[The safety and efficacy of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia in children].

To explore the safety and efficacy of blinatumomab in the treatment of CD19 positive (CD19+) B-cell acute lymphoblastic leukemia (B-ALL) in children. A retrospective analysis was conducted on the clinical data of pediatric B-ALL patients who received blinatumomab treatment from Hematology & Blood Diseases Hospital of Chinese Academy of Medical Sciences from August 2021 to October 2023. Based on their disease status, the patients were divided into refractory/relapsed(RR) group, minimal residual disease clearance (MC) group, and chemotherapy intolerance (IC) group. Clinical data of the children were collected to evaluate the adverse drug reactions, therapeutic efficacy and survival of the children. In total, 35 patients were included, with 20 males and 15 females, aged from 0.6 to 16.4 (9.9±4.2) years old. There were 10 cases in the RR group, 20 cases in the MC group and 5 cases in the IC group. A total of 56 cycles of infusion were completed, with one cycle in 24 cases, two cycles in 5 cases, three cycles in 2 cases and four cycles in 4 cases. The median infusion time [M (Q1, Q3)] from the first to the fourth cycle was 14 (14, 28) days, 28 (28, 28) days, 28 (28, 28) days and 28 (26, 28) days, respectively. In terms of adverse reactions, the incidence of grade 1-2 cytokine release syndrome(CRS) was 57.1% (32/56), with grade 1 CRS accounting for 84.4% (27/32). The incidence rate of immune effector cell-associated neurotoxicity syndrome(ICANS) (grade 4) was 1.8% (1/56). In the RR group, 6 cases were treated effectively, and minimal residual disease(MRD) turned negative, before treatment, MRD levels were all less than 20%. Among them, 3 cases had MRD turning positive again 14 to 42 days after discontinuation of Belintoumab. Four cases were treated ineffectively, with MRD >20% before treatment. All MRD positive cases in MC group turned negative and all MRD negative cases in the IC group remained negative after treatment. The median follow-up time of RR group was 5.7 (3.8, 9.4) months, and 1 year median survival rate and event-free survival rate were 40.0%±21.9% and 33.3%±19.2%, respectively. The median follow-up time for MC and IC group patients was 6.7 (5.2, 12.5) months and 7.1 (5.1, 7.6) months, respectively, with an event free survival rate of 100%. The safety and efficacy of using belintoumab in partial RR, MRD clearance, and chemotherapy intolerance are good.

[Clinical and prognostic characteristics of pediatric acute myeloid leukemia with myelodysplasia-related changes under different diagnostic criteria].

Objective: To evaluate the clinical and prognostic differences in acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) children under different diagnostic criteria (World Health Organization (WHO) 2016 and WHO 2022 criteria). Methods: In this retrospective cohort study, clinical characteristics and prognosis information of 260 acute myeloid leukemia (AML) children admitted to Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from August 2017 to August 2021 were analyzed retrospectively. According to WHO 2016 and WHO 2022 diagnostic criteria, patients were divided into AML-MRC group and non-AML-MRC group, the prognostic and genetic differences between two groups were compared respectively. Meanwhile, the characteristics of children with 8 MRC-related genes defined in WHO 2022 diagnostic criteria were described. Mann-Whitney U test, chi-square test were used for comparison between groups. Survival curve was plotted by Kaplan-Meier method, and comparison between groups was performed by Log-Rank method. Results: Among the 260 children, there were 148 males and 112 females. The follow-up time was 26 (16, 38) months. A total of 28 children (10.8%) were diagnosed with AML-MRC according to the WHO 2016 diagnostic criteria. Compared with non-AML-MRC children, the frequency of PTPN11, RUNX11, SH2B3, MPL and STAG2 mutations was higher in AML-MRC children (25.0% (7/28) vs. 4.3% (10/232), 14.3% (4/28) vs. 3.9% (9/232), 10.7% (3/28) vs. 2.2% (5/232), 10.7% (3/28) vs. 2.2% (5/232), 10.7% (3/28) vs. 0.9% (2/232), all P<0.05). The 2-year overall survival (OS) and events free survival (EFS) rate of 28 AML-MRC children under WHO 2016 diagnostic criteria were worse than those of 232 non-AML-MRC children ((62.1±10.8)% vs. (94.5±1.6)%, χ2=22.1,P<0.001;(48.0±10.6)% vs. (70.9±3.2)%, χ2=6.33,P=0.012). Twenty-seven children (10.4%) were eventually diagnosed with AML-MRC according to WHO 2022 criteria, their 2-year OS rate were worse than 233 non-AML-MRC children ((60.8±11.1)% vs. (94.5±1.6)%, χ2=24.49,P<0.001), and there was no statistically significant difference in EFS rate between two groups at 2 years ((55.1±10.8)% vs. (70.1±3.2)%, χ2=2.44, P=0.119). Conclusions: Compared with the 2022 WHO diagnostic criteria, the survival rates of children with AML-MRC under the 2016 WHO diagnostic criteria were worse than that of children without MRC.The new version of the AML-MRC diagnostic criteria emphasizes the importance of genes.

