Supportive care interventions used to manage chemotherapy‐induced myelosuppression (CIM), including granulocyte colony‐stimulating factors (G‐CSFs), erythropoiesis‐stimulating agents (ESAs), and red blood cell (RBC) transfusions, are burdensome to patients and associated with greater costs to health care systems. We evaluated the utilization of supportive care interventions and their relationship with the myeloprotective agent, trilaciclib.Data were pooled from three independent randomized phase 2 clinical trials of trilaciclib or placebo administered prior to chemotherapy in patients with extensive‐stage small cell lung cancer (ES‐SCLC). The impact of supportive care on the duration of severe neutropenia (DSN), occurrence of severe neutropenia (SN), and occurrence of RBC transfusions on/after week 5 was analyzed across cycles 1–4. Concordance and association between grade 3/4 anemia, RBC transfusions on/after week 5, and ESA administration was also evaluated.p < 0.0001; 3.3% vs. 11.8%, p = 0.0254; and 14.6% vs. 26.1%, p = 0.0252, respectively). Compared with placebo, trilaciclib significantly reduced DSN and SN, irrespective of G‐CSF administration. RBC transfusions and ESAs were most often administered in patients with grade 3/4 anemia; however, patients typically received RBC transfusions over ESA administration.The use of G‐CSFs, ESAs, or RBC transfusions on/after week 5 was significantly lower among patients receiving trilaciclib versus placebo . Compared with placebo, administering trilaciclib prior to chemotherapy reduces chemotherapy‐induced neutropenia and anemia, with a reduction in the use of hematopoietic growth factors and red blood cell transfusions. By improving key myelosuppressive endpoints and reducing the need for associated supportive care, trilaciclib has the potential to reduce both the societal and economic burden of chemotherapy‐induced myelosuppression on patients with extensive‐stage small cell lung cancer. When people who are treated with chemotherapy develop a condition called myelosuppression, they often receive supportive care treatments that help the body produce new blood cells. However, some supportive care treatments have negative side effects, and many are quite expensive. Researchers found that people who were given a new drug (trilaciclib) before they were treated with chemotherapy did not need as much supportive care as people who were given placebo (an inactive drug). The researchers concluded that trilaciclib may help to reduce the burden of myelosuppression on patients, caregivers, and health care systems.1Standard chemotherapy‐based regimens for extensive‐stage small cell lung cancer (ES‐SCLC), including carboplatin plus etoposide (E/P), E/P plus atezolizumab (E/P/A), E/P plus durvalumab, and second‐line lurbinectedin and topotecan, often lead to clinically significant myelosuppression.Chemotherapy‐induced myelosuppression is typically managed with chemotherapy dose delays and/or reductions, along with supportive care involving growth factor administration (granulocyte colony‐stimulating factors [G‐CSFs] or erythropoiesis‐stimulating agents [ESAs]), as well as red blood cell (RBC) and platelet transfusions.For example, although G‐CSFs reduce infectious complications related to febrile neutropenia (FN), their use is commonly associated with bone pain.Trilaciclib is an intravenous cyclin‐dependent kinase (CDK)4/6 inhibitor that, when administered prior to chemotherapy, transiently arrests CDK4/6‐dependent hematopoietic stem and progenitor cells (HSPCs) in the G1 phase of the cell cycle during chemotherapy exposure, thus protecting them from chemotherapy‐induced damage (myeloprotection).The myeloprotective effects of trilaciclib have been investigated in three independent, randomized, placebo‐controlled, double‐blind phase 2 clinical trials in patients with ES‐SCLC, with results demonstrating that administering trilaciclib prior to chemotherapy was effective at reducing the incidence of multilineage CIM. Compared with placebo, administering trilaciclib prior to chemotherapy also resulted in the need for fewer dose modifications, fewer supportive care interventions, and improved quality of life.In this analysis, data from all three trials were pooled to evaluate the utilization of G‐CSFs, ESAs, and RBC transfusions. The relationship between supportive care interventions and the myeloprotective benefits of trilaciclib was also explored.22.1This retrospective analysis used pooled data from the phase 2 portions of three randomized, double‐blind, placebo‐controlled clinical trials in which trilaciclib or placebo was administered prior to chemotherapy Table : G1T28‐0In each trial, administration of ESAs and primary prophylaxis with G‐CSFs was prohibited in cycle 1, although therapeutic G‐CSF was allowed. In cycles 2 and higher, supportive care measures, including ESAs and G‐CSFs (prophylactic or therapeutic), were permitted per standard of care guidelines. RBC and platelet transfusions were allowed per investigator discretion throughout the treatment period.All trials were designed and conducted in accordance with the Declaration of Helsinki and International Council for Harmonization Good Clinical Practice guidelines. The protocols and study‐related materials were approved by the institutional review board or independent ethics committee of each participating site. All patients provided written informed consent.2.29/L to the date of first ANC value ≥0.5 × 109/L, with no subsequent ANC values <0.5 × 109/L in that cycle. For patients without SN in cycle 1, DSN was set to 0.Neutrophil and RBC‐related endpoints were assessed, including: duration of severe neutropenia (DSN) in cycle 1 (whereby severe neutropenia [SN] was defined as an absolute neutrophil count [ANC] <0.5 × 10⁹ cells/L), occurrences of SN, G‐CSF administration, grade 3/4 anemia, and ESA administration, and occurrence and number of RBC transfusions on/after week 5. DSN in cycle 1 was defined as the number of days from the date of first ANC value <0.5 × 10Granulocyte colony‐stimulating factors use was classified as prophylactic (pegfilgrastim or filgrastim), therapeutic (filgrastim), or other Table .Red blood cell transfusions before week 5 were excluded to ensure that analyses of potential benefit were not confounded by the residual effect of previous treatment. RBC transfusions were considered clinically appropriate for hemoglobin <8.0 g/dl; hemoglobin ≥8.0 and <9.0 g/dl, with a history of atherosclerosis; and/or symptomatic anemia or hospitalization for a life‐threatening event, regardless of hemoglobin value.2.3t‐test. Treatment effect for occurrence of SN and other binary endpoints was assessed using a modified Poisson model with the adjusted relative risk , its 95% CI, and two‐sided p‐value reported. For endpoints that captured the number of events, treatment group differences were evaluated using a negative binomial regression model, and the aRR, its 95% CI, and two‐sided p‐value reported. To account for potential variability among patients and studies when assessing treatment effect, ECOG PS (0/1 or 2), presence of brain metastases (yes or no), and trial  were used as common factors in all statistical models. Corresponding baseline values were included as covariates where appropriate.Data from the three trials were pooled and analyses conducted using intention‐to‐treat (ITT) principles. Treatment effect on DSN in cycle 1 was evaluated using a nonparametric analysis of covariance model, with treatment group difference in mean DSN and its 95% confidence interval (CI) generated using a Satterthwaite pinteraction <0.20.To assess the impact of the potential confounding factor of G‐CSF administration, treatment effect on DSN in cycle 1 and occurrence of SN in cycle 1 were analyzed with an additional term of G‐CSF administration in the model. Occurrence of SN was assessed in cycle 1 rather than across the treatment period because G‐CSF administration was a cycle‐based decision. DSN and occurrence of SN for patients with and without G‐CSF use were also analyzed by cycle for the first four cycles of treatment . ITT patients were included in the cycle 1 data analysis, while analyses of later cycles only included patients who started the specific cycle. Summary statistics for DSN and occurrence of SN at each cycle for patients with or without G‐CSF administration were provided. The treatment effect on DSN in cycle 1 in each subgroup was tested using the nonparametric model, and the group difference in occurrence of SN in cycles 1 and 2  was tested using the modified Poisson model. Consistency of treatment effects on occurrence of SN between patients with or without G‐CSF use at cycles 1 or 2 was tested using a separate modified Poisson model with additional terms of G‐CSF administration and treatment by G‐CSF interaction, whereby statistically significant interaction was defined as The number and percent of patients in each category of G‐CSF use  were summarized by treatment group at each cycle.Subgroup analyses to evaluate the impact of ESA administration on each RBC‐related endpoint were conducted using the same models described above, with additional terms of ESA administration and treatment by ESA interaction. The proportion of patients with RBC transfusions on/after week 5 was summarized by cycle for the first four cycles of treatment for each treatment group. To evaluate relationships among occurrence of grade 3/4 anemia, RBC transfusions, and ESA administration within a treatment group, Cohen's unweighted kappa was calculated to test concordance between two variables, and a chi‐square test performed to assess the association between them.33.1n = 123; placebo, n = 119; ITT analysis set). As described previously, patient demographics and baseline disease characteristics were generally comparable but with slightly higher proportions of male patients and current smokers in the trilaciclib group than in the placebo group.Overall, 242 patients were randomized . Throughout the treatment period, 14 patients (11.4%) in the trilaciclib group and 63 patients (52.9%) in the placebo group had SN .DSN in cycle 1 was significantly shorter in the trilaciclib group than in the placebo group .p < 0.0001). Across cycles 1 to 4, trilaciclib reduced mean DSN and occurrence