Deep DNA sequencing of MGMT, TP53 and AGT in Mexican astrocytoma patients identifies an excess of genetic variants in women and a predictive biomarker.

PURPOSE: Astrocytomas are a type of malignant brain tumor with an unfavorable clinical course. The impact of AGT and MGMT somatic variants in the prognosis of astrocytoma is unknown, and it is controversial for TP53. Moreover, there is a lack of knowledge regarding the molecular characteristics of astrocytomas in Mexican patients. METHODS: We studied 48 Mexican patients, men and women, with astrocytoma (discovery cohort). We performed DNA deep sequencing in tumor samples, targeting AGT, MGMT and TP53, and we studied MGMT gene promoter methylation status. Then we compared our findings to a cohort which included data from patients with astrocytoma from The Cancer Genome Atlas (validation cohort). RESULTS: In the discovery cohort, we found a higher number of somatic variants in AGT and MGMT than in the validation cohort (10.4% vs < 1%, p < 0.001), and, in both cohorts, we observed only women carried variants AGT variants. We also found that the presence of either MGMT variant or promoter methylation was associated to better survival and response to chemotherapy, and, in conjunction with TP53 variants, to progression-free survival. CONCLUSIONS: The occurrence of AGT variants only in women expands our knowledge about the molecular differences in astrocytoma between men and women. The increased prevalence of AGT and MGMT variants in the discovery cohort also points towards possible distinctions in the molecular landscape of astrocytoma among populations. Our findings warrant further study.

Analysis of cancer mortality in Mexico using the results of the Global Burden of Disease 2021 study.

BACKGROUND: In Mexico, cancer mortality rates have undergone changes over the past decades. OBJECTIVE: To analyze the evolution of cancer mortality rates in Mexico between 1990 and 2021. MATERIALS AND METHODS: Based on the Global Burden of Disease study, the mortality rates for the 10 most prevalent types of cancer in Mexico were obtained, at the national and regional level and by states, considering different age groups and gender. Global Burden of Disease reassigns misclassified causes and distributes them among different types of cancer; subsequently, it models and adjusts the causes to the total number of deaths with a model of a set of causes of death and a cause corrector, which corrects INEGI's mortality records. RESULTS: The cancer mortality rate went from 117.87 in 1990 to 84.18 in 2021. In women, breast, cervical, stomach and lung cancers were the most frequent. In men, the most common were prostate, stomach, lung, and colon and rectum cancer. The decrease in cancer mortality for men and women stood out, particularly from lung and cervical cancer. CONCLUSIONS: The results provide information for the development of health policies and specific prevention and control strategies to address the impact of cancer in Mexico.

ANTECEDENTES: En México, las tasas de mortalidad por cáncer han experimentado cambios a lo largo de las últimas décadas. OBJETIVO: Analizar la evolución de las tasas de mortalidad por cáncer en México entre 1990 y 2021. MATERIALES Y MÉTODOS: Del Global Burden of Disease (GBD) se obtuvieron las tasas de mortalidad de los 10 tipos de cáncer más predominantes en México, en el ámbito nacional y por estados, considerando distintos grupos etarios y el sexo. En el GBD se reasignan las causas mal clasificadas y se distribuyen entre los distintos cánceres; posteriormente se modelan y ajustan las causas al total de fallecimientos con un modelo de conjunto de causas de muerte y un corrector de causas, con lo cual se corrigen registros de mortalidad del INEGI. RESULTADOS: La tasa de mortalidad por cáncer pasó de 117.87 en 1990 a 84.18 en 2021. En las mujeres, los cánceres de mama, cervicouterino, estómago y pulmón fueron los más frecuentes; en los hombres, de próstata, estómago, pulmón, colon y recto. Destacó la disminución de la mortalidad por cáncer en hombres y mujeres, en particular por cáncer de pulmón y cérvix uterino. CONCLUSIONES: Los resultados ofrecen información para desarrollar políticas de salud y estrategias de prevención y control específicas para enfrentar el impacto del cáncer en México.