[Clinical features and prognostic analysis of testicular relapse in pediatric acute lymphoblastic leukemia].

Objective: To investigate the clinical features and prognosis of testicular relapse in pediatric acute lymphoblastic leukemia (ALL). Methods: Clinical data including the age, time from initial diagnosis to recurrence, relapse site, and therapeutic effect of 37 pediatric ALL with testicular relapse and treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences between November 2011 and December 2022 were analyzed retrospectively. Patients were grouped according to different clinical data. Kaplan-Meier analysis was used to evaluate the overall survival (OS) rate and event free survival (EFS) rate for univariate analysis, and Cox proportional-hazards regression model was used to evaluate the influencing factors of OS rate and EFS rate for multivariate analysis. Results: The age at initial diagnosis of 37 pediatric testicular relapse patients was (5±3) years and the time from initial diagnosis to testicular recurrence was (37±15) months. The follow-up time was 43 (22, 56) months. Twenty-three patients (62%) were isolated testis relapse. The 5-year OS rate and EFS rate of the 37 relapsed children were (60±9) % and (50±9) % respectively. Univariate analysis showed that the 2-year EFS rate in the group of patients with time from initial diagnosis to testicular recurrence >28 months was significantly higher than those ≤28 months ((69±10)% vs. (11±11)%, P<0.05), 2-year EFS rate of the isolated testicular relapse group was significantly higher than combined relapse group ((66±11)% vs. (20±13) %, P<0.05), 2-year EFS rate of chimeric antigen receptor T (CAR-T) cell treatment after relapse group was significantly higher than without CAR-T cell treatment after relapse group ((78±10)% vs. (15±10)%, P<0.05). ETV6-RUNX1 was the most common genetic aberration in testicular relapsed ALL (38%, 14/37). The 4-year OS and EFS rate of patients with ETV6-RUNX1 positive were (80±13) % and (64±15) %, respectively. Multivariate analysis identified relapse occurred≤28 months after first diagnosis (HR=3.09, 95%CI 1.10-8.72), combined relapse (HR=4.26, 95%CI 1.34-13.52) and CAR-T cell therapy after relapse (HR=0.15,95%CI 0.05-0.51) were independent prognostic factors for 2-year EFS rate (all P<0.05). Conclusions: The outcome of testicular relapse in pediatric ALL was poor. They mainly occurred 3 years after initial diagnosis. ETV6-RUNX1 is the most common abnormal gene.Patients with ETV6-RUNX1 positive often have a favorable outcome. Early relapse and combined relapse indicate unfavorable prognosis, while CAR-T cell therapy could significantly improve the survival rate of children with testicular recurrence.

[Clinical features and long-term prognostic analysis of relapsed pediatric acute lymphoblastic leukemia].

