Mesenchymal Hamartoma of the Liver and DICER1 Syndrome.

Mesenchymal hamartoma of the liver (MHL) is a benign tumor affecting children that is characterized by a primitive myxoid stroma with cystically dilated bile ducts. Alterations involving chromosome 19q13 are a recurrent underlying cause of MHL; these alterations activate the chromosome 19 microRNA cluster (C19MC). Other cases remain unexplained. We describe two children with MHLs that harbored germline DICER1 pathogenic variants. Analysis of tumor tissue from one of the children revealed two DICER1 "hits." Mutations in DICER1 dysregulate microRNAs, mimicking the effect of the activation of C19MC. Our data suggest that MHL is a new phenotype of DICER1 syndrome. (Funded by the Canadian Institutes of Health Research and others.).

Risk Factors of Clinical Relapses in Pediatric Luminal Crohn's Disease: A Retrospective Cohort Study.

INTRODUCTION: There is currently little knowledge on factors associated with the relapse of Crohn's disease (CD) in children. The aims of this study were to describe the risk factors associated with relapse in pediatric CD and the changes in the relapse rate over the past decade. METHODS: Patients younger than 18 years and diagnosed between 2009 and 2019 were included in this retrospective cohort study. Clinical, endoscopic, histological, and laboratory data, as well as induction and maintenance treatments, were collected from the medical records. Survival analyses and Cox regression models were used to assess the impact of these risk factors on relapse. RESULTS: Six hundred thirty-nine patients were included. There was a decrease in the clinical relapse rate over the past decade: 70.9% of the patients diagnosed between 2009 and 2014 relapsed as compared with 49.1% of the patients diagnosed between 2015 and 2019 (P < 0.0001). The following variables were associated with clinical relapse: female sex (adjusted hazard ratio [aHR] = 1.52, P = 0.0007), exposure to oral 5-ASA (aHR = 1.44, P = 0.04), use of immunomodulatory agents compared with tumor necrosis factor-alpha inhibitors (methotrexate aHR = 1.73, P = 0.003; thiopurines aHR = 1.63, P = 0.002), presence of granulomas (aHR = 1.34, P = 0.02) and increased eosinophils on intestinal biopsies (aHR = 1.36, P = 0.02), high levels of C-reactive protein (aHR = 1.01, P < 0.0001) and fecal calprotectin (aHR = 1.08, P < 0.0001), and low serum infliximab levels (aHR = 2.32, P = 0.001). DISCUSSION: Relapse of pediatric CD has decreased in the past decade. The risk of relapse is significantly associated with clinical, endoscopic, histological, and laboratory variables and treatment strategies.

Characterization of the Transition Zone in Short Segment Hirschsprung Disease Using Calretinin Immunostaining.

Introduction: The detailed expression pattern of calretinin immunohistochemistry in the transition zone (TZ) of Hirschsprung disease (HSCR) has not yet been reported. This study aims to examine the value of calretinin immunohistochemistry for more accurately determining the distal and proximal border of the TZ in short segment HSCR. Methods: Specimens of pull-through surgery from 51 patients with short form of HSCR were analyzed on two longitudinal strips using hematoxylin and eosin (H&E) staining and calretinin immunohistochemistry. Results: In all but two patients, the first appearance of calretinin expression was seen on mucosal nerve fibers before the appearance of any ganglion cells, indicating the distal border of the TZ. The maximum distance between the distal border of the TZ and the proximal border of the TZ, defined by ganglion cells in a normal density on H&E stained sections, a strong calretinin expression on mucosal nerve fibers and in >80% of submucosal and myenteric ganglion cells, with no nerve hypertrophy and absence of ganglionitis was 60 mm. Conclusion: The distal border of the TZ is characterized by calretinin positive intramucosal neurites in nearly all of short form of HSCR and not by calretinin expression on ganglion cells.