Landscape of Germline Genetic Variants in AGT, MGMT, and TP53 in Mexican Adult Patients with Astrocytoma.

Astrocytoma is the most common type of primary brain tumor. The risk factors for astrocytoma are poorly understood; however, germline genetic variants account for 25% of the risk of developing gliomas. In this study, we assessed the risk of astrocytoma associated with variants in AGT, known by its role in angiogenesis, TP53, a well-known tumor suppressor and the DNA repair gene MGMT in a Mexican population. A case-control study was performed in 49 adult Mexican patients with grade II-IV astrocytoma. Sequencing of exons and untranslated regions of AGT, MGMT, and TP53 from was carried in an Ion Torrent platform. Individuals with Mexican Ancestry from the 1000 Genomes Project were used as controls. Variants found in our cohort were then assessed in a The Cancer Genome Atlas astrocytoma pan-ethnic validation cohort. Variants rs1926723 located in AGT (OR 2.74, 1.40-5.36 95% CI), rs7896488 in MGMT (OR 3.43, 1.17-10.10 95% CI), and rs4968187 in TP53 (OR 2.48, 1.26-4.88 95% CI) were significantly associated with the risk of astrocytoma after multiple-testing correction. This is the first study where the AGT rs1926723 variant, TP53 rs4968187, and MGMT rs7896488 were found to be associated with the risk of developing an astrocytoma.

Cervical Cancer in Young Women: Do They Have a Worse Prognosis? A Retrospective Cohort Analysis in a Population of Mexico.

BACKGROUND: Cervical cancer (CC) is a global problem; it is among the five leading causes of cancer death in women. Several studies have examined the association between age and disease prognosis; however, controversy still exists. The objective of the present study is to determine if age at diagnosis has an impact on overall survival (OS) and disease-free survival (DFS). MATERIALS AND METHODS: Retrospective cohort of 2,982 patients with CC treated at the National Cancer Institute of Mexico from 2005 to 2015. We collected demographic, clinical, and treatment data, as well as current status, of 2 groups: women under and over 40 years of age. We calculated OS and DFS rates with Kaplan-Meier estimates. Cox proportional hazards modeling was used to determine risks. RESULTS: The median follow-up time was 26.5 months (percentile [P]25 -P75 , 11-60.23). When comparing DFS, OS, stage, and histologic subtype between young patients <40 and adult patients >40, we did not observe any difference. We found that in both groups, locally advanced and advanced stage, neuroendocrine subtype, hydronephrosis, and positive inguinal lymph nodes increased the risks of death and recurrence. Having been pregnant was identified as protective factor in DFS (hazard ratio, 0.54; 95% confidence interval, 0.04-0.71). CONCLUSION: We corroborated that age at diagnosis is not a prognostic factor for decreased or increased OS or DFS, and in both groups, the stage, histologic subtype, hydronephrosis, and node involvement were identified as factors adverse to OS and DFS, and pregnancy history was a protective factor in DFS. IMPLICATIONS FOR PRACTICE: The present study directly affects everyday clinical practice because it allows us to focus on the most relevant prognostic factors in patients with cervical cancer. When planning treatment and follow-up, clinicians should focus on stage at diagnosis, histologic subtype, hydronephrosis, and distant metastasis instead of patients' age. They should also be aware of any previous pregnancies and poor response, or nonresponse, to treatment, which results in disease progression and persistence. Paying attention to these factors affecting overall survival and disease-free survival will help treat patients better and increase their chances of survival and improve their quality of life.

[Prognostic factors in patients with breast cancer and brain metastasis as the first site of recurrence]. / Factores pronósticos en pacientes con cáncer de mama y metástasis cerebral como primer sitio de recurrencia.