Objective: To investigate the clinical characteristics and long-term prognostic factors of relapsed pediatric acute lymphoblastic leukemia (ALL). Methods: Clinical data including the age, time from initial diagnosis to relapse, relapse site, and molecular biological features of 217 relapsed ALL children primarily treated by the Chinese Children's Leukemia Group (CCLG)-ALL 2008 protocol in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences between April 2008 and April 2015 were collected and analyzed in this retrospective cohort study. Kaplan-Meier analysis was used to evaluate the overall survival (OS) rate and event free survival (EFS) rate for univariate analysis, and Cox proportional-hazards regression model was used to evaluate the influencing factors of OS rate and EFS rate for multivariate analysis. Results: The age at initial diagnosis of 217 relapsed patients was 5 (3, 7) years. There were 135 males and 82 females. The time from initial diagnosis to relapse of 217 children was 22 (10, 39) months. After relapse, 136 out of 217 children (62.7%) received treatment and the follow-up time was 65 (47, 90) months. The 5-year OS rate and EFS rate of the 136 relapsed children were (37±4) % and (26±4) %, respectively. The predicted 10-year OS rate and EFS rate were (35±5) % and (20±4) %, respectively. Univariate analysis showed that the 5-year OS rate in the group of patients with late relapse (43 cases) was significantly higher than those with very early (54 cases) and early relapse (39 cases) ((72±7)% vs. (16±5)%, (28±8)%, χ2=35.91, P<0.05), 5-year OS rate of the isolated extramedullary relapse group (20 cases) was significantly higher than isolated bone marrow relapse group (102 cases) and combined relapse group (14 cases) ((69±11)% vs. (31±5)%, (29±12)%, χ2=9.14, P<0.05), 5-year OS rate of high-risk group (80 cases) was significantly lower than standard-risk group (10 cases) and intermediate-risk group (46 cases) ((20±5)% vs. (90±10)%, (54±8)%, χ2=32.88, P<0.05). ETV6::RUNX1 was the most common fusion gene (13.2%, 18/136). The predicted 10-year OS rate of relapsed children with positive ETV6::RUNX1 was significantly higher than those without ETV6::RUNX1 (118 cases) ((83±9)% vs. (26±5)%, χ2=14.04, P<0.05). The 5-year OS for those accepted hematopoietic stem cell transplantation (HSCT) after relapse (42 cases) was higher than those without HSCT (94 cases) ((56±8)% vs. (27±5)%, χ2=15.18, P<0.05). Multivariate analysis identified very early/early relapse (HR=3.91, 95%CI 1.96-7.79; HR=4.15, 95%CI 1.99-8.67), bone marrow relapse including isolated bone marrow relapse and combined relapse (HR=6.50, 95%CI 2.58-16.34; HR=5.19, 95%CI 1.78-15.16), with ETV6::RUNX1 (HR=0.23, 95%CI 0.07-0.74) and HSCT after relapse (HR=0.24, 95%CI 0.14-0.43) as independent prognostic factors for OS (all P<0.05). Conclusions: Relapsed pediatric ALL mainly occurs very early and often affects bone marrow, which confer poor outcome. ETV6::RUNX1 is the most common genetic aberration with a favorable outcome. HSCT could rescue the outcome of relapsed children, though the survival rate is still poor.

[Genomics of next generation sequencing in pediatric B-acute lymphoblastic leukemia and its impact on minimal residual disease].

Objective: To describe the gene mutation profile of newly diagnosed pediatric B-acute lymphoblastic leukemia (B-ALL) and analyze its effect on minimal residual disease (MRD). Methods: A total of 506 newly diagnosed B-ALL children treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from September 2018 to July 2021 were enrolled in this retrospective cohort study. The enrolled children were divided into MRD ≥1.00% group and <1.00% group according to MRD results on the 19th day since chemotherapy, and MRD ≥0.01% group and <0.01% group according to MRD results on the 46th day. Clinical characteristics and gene mutations of two groups were compared. Comparisons between groups were performed with chi-square test or Fisher's exact test. Independent risk factors of MRD results on the 19th day and the 46th day were analyzed by Logistic regression model. Results: Among all 506 patients, there were 318 males and 188 females. On the 19th day, there were 114 patients in the MRD ≥1.00% group and 392 patients in the MRD <1.00% group. On the 46th day, there were 76 patients in the MRD ≥0.01% group and 430 patients in the MRD <0.01% group. A total of 187 gene mutations were detected in 487 (96.2%) of 506 children. The most common gene mutations were signal transduction-related KRAS gene mutations in 111 cases (22.8%) and NRAS gene mutations in 99 cases (20.3%). Multivariate analysis showed that PTPN11 (OR=1.92, 95%CI 1.00-3.63), KMT2A (OR=3.51, 95%CI 1.07-11.50) gene mutations and TEL-AML1 (OR=0.48, 95%CI 0.27-0.87), BCR-ABL1 (OR=0.27, 95%CI 0.08-0.92) fusion genes and age >10 years (OR=1.91, 95%CI 1.12-3.24) were independent influencing factors for MRD ≥1.00% on the 19th day. BCORL1 (OR=2.96, 95%CI 1.18-7.44), JAK2 (OR=2.99, 95%CI 1.07-8.42) and JAK3 (OR=4.83, 95%CI 1.50-15.60) gene mutations and TEL-AML1 (OR=0.43, 95%CI 0.21-0.87) fusion gene were independent influencing factors for MRD ≥0.01% on the 46th day. Conclusions: Children with B-ALL are prone to genetic mutations, with abnormalities in the RAS signaling pathway being the most common. Signal transduction related PTPN11, JAK2 and JAK3 gene mutations, epigenetic related KMT2A gene mutation and transcription factor related BCORL1 gene mutation are independent risk factors for MRD.