Gray Zone Lymphoma Arising in the Neck of a Teenager With a Germline Mutation in TP53.

Gray zone lymphoma is an aggressive disease for which appropriate management is still debated. We report a 15-year-old girl with a cervical mass, an enlarged ipsilateral tonsil, and anemia. Both sites showed hypermetabolism on F18-FG positron emission tomography/CT. Surgical resection was diagnostic of Epstein-Barr virus-negative gray zone lymphoma cervical and tonsillar involvement. No abnormality was found in cytogenetic analysis on tumor cells. However, exome sequencing in peripheral blood DNA revealed a germline mutation in TP53. Complete response was achieved after surgery and 6 cycles of rituximab with dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin regimen.

Multiple DICER1-related tumors in a child with a large interstitial 14q32 deletion.

Germ-line interstitial deletions involving the 14q32 chromosomal region, resulting in 14q32 deletion syndrome, are rare. DICER1 is a recently described cancer-predisposition gene located at 14q32.13. We report the case of a male child with a ∼5.8 Mbp 14q32.13q32.2 germ-line deletion, which included the full DICER1 locus. We reviewed available clinical and pathological material, and conducted genetic analyses. In addition to having congenital dysmorphic features, the child developed multiple DICER1 syndrome-related tumors before age 5 y: a pediatric cystic nephroma (pCN), a ciliary body medulloepithelioma (CBME), and a small lung cyst (consistent with occult pleuropulmonary blastoma Type I/Ir cysts seen in DICER1 mutation carriers). He also developed a cerebral spindle-cell sarcoma with myogenous differentiation. Our investigations revealed that the deletion encompassed 31 protein-coding genes. In addition to the germ-line DICER1 deletion, somatic DICER1 RNase IIIb mutations were found in the CBME (c.5437G > A, p.E1813K), pCN (c.5425G > A, p.G1809R), and sarcoma (c.5125G > A, p.D1709N). The sarcoma also harbored a somatic TP53 mutation: c.844C > T, p.R282W. Additional copy number alterations were identified in the CBME and sarcoma using an OncoScan array. Among the 8 cases with molecularly-defined 14q32 deletions involving DICER1 and for whom phenotypic information is available, our patient and one other developed DICER1-related tumors. Biallelic DICER1 mutations have not previously been reported to cause cerebral sarcoma, which now may be considered a rare manifestation of the DICER1 syndrome. Our study shows that DICER1-related tumors can occur in children with 14q32 deletions and suggests surveillance for such tumors may be warranted.

Genomic study of severe fetal anomalies and discovery of GREB1L mutations in renal agenesis.

PURPOSE: Fetal anomalies represent a poorly studied group of developmental disorders. Our objective was to assess the impact of whole-exome sequencing (WES) on the investigation of these anomalies. METHODS: We performed WES in 101 fetuses or stillborns who presented prenatally with severe anomalies, including renal a/dysgenesis, VACTERL association (vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula, renal anomalies, and limb abnormalities), brain anomalies, suspected ciliopathies, multiple major malformations, and akinesia. RESULTS: A molecular diagnosis was obtained in 19 cases (19%). In 13 of these cases, the diagnosis was not initially suspected by the clinicians because the phenotype was nonspecific or atypical, corresponding in some cases to the severe end of the spectrum of a known disease (e.g., MNX1-, RYR1-, or TUBB-related disorders). In addition, we identified likely pathogenic variants in genes (DSTYK, ACTB, and HIVEP2) previously associated with phenotypes that were substantially different from those found in our cases. Finally, we identified variants in novel candidate genes that were associated with perinatal lethality, including de novo mutations in GREB1L in two cases with bilateral renal agenesis, which represents a significant enrichment of such mutations in our cohort. CONCLUSION: Our study opens a window on the distinctive genetic landscape associated with fetal anomalies and highlights the power-but also the challenges-of WES in prenatal diagnosis.