of SN irrespective of G‐CSF administration , and statistically significant reductions in DSN in cycle 1 and occurrence of SN were observed for patients with and without G‐CSF use . For patients receiving trilaciclib versus placebo, use of ESAs or RBC transfusions on/after week 5 was 3.3% versus 11.8% (p = 0.0254) and 14.6% versus 26.1% (p = 0.0252), respectively.The proportion of patients with grade 3/4 anemia was significantly lower in the trilaciclib group than in the placebo group , rejecting the hypothesis of no association. Concordance between grade 3/4 anemia and ESA administration was lower; however, the chi‐square p‐value was significant . Among patients with grade 3/4 anemia, concordance between ESA administrations and RBC transfusions on/after week 5 was low .Overall, there was high concordance between grade 3/4 anemia and RBC transfusions on/after week 5, regardless of treatment group. The chi‐square Overall, 49 patients (20.2%) had a total of 87 RBC transfusions on/after week 5. Of these, 43 patients (87.8%) had at least 1 RBC transfusion that was classified as clinically appropriate, and 11 patients (22.4%) had at least 1 transfusion event classified as inappropriate.4Neutropenia and anemia are common side effects of myelosuppressive chemotherapy that add to the total burden borne by patients with cancer and their families.In this retrospective pooled analysis of data from three global, independent, randomized, double‐blind, placebo‐controlled, multicenter phase 2 trials in patients with ES‐SCLC, administering trilaciclib prior to chemotherapy significantly reduced DSN in cycle 1 and occurrence of SN across cycles 1 to 4 regardless of G‐CSF administration. Administering trilaciclib prior to chemotherapy significantly reduced the use of G‐CSF to approximately half that observed in patients receiving placebo. The reduction was apparent for both therapeutic and prophylactic administration. “Other” G‐CSF use, which was generally the largest category in the trilaciclib group, was more balanced between groups, suggesting that physicians frequently base G‐CSF use on subjective clinical judgment rather than objective data such as ANC counts and fever, with such cases occurring relatively evenly in both treatment groups.Historically, consensus guidelines have recommended primary prophylactic use of G‐CSFs when the risk of FN is high (>20%) on the basis of chemotherapy and patient risk factors.Despite the negative impact of chemotherapy‐induced anemia, its occurrence is frequently underestimated and its management is often delayed.p‐value was below the statistical threshold for evidence of a differential treatment effect. However, the observed difference in RBC transfusions between patients who did or did not receive ESA administration should be regarded with caution given the very low patient numbers and number of ESA administrations. For grade 3/4 anemia, there was no differential treatment effect between patients who did or did not receive ESA administration.Analyses of the occurrence of grade 3/4 anemia or the use of RBC transfusions by ESA administration were restricted by the limited number of patients who received ESAs. Among patients who did not receive ESAs, both the occurrence of grade 3/4 anemia and the use of RBC transfusions were significantly reduced with trilaciclib compared with placebo. For RBC transfusions, the interaction There was a strong concordant relationship between RBC transfusions on/after week 5 and grade 3/4 anemia, indicating that most patients who received a transfusion had severe anemia and vice versa. Although grade 3/4 anemia did not frequently result in ESA administration, when ESAs were given, they were usually administered to patients with grade 3/4 anemia.Although initial approval of ESAs in the United States reduced the need for RBC transfusions, subsequent studies suggested that ESA use was associated with decreased survival and/or tumor progression or recurrence, leading to changes in US prescribing practices.p = 0.0088).In addition to placing a considerable humanistic burden on patients and their caregivers,5The results from this pooled analysis show clear benefits associated with the administration of trilaciclib prior to chemotherapy in patients with ES‐SCLC. Compared with placebo, trilaciclib consistently reduced the duration and occurrence of SN, regardless of G‐CSF administration. This translated into a reduction in G‐CSF administration compared with placebo. Trilaciclib also consistently reduced the occurrence of chemotherapy‐induced anemia, which was reflected in the reduction of RBC transfusions on/after week 5 and ESA use. By improving key myelosuppressive endpoints and reducing the need for associated supportive care, trilaciclib has the potential to reduce both the societal and economic burden of CIM.Outside of the submitted work, Renata Ferrarotto has received personal fees from Ayala Pharma, Carevive, Cellestia Biotech, Klus Pharma, Medscape, Prelude, and Regeneron‐Sanofi, and has received grants from AstraZeneca, Genentech, Inc., Merck, Oropharynx Program Stiefel clinical trials, Pfizer, the ASCO Career Development Award, and the MD Anderson Khalifa Award; William Edenfield serves as a consultant for Chimerix Corp, and Jennifer M. Johnson has received research funding from AstraZeneca, Bristol Myers Squibb, and Merck, and has served as a consultant for Rakuten Medical and Foundation Medicine. Yili Pritchett and Rajesh K. Malik are employees and shareowners of G1 Therapeutics, Inc. Shannon R. Morris was an employee of G1 Therapeutics, Inc., at the time of study. Ian Anderson, Balazs Medgyasszay, Maria Rosario García‐Campelo, Trevor M. Feinstein, Sujith Kalmadi, Philip Lammers, A. Sanchez‐Hernandez, and Tibor Csőszi have no conflicts of interest to declare.Renata Ferrarotto: Investigation, writing—review and editing; Ian Anderson: Investigation, writing—review and editing; Balazs Medgyasszay: Investigation, writing—review and editing; Maria Rosario García‐Campelo: Investigation, writing—review and editing; William Edenfield: Investigation, writing—review and editing; Trevor M. Feinstein: Investigation, writing—review and editing; Jennifer M. Johnson: Investigation, writing—review and editing; Sujith Kalmadi: Investigation, writing—review and editing; Philip Lammers: Investigation, writing—review and editing; A. Sanchez‐Hernandez: Investigation, writing—review and editing; Yili Pritchett: Conceptualization, methodology, formal analysis, writing—review and editing; Shannon R. Morris: Conceptualization, methodology, formal analysis, writing—review and editing; Rajesh K. Malik: Conceptualization, methodology, formal analysis, writing—review and editing; Tibor Csőszi: Investigation, writing—review and editing.All trials were designed and conducted in accordance with the Declaration of Helsinki and International Council for Harmonisation Good Clinical Practice guidelines. The protocols and study‐related materials were approved by the institutional review board or independent ethics committee of each participating site. All patients provided written informed consent.The authors declare that they have no competing interests.Table S1‐S3Click here for additional data file.


The tumor reduction effect of pembrolizumab is extremely high compared to standard chemotherapy and might show prolonged survival. Therefore, the MSI status should be examined in patients with cholangiocarcinoma. Contrast‐enhanced computed tomography (CT) revealed a primary tumor (black arrow) before the initiation of pembrolizumab (A). After three cycles of pembrolizumab, the primary tumor showed a 32% reduction (B). After 14 cycles, the primary tumor shoed a 79% reduction (C). Here, we report a case of MSI‐high cholangiocarcinoma effectively treated with pembrolizumab. MSI status should be actively evaluated in patients with cholangiocarcinoma.Cholangiocarcinoma is considered to be intractable and has a poor prognosis. Gemcitabine plus cisplatin is recommended as first‐line chemotherapy for patients with advanced cholangiocarcinoma based on an ABC‐02 study.Recently, immune checkpoint inhibitors (ICIs) have shown promising responses in cancer treatment, regardless of primary site.Here, we present a case of MSI‐high intrahepatic cholangiocarcinoma (ICC) in which the tumor was markedly reduced by pembrolizumab treatment.2A 50‐year‐old man presented with epigastric and back pain and was admitted to hospital. Laboratory data revealed alkaline phosphatase (ALP) 824 U/L 106‐322 U/L), ɤ‐GTP 401 U/L (5‐50 U/L) and carcinoembryonic antigen (CEA) 21.1 ng/mL . An analysis of liver tumor biopsy specimens showed a high MSI status; the patient was administered pembrolizumab . After three cycles of pembrolizumab, the CEA level declined to within normal range (2.3 ng/mL), with CT showing a reduction of 69mm and 32% primary tumor reduction Figure , leading3We report a case of advanced cholangiocarcinoma treated with ICI that showed a high response to pembrolizumab monotherapy after standard chemotherapy. Pembrolizumab, is an anti–PD‐1 monoclonal antibody that has shown antitumor activity in various types of cancers, including nonsmall‐cell lung cancer, gastric cancer, urothelial cancer, and melanoma.Only three case reports exist of MSI‐high advanced cholangiocarcinoma treated with pembrolizumab.In our case, pembrolizumab monotherapy for ICC refractory to GCS combination therapy was found to be effective, with a PR maintained 13 months after treatment. The frequency of MSI‐high in all cholangiocarcinomas is only 2%,4MSI‐high cases are rare among patients with advanced cholangiocarcinoma; however, the potential therapeutic effect of pembrolizumab is extremely high. Therefore, the MSI status of advanced cholangiocarcinoma cases should be actively examined since some patients can benefit from pembrolizumab treatment.YI and MO: collected data and wrote and edited the manuscript. GO, SA, MY, TA, and SF: involved in the patient's care. MF: Pathologist who diagnosed this disease. MM: supervised this study.The authors declare that they have no conflicts of interest.YI and MO: collected data and wrote and edited the manuscript. GO, SA, MY, TA, and SF: involved in the patient's care. MF: Pathologist who diagnosed this disease. MM: supervised this study.