[A long-term follow-up study of 82 children with acute myeloid leukemia].

Objective: To investigate the efficacy and the prognostic factors of Chinese Academy of Medical Sciences 2005 (CAMS-2005) regimen in the treatment of pediatric acute myeloid leukemia (AML). Methods: Eighty-eight cases of newly-diagnosed AML patients, who were treated with the CAMS-2005 regimen from April 2005 to July 2009, were enrolled in this case observational study. Clinical characteristics, long-term prognosis and prognostic factors were analyzed retrospectively. Overall survival (OS) and event free survival (EFS) rates were estimated by the Kaplan-Meier method. Rates of survival between the groups were compared by the Log-rank test. Prognostic factors were evaluated by COX regression analysis. Results: A total of 82 cases were enrolled in this study, including 34 core binding factor(CBF)-AML patients and 48 non-CBF-AML patients. There were 45 males and 37 females. The median age at diagnosis was 8.0 (0.7-16.0) years. During the induction therapy, 3 patients (4%) developed treatment-related early-death, while 63 patients (77%) achieved complete remission (CR) and 53 patients (65%) achieved CR after 1 course. Twenty-one patients (33%) had relapsed disease. The CR rates of CBF-AML patients and non-CBF-AML patients were 91% (31/34) and 67% (32/48) (χ(2)=5.410, P=0.020) , while the relapse rates were 29% (9/31) and 38% (12/32) (χ(2)=0.508, P=0.476) . The 8-year OS and EFS rates of all 82 patients were 59%(48/82) and 51%(42/82). The 8-year OS rates of CBF-AML patients and non-CBF-AML patients were 74% (25/34) and 48%(23/48) (χ(2)=5.812, P=0.016), while the 8-year EFS rates of CBF-AML patients and non-CBF-AML patients were 71%(24/34) and 38%(18/48) (χ(2)=8.682, P=0.003). There were statistically significant differences between groups. The 8-year OS rates of patients who achieved CR after 1 course and other patients were 68% (36/53) and 46% (12/26) (χ(2)=9.606, P=0.002), while the 8-year EFS rates were 66% (35/53) and 27% (7/26) (χ(2)=19.471, P=0.000), the differences were all statistically significant. COX multivariate analysis showed that CBF-AML or non-CBF-AML and whether achieved CR after 1 course were independent prognostic factors of OS rates (relative risk: 2.538, 2.561) and EFS rates (relative risk: 3.050, 3.686) (P <0.05). Conclusions: The efficacy of the CAMS-2005 regimen in the treatment of AML patients was well. CBF-AML or non-CBF-AML and whether achieved CR after 1 course were independent prognostic factors for pediatric AML patients.

[Heterogeneity and clonal evolution in pediatric ETV6-RUNX1(+) acute lymphoblastic leukemia by quantitative multigene fluorescence in situ hybridization].