A case of neuroblastoma in DICER1 syndrome: Chance finding or noncanonical causation?

DICER1 syndrome is an inherited disorder associated with at least a dozen rare, mainly pediatric-onset tumors. Its characterization remains incomplete. Some studies suggested that neuroblastoma (NB) may be involved in this syndrome. Here, we describe the case of a 14-year-old female presenting with a multinodular goiter (MNG) and a collision tumor composed of NB and cystic nephroma (CN). She is a carrier of a deleterious germline mutation in exon 23 of DICER1 and harbored different somatic mutations in the CN and MNG. However, no second hit was found in the NB, questioning its status as a DICER1-related tumor.

Sequencing of DICER1 in sarcomas identifies biallelic somatic DICER1 mutations in an adult-onset embryonal rhabdomyosarcoma.

BACKGROUND: Sarcomas are rare and heterogeneous cancers. We assessed the contribution of DICER1 mutations to sarcoma development. METHODS: The coding region of DICER1 was sequenced in 67 sarcomas using a custom Fluidigm Access Array. The RNase III domains were Sanger sequenced in six additional sarcomas to identify hotspot DICER1 variants. RESULTS: The median age of sarcoma diagnosis was 45.7 years (range: 3 months to 87.4 years). A recurrent embryonal rhabdomyosarcoma (ERMS) of the broad ligament, first diagnosed at age 23 years, harboured biallelic pathogenic somatic DICER1 variants (1 truncating and 1 RNase IIIb missense). We identified nine other DICER1 variants. One somatic variant (p.L1070V) identified in a pleomorphic sarcoma and one germline variant (c.2257-7A>G) may be pathogenic, but the others are considered to be benign. CONCLUSIONS: We show that deleterious DICER1 mutations underlie the genetic basis of only a small fraction of sarcomas, in particular ERMS of the urogenital tract.

Intestinal Metaplasia of the Esophagus in Children With Esophageal Atresia.

OBJECTIVES: Patients with esophageal atresia/tracheoesophageal fistula (EA-TEF) can develop Barrett esophagus as a long-term consequence of their condition. Intestinal metaplasia (IM), a risk factor for developing adenocarcinoma of the esophagus, has not been well characterized in the pediatric population. METHODS: Retrospective review of patients with EA-TEF followed at 3 academic pediatric centers between the years 1997 and 2014. RESULTS: Among 542 children and adolescents, 1.3% (7 patients, 5 girls) were diagnosed with IM based on endoscopy and pathology. Six of the patients had EA-TEF type C, whereas the last patient had a "long gap" type A atresia. Patients were diagnosed with gastric metaplasia either before the IM diagnosis in 4 patients or concomitantly in 3. The median (range) age of diagnosis for gastric metaplasia was 7.9 (range 2-17.2) and for IM 10.9 (2-17.2) years. Gastroesophageal reflux (GER) symptoms were nonspecific. Five patients were on proton pump inhibitor therapy for symptomatic GER at the time of diagnosis of IM. 2 of the 7 patients had previously undergone Nissen fundoplication. One patient, who had undergone a Nissen fundoplication, was restarted on proton pump inhibitor once the diagnosis of IM was made. All patients had repeated endoscopy and dysplasia was not observed with a median follow-up of 1.7 (range 1-4.9) years. CONCLUSIONS: IM occurs in patients with EA-TEF, some as young as 2 years. Therefore, early endoscopic surveillance should be considered in this GER-prone population.

High-sensitivity sequencing reveals multi-organ somatic mosaicism causing DICER1 syndrome.