Contradictory opinions about whether early correction and fusion surgeries should be performed for congenital scoliosis (CS) patients at a young age exist. The objectives of this study were to analyze the association between patient characteristics and fusion-surgery outcomes in CS patients treated with spinal correction and fusion surgeries and to report risk factors for extended length of stay (LOS), more estimated blood loss (EBL), longer fused segments and higher medical costs.We analyzed data of 1,207 CS inpatients treated with fusion surgeries in our institute from January 2010 - December 2019. All patients underwent spinal X-ray, CT, MRI, echocardiogram and urogenital ultrasound. We analyzed demographic and clinical information and outcome measures, including LOS, EBL, fused segments and medical costs.p < 0.001), musculoskeletal defects  and thoracic deformity  were risk factors for extended LOS. Age at fusion , male sex , mixed defects  and failure of formation  were risk factors for more EBL. Age at fusion  was a risk factor for longer fused segments. Age at fusion  and thoracic deformity  were risk factors for higher medical costs.Age at fusion  and sex  for more EBL. Congenital scoliosis (CS) is the lateral curvature of the spine resulting from vertebral deformities that develop during the 4th to 6th weeks of gestation and are present at birth. CS was reported to have an incidence of approximately 1 in every 1,000 live births . Echocardiogram; urogenital ultrasound; spine imaging; including MRI, X-ray and CT; and systemic physical examination were undertaken in all patients to screen and diagnose associated anomalies. Primary outcomes, including the length of stay, estimated blood loss, fusion segments , medical costs and complications, were extracted from medical records and analyzed.P-value < 0.1 were included as potential risk factors in multiple logistic regression analysis to determine significant independent risk factors. A P-value < 0.05 was considered statistically significant in multivariate analysis. All graphs were plotted by GraphPad Prism 8 .All statistical analyses were performed using SPSS Statistics Version 23 . An independent Student’s t test was used to analyze continuous data with a normal distribution. A nonparametric test was used to analyze continuous data with a non-normal distribution. The chi-square test was used to analyze enumerated data. In the univariate analysis, factors with a p < 0.001). 564 patients (46.7 %) were male, while 643 patients (53.3 %) were female. 50 % of patients (n = 604) had failure of formation, 18.5 % of patients (n = 223) had failure of segmentation and the remaining 31.5 % of patients (n = 380) had mixed defects. Regarding vertebral anomaly location, cervical, thoracic and lumbar vertebral anomalies were found in 67 patients (5.6 %), 930 patients (77.1 %) and 423 patients (35.1 %), respectively , 15.5 % (n = 187), 13.7 % (n = 165), 3.6 % (n = 44) and 5.8 % (n = 70), respectively. Twenty-three point nine percent (n = 288) of patients had an extended length of stay (defined as LOS > 14 days), 20.2 % (n = 244) of patients had more estimated blood loss (defined as EBL > 800 ml), 48.6 % (n = 587) of patients had more than 8 levels fused and 22.0 % (n = 266) of patients had medical costs greater than 20,000 USD , musculoskeletal defects  and thoracic deformity  were found to be independent risk factors for extended LOS in multivariate analyses , male , CS types of mixed defects  and failure of formation  were found to be independent risk factors for estimated blood loss greater than 800 ml , thoracic deformity , intraspinal defects , CS types of failure of segmentation , mixed defects  and cervical deformity  were identified as independent risk factors for more than 8 levels fused , different CS type distribution (p < 0.001), more patients with thoracic deformity (p < 0.001) and more patients with intraspinal (p = 0.009) and urogenital defects (p = 0.024) in univariate analysis. Although lumbar deformity had a P-value < 0.1 in univariate analysis, we excluded it from multivariate analysis, as it appeared to be a protective factor for higher medical costs. After multivariate analysis, age at operation  and thoracic deformity  were identified as independent risk factors for medical costs greater than 20,000 USD  (Table p = 0.075) and medical costs > 20,000USD  and higher incidences of EBL > 800ml .The total osteotomy rate in our CS cohort is 56.7 % (n = 159) and complication rate in patients of 0–3 years old and in patients of more than 3 years old showed no statistically significance . Rates for specific complications were 2.0 % for implantation-related complications, 2.0 % for adding-on and decompensation, 1.3 % for incision-related complications, 1.1 % for infections, 3.3 % for pleural effusion and hemothorax, 2.0 % for neurologic complications and 0.4 % for CSF leakage.As for the analysis of complication information Table , we founThe spine undergoes a bimodal growth spurt: once during the first 3 to 5 years of life and again during puberty [The most prevalent concern about early fusion is damaged pulmonary function. CS patients treated with early spinal fusion have lower forced vital capacity, forced expiratory volume, vital capacity and total lung capacity than healthy children, and the impairment of pulmonary function is more severe in patients with thoracic fusions . Early sAnother concern is that pedicle screws have to pass through the neurocentral cartilage (NCC), which is responsible for the growth of the pedicles and the posterior vertebral body, and the NCC begins to fuse at 3 to 5 years of age . Thus, tBy directly analyzing the interrelationship between age at fusion and patient outcomes, Wu et al.  found thTheoretically speaking, early resection of congenital vertebral deformities and correction of scoliosis are beneficial to prevent the development of severe local deformities and secondary curves. Once secondary curves become structured, the extension of instrumented fused segments to comprise these curves is unavoidable. Earlier surgeries lead to milder deformities, shorter fusion segments and better outcomes. This strategy of early surgery is effective, particularly for CS patients with a single hemivertebra, in which case the hemivertebra is the only cause of deformity. As long as hemivertebrae are completely resected and local scoliosis is appropriately corrected, there was no risk of further progression and the formation of structured secondary curves . This prChang et al.  demonstrWe also found that thoracic deformity was a risk factor for extended LOS, longer fused segments and higher medical costs, with an odds ratio from 1.67 to 11.30 Figs. . In our Other identified risk factors include musculoskeletal defects for extended LOS Fig.  and CS tGenerally, the strategy of early surgery is effective and safe for CS patients with local congenital vertebral malformations to minimize deformities at surgery, fusion segments, surgical trauma and medical costs. In our clinical practice, these patients, who underwent early osteotomy and short-segment fusion, were observed to have growth potential that was similar to that of their healthy peers. Thus, for CS patients with local congenital vertebral malformation and limited scoliosis, we recommended that spinal surgery be performed as early as 1.5 to 2 years of age to eliminate most forces that lead to the progression of spinal deformity. Otherwise, due to its natural history, deformities will become more severe as CS patients age and require greater surgeries and longer fusion segments, which will lead to more EBL, higher costs and longer LOS.In summary, our study has two major strengths. First, to our knowledge, this is one of the largest single-center CS cohorts to date. Second, we are the first to comprehensively analyze the association between demographic and clinical information and outcome measures in a large CS cohort treated in a single center. However, as this is a retrospective study of data extracted from medical records, the quality and reliability of the results of this study were largely influenced by the recorders, imaging interpreters and medical record quality.We found that older age at surgery was a risk factor for extended LOS, more EBL, longer fused segments and higher medical costs, with the risk increasing by 5–21 % for each year increase in patient age at the time of fusion surgery. Thoracic deformity is a risk factor for extended LOS, longer fused segments and higher medical costs. Other identified risk factors include musculoskeletal defects for extended LOS, CS type (FF and MD) and sex  for more EBL.


In different cancer entities, several studies have shown the adverse effects of cancer on mental health, psychological well-being and the increased risk of high emotional distress in cancer patients. This study aims to analyze psychosocial distress levels and their relationship between sociodemographic parameters and selected items on the Distress Thermometer (DT) Problem List in head and neck squamous cell carcinoma (HNSCC) patients.We assessed a total of 120 HNSCC patients using the Distress Thermometer (DT) Problem List. Distress scores (DTS) of 90 patients were available. A DTS of ≥ 5 on the visual analogue scale represents clinically relevant distress. Data analysis consisted of descriptive statistics, comparison of mean values for different DTS subcategories and correlation between DTS scores and parameters of tumor classification, sociodemographic variables and selected problems.Distress was present in 57.7% of the sample, with a total of 52 patients with a DTS  ≥ 5. The mean DTS was 4.7 (SD 2.4). Patients with newly diagnosed HNSCC had significantly higher DTS. Distress levels were significantly associated with sadness, general worries, anxiety, nervousness, sleeping disorders, mouth sores and fever. Out of the total sample, 6 patients and out of these 6 individuals, 5 patients with a DTS ≥ 5 requested referrals to psycho-oncological service.High distress levels were common in HNSCC patients but only few patients desired psycho-oncological care. Addressing patients’ supportive care needs in routine clinical practice is essential to meet unmet needs of HNSCC patients and thus improve cancer care. Patients with head and neck squamous cell carcinoma (HNSCC) represent a particular challenge from a psycho-oncological point of view. Compared to patients with other tumor entities, HNSCC patients have been found to show elevated levels of psychological distress including anxiety and depression, higher subjective burden of mental illness , mental In all, only about 30% of the patients identified as having significant psychological distress received a recommendation or referral over the course of their medical treatments , 7.Using other diagnostic tools, a high willingness to participate in psycho-oncological intervention could already be shown for cancer patients over 60 years of age . One insDistress is defined by the NCCN as “a multifactorial, unpleasant, emotional experience of a psychological, social, and/or spiritual nature that may interfere with the ability to cope effectively with cancer, its physical symptoms, and its treatment” (NCCN 2007). In HNSCC, patients’ pain and high levels of distress are reported rather early, before their further assessment and therapy is determined . FurtherThe aim of the present study was to estimate the frequency of patients with high level of distress (DT score ≥ 5 –16) amonn = 120 patients.The calculation of our aimed sample size was performed on the basis of an expected rate of approximately 34.5% based on reported 34.5% in NCLC , 34.0% iThe study population was composed of 120 consecutively accrued HNSCC patients presented to the Department of Otolaryngology, Head and Neck Surgery for the assessment and management of their cancer from January 2018 to September 2020. The study was approved by our institutional review board . After providing informed consent, the patients were asked to complete the DT and the problem list questionnaire on the day of admission as screening procedure for assessing the need for psycho-oncological intervention as recommended in the German S3-guideline for psycho-oncology. All participating patients had sufficient