Objective: To evaluate heterogeneity and clonal evolution in pediatric ETV6-RUNX1(+) acute lymphoblastic leukemia (ALL) in China. Methods: Totally 48 children (<14 years) with newly diagnosed ETV6-RUNX1(+) ALL in Institute of Hematology and Blood Disease Hospital, CAMS and PUMC, from February 2006 to June 2011 were included. The copy number variations were analyzed by quantitative multigene fluorescence in situ hybridization (QM-FISH) in 48 patients. Non-normal distribution of measurement data were shown with Median (range) , count data were shown with percent (%) . Overall survival and event-free survival were estimated by the Kaplan-Meier method and compared with the log-rank test. Results: Forty-eight patients were tested by QM-FISH. Of 48 patients, 70.8% harbored one clone, 18.8% two subclones, and 10.4% three or more subclones. The clone heterogeneity was detected by two different models: the linear succession model and the branching evolution model. ETV6-RUNX1(+) ALL relapse evolved from an ancestral clone or a new clone. The patients relapsed from a new clone got the worse outcome. Conclusion: The clone evolution was detected in pediatric ETV6-RUNX1(+) ALL in China. QM-FISH might be helpful to evaluate the outcome of relapsed patients. A new clone was associated with a poorer outcome.

Preliminary evidence that cell death may contribute to separation of the trachea from the primitive foregut in the rat embryo.

BACKGROUND/PURPOSE: The embryogenesis of many congenital tracheoesophageal abnormalities has been elucidated poorly, mainly because of an incomplete understanding of the normal mechanism of separation of the primitive foregut into the trachea and esophagus. There has been controversy about the existence and significance of the so-called tracheoesophageal septum. This study investigates the normal mechanism of tracheoesophageal separation in the rat. METHODS: Timed-pregnant Sprague-Dawley rats were killed on gestational days 11 to 16, respectively (the day on which the vaginal smear showed sperm was considered day 0 of gestation). Thirty-six embryos (six embryos from each age group) were fixed in 10% formalin. Fixed embryos from gestational day 13, 12, and 11 were sandwiched in melted agar to facilitate proper orientation before being processed. Half of the fetuses from each age group were sectioned serially in a transverse plane and the other half in a sagittal plane. The histological sections were stained with H&E. RESULTS: On gestational day 11, the foregut appears as a ventrodorsal slit in transverse section. Below the primitive pharynx, the epithelial cells on the ventral part of the foregut are actively proliferating, producing two lateral epithelial bulges (primitive bronchial buds). The epithelial cells of the dorsal aspect of the foregut show no sign of active growth. On day 12, the proximal trachea still shares a common lumen with the foregut. The distal trachea, the tracheal bifurcation, and both primary bronchi are recognized easily. At the point of tracheoesophageal separation, there is obvious debris of dead epithelial cells and condensed nuclei. These appearances were specifically located around the so-called tracheoesophageal septum and nearby grooves, resulting in enfolding cristae. There was no cell proliferation or inflammatory response in this region. On gestational day 13, the separation of the trachea and esophagus was almost complete. CONCLUSION: The present study demonstrates that a specific and consistent pattern of cell death in the region of the "tracheoesophageal septum" produced enfolding cristae that may be part of the mechanism of separation of the trachea and esophagus.

Relationship of the notochord to foregut development in the fetal rat model of esophageal atresia.

BACKGROUND/PURPOSE: The notochord (Nt) is thought to act as a primary organizer for adjacent axial embryonic organs. The current study used the Adriamycin-induced fetal rat model of esophageal atresia and tracheoesophageal fistula (EA-TEF) to determine whether anomalies of the foregut (FG) were associated with an abnormal Nt. METHODS: Eight experimental female Sprague-Dawley rats received intraperitoneal injection of Adriamycin (1.75 mg/kg) on gestational days 6 to 9 inclusive, and 4 control rats received saline injection only. Their embryos were harvested on gestational days 11, 12, 13, and 14. Embryos from each age subgroup were serially sectioned and stained with H&E. The FG and Nt were traced from the primitive pharynx to the level of the stomach. RESULTS: By day 11, the Nt of control embryos had completely separated from the FG and was located immediately ventral to the neural tube. On gestational day 12, the Nt detached from the neural tube, and the trachea and esophagus were separating. On day 11, in the Adriamycin-treated embryos, the Nt was still attached to an FG that was narrowed or occluded. On day 12, the Nt remained adherent to the FG from the primitive pharynx to the level above the primitive respiratory buds, at which point it became thicker and branched sagittally, with the anterior branch contacting or merging with the FG. The FG usually loses its lumen or continuity when in contact with the Nt. CONCLUSIONS: Exposure of rat embryos to Adriamycin leads to abnormal development of the Nt, including prolonged attachment to or fusion with the FG, and abnormal branching. Traction on the FG by the Nt produces occlusion of its lumen and may result in its complete interruption. Separation of the Nt from the FG would appear to be a prerequisite for the normal development of the FG into its derivatives: the esophagus and trachea.

