[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 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.

[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.

The development of the proximal oesophageal pouch in the adriamycin rat model of oesophageal atresia with tracheo-oesophageal fistula.

This study examined the morphological development of the proximal oesophagus in the Adriamycin-induced rat model of oesophageal atresia. The proximal oesophageal segment in oesophageal atresia with tracheo-oesophageal fistula (OA\TOF) has been assumed to be of similar embryological origin to the distal oesophagus. However, recent research using the Adriamycin model of OA\TOF has indicated that these structures may have a different origin. Time-mated Sprague-Dawley rats were administered either Adriamycin intraperitoneally or saline of an equivalent volume between days 6-9 of gestation. The rats were sacrificed between days 11-19 of gestation, their embryos removed and histologically sectioned. These sections were analysed to observe the morphological changes occurring in the proximal foregut. The proximal oesophageal pouch first appeared on day 15.25 as a dorsal outpouching of the proximal foregut immediately cranial to an area of apoptosis in the dorsal epithelium of the distal pharynx. It elongated through a process of cellular proliferation until it was clearly formed on day 16. Relatively little growth occurred from days 17-19. In the rat developing oesophageal atresia, the proximal oesophageal pouch has an origin different to that of the distal oesophagus. This study may explain the difference in immunohistological properties and intrinsic nervous supply between the proximal and distal oesophageal segments in oesophageal atresia.

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.

Evidence of a common pathogenesis for foregut duplications and esophageal atresia with tracheo-esophageal fistula.

The pathogenesis of the alimentary tract duplications, including foregut duplications (FgD) remains speculative. The accidental finding of FgD in fetal rats with esophageal atresia and tracheoesophageal fistula (EA-TEF) induced by Adriamycin provided an animal model to investigate a possible relationship between these two entities. Timed-pregnant rats were intraperitoneally injected with Adriamycin (1.75 mg/kg) on gestational Days 6 to 9. Their embryos were harvested by Caesarean section from gestational Days 14 to 21. Forty-six of embryos were processed and serially sectioned in the transverse or sagittal planes. EA-TEF occurred in 43/46 (93%) embryos of which 11 (24%) were found to have an associated FgD located at the level where the esophagus was absent. Six FgDs communicated with the foregut or the trachea. Five noncommunicating FgDs were located between the foregut and the vertebral column. In the control embryo, the notochord was located in the centre of the vertebral column from Day 11 of the gestation. In Day 14, 15 and 16, however, embryos exposed to Adriamycin, an abnormal notochord or branch frequently was located within the mesenchyme of the maldeveloped foregut or attached to the duplication cyst. In some, it appeared that the notochord was drawing the cyst-like structure away from the foregut. The present study confirms that duplications adjacent to the esophagus arise from the foregut and that failure of the foregut to detach from the notochord at the normal time may contribute to the development of foregut duplications.

Demonstration of abnormal notochord development by three-dimensional reconstructive imaging in the rat model of esophageal atresia.

The notochord (Nt) is believed to have a role in the development of axial organs. This study was undertaken to reconstruct in three dimensions (3D) the relationship of the Nt to abnormal development of the foregut (Fg) in the adriamycin-induced rat model of esophageal atresia (EA). Pregnant Sprague-Dawley rats were given 1.75 mg/kg adriamycin intraperitoneally on gestational days 6 9 inclusive; control rats received i.p. saline of equal volume, or no injection. Rats were killed between days 11 and 14 and their embryos harvested, histologically sectioned serially, and stained with hematoxylin and eosin. Digitized photographs were taken of serial transverse sections; these photos were traced and used as the basis for 3D reconstruction. From day 11 the normal Nt is no longer in contact with the respiratory or Fg mesenchyme. In adriamycin-treated embryos the Nt branches abnormally as it enters the Fg mesenchyme. Adherence of the Nt to the mesenchyme of the Fg exerts mechanical traction pulling the upper Fg dorsally. The severity of the Fg abnormalities correlates with the length of the ventral extension of the Nt within the Fg mesenchyme: the embryo develops atresia of the esophagus or trachea when the Nt is grossly abnormal. The Nt undergoes reactive thickening in the absence of Fg structures ventral to it. Thus, structural lesions of the Fg (e.g., atresias) are associated with abnormalities of the Nt. The relationship of the Nt to the Fg mesenchyme determines the severity of the abnormality induced by adriamycin: extensive adherence produces tracheal agenesis and EA.