OBJECTIVE: To evaluate the prognostic factors (clinicalpathological characteristics and treatments) in patients with breast cancer and metastasis to central nervous system (CNS) as the first site of the disease. MATERIAL AND METHODS: Kaplan-Meier method and life tables were used to estimate overall survival time over a retrospective cohort of 125 breast cancer patients treated at the Instituto Nacional de Cancerología (INCan) during 2007-2015, who presented metastasis to the CNS as the first site of extension of the disease. The cox proportional hazards model was used to determine the prognosis factors. RESULTS: The median overall survival time was 14.2 months (IC95%: 11.83-26.93). Patients with triple negative (TN), according to inmunohistochemistry analysis classification, had lower survival times (p=0.0004) and had a risk of dying two times (p=0.037) higher than patients with a different immunophenotype (HR: 2.77. 95%CI: 1.10-6.99). The degree of intermediate SBR increases the risk of dying in patients with metastasis (HR 2.76, 95% CI: 1.17-6.51). CONCLUSIONS: CNS metastasis continues to be a poor prognostic factor that reduces survival and affects quality of life. It is recommended to monitor the early presence of clinical neurological manifestations during follow-up for prompt treatment. TN patients have worse prognosis and HER2+ a better control.

OBJETIVO: Evaluar los factores pronósticos (características clínico-patológicas y tratamientos) en las pacientes con cáncer de mama y metástasis al sistema nervioso central (SNC) como primer sitio de afección. MATERIAL Y MÉTODOS: Cohorte retrospectiva, formada por 125 pacientes con cáncer de mama atendidas en el Instituto Nacional de Cancerología durante 2007-2015, quienes presentaron afección en el SNC como primer sitio de metástasis. A través del método Kaplan-Meier y tablas de vida se estimó la supervivencia global. El modelo de riesgos proporcionales de Cox fue utilizado para determinar los factores pronósticos. RESULTADOS: La mediana de supervivencia global fue de 14.2 meses (IC95% 11.8-26.9). Pacientes clasificadas por inmunohistoquímica como triple negativo (TN) presentaron tiempos de supervivencia más cortos (p<0.004) y con dos veces más riesgo de fallecer, en comparación con los otros inmunofenotipos (HR= 2.77; IC95% 1.10-6.99); asimismo, se identificó que un grado intermedio en la escala Scarff-Bloom-Richardson incrementa el riesgo de morir en pacientes con metástasis (HR=2.76; IC95% 1.17-6.51). CONCLUSIONES: La metástasis al SNC continúa siendo un factor de mal pronóstico que reduce la supervivencia y afecta la calidad de vida. Se recomienda vigilar puntualmente la presencia de manifestaciones clínicas neurológicas durante el seguimiento, para una rápida intervención. Las pacientes TN tienen peor pronóstico, y las HER2+ (es decir, con resultado positivo para el receptor 2 del factor de crecimiento humano epidérmico), mejor control a mediano plazo.

Molecular Biology In Young Women With Breast Cancer: From Tumor Gene Expression To DNA Mutations.

Young women with breast cancer (YWBC) represent roughly 15% of breast cancer (BC) cases in Latin America and other developing regions. Breast tumors occurring at an early age are more aggressive and have an overall worse prognosis compared to breast tumors in postmenopausal women. The expression of relevant proliferation biomarkers such as endocrine receptors and human epidermal growth factor receptor 2 appears to be unique in YWBC. Moreover, histopathological, molecular, genetic, and genomic studies have shown that YWBC exhibit a higher frequency of aggressive subtypes, differential tumor gene expression, increased genetic susceptibility, and specific genomic signatures, compared to older women with BC. This article reviews the current knowledge on tumor biology and genomic signatures in YWBC.

Radiotherapy for Metastatic Breast Cancer in Mexico: Results from the 2015 National Survey.

BACKGROUND: Radiation therapy is a keystone to improve survival and quality of life in breast cancer patients. In Mexico, however, scarce information is available on the obstacles faced by radio-oncologists to provide appropriate treatment. OBJECTIVE: To determine the most frequent issues faced by physicians to provide radiation therapy for metastatic breast cancer in Mexico. METHODS: A survey of 16 multiple-choice questions to be answered electronically by 167 radio-oncologists currently working in Mexico was designed and thereafter analyzed for differences between private and public practices, based on the responses from the surveyed participants. RESULTS: 98.5% of surveyed responders attended patients with breast cancer. We observed a significant difference between private vs. public practice for the main difficulties in providing radiation therapy, with an increased frequency (85.8%) of "treatment cost by itself" in private practice vs. 50.7% in public practice (p < 0.05). Significant differences were observed in the "Time to initiate treatment" question, with "Less than one week" as the response in 86% of those physicians in private practice vs. 50% for those in public practice (p < 0.001). CONCLUSIONS: Using a survey targeted at radio-oncologists, we analyzed the most important obstacles for accessing radiation therapy for metastatic breast cancer in Mexico. This information may be useful for healthcare decisions related to radiation therapy in women with breast cancer in Mexico.