Apoptosis during regression of the tailgut and septation of the cloaca.

PURPOSE: Apoptosis is involved in the embryonic morphogenesis of many organs. The current study was undertaken to ascertain the role of apoptosis during cloacal development in the rat. METHODS: One hundred five rat embryos, ranging from gestational days 11 to 16, were sectioned serially in the transverse or sagittal planes and stained with H&E. The cloaca, urorectal septum, rectum, urogenital sinus, Wolffian ducts, and tailgut (TG) were examined consecutively in temporospatial sequence. RESULTS: The tailgut immediately distal to the hindgut starts to regress by apoptosis on day 12 of gestation in a craniocaudal direction and has regressed completely by day 13.5. A large number of apoptotic cells and debris can be identified in the urorectal septum during cloacal septation. Vacuoles are formed by coalescence of apoptotic cells at the tip of urogenital sinus from day 15 to 16, and, at the same time, sporadic apoptotic bodies in the anal membrane contribute to its thinning. CONCLUSION: Results of the current study confirm that apoptosis occurs in a specific temporo-spatial sequence in the hindgut and cloaca and appears to be an important mechanism in TG regression, uro-rectal separation, urethral opening, and rupture of the anal membrane.

Temporospatial aberrations of apoptosis in the rat embryo developing esophageal atresia.

BACKGROUND/PURPOSE: Recent work has shown that apoptosis is a key component of the normal development of the foregut. This study was designed to compare the patterns of apoptosis in the normal foregut with those in the fetus developing esophageal atresia and tracheoesophageal fistula (EA-TEF) using 3-dimensional reconstructive techniques. METHODS: Timed pregnant rats that received no treatment (control group) or received Adriamycin intraperitoneally (experimental group) had their embryos removed between days 11 and 14 of gestation. The embryos were sectioned serially and stained with H&E. Three-dimensional reconstructions were made of the foregut and areas of apoptosis were marked on them to facilitate analysis of apoptotic patterns. RESULTS: Apoptosis was evident in control embryos in the region in which tracheoesophageal separation occurs from days 12 and 12.5. Experimental embryos showed no apoptosis until day 13 when apoptosis was observed immediately posterior to the foregut within the esophageal mesenchyme and in the laryngeal mesenchyme ventral to the foregut. CONCLUSIONS: The pattern, timing and location of apoptosis in rats developing EA-TEF is abnormal. Our work indicates that it is actually a complete lack of apoptosis at the crucial stage of development that leads to this abnormality rather than an alteration in the patterning of apoptosis at this crucial time. The observation of apoptosis only within the mesenchyme raises the possibility that apoptosis in the foregut developing EA-TEF may be a reaction to that abnormal development rather than its cause.

Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo.

BACKGROUND/PURPOSE: The normal process of division of the cloaca into a rectum and urogenital tract is still not fully understood. The main controversies relate to how the urorectal septum (URS) divides the cloaca and whether the URS fuses with the cloacal membrane. This study used a 3-dimensional reconstruction technique, combined with histologic correlation, to observe the developmental and septational processes of the cloaca of the normal rat embryo from gestational days 11 to 16. METHODS: Normal rat embryos from gestational days 11 to 16 were sectioned serially both transversely and sagittally and stained with H&E. 3-dimensional reconstructions were performed on embryos younger than day 13.5. The relevant structures were examined in a temporo-spatial sequence. RESULTS: The tailgut started to regress by apoptosis on day 12 in a cranio-caudal direction. The URS, first evident in day-12.5 embryos, extended and fused with the cloacal membrane on day 15 of gestation, completing the separation of the cloaca into rectum and bladder. Regression of the tailgut and ventral protrusion of the urogenital sinus markedly changed the configuration of the cloaca. The cloacal membrane did not break down until after it had fused with the URS. CONCLUSIONS: These findings clarify the relative contributions made by active septation of the cloaca by the URS and configurational changes of the cloaca to produce a rectum and bladder. The URS fuses with the cloacal membrane before the anal and urogenital membranes break down.