knowledge of the German language in order to complete the questionnaire on their own. According to the study protocol, we finished the enrollment after recording of the 120th patient in September 2020, and started retrospective analyses of questionnaires. Patient selection is shown in Fig. The Distress Thermometer (DT) is a brief, validated screening measure to identify the level of distress and the sources of distress . We usedThe demographic parameters “Sex”, “Age”, “Living with a partner”, “Tumor site” and “Tumor stage” were chosen to investigate their potential influence on distress of HNSCC patients, as previous studies in other malignancies were able to show that distress was significantly higher in patients being female, being at a specific age group and having different tumor sites . On the Student’s t test or Mann-Whitney U test and analysis of variance (ANOVA) were used to analyze differences in mean values. Effect sizes were analyzed with Cohen’s d. Spearman correlation was performed to analyze the relationship between distress and age or parameters of tumor classification. All methods were carried out in accordance with relevant guidelines and regulations.The statistical analysis was performed using the SPSS software package . Descriptive analyses were used for sociodemographic characteristics and to report the results obtained from the DT. The total sample size was 120 patients, of which 83.3% were male. The mean age was 62.6 (SD 8.6) and ranged from 41 to 85 years. The patients presented with newly diagnosed , recurrent  or after  curative therapy of HNSCC. In all, fifty patients (41.7%) answered the questionnaire after they had received therapy for HNSCC in the past. Seventy-four patients (61.7%) were married or cohabited with a partner. The tumors were mainly located in the oropharynx (43.3%) followed by the larynx (21.7%) and oral cavity (20.8%). Twenty-seven patients (22.5%) received surgery only, 49 (40.8%) received surgery and adjuvant radio(chemo)therapy, 39 (32.5%) received curative radio(chemo)therapy and 5 patients (4.2%) underwent palliative radio(chemo)therapy. Chi-Squared-Test revealed a significant relationship among patients with distant metastases, as patients with distant metastases where significantly more likely to have a DTS ≥ 5. Further patient characteristics are shown in Table n = 90, SD 2.4, 30 DTS missing). Fifty-two patients (57.7%) were at or above the cut-off score ≥ 5. Patients with newly diagnosed HNSCC (before therapy) had significantly higher distress scores  than patients with recurrent HNSCC or after cure . There were no differences between mean DTS for tumor sites  = .75, p = .53), gender (p = .10), or relationship status (p = .99). No significant correlation was found between the DTS and age (p = .07), tumor size (p = .53) or presence of metastases in lymph nodes (p = .54).The mean DTS was 4.7  in DTS between the two groups of patients with newly diagnosed HNSCC (47 DTS available) and recurrent disease only (10 DTS available).Mann-Whitney Among patients with a DTS ≥ 5, 92.3% (48 patients) selected at least one item in the physical and 61.5% (28 patients) in the emotional domain. The rates of endorsement of at least one item for the other domains were 7.7% (4 patients) for the practical domain, 1.9% for the family domain (1 patient) and 3.8% (2 patients) for the spiritual domain . Furthermore, there was a significant positive correlation between DTS and the number of problem items selected .There was a significant difference in the number of problem items selected between those with DTS ≥ 5  and those with DTS < 5 . The calculation showed significantly higher DTS, compared to individuals without these complaints, for anxiety, sadness, nervousness, sleeping disorders, mouth sores and newly diagnosed HNSCC Table . The calPsychological support was demanded by 6 individuals out of a total of 104 patients . Out of those 6 individuals, 5 had a DTS ≥ 5 (1 DTS missing).Coping with diagnosis and outcome of treatment means a significant physical and psychological burden for HNSCC patients, especially as HNSCC and its treatment may result in disfigurement and dysfunction. Unmet psychosocial needs can complicate the treatment course and recovery of HNSCC patients as well as their survival . In the Patients in our study showed high levels of unmet needs in the emotional and physical domain. In the emotional domain, the present analysis confirms the importance of anxiety and worries as common problems in about two thirds of the patients. In the physical domain “mouth sores” was the most often mentioned problem 46.7%), followed by “sleep” (35.8%) and “pain” (34.2%), which also were the most often selected items in the patient group with significant distress. Similar to the study of Schell et al. the DTS neither correlated with age, nor with tumor size 6.7%, fol.d = .56 furthermore indicates a relevant effect for these findings [ρ = .38 shows a moderate correlation between these variables. In combination however, these results suggest that individuals, who tend to report more problems, also have higher distress or vice versa. Future prospective studies could investigate whether the number of selected items on the problems list predicts patients’ distress as valid as the DTS itself. On this basis, a second cut-off score could possibly be established to include the problems list for the evaluation of the distress thermometer, making the evaluation process more independent of the DTS alone.In our cohort, patients with significant distress chose a significantly higher number of problems on the items list than patients with a DTS < 5. The total number of selected items also showed a significant positive correlation with the DTS. An effect size of findings , whereasd = .76 was found for patients, who stated “mouth sores” as problem, which has to be interpreted as a relevant effect [d = .46 [Patients who chose the items “mouth sores”, “worries”, “anxiety”, “sadness, “nervousness”, “fever” and “sleep” on the problems list showed significantly higher DTS than patients, who did not report these problems. Patients selecting “sadness” as a problem, interestingly showed the highest mean DTS . The highest effect size t effect . These ft effect  were als[d = .46 , compare[d = .46 . However[d = .46 . The bes[d = .46 . Various[d = .46 , 30. Can[d = .46 –33, fema[d = .46 , 34 and [d = .46 . In our [d = .46  potentia[d = .46 . The pre[d = .46 . It also[d = .46 . However[d = .46 . In HNSC[d = .46 .Given the retrospective character of the acquired data there are several limitations of this study. It consists of a relatively small number of patients and there was variability in time elapsed since cancer-diagnosis. Inherent to the design we have to deal with some missing data, which could have impacted the presented results. We cannot provide information about the presence or absence of a caregiver, which could possibly have influenced patients DTS, as this information is not routinely assessed. Any ongoing care for the HNSCC patients by a psychologist independent from our hospital cannot be excluded as we did not ask explicitly for this potentially relevant factor that consequently could not be considered in the data analysis.HNSCC patients suffer from high distress levels, but the HNSCC patients’ supportive care needs must be routinely assessed to provide a basis to better understand how distress management can be improved. In the present study, HNSCC patients, who suffered from newly diagnosed HNSCC and sadness, general worries, anxiety, nervousness, sleeping disorders, mouth sores or fever on the problem list of the DT were at higher risk for significant distress compared to HNSCC patients without those complaints. Distress screening enhances the awareness of psycho-oncological issues, but its use in HNSCC patients is associated with some difficulties as only a small percentage of our cohort, regardless of being at the stage before or after therapy, desired psycho-oncological support. Further research is needed to investigate to what extent and at which point of time HNSCC patients desire psychosocial care and which activities raise the acceptance of psycho-oncological support in these patients. Addressing HNSCC patients’ supportive care needs by offering visiting a psycho-oncologist routinely to each patient might be a first step to do so.


Rangifer tarandus caribou) population, moose (Alces americanus) and their incidental predators, the black bear (Ursus americanus) and coyote (Canis latrans), and to quantify the extent to which these predators consumed caribou. Bayesian isotopic analysis showed a small overlap in trophic niche for the two sympatric ungulates suggesting a low potential for resource competition. Our results also revealed that caribou occupied a larger isotopic niche area than moose, suggesting a greater diversity of resources used by caribou. Not surprisingly, coyotes consumed mainly deer (Odocoileus virginianus), moose, snowshoe hare (Lepus americanus), and occasionally caribou, while bears consumed mainly vegetation and, to a lesser extent, moose and caribou. As coyotes and bears also feed on plant species, we documented trophic niche overlap between caribou and their predators, as searching for similar resources can force them to use the same habitats and thus increase the encounter rate and, ultimately, mortality risk for caribou. Although the decline in the Gaspésie caribou population is mostly driven by habitat‐mediated predation, we found evidence that the low level of resource competition with moose, added to the shared resources with incidental predators, mainly bears, may contribute to jeopardize the recovery of this endangered caribou population. Highlighting the trophic interaction between species is needed to establish efficient conservation and management strategies to insure the persistence of endangered populations. The comparison of trophic niches of species sharing the same habitat or resources is fundamental to evaluate the mechanisms of coexistence or competition and eventually predict the consequences of ecosystem changes in the community.Documenting trophic niche partitioning and resource use within a community is critical to evaluate underlying mechanisms of coexistence, competition, or predation. Detailed knowledge about foraging is essential as it may influence the vital rates, which, in turn, can affect trophic relationships between species, and population dynamics. The aims of this study were to evaluate resource and trophic niche partitioning in summer/autumn between the endangered Atlantic‐Gaspésie caribou ( We have reconstructed the trophic niches of two ungulate prey, moose and Atlantic‐Gaspésie caribou, and of two incidental predators, coyote and black bear, living in sympatry in a protected area surrounded by intensively disturbed habitats. Using stable isotopes, we showed a small overlap in dietary niche for the two sympatric ungulates, suggesting a low potential for resource competition. However, we highlighted a trophic niche overlap between caribou and their predators, which can increase the encounter rate and associated mortality risk for caribou, an endangered species according to the Canadian Species at Risk Act. The perfundamental ecological niche describes the range of optimal conditions wherein a species is able to persist in the absence of competition and predation, whereas the realized ecological niche considers all the constraints to which an animal is exposed, including competition and predation  moved from open meadows toward forest edges that provide lower‐quality forage but better protection from wolf (Canis lupus) predation. Characterizing the partitioning and level of overlap of trophic niches between prey and its conspecific competitors is critical to inform possible ecological relationships. This type of analysis can also be applied to prey that share resources with incidental, omnivorous predators which thus act simultaneously as predator and competitor. In such complex interactions, searching for similar resources in the same area can increase the encounter rate between prey and an opportunistic predator.The trophic niche of an individual or a species belongs to the ecological niche, but it is built using a subset of variables