"Joven & Fuerte": Program for Young Women with Breast Cancer in Mexico - Initial Results.

Despite the high rates of breast cancer among young Mexican women, their special needs and concerns have not been systematically addressed. To fulfill these unsatisfied demands, we have developed "Joven & Fuerte: Program for Young Women with Breast Cancer in Mexico," the first program dedicated to the care of young breast cancer patients in Latin America, which is taking place at the National Cancer Institute of Mexico and the two medical facilities of the Instituto Tecnológico y de Estudios Superiores de Monterrey. The program was created to optimize the complex clinical and psychosocial care of these patients, enhance education regarding their special needs, and promote targeted research, as well as to replicate this program model in other healthcare centers across Mexico and Latin America. From November 2013 to February 2017, the implementation of the "Joven & Fuerte" program has delivered specialized care to 265 patients, through the systematic identification of their particular needs and the provision of fertility, genetic, and psychological supportive services. Patients and families have engaged in pedagogic activities and workshops and have created a motivated and empowered community. The program developed and adapted the first educational resources in Spanish dedicated for young Mexican patients, as well as material for healthcare providers. As for research, a prospective cohort of young breast cancer patients was established to characterize clinicopathological features and psychosocial effects at baseline and during follow-up, as a guide for the development of specific cultural interventions addressing this vulnerable group. Eventually, it is intended that the program's organization and structure can reach national and international interactions and serve as a platform for other countries.

SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

The importance of registries in cancer control.

Cancer is one of the major causes of morbidity and mortality in the world, with 14.1 million new cases and 8.2 million deaths annually. A marked disparity exists between developed countries and developing countries, with 57% of new cases and 65% of deaths in 2012 occurring in developing countries. This global picture can only be obtained because of data obtained from population-based cancer registries, which allow cancer estimations for different geographic areas. Our objective is to perform a review of different types of registries and their role in the control of cancer. These types of registries are lacking in developing countries. In Central and South America, only 6% of the population is included in cancer registries versus 83% in North America. It is necessary to increase the coverage of cancer registries to obtain more reliable data that will more appropriately guide control programs.

Estimating the indirect costs associated with the expected number of cancer cases in Mexico by 2020.

OBJECTIVE: To estimate the indirect costs generated by adults with cancer in Mexico from 2002-2020. MATERIALS AND METHODS: Using information from national sources and the national cancer incidence from GLOBOCAN, we estimated income lost due to premature death (ILPD), short-term benefits (STBs), disability pensions (DPs), and opportunity costs for the carer (OCCs) generated by patients with cancer. Amounts were reported in Mexican pesos. RESULTS: We estimated 23 359 deaths and 216 679 new cases of cancer by 2020, which would be associated with a total indirect cost of 20.15 billion Mexican pesos. Men are expected to generate 54.9% of these costs. ILPD is expected to comprise the highest percentage of the cost (60%), followed by OCCs (22%), STBs (17%) and DPs (1%). CONCLUSIONS: From an economic perspective, the results emphasize the need to strengthen preventive interventions and early detection of cancer among adults to reduce its effect on the productivity of Mexico.

Integrated oncology and palliative care: five years experience at the National Cancer Institute of Mexico.