Tracheomalacia with esophageal atresia and tracheoesophageal fistula in fetal rats.

BACKGROUND: Many patients who have esophageal atresia and tracheoesophageal fistula (EA-TEF) have associated tracheomalacia, which is thought to be one of the reasons for respiratory complications after surgical correction of the abnormality. METHODS: In this study, tracheas from Adriamycin-induced EA-TEF fetal rats were examined histologically and relevant cross-sectional parameters of the tracheas were measured. RESULTS: The tracheal lumen in tracheomalacia was small and irregular, losing its normal "D" shape. In most rats, the cartilaginous ring was broken into two to four segments, making the trachea lose its rigid support. The submucosa was thickened with prominent bulging of its membranous part into the tracheal lumen. The ratio of the inner luminal cross-sectional area to the outer tracheal cross-sectional area in EA-TEF rats was 15.7%, compared with a control ratio of 47.2%. In EA-TEF rats, the length of the cartilaginous ring was significantly shortened (P < .001), but not the length of membranous trachea, thus resulting in a cartilaginous/membranous (C/M) ratio of 1.55:1, markedly lower than that of normal rats (4.34:1, P < .001). The reduction of anterior-posterior diameter of the tracheal lumen was more marked than that of the transverse diameter. CONCLUSIONS: These observations suggest that the trachea in EA-TEF rats has a smaller lumen and is more flaccid than normal, making it prone to airway obstruction. The fact that tracheomalacia developed only in fetuses who had EA-TEF indicates that the factors that result in EA-TEF also cause tracheomalacia.

The vagus and recurrent laryngeal nerves in the rodent experimental model of esophageal atresia.

BACKGROUND: After surgical correction of their esophageal atresia and tracheoesophageal fistula (EA-TEF), many patients exhibit evidence of esophageal dysmotility. Controversy exists as to whether the esophageal motility disorders result from denervation caused by surgery or from an inherent abnormal innervation of the esophagus. METHODS: The present study used an Adriamycin-induced EA-TEF fetal rat model to trace the course and branching of both the vagus and recurrent laryngeal nerves. Abnormalities observed in EA-TEF rat fetuses include: (1) fewer branches from both recurrent laryngeal nerves; (2) deviation of the left vagus from its normal course below the aorta, passing behind the fistula to approach and join with the right vagus to form a single nerve trunk on the right side of the esophagus; (3) relatively few branches from the single vagal nerve trunk (composed of fibers of the left and the right vagus) on the surface of the lower esophagus. CONCLUSIONS: Fetuses affected by EA-TEF have inherent abnormalities in the course and branching pattern of the vagus nerves as they descend through the thorax, culminating in a deficient extrinsic nerve fiber plexus in the lower esophagus. These observations may account for the esophageal motility disorders seen in patients who have EA-TEF even before surgical intervention.

Stages of normal tracheo-bronchial development in rat embryos: resolution of a controversy.

The embryonic events surrounding tracheo-esophageal separation remain controversial. The present study was undertaken to clarify early tracheo-bronchial development in the rat embryo at a critical period of organogenesis. Twenty-seven timed-mated Sprague-Dawley rats were divided into nine groups of three rats. Their embryos were harvested on gestational days 11-15 at intervals of 8 h, processed and sectioned transversely. The sections were stained with hematoxylin and eosin and examined serially. The foregut is a single tube on gestational day 11. During the following 16 h, there is localized and rapid growth of the respiratory epithelium and a laterocaudal expansion to form the bronchial buds and a protuberance on the ventral wall of the foregut (future tracheal carina). From gestational days 12-12 + 8, cellular debris and apoptotic epithelial cells are specifically located in the tracheo-esophageal groove, resulting in collapse and fusion of the lateral walls of the foregut, effectively separating the trachea and esophagus. Afterwards, the epithelial proliferation dominates the process of tracheo-esophageal separation until it reaches the caudal end of the laryngeal epithelial lamina on gestational day 15. The present study shows that separation of the trachea from the esophagus involves three consecutive stages: (i) epithelial proliferation resulting in the formation of bronchial buds and the tracheal carina; (ii) epithelial apoptosis leading to separation of the trachea and esophagus; and (iii) epithelial proliferation to complete the separation process.

Understanding tracheomalacia.