related to trophic resources Figure . The tro15N) occur from one trophic level to the next (+3–4‰), making them useful indicators of trophic position  is particularly useful for delineating carbon sources and foraging locations , and black bear (Ursus americanus).Diet composition is often inferred from the analysis of prey remains in scats and stomach contents of consumers  populations are declining in Canada and some small, isolated herds are particularly at risk  density and was paralleled with an increase in density of black bears and coyotes (Canis latrans)  – white birch (Betula papyrifera) bioclimatic domain. It is located in the Gaspésie National Park and the surrounding Matane, Dunière, and Chic‐Chocs Wildlife Reserves, and Casault controlled harvesting zone (hereafter referred to as ZEC Casault)  Figure . The Gas) Figure . Populat) Figure . A small) Figure . Intensi) Figure . These c Dorais, . This in Dorais,  that has Dorais,  and adul Dorais,  via an a2.2We captured 44 caribou in the winter of 2013 and 2014 across the Gaspésie caribou range using a net gun fired from a helicopter. We collected caribou hair samples from the rump of the animal, and dried and stored them in paper bags at ambient temperature until processing. We used the same sampling and conservation protocol for hair samples collected from 90 moose, 127 coyote, and 57 black bear carcasses across the three Wildlife Reserves that overlap the Gaspésie National Park Figure . Moose w2.3Odocoileus virginianus), 1 snowshoe hare (Lepus americanus), 2 large rodents , 8 Canada lynx (Lynx canadensis), and 7 moose (Alces americanus). We also collected hairs from 22 individuals belonging to 6 species that were harvested by sport trappers or opportunistically collected in the ZEC Casault during summer, including 5 different species of rodents  and 1 species of large rodent . We also collected the feathers of three ruffed grouses (Bonasa umbellus) and hair from four snowshoe hares which were harvested by sport hunters in the ZEC Casault. We dried and stored hair and feathers in paper bags at ambient temperature until processing.We collected samples opportunistically from all potential food sources consumed by moose, coyotes, and bears to describe the composition of their diet. We collected hair samples from 22 individuals belonging to 6 different species that were accidentally trapped during the predator control program, including 4 white‐tailed deer (n = 93) and Mount Logan (n = 72) in the Gaspésie National Park and of Petit Mount Ste‐Anne (n = 53) in the Chic‐Chocs Wildlife Reserves  and hymenoptera orders (three ants and two wasps). We also collected plant samples in July 2017 belonging to 46 different species in the montane boreal forest area of Mount Albert  and δ15N (no lipid‐extracted) values separately to account for lipid effect on stable isotope signatures: one part of the subsamples received no further treatment prior to nitrogen isotope analysis, and the second part was lipid‐extracted prior to carbon isotope analysis  for caribou hair and plant tissues, and in the Stable Isotope in Nature Laboratory  for other animal tissues and insects. By convention, 13C and 15N isotope abundances are expressed in delta notation (‰), as δX = [(Rsample/Rstandard) −1] × 1000, where X is 13C or 15N, and Rsample is the corresponding ratio 13C/12C or 15N/14N; Rstandard represents the ratios of the respective standards: Vienna Peedee Belemnite (PDB) and atmospheric nitrogen (AIR). We evaluated the accuracy of our isotopic analysis using three commercially certified materials  and the precision of measurement by randomly duplicating a subset of our samples. Replicates using certified B2151 materials (n = 31) indicated a systematic error of ±0.22 for δ13C and ±0.24‰ for δ15N, whereas replicates using certified Acetanilide and Nicotinamide materials (n = 36) indicated a systematic error of ±0.08‰ for δ13C and ±0.10‰ for δ15N. The average deviations observed between replicates of hair, fish muscle, insect, and plant samples (n = 97) indicated an analytical error of 0.16‰ for δ13C and 0.20‰ for δ15N.We weighed 0.500–0.700 mg (± 0.001 mg) subsamples of powdered caribou hair and plant tissues, and 1.000–1.200 mg (± 0.001 mg) of other animal tissues and insects into a tin capsule. We analyzed samples to assess δ2.6x% to y% of the assimilated diet   as prior distributions into our SIMM and added prior distributions for each source  and the 95% CI in the bi‐dimensional isotopic space of δ13C and δ15N values using the SIBER library  by estimating the Bayesian standard ellipse area , graminoids, fruits (89.4% [62.0 to 96.8]), and willow , but also evergreen trees (28.8% [5.4 to 42.3]) and shrubs  was larger for caribou  than for moose (1.25‰2 [1.04–1.54]), coyotes (1.21‰2 [1.02–1.44]), and bears (1.18‰2 [0.89–1.53])  than for moose, coyotes, and bears. SDNND was larger for moose (0.33‰) than for caribou, coyotes, and bears , suggesting that the quest for similar plant species can increase the encounter rate probability between these incidental predators and caribou, explaining partially the increased mortality risk and accelerated decline for this endangered population.4.1Caribou consumed a great diversity of resources in summer/autumn, and we found a high interindividual difference in diet, potentially attributed to different niche habits. In a companion study , we detailed caribou diet using the same tissue samples, and showed that caribou consumed mainly lichens, deciduous trees, and shrubs, but also mosses, evergreen trees, ferns, and horsetails. These results are supported by findings obtained by Christopherson et al.  in the sSimilar to Christopherson et al. , we founOdocoileus hemionus, moose, and elk: Hodder et al., Some studies highlighted that resources partitioning between ungulates may be a result of past competition  clear‐cuts , which are opportunistic omnivores, suggesting that cross‐trophic competition might be at play between these three species. While previous studies have shown that caribou, coyotes, and bears are spatially distributed in different elevation zones  in Québec ; Formal analysis (lead); Investigation (lead); Methodology (lead); Writing – original draft (lead). Fanie Pelletier: Conceptualization ; Formal analysis (supporting); Funding acquisition (supporting); Supervision (supporting); Writing – original draft (supporting); Writing – review & editing . Martin‐Hugues St‐Laurent: Conceptualization ; Data curation (lead); Formal analysis (supporting); Funding acquisition (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing – original draft (supporting); Writing – review & editing .Appendix S1Click here for additional data file.


Immune checkpoint inhibitors (ICIs) have been used for various carcinomas. However, immune‐related adverse events have been observed. There have been few reports of treatment with biologics for severe bronchial asthma induced by ICI; therefore, their efficacy is unknown. We report two cases of severe bronchial asthma requiring systemic steroid administration while using anti‐programmed death‐ligand 1 (PD‐L1) antibody for advanced non‐small‐cell lung cancer. The anti‐interleukin‐5 antibody, mepolizumab, was introduced, resulting in the discontinuation of systemic prednisolone and good asthma control. These reports suggest that treatment with biologics may be effective in severe cases of poorly controlled bronchial asthma during ICI therapy. We report two cases of severe bronchial asthma requiring systemic steroid administration while using anti‐programmed death‐ligand 1 (PD‐L1) antibody for advanced non‐small cell lung cancer. In each case, mepolizumab was introduced, resulting in discontinuation of systemic prednisolone and good control of asthma. Immune checkpoint inhibitors (ICIs) have been used for various carcinomas and have become the mainstay of chemotherapy. Anti‐programmed death‐1 (PD‐1) protein and anti‐programmed death‐ligand 1 (PD‐L1) antibodies activate anti‐tumour immunity by inhibiting the binding of PD‐L1 of tumour cells to PD‐1 of immunocytes.Steroids are the main treatment for irAE and do not influence the prognosis of lung cancer.We report two cases of severe bronchial asthma requiring systemic steroid administration while using anti‐PD‐L1 antibody for advanced non‐small cell lung cancer. In each case, mepolizumab was introduced, resulting in discontinuation of systemic prednisolone (PSL) and good control of asthma.2, day 1) and tegafur/gimeracil/oteracil (TS‐1)  as the first‐line treatment. No granulocyte colony‐stimulating factor (GCSF) drugs were used during the initial chemotherapy course, and the eosinophil count did not exceed 300. The primary tumour was enlarged, and the diagnostic evaluation was progressive disease (PD). Atezolizumab, an anti‐PD‐L1 antibody , was started as the second‐line therapy. The tumour shrank, and the best effect was a partial response (PR). However, from the 19th course, daily coughing, wheezing and nocturnal symptoms appeared; therefore, fluticasone furanecarboxylate (200 μg)/umeclidinium bromide (62.5 μg)/vilanterol (25 μg) and a leukotriene receptor antagonist (LTRA) were administered. However, the eosinophil count, which had been elevated before treatment  of 1.59 L, FEV1/forced vital capacity (FVC) of 64.37% and fractional exhaled nitric oxide (FeNO) of 78 ppb . After a diagnosis of severe asthma and initiation of mepolizumab , these symptoms resolved. At the fourth dose, the symptoms were almost completely resolved and the respiratory function tests showed FEV1 of 1.88 L, FEV1/FVC of 54.49% and FeNO of 25 ppb. Atezolizumab was continued with mepolizumab until the 30th course of PD and is ongoing.The patient was a 70‐year‐old man with a 10 pack‐year history of smoking. He had a history of allergic rhinitis and bronchial asthma, but his symptoms were stable without use of inhaled corticosteroids (ICS). He was diagnosed with squamous cell carcinoma of the lung cT4N3M1a stage IVA. The epidermal growth factor receptor mutation was negative, with PD‐L1 < 1%, and he received two courses of cisplatin  and low doses of budesonide 320 μg)/formoterol (9 μg). He was diagnosed with squamous cell carcinoma of the lung cT4N1M0 stage IIIA, with PD‐L1 1%, and underwent chemoradiotherapy. Chemotherapy consisted of cisplatin (day 1) and TS‐1 (days 1–14) every 4 weeks for 2 cycles. Once‐daily thoracic radiotherapy was administered at a total dose of 60 Gy, 5 days a week, in 30 fractions. No GCSF agents were used during chemoradiation therapy, and the eosinophil count never exceeded 300. As the treatment effect was PR, durvalumab was then administered as maintenance therapy. After the start of durvalumab, the number of eosinophils in the peripheral blood gradually increased from 50 to approximately 300 , 177, 181, 189, 355, 407 and 426 trials, bronchial asthma as an adverse drug reaction was observed in two of 5339 patients and there were no serious cases (Reference from the drug interview form for pembrolizumab in Japan). Bronchial asthma is thus a very rare side effect of the use of ICIs. Nevertheless, similar to the cases presented, there are several reports of bronchial asthma onset and exacerbation during the use of ICIs.On the other hand, eosinophils are also components of the tumour immune microenvironment. They are a source of anti‐tumour  and pro‐tumour  molecules. Eosinophils play an anti‐tumour role in some neoplasms , whereas they are associated with poor prognosis in others .1 over time,In bronchial asthma, exacerbations increase the decline in FEVIn conclusion, two patients with severe bronchial asthma caused by ICI were treated with mepolizumab and their asthma symptoms improved. Treatment with biologics may be effective in severe cases of poorly controlled bronchial asthma during ICI therapy. We have reported these adverse events to the pharmaceutical companies for pharmacovigilance purposes.None declared.Conceptualization: Toshiyuki Sumi. Investigation: Toshiyuki Sumi, Yuta Nagahisa, Kengo Matsuura, Motoki Sekikawa and Yuichi Yamada. Writing—original draft: Toshiyuki Sumi. Writing—review and editing: Hisashi Nakata and Hirofumi Chiba.The authors declare that appropriate written informed consent was obtained for the publication of this case report and accompanying images.