Under the national plan for addressing cancer, prevention and detection play important roles. However, the cost of treatments and late diagnosis represent a significant burden on health services. At the National Cancer Institute, more than half of patients present with tumors in advanced stages, and approximately 10% of patients seen for the first time exhibit terminal-stage malignancies, where there are no feasible cancer treatment options, and the patients are instead admitted to the hospital exclusively for palliative symptomatic management. In 2010, the National Cancer Plan began implementing a model of integrative management of palliative care in oncology that has gradually come to include symptomatic palliative care, involving ambulatory, distant and hospitalized management of patients with cancer, in its final stages and, more recently, in earlier stages.

Challenges in the development and implementation of the National Comprehensive Cancer Control Program in Mexico.

Chronic noncommunicable diseases (NCDs), including cancer, have become the leading cause of human morbidity and mortality. In Mexico, cancer is the third leading cause of death, with a high incidence among the economically active population, a high proportion of advanced stages at diagnosis and limited care coverage for patients. However, no public policy aimed at managing this important public health problem has been developed and implemented to date. This manuscript describes the first interinstitutional proposal of a National Program for Cancer Control, considering the known risk factors, early detection, treatment, palliative care and patient rehabilitation. This manuscript also outlines a series of thoughts on the difficulties and needs that the Mexican health system faces in achieving the main objectives of the program: to decrease the incidence of cancer, to increase survival and to improve the quality of life for this group of patients.

Utilization of hospital services for cancer care in Mexico.

OBJECTIVE: To analyze the utilization of hospital services for cancer care by location, sex, age group and care institution in Mexico from 2004-2013. MATERIALS AND METHODS: Time series study from 2004-2013, based on administrative records of hospital discharges for cancer in the health sector, including the private sector. RESULTS: The utilization rate increased significantly from 290 to 360 per 100 000 inhabitants. A total of 62% of hospital discharges related to malignant tumors were concentrated in eight types of cancer. Leukemia, breast and colorectal cancers almost doubled in the period. While lung cancer showed a decline among men, it increased among women. A total of 63.1% of cancer patients were women, and 81% of cases occurred in the public sector. From 2011, the Ministry of Health was the main provider of hospital services for cancer care. CONCLUSIONS: Increases in utilization were mainly found in the Ministry of Health, quite possibly as a result of the implementation of universal insurance.

Estimation of the Cost-Effectiveness of Breast Cancer Screening Using Mammography in Mexico Through a Simulation.

BACKGROUND: Currently, breast cancer is the most prevalent tumor among Mexican women. Screening methods such as mammography could potentially reduce the health and economic burden of breast cancer; however, its risk-benefit balance is still unclear. OBJECTIVE: To estimate the cost-effectiveness of different breast cancer screening programs using mammography in Mexico and to contribute to the decision-making process on this preventive measure. METHODS: A simulation study was performed using population data and incidence rates. Several screening programs were assessed using the cost-effectiveness methodology recommended by the World Health Organization. RESULTS: The feasible recommended screening program has an examination schedule periodicity of every three years, with a population coverage of 0, 15, 18, 20, 25, 20, 18, and 0% for the age groups of 25-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, and 70-75 years, respectively. CONCLUSIONS: Given the present coverage in Mexico, it is necessary to optimize our resource allocation to improve the country's breast cancer prevention policy.

A study of the impact of the 21-gene breast cancer assay on the use of adjuvant chemotherapy in women with breast cancer in a Mexican public hospital.

BACKGROUND: The majority of breast cancer patients in Mexico are treated through the public health system and >80% receive adjuvant chemotherapy. The aim of this prospective study was to characterize the impact of the Oncotype DX assay on adjuvant therapy decision making and the confidence in those decisions amongst public sector physicians in Mexico. METHODS: Ninety-eight consecutive patients with ER+, HER2-, stage I-IIIa, N0/N1-3 node-positive breast cancer from the Instituto Nacional de Cancerología were eligible for the study. The primary endpoint was the overall change in treatment recommendations after receiving the assay results. RESULTS: Of 96 patients, 48% received a chemohormonal therapy recommendation prior to testing. Following receipt of results, treatment decisions changed for 31/96 (32%) patients, including 17/62 (27%) node-negative patients and 14/34 (41%) node-positive patients. The proportion of patients with a chemotherapy-based recommendation decreased from 48% pre- to 34% post-assay (P=0.024). 92% of physicians agreed that they were more confident in their treatment recommendation after ordering the assay. CONCLUSIONS: These results suggest that use of the 21-gene assay in the Mexican public health system has a meaningful impact on adjuvant treatment recommendations that may reduce the overall use of chemotherapy.