Abnormalities of the tracheal cartilage in the rat fetus with tracheo-oesophageal fistula or tracheal agenesis.

Many infants with oesophageal atresia and tracheo-oesophageal fistula (OA/TOF) have associated tracheomalacia (TM), which is one of the reasons for respiratory complications after surgical correction of the atresia. OA/TOF was induced in the offspring of pregnant rats by intraperitoneal injection of adriamycin. Fetuses were harvested by caesarean section. The trachea, oesophagus, lungs, and stomach were removed en bloc and stained for cartilage using Alcian blue. The tracheas were examined, photographed, and relevant parameters pertaining to the tracheal cartilage were measured. Exposure to adriamycin resulted in a range of anatomical defects including OA/TOF (47%) and tracheal agenesis (TA) (41%). Adriamycin-treated fetuses were smaller (P < 0.01), yet had longer tracheas (P < 0.001) than control fetuses. The OA/TOF fetuses had more tracheal cartilage rings than controls (P < 0.01), whereas TA fetuses had fewer (P < 0.001). Both OA/TOF and TA fetuses had more malformed tracheal cartilage rings than controls (P < 0.001 and P < 0.05, respectively). Cartilage in the proximal part of the trachea was most frequently and severely affected (P < 0.05). These observations clarify the structural abnormalities of tracheal cartilage that occur in rat fetuses with OA/TOF or TA induced by adriamycin, and may explain the functional disturbances of TM seen in OA/TOF.

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.

The contribution of the adriamycin-induced rat model of the VATER association to our understanding of congenital abnormalities and their embryogenesis.

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 the VATER association. Most interest has been centered on the foregut, where the model has clarified some aspects of the development of esophageal atresia (EA), tracheal agenesis, and other communicating bronchopulmonary foregut malformations. It has demonstrated aberrations in the nerve supply to the esophagus in EA and allowed the study of tracheomalacia. A relationship between an abnormal notochord, foregut abnormalities, and vertebral defects has been shown, and the model has reignited interest in the role of the notochord as a regional organizer of axial development. The normal temporospatial characteristics of apoptosis during fore- and hindgut development is disturbed in this model, resulting in abnormal morphology. The indications are that this model will continue to clarify the processes that lead to many of the structural congenital abnormalities that are seen in infants born with the VATER association.

Does the urorectal septum fuse with the cloacal membrane?

PURPOSE: Traditional theories of cloacal embryogenesis assume that the urorectal septum fuses with the cloacal membrane before the anal membrane disintegrates. However, recent observations in humans and other species raise doubt about this assumption. We determined whether urorectal septum fusion occurs in rats. MATERIALS AND METHODS: Rat embryos were harvested at specific times between days 11 and 16 of gestation. We evaluated the morphology, growth and relationship of the urorectal septum to the cloacal membrane on serial histological sections. RESULTS: The urorectal septum consistently fused with the cloacal membrane on day 15 of gestation before the cloacal membrane began to disintegrate. CONCLUSIONS: In rats the urorectal septum fuses with the cloacal membrane, after which the urogenital membrane and anal membrane disintegrate by a process of apoptosis.

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.

Development of the bones and synovial joints in the rat model of the VATER association.

The adriamycin-induced rat model of the Vertebral, Anorectal, Tracheo-Esophageal, Radial and Renal (VATER) association produces a variety of vertebral, rib, and limb abnormalities. This study was designed to document accurately the nature of these abnormalities and to determine whether synovial joints are affected. Fetuses from pregnant Sprague Dawley rats that had received intraperitoneal injections of 1.75 mg/kg of adriamycin on days 6-9 or 10-13 of gestation were harvested. Double-stained skeletal preparations and histological sections were examined for vertebral, rib, and limb anomalies. The incidence of anomalies was high in the group treated on gestational days (GD) 6-9, while it was low in the GD 10-13 group. The length and thickness of the long bones were reduced, with bowing and reduction in their endochondral ossification. Sirenomelia occurred in the group treated on GD 6-9, and was often associated with a short tail and anal atresia. The joint cavities, and intra-articular structures such as menisci and the cruciate ligaments developed normally from the mesenchymal interzone. These data indicate that adriamycin inhibits skeletal growth and differentiation without any interference in the differentiation of the mesenchymal interzone, thus producing normal synovial joints.

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.

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.

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.