Gonatoxia Karsch, 1889 species are fully alate Phaneropterinae, which are perfectly adapted to the foliage of forests. We examined five species using combined cytogenetic and molecular data to determine the inter- and intraspecific genetic diversity. The variation in the diploid number of chromosomes in males ranged from 2n = 28 + X0 and 26 + X0 to 2n = 6 + X0. Fluorescence in situ hybridization showed from one to many 18S rDNA loci as well as interstitial sequences, especially in G. helleri. 18S rDNA loci coincided with active NOR and C-banding patterns. The isolation of populations of the species explains differences in the number of chromosomes (G. maculata), chromosomal polymorphism and chromosomal heterozygosity (G. helleri). Our molecular phylogeny based on the COI locus supported the monophyly of the genus Gonatoxia and separateness of the five examined species in accordance with their morphological features and chromosome numbers as well as the species’ distribution.East Africa is a hotspot of biodiversity of many orthopteran taxa, including bushcrickets.  In closely related taxa that differ in many structural features of their chromosomes, either numerically or morphologically, these changes may reveal reproductive isolation4. Variation in chromosome number may indicate that a genus/species underwent speciation processes concomitantly with chromosomal rearrangements (CRs), which play a role in speciation via reproductive isolation5. Recent theoretical and empirical studies have suggested that CRs can alter the rate and pattern of gene flow within or between species, through a reduction in the fitness of chromosomal hybrids, or by reducing recombination rates in rearranged areas of the genome, according to the suppressed-recombination model of chromosome speciation9. CRs have been shown to play a causative role in isolating species or populations in various insect genera16 including Orthoptera e.g.24. An interesting example of the association CRs with evolution and adaptation to changing environments is Trimerotropis. This American genus is characterised by a high incidence of inversions and inversion polymorphisms, and exhibit a clinal distribution of inversions polymorphism and evolutionary significance of inversion clines24.Differences in chromosome number (karyotype variability) or chromosomal polymorphism within species are seen in many plant and animal groups, and may be involved in adaptation e.g.25, although this is not always the case. Extreme cases of rapid chromosome evolution are found in insects e.g.28.The analysis of karyotypes is a useful tool for systematic and evolutionary studies because closely related species tend to have more similar karyotypes than more distinctly related ones7. Fluorescence in situ hybridization (FISH) paired with classical cytogenetics, provides more detailed comparisons between the karyotypes of different taxa, and accurately identify CRs between species and/or genera. The number and location of rDNA signals (FISH) with distribution of heterochromatin  are useful to study karyotype evolution, identification of genus- and species-specific patterns as well as describing and delimiting new taxa within tettigoniid bushcrickets (e.g.32).Different banding techniques are the most frequently used methods to identify the presence of structural rearrangements that contribute to speciation36).To better understand the role of chromosomal changes in species diversification, a correlation between chromosomal and genetic variability needs to be examined in the context of species’ geographical distributions, especially comparing endemic with widespread species , but also in fully alate and thus mobile taxa (e.g. in Eurycorypha37 or Amytta62). Orthoptera were also first forest dwellers and later adapted to open land habitats in Africa due to the aridification of Africa beginning from 8 mya51.Mountainous East Africa is known for its high degree of biodiversity and endemism, especially along the so-called Eastern Arc Mountains, stretching through Tanzania and southern Kenya. East Africa is a hotspot of biodiversity of many orthopteran taxa, including bushcricketsParapyrrhicia Brunner von Wattenwyl, 189163 and in Plangia Stål, 187343, and 2n♂ = 28 + X0 in Eurycorypha Stål, 187337. In contrast to this apparent uniformity, an extreme case of rapid chromosome evolution was detected in Gonatoxia Karsch, 1889 species46. Gonatoxia is a very fascinating African genus characterized by a high chromosome variability rarely observed in tettigoniids  and chromosomal polymorphism within and between species. These bushcrickets are fully alate Phaneropterinae, perfectly adapted to the foliage of forests. Only in the last couple of years more information on the bioacoustics64 and cytogenetics3 of this genus became available, and new species were described46. Gonatoxia maculata Karsch, 1889 (2n♂ = 28 + X0 and 2n = 26 + X0), a species of savanna woodlands and deciduous dry forests, has the most widespread distribution of the genus, with individuals recorded from Somalia, Kenya and Tanzania while G. furcata Hemp, 2016 (2n♂ = 26 + X0) and G. immaculata Karsch, 1889 (2n♂ = 26 + X0) are endemics occurring in wet lowland and submontane forests and coastal and lowland wet forests, respectively. Gonatoxia immaculata is only known from localities in the East Usambara Mountains and might also occur e.g. in the Nguru Mountains further south, while G. furcata is at present known only from the Udzungwa Mountains. Gonatoxia helleri Hemp, 2016 (2n♂ = 6 + X0) was found at many localities syntopically with G. immaculata and G. furcata, overlapping with their ranges  both within its genus and among the Phaneropterinae. Thus, even the most basic characteristics of karyotypes seem to be useful tools to apply in the taxonomy of Gonatoxia46.Chromosome diversity of phaneropterine bushcrickets from Eastern Africa shows that most genera are characterized by karyotypic constancy among species, in particular by absence or only a low level of interspecific variability of chromosome numbers. Genomes of three Phaneropterinae genera have uniform karyotypes, with respective chromosome numbers of 2n♂ = 30 + X0 in Gonatoxia today. Chromosomal painting was useful for the definition of structural rearrangements, and molecular cytogenetics allowed us to detect homologies between related taxa. We mapped the chromosomal data obtained here, including previous data of G. helleri3 to test chromosomal evolution in the whole genus. Additionally, we have produced and used a phylogenetic tree based on cytochrome c oxidase subunit I (COI) sequences. The mitochondrial COI gene is unique enough to detect and delimit species of invertebrates69. The combination of chromosomal and DNA barcode data in comparative phylogenetic studies has been already used to clarify the species position (e.g. Lepidoptera and Diptera71). This enabled two major hypotheses to be tested: (1) chromosome numbers are correlated with phylogenetic lineages, (2) different types of characters (karyotypes and DNA sequences) exhibit geographical differences related to species distribution.The aim of this study was to describe the evolutionary relationships and chromosomal changes that created the karyotypes observed in Gonatoxia taxa revealed differences between their karyotypes. At the species level and between localities, the variation in the diploid number in males ranged from 2n = 28 + X0 and 26 + X0 to 2n = 6 + X0, in all cases with an X0 sex chromosome system (Table 3) and active nucleolus organizer regions (NORs) including those described previously46 and in the present study, revealed differences between species as well as between and within populations  and Gonotaxia sp.  is characterized by 2n = 28 + X0, FN = 29 in the male and 28 + XX, FN = 30 in the female  , the chromosome number was reduced to 2n = 26 + X0/26 + XX, FN = 27/28  , G. sp. (Ka: CH8046♂) and G. immaculata (Ni: CH8244♂) (Table G. maculata Uc: CH7963♂ and G. sp. Ka: CH8552♂), showed structural rearrangements such as end-to-end association  had a chromosome number of 2n = 26 + X0/26 + XX, FN = 29/30 ; in this karyotype one medium/small-sized pair of chromosomes was metacentric, while others were acrocentric, and the sex chromosome (X) was the largest element in the karyotype , FN = 10–13 in the male, and 2n = 8 (6 + XX), FN = 11–14 in the female. In this karyotype, the first long pair of autosomes was metacentric, whereas the second long and small third pairs were polymorphic in respect to the morphology of homologous chromosomes in specimens from the analyzed localities  in the interstitial region   showed signals at the distal end of each chromosome in the analyzed species. Sometimes the size of clusters on the ends of homologous chromosomes could also vary  in the paracentromeric and distal regions. In the analyzed specimens of G. maculata, G. sp. and G. furcata, DAPI- (negative) and weakly stained by CMA3 + (GC-rich) signals were localized interstitially in the 6/7th bivalent, and distally in small and long bivalents in G. immaculata. These CMA3 + bands coincided with one, two or three active NORs and rDNA/FISH signals  (for 1 specimen) and G. immaculata from Nilo (Ni) (for 2 specimens), Sigi (Si) (for 1 specimen) and Amani (Am) (for 1 specimen). The aligned COI dataset had a length of 620 bp, with a total of 255 polymorphic characters, of which 237 were parsimony-informative. The model of substitution was the symmetrical gamma distribution model (SYM + G). The obtained BI and ML topologies were congruent  = 1.00 and bootstrap value (BV) = 100%, Fig. Gonatoxia furcata from the Udzungwa Mountains  with the diploid chromosome number (2n) = 26 + X0 was the sister taxon to all other Gonatoxia species (PP ≤ 0.94 and BV ≤ 94%). Gonatoxia immaculata from the East Usambara Mountains  with 2n = 26 + X0 was the sister taxon to G. sp. from Kazimzumbwi Forest Reserve (Ka) with 2n = 28 + X0 (PP ≤ 0.94 and BV ≤ 94%) and these two were the sister taxa to G. maculata from Mt. Kilimanjaro  with 2n = 28 + X0 and the North Pare Mountains and G. helleri from various localities  with 2n = 6 + X0 (PP = 0.99–0.95 and BV = 99–95%). Small molecular differences at the population level could be detected between G. helleri specimens from the East Usambara, Udzungwa and Uluguru Mountains, despite this they formed one subclade within the Gonatoxia group.Analyzed specimens of G. helleri.Mapping the chromosomal characters onto the phylogenetic tree showed three groups with different degrees of polymorphism in chromosome number and the number of FISH-rDNA loci Fig. . HoweverGonatoxia, on a population as well as the species level. We wanted to examine the mechanisms of karyotype evolution in a phylogenetic context. This study presents the preliminary results of molecular investigations, in an attempt to discover species boundaries in the chromosomally diverse taxa of the genus Gonatoxia.The examination of karyotypes presented in this study provides insights into a hidden variation in 46 and shows that Gonatoxia exhibits a high chromosome differentiation between species and complicated cytogenetic systems (polymorphism), which are unusual in tettigoniids. Despite this, differences between karyotypes of populations in the same species/taxa and between species may be concealed by the distribution of specific chromosomal markers, the extensively investigated major rDNA genes. The observed diversity of chromosome numbers in this genus could theoretically be the result of chromosomal rearrangements (CRs) emerging from the ancestral/model karyotype (2n = 30 + X0) found in most tettigoniids (e.g.71) and most other African Phaneropterinae genera63. However, this model chromosome number was not found in Gonatoxia. For the karyotype 2n = 28 + X0 of G. maculata  and G. sp. , it is hypothesized that the autosome number was reduced by one tandem fusion. This number of chromosomes, including only acrocentric chromosomes, has been also reported for African species of Eurycorypha and Plangia43. However, the same number is also found in more distantly related genera72, indicating several independent reductions in this subfamily. On the other hand, a reduced chromosome number of 2n = 26 + X0, probably caused by two fusions  in one specimen of G. maculata from Lembeni (North Pare Mountains) and G. immaculata from all localities , might imply that a chromosomal mutation happened probably independently in individuals. The chromosome number variation between the populations of G. maculata , which most likely are different chromosomal races, suggests ongoing differentiation in isolated populations (Table Steropleurus martorelli complex (Bradyporinae), a coastal species endemic to the Iberian Peninsula with diploid chromosome numbers in the male ranging from 29 to 2373. In G. furcata (2n = 27) from the Udzungwa Mountains, however, a different mechanism in reducing the chromosome number must be assumed. This reduction was probably caused by one fusion and an additional Robertsonian translocation (i.e. fusion-fission cycle) between two small pairs of autosomes. Besides these changes in chromosome number, it should be noted that in some individuals of G. maculata, G. sp. and G. immaculata, variation of the