Tracheomalacia is an abnormality of the trachea that probably is present to some degree in all infants and children with oesophageal atresia. It causes the trachea to collapse during breathing, leading to obstruction of the upper airway. Our knowledge of the structural abnormalities underlying tracheomalacia is limited, mainly because patients with oesophageal atresia usually survive. Recently, the Adriamycin-induced rat model of oesophageal atresia and tracheomalacia has clarified some aspects of its pathology and the factors which may influence its development. The rat model suggests that the same detrimental factors that cause oesophageal atresia might also affect the development of the trachea, and that anomalous great vessels may exacerbate the severity of tracheomalacia locally.

Pathohistological study of adriamycin-induced tracheal agenesis in the fetal rat.

There have been few studies of tracheal agenesis (TA) because it is an extremely rare condition and is invariably fatal. However, it is recognised to be associated with a variety of oesophageal abnormalities, including oesophageal atresia (OA) and communicating bronchopulmonary foregut malformations (CBPFM). Except for some gross pathological reports, there has been no detailed histological investigation of this congenital abnormality. Adriamycin-induced TA in fetal rats provides a means of studying this uncommon condition, including its relationship to oesophageal abnormalities. TA was induced in rat features by intra-peritoneal injection of adriamycin (2 mg/kg) into timed-pregnant rats on days 6-9. Their 33 fetuses were removed by caesarean section, cut transversely, and stained after which the slides were examined. OA developed in 27/33 adriamycin-treated fetuses, TA occurred in 5. Four had type III while the 5th had type II TA. The oesophagus of fetuses with TA acquired tracheal features in the region where both ectopic bronchi originated. Two fetuses with TA also had OA, and 4 had a CBPFM. Other abnormalities detected involved the gastrointestinal, urinary and cardiovascular systems. The association of TA and oesophageal abnormalities suggests that there may be a spectrum of abnormalities comprising a number of variants, which may have a common aetiology. The other abnormalities co-existing in this animal model mirror those found in reported cases of human TA. Ultimately, the rat model of OA and TA may give insight into the embryogenesis of these malformations.

Communicating bronchopulmonary foregut malformations in the adriamycin-induced rat model of oesophageal atresia.

BACKGROUND: Communicating bronchopulmonary foregut malformations (CBPFM) are rare abnormalities of the development of the primitive foregut that result in an abnormal communication between the upper gastrointestinal tract and pulmonary tissue. They usually occur in isolation, but sometimes are seen in association with oesophageal atresia (OA). METHODS: Communicating bronchopulmonary foregut malformations were induced in the offspring of pregnant rats by intraperitoneal injection of Adriamycin (Delta West Pty Ltd, Bentley, Western Australia, Australia). Fetuses harvested by caesarean section were fixed in 10% formalin, transversely sectioned and stained with haematoxylin and eosin. Serial examination of the slides allowed three-dimensional reconstruction of the anatomy of the pulmonary system and the oesophagus. RESULTS: Communicating bronchopulmonary foregut malformations occurred in nine (30%) of fetuses with OA. Three types of CBPFM were produced: an isolated pulmonary structure (accessory lung) attached to the lower oesophagus via a patent bronchus (6 fetuses); an anomalous bronchus from the lower oesophagus to the lower part of the left lung (two fetuses); and atresia of the trachea (one fetus). CONCLUSIONS: These observations are consistent with the assertion that CBPFM and OA are variations of a spectrum of abnormalities and may have a similar aetiology. In the rat model it would appear that Adriamycin interferes with the timing and progression of lung bud differentiation at a time when the primitive foregut is developing rapidly. Ultimately, this model may shed light on the embryogenesis of both anomalies.

Hedgehog in the human: a possible explanation for the VATER association.

Foregut malformations are relatively common anomalies, occurring in 1 in 2000-5000 live births. The adriamycin-induced rat model of the VATER association has provided a means of studying the morphogenesis of a variety of major congenital structural abnormalities similar to those seen in humans with VATER association. The secreted glycoprotein, Sonic hedgehog (Shh), may act as an endodermal signal that controls gut and lung patterning. Mice with targeted deletion of Shh have foregut defects that are consistent with those produced by administration of adriamycin. It is possible that mutations induced by adriamycin may result from the breakdown of the Shh signalling pathway.