from 10 animals per group, we were able to detect an inter-arm standardized effect size of 1.33 with 80% power using a two-sided, two-sample