[In situ and invasive carcinoma identified through an opportunistic screening mammography in asymptomatic women in Mexico City]. / Carcinoma in situ e infiltrante identificado por tamizaje mamográfico oportunista en mujeres asintomáticas de la Ciudad de México.

OBJECTIVE: To describe the mammographic findings and carcinoma detection rate in asymptomatic women of Mexico City, that participated in an opportunistic screening program. MATERIALS AND METHODS: 39 491 participants were included, with mammograms performed and interpreted in the National Cancer Institute, from 2008 to 2011. The mammographic findings, type of lesion and true positives (TP), are described by age groups. We calculated the crude effect of age on the classification BIRADS (Breast Imaging Reporting and Data System) 0 and the type of lesion. RESULTS: The median age was 50 (45-57) years. 80.5% were classified as BIRADS 2, 11.4%(0), 4.1%(1), 3.5%(3), 0.5%(4) y 0.1%(5). Malignant lesions were detected in 1.3 and 3.3 per 1000 and the proportion of true positives (TP) was 8.2% and 20.6%, in women of 41-50 and 51-70 years, respectively. CONCLUSIONS: Although some cases are detected in women 40 to 50 years, in women over 50 years the screening by mammography is more efficient, with a higher proportion of cases detected and fewer false positives.

[Evaluation of breast cancer treatment at a tertiary-level institution with Popular Health Insurance in Mexico]. / Evaluación del tratamiento del cáncer de mama en una institución del tercer nivel con el Seguro Popular, México.

BACKGROUND: In our country breast cancer represents a major health problem. Only 45% of all population has access to health services, the consequence is delay in diagnosis and treatment. In Mexico, 66% of all new cases of breast cancer are diagnosed in locally advanced stages. From May 2007 the Health System Protection Against Catastrophic Expenses, called Seguro Popular (SP), breast cancer was included in covering the treatment of this neoplasm in any patient without access to social security. OBJECTIVE: To evaluate the results and impact of SP in the adjuvant and neoadjuvant treatment of a group of patients diagnosed with breast cancer at an institution of national reference. MATERIAL AND METHODS: We analyzed a group of 259 patients in stages (I-IIIC). The clinical stages I and II (55 patients) were treated with adjuvant chemotherapy FAC -T (fluorouracil 500 mg/m2, adriamycin 50 mg/m2 and cyclophosphamide 500 mg/m2 (FAC) followed by 12 weeks of paclitaxel 80 mg/m2 +/- trastuzumab loading dose of 4 mg/kg followed by 2 mg/kg); 204 patients in locally advanced stages (IIB-IIIC) received FAC-T +/- trastuzumab followed by surgery. Adjuvant treatment consisted of endocrine therapy for hormone-sensitive patients and radiotherapy 50 cGy according to international standards. RESULTS: The age at diagnosis was 47 years (range 23-68). 80% of them were locally advanced stages (IIB-IIIC) and were treated in a neoadjuvant setting, 20% was in early stages, treated with surgery and adjuvant chemotherapy The disease-free survival and overall survival at 30 months was 85.7 and 90% respectively. Overall pathologic complete response was obtained in 15% of cases. In the subgroup analysis showed that 41% of patients HER2 (+), 29% of triple-negative patients and 9% of hormone-sensitive tumors achieved complete pathological response (p = 0.0001). CONCLUSION: This is the first analysis of efficacy of adjuvant and neoadjuvant treatment in breast cancer since the introduction of popular secure non-entitled population. It is clear that treatment efficacy is similar to that reported in the literature, with 15% of pRC and survival to 30 months in 94-80%. The coverage of health expenditures treats a larger number of patients optimally. Along with this, efforts should be made to reduce the high frequency of diagnosis at advanced stage.