chromosome number in the same individual (instability/mosaic cells) in mitotic division, and irregularities in meiotic cell division, such as translocation, asynapsis, loops or additional elements, were observed—although rarely. As a consequence of these mechanisms, an aneuploid-dysploid mosaicism of the chromosome complement cannot be excluded. Diploid chromosome rearrangements (pseudoaneuploidy) are often assumed to be the dominant mechanism of a basic chromosome number reduction in grasses (e.g.74). In turn, the main characteristics of the karyotype of G. helleri, with only seven large chromosomes (2n = 6 + X0), are a reduced chromosome number and the asymmetrical karyotype (karyomorphs) rarely being found in tettigoniids. Additionally, in this species irregularities in the course of meiosis compared to other Gonatoxia species were observed. This reflects the derivation by multiple rearrangements of the ancestral karyotype, probably by Robertsonian translocations and/or tandem fusions and inversions. Additionally, in some individuals, a diploid number was observed with intra-individual variability among cells, with 14  and 16  pairs of chromosomes, probably corresponding to tetraploid levels3. In this species, an incompatible meiotic configuration and a level of heterozygosity in some populations may have been a precondition of colonizing new habitats and might be a case of adaptive diversification in G. helleri3.Our cytogenetic examination confirms previous study findingsns Table . In tettGonatoxia exhibits a great variation in its karyotypic macrostructure e.g. the number of chromosomes and the morphology and the distribution of specific chromosomal markers, such as in the extensively investigated tettigoniid major rRNA genes (18S), telomeric DNA and constitutive heterochromatin. The application of different staining techniques  provides useful markers for the characterization of the chromosomes of phaneropterines and other tettigonids32, and is important for the development of genus or species-specific models of karyotypes. The karyotypes of individuals of G. maculata, G. sp. and G. furcata (regardless of the number of chromosomes), have a single 18S rDNA/active NOR-bearing chromosome pair, which is typical of most African and European phaneropterid taxa examined so far. Generally, rDNA loci in Phaneropterinae were situated in the pericentromeric region, which would imply that this chromosomal feature may be plesiomorphic, or is found rarely in the interstitial region (e.g.32). Individuals of G. maculata, G. sp. and G. furcata, similar detected in one Parapyrrhicia63 and many Eurycorypha species37, have 18S rDNA clusters placed in an interstitial position, which may have resulted from CRs , transposable element insertions or ectopic recombinations75. In contrast, G. immaculata shows inter- and intraspecific polymorphism including the number (two or rarely three) and position  of 18S rDNA sites. Two pairs of chromosomes bearing DNA clusters appear to be potentially useful cytogenetic markers for distinguishing this species among other Gonatoxia species with 29/27 chromosomes in their karyotypes. Interestingly, the occurrence of three DNA clusters in G. immaculata was connected with intraspecific variation. This genetic variance and instability may be the result of rapid recent and/or ongoing diversification and/or frequent hybridization. The diversity of the number and distribution of 18S rDNA between Gonatoxia species may be explained by (i) ectopic recombination, as described for the scorpion Tityus obscurus45; (ii) translocations, considering that heterochromatic regions rich in rDNA are also considered hotspots for chromosome breaks76; or (iii) transposition of active transposable elements78.Our cytogenetic data show that the East African genus 63). However, compared to phaneropterines and other tettigoniids, the chromosomal distribution of major 18S rDNA signals in G. helleri is unique. Specifically, there are differences in the location , a high number of sites with differing intensity of hybridization signals, and the presence of interstitial telomeric sequences (ITSs) near the pericentromeric and interstitial regions and/or near the telomeric region. The presence of several sites with ITSs  in G. helleri3, and a single ITS near the secondary construction in the X chromosome of G. maculata, confirm the hypothesis that this karyotype/these karyomorphs could be the result of telomere-telomere fusions of the chromosomes, inversions , unequal crossing over, or the insertion of telomeric DNA into unstable sites during the repair of double-strand breaks79. ITSs have been regarded as remnants of chromosomal fusions promoted by repetitive subtelomeric elements as well as inversions81.Generally, our results demonstrate a coincidence of the locations of rDNA loci and an active NOR/s, as well as C-positive and G–C rich heterochromatin regions, similar to other African phaneropterids e.g.Gonatoxia species form a monophyletic group. Gonatoxia furcata, endemic to the Udzungwa Mountains (Ma), clusters at the base of Gonatoxia, being the sister group to all other species of this genus. Gonatoxia immaculata from the East Usambara Mountains (Mg) and Gonatoxia sp. from Kazimzumbwi Forest Reserve (Ka) further south at the coast formed one sister clade. Gonatoxia maculata, probably a widespread species in East Africa, and G. helleri , another widespread species, formed the sister clade. Observed chromosome numbers agree well with the molecular information gained from the COI gene and their habitats as well as their distribution. Gonatoxia furcata, a rare species only found in low numbers in the Udzungwa Mountains, could have a reduced chromosome set due to habitat constriction, individuals of this species having a small area of occurrence. Local adaptation could explain the evolution of many chromosome fusions, which are some of the most common chromosome rearrangements in nature82. This mechanism of chromosome reduction, in connection with a restricted habitat, has already been discussed for the hexacentrine genus Aerotegmina38, which includes species with a mix of an ancestral number (2n = 32 + X0) and a reduced number of chromosomes (2n = 26 + X0). Similar events have been suggested to have affected the speciation of African mole-rats (Rodentia) of the genera Cryptomys and Fukomys, endemic to sub-Saharan regions83, or Arvicanthis84, which are characterized by extreme chromosomal variation.In our phylogenetic reconstruction, the five analyzed Gonatoxia furcata with a reduced chromosome number (2n = 26 + X0) exhibits the ancestral (plesiomorphic) condition, whereas most other taxa had a higher number of chromosomes (2n = 28 + X0). Only G. helleri, probably the most recently evolved species, with a wide distribution and occupying a wide ecological niche from lowland to montane forests, had an extremely reduced chromosome number. Additionally, small molecular differences were detected between several specimens of G. helleri from different localities and habitats (lowland wet forest in the East Usambaras to lowland wet forest in the Udzungwas and montane forest in the Ulugurus). Specimens from the Udzungwa and Uluguru Mountains clustered together, showing the geographical proximity of these mountain ranges, while specimens from the further north located East Usambara Mountains formed a separate cluster at the base of the clade of G. helleri. These clusters of G. helleri also suggest that populations are isolated from each other and that speciation is currently in progress in this species.Gonatoxia and other African Phaneropterinae. These data showed to be convincing tools, supporting systematic and taxonomic research and uncovering hidden biodiversity. Our results show that Gonatoxia represents a genetically diverse group, showing variation in terms of the number and morphology of chromosomes and the distribution of DNA clusters. Considering the current geographical range, the isolation of populations of one species and their respective karyotypes, differences in rDNA/NOR numbers , and the abundance of chromosomal heterozygotes (G. helleri), this probably represents a case of preconditioning to successfully colonize new habitats and to cope with changing environments. The fixation and/or heteromorphism of different CRs, by geographic isolation and genetic drift could have been triggered by climatic events that changed the environment, leading CRs to play some role in lineage diversification. The observed chromosome numbers and their distribution agree well with the molecular information gained from the COI gene. The knowledge compiled here provided useful information for understanding the speciation processes and faunal formation of African insects. Thus, we here presented data on the genus Gonatoxia serving as a model to understand biogeographic patterns in East Africa through a combination of analysing chromosome numbers and structures, and combining them with molecular methods and information on the habitat.Our integrative approach, combining the cytogenetic and phylogenetic methods presented herein, constitutes a step towards a better understanding of chromosomal organization and relationships within Gonatoxia . The sampled bushcrickets were taken in the field from 10 populations in northern Tanzania , and squashed in 45% acetic acid. Cover slips were removed using a dry ice procedure and the slides were air dried. To determine the distribution of heterochromatin Giemsa C-banding was carried out using the methods described in85, the GC/AT-rich chromosomal segments were stained with fluorochromes CMA3 (chromomycin A3) and DAPI 86. The silver staining method (AgNO3) for localization of NORs was performed as previously reported87. FISH experiments were performed following the protocol of88. The rDNA gene was amplified and labeled using primers 18S forward and 18S reverse89, and biotin-16-dUTP  by polymerase chain reaction (PCR). Telomere probes were PCR-amplified and labeled using the primers TTAGG_F and TTAGG_R89 and digoxigenin-11-dUTP . FISH signals were detected using avidin-FITC  and anti-digoxigenin rhodamine . Preparations were counterstained with ProLong Gold antifade reagent containing DAPI . Images were captured using a Nikon Eclipse 400 fluorescence microscope equipped with a CCD DS-U1 camera and the NIS-Elements BR 3.0 software package. Information was based on analysis of mitotic metaphase  and/or meiotic divisions using both rDNA and telomeric DNA . In each species/individual, karyograms were reconstructed by arranging homologous chromosomes in decreasing size.Testes, ovaries, and somatic hepatic caeca were dissected, incubated in hypotonic solution (0.9% sodium citrate), fixed in ethanol—acetic acid  was performed in 20 µl of reaction volume consisting of 2 µl reaction buffer, 25 mM MgCl2, 10 mM dNTP mixture, 10 µM forward and reverse primers, 1–3 µl of genomic DNA, 5 U of Gold Taq DNA polymerase , and sterile deionized water. To amplify COI, the following PCR protocol was used: 36 cycles at 94 °C for 1 min, 48 °C for 1 min and 72 °C for 2 min, with the final extension at 72 °C for 7 min. The PCR products were purified with EPPiC Fast . Sequencing reactions were carried out in 10 µl reactions containing: 1.5 µl of sequencing buffer, 1 µl of the BrilliantDye v3.1 Terminator Cycle Sequencing Kit , 1 µl of primer (forward or reverse), 3.0 µl of the purified products and 3.5 µl of sterile water. The sequencing reaction was as follows: 3 min at 94 °C, 25 cycles of 10 s at 96 °C, 5 s at 55 °C and 90 s at 60 °C. The 29 individuals which gave positive results on PCR amplification of the COI gene of Gonatoxia, Eurycorypha, Plangia and Parapyrrhicia were successfully sequenced. The nucleotide sequences of the analyzed specimens were deposited in GenBank under the accession numbers provided in Table Genomic DNA extraction was carried out from 34 single specimens  with default parameters. Sequences were checked for protein-coding frame shifts to detect pseudogenes using MEGA v. X91, and compared with sequences from GenBank through a BLAST search. Phylogenetic trees were estimated using Bayesian Inference (BI) and maximum likelihood (ML). The substitution model of evolution was estimated in MrModeltest v. 2 software92 using the Akaike Information Criterion (AIC). BI was implemented in MrBayes v. 3.293 with four independent runs, each having one cold and three heated Markov chains. The analysis was run for 6 million generations, with trees sampled every 100 generations. The first 25% of each run was discarded as burn-in. Convergence among the runs was assessed using Tracer v. 1.794. The ML analysis was conducted in IQ-TREE v. 195. Nodal support was assessed using a nonparametric bootstrap with 1000 replicates. To visualize the patterns of evolution in Gonatoxia, chromosomal characters, habitats and distribution were mapped according to parsimony criteria in a pruned phylogenetic tree from Fig. 96. To project a tree onto a geographic map we used “phytools” package v. 0.7-7097 in R statistical software v. 4.0.398.The obtained nucleotide sequences were edited, assembled into contigs and aligned in CodonCode Aligner v. 8.0 (CodonCode Corporation; Supplementary Information.


MW1 = 13.15; SD = 1.09; 49% girls) from 7th to 9th grade. The European Cyberbullying Intervention Project Questionnaire and the Anger Rumination Scale were administered in four waves with 6 months intervals over a total period of 18 months. The associations between the variables were analyzed with a cross‐lagged model. We found that: cybervictimization predicted anger rumination and cyberaggression; anger rumination was associated with later increases in both cybervictimization and cyberaggression: but involvement in cyberaggression predicted neither subsequent involvement in cybervictimization, nor in anger rumination. In addition, cybervictimization was found to mediate the association between anger rumination and cyberaggression. This study expands the understanding of the factors associated with cybervictimization and cyberaggression, and its results indicate that intervention programs should focus on boosting self‐control to decrease impulsive behavior and protocols to prevent and intervene in cyberbullying.Adolescents' involvement in cyberbullying has been a growing public health concern for some time. Cybervictimization and cyberaggression are two phenomena that previous research has often shown to be associated. However, longitudinal research into these associations and also into potential risk factors for these phenomena is less common. Anger rumination is a proven risk factor for aggressive behavior, but the relationship between anger rumination and victimization is not clear. The present longitudinal study investigated the associations between cybervictimization, anger rumination and cyberbullying in a sample of 3017 adolescents ( Individuals who engage in anger rumination may be more inclined to focus on the negative feelings caused by the stressful event, rather than on addressing the problem. Previous research suggests that such emotion‐focused coping is associated with an increase in anxiety and depression  ; involvement in cyberaggression does not lead to a later increase or reduction in cybervictimization (Hypothesis 1b); cybervictimized adolescents show an increase in anger rumination (Hypothesis 2a), and anger rumination predicts an increase in cybervictimization (Hypothesis 2b); anger rumination predicts an increase in cyberaggression (Hypothesis 3a), but there is no reverse relationship (Hypothesis 3b); Finally, we expect that boys and girls do not differ in the associations between cybervictimization, anger rumination and cyberaggression (Hypothesis 4a), and that, due to the narrow age range of the study's participants  there are no differences in the associations between early and middle adolescents (Hypothesis 4b).The possible indirect effects were analyzed. We hypothesized that anger rumination would mediate the association between cybervictimization and cyberaggression (Hypothesis 5a). This is consistent with previous studies which found the mediating role of anger rumination as a risk mechanism of aggression with trait self‐control, trait anger and hostile attribution bias as predictors ; Wave 2 (W2) in May 2018 included 2553 ; Wave 3 (W3) in November 2018 included adolescents 2362 ; and Wave 4 (W4) in May 2019 included 2361 adolescents . Of the total sample, 59% participated in all four waves, 22% participated at three time points, 11% at two time points, and 7% took part at only one time point.The participants were drawn from a large longitudinal study into personal and ecological developmental risks and protective factors during adolescence. The convenience sample comprised 3,017 adolescents  between 11 and 16 years old, attending Grades 7–9, and included 115 classes from 13 middle schools in Southern Spain. In this study, we analyzed four waves of data collected during the years 2017–2019 at 6‐month intervals. At each data collection point the sample varied due to temporary absence or changes of school. Wave 1 (W1 hereafter) in November 2017 included 2790 adolescents  to 4 . Responses to the items were averaged within each dimension. Higher scores correspond to higher levels of cybervictimization and cyberaggression. The internal consistency of the scale in our study is presented in the Results section.We measured cybervictimization and cyberaggression using the European Cyberbullying Intervention Project Questionnaire  and age (1 = early adolescents: 2 = middle adolescents) differences. The internal consistency of the scales was assessed with Cronbach's alpha. Longitudinal measurement invariance was analyzed to verify the consistency of the constructs over time ; (b) cross‐lagged paths between different variables in adjacent waves ; and (c) covariances between different variables measured at the same wave , from W2 to W4, the covariances are based on the residual variances. To allow an efficient and systematic interpretation of the associations, we compared several models with decreasing constraints imposed on the estimation of abovementioned paths. Models were built in four steps: in Model 1 the cross‐lagged paths, autoregressive paths, and the residual covariances between the variables in the same wave were constrained to be equal over time (from W2 to W4); in Model 2 the residual covariances were freely estimated; in Model 3 the residual covariances and cross‐lagged paths were also freely estimated; and in addition, in Model 4 the residual covariances, cross‐lagged paths and autoregressive paths were freely estimated. The Scaled χ2 Difference test  autoregressive paths within the same variable over adjacent waves . Missing data character was explored though the Missing Completely at Random test (MCAR). Although Little's MCAR test provided a significant result (p < .001), correction of this result for sensitivity to sample size through the normed χ2 (χ2/df = 1.34)  and middle (14–16 years) adolescence. Middle adolescents reported more cybervictimization, anger rumination, and cyberaggression than early adolescents. Following Cohen  = 165.090, p < .001; CFI = 0.979, TLI = 0.975; and RMSEA = 0.025, 90% CI [0.021–0.030]. After the covariances between variables in the same wave were allowed to vary over time, Model 2 did not reveal fit better than Model 1, consequently Model 1 was retained: (51) = 157.135, p < .001; CFI = 0.979, TLI = 0.973; and RMSEA = 0.026, 90% CI [0.022–0.031], ∆χ² (6) = 9.24, p > .05. Model 3, with additional unconstraint to the cross‐lagged paths, again showed good fit: χ² (40) = 136.671, p < .001; CFI = 0.981, TLI = 0.969; and RMSEA = 0.028, 90% CI [0.023–0.034]. Fit indices did not improve significantly from Model 1, ∆χ² (11) = 20.31, p > .05. Finally, Model 4, in which autoregressive paths were allowed to vary over time once more showed good fit: χ² (34) = 118.020, p < .001; CFI = 0.984, TLI = 0.968; and RMSEA = 0.029, 90% CI [0.023–0.035]. Model fit did not improve in comparison with Model 1, ∆χ² (23) = 33.1, p > .05. Given the lack of significant differences between the models, the Model 1 was used to analyze the associations between the variables.We estimated and compared hierarchical cross‐lagged models to which constraints were introduced in a stepwise manner. Model 1  had good model fit: ps > .05 for all Wald tests), indicating an absence of gender and age differences with regard to the associations. Based on the results found, the INDIRECT models were added to analyze the possible mediations between variables. The statistically significant mediated paths are presented in Table The results of the cross‐lagged model are shown in Figure 4We looked at the longitudinal associations between anger rumination, cybervictimization and cyberaggression, as the possible mediation effects.In previous research, cybervictimization and cyberaggression were strongly associated . Finally, the students in this study all fell into a relatively narrow age range, which limits the ability to generalize our results to other age groups. Future studies may also want to assess whether the associations between cybervictimization, anger rumination and cyberaggression differ between those just starting to use technology as pre‐adolescents and later adolescents or emergent adults with more experience in cyberspace.In spite of these limitations, this study offers a contribution to the growing body of research into factors associated with cybervictimization and cyberaggression among adolescents. As an empirical contribution, our findings show that anger rumination predicts an increase in later involvement in cybervictimization and cyberaggression, and that cybervictimization experiences and the anger they cause will in some adolescents lead to (higher levels) of anger rumination and cyberaggression. Finally, the study contributes to further insights into the association between anger rumination and cyberaggression by highlighting cybervictimization as a mediating mechanism between both.The results of our study also have practical implications; they highlight the importance to develop cognitive strategies that improve self‐control to decrease impulsive and risky behavior and consequently victimization and aggression online. Denson et al.  show howThe authors declare that there are no conflict of interests.