BACKGROUND: Somatic mosaicism is being increasingly recognised as an important cause of non-Mendelian presentations of hereditary syndromes. A previous whole-exome sequencing study using DNA derived from peripheral blood identified mosaic mutations in DICER1 in two children with overgrowth and developmental delay as well as more typical phenotypes of germline DICER1 mutation. However, very-low-frequency mosaicism is difficult to detect, and thus, causal mutations can go unnoticed. Highly sensitive, cost-effective approaches are needed to molecularly diagnose these persons. We studied four children with multiple primary tumours known to be associated with the DICER1 syndrome, but in whom germline DICER1 mutations were not detected by conventional mutation detection techniques. METHODS AND RESULTS: We observed the same missense mutation within the DICER1 RNase IIIb domain in multiple tumours from different sites in each patient, raising suspicion of somatic mosaicism. We implemented three different targeted-capture technologies, including the novel HaloPlex(HS) (Agilent Technologies), followed by deep sequencing, and confirmed that the identified mutations are mosaic in origin in three patients, detectable in 0.24-31% of sequencing reads in constitutional DNA. The mosaic origin of patient 4's mutation remains to be unequivocally established. We also discovered likely pathogenic second somatic mutations or loss of heterozygosity (LOH) in tumours from all four patients. CONCLUSIONS: Mosaic DICER1 mutations are an important cause of the DICER1 syndrome in patients with severe phenotypes and often appear to be accompanied by second somatic truncating mutations or LOH in the associated tumours. Furthermore, the molecular barcode-containing HaloPlex(HS) provides the sensitivity required for detection of such low-level mosaic mutations and could have general applicability.

Revisiting Pathological Criteria for Earlier Diagnosis of Coeliac Disease.

OBJECTIVES: The diagnosis of coeliac disease (CD) remains sometimes difficult because the histological criteria are not fully met. The aim of this study was to refine histological diagnostic criteria of CD. METHODS: One hundred seventy-five duodenal bulb D1 (n = 79) and duodenal D2 (n = 96) biopsies of 96 patients with CD (58 girls, mean age 7 years), 135 normal D2 biopsies (69 girls, mean age 12 years), and 64 D2 biopsies of other digestive disorders (DDs) (39 girls, mean age 13 years) obtained from children during a period of 4 years were reviewed. RESULTS: Interobserver agreement was greater for the classification of Corazza-Villanacci than for Marsh-Oberhuber (κ = 0.812 vs κ = 0.409, respectively). Between 40 and 70 intraepithelial lymphocytes (IELs) per 100 epithelial cells (ECs), 32% of patients were CD, whereas 50% had other DD. Above 70 IELs per 100 EC, 53% were CD, and only 6% had other DD. In CD, IELs were significantly located above EC nuclei compared with other DD, (12 IELs/100 EC vs 2 IELs/100 EC, respectively). In 21% of CD cases, D2 were normal and the diagnosis could only be made on D1. Finally, 6% of CD cases showed isolated increase of IELs in D1 without architectural modification. CONCLUSIONS: D1 allowed diagnosis of CD in 21% of cases and IEL >70 per 100 EC correlated strongly with CD. Between 40 and 70 IELs per 100 EC, CD is very likely but other DD must be considered. Finally, the preferential localisation of IELs above EC nuclei favours CD, and increased IEL in D1 may be the sole abnormality.

Intestinal congenital/infantile fibrosarcoma: a new clinico-pathological entity?

Congenital/infantile fibrosarcoma (IFS) is a relatively rare form of fibrosarcoma diagnosed at birth or during early years of life and that differs from its adult counterpart because of a more favorable behavior. IFS is also known as cellular congenital mesoblastic nephroma, when it affects the kidney and is often but not always characterized by the ETV6-NTRK3 fusion transcript. We report herein the first series of an exceptional tumor of the small intestine occurring in newborns. The four patients shared a stereotyped clinico-pathological presentation with early and acute onset, intestinal perforation, and an infiltration by a highly cellular spindle cell tumor within the dilated intestinal wall exhibiting pathologic features typical of IFS. Molecular studies for the ETV6-NTRK3 translocation were negative in the three cases tested. Patients were treated by surgical wide resection alone and are alive and well (follow-up: 36 months-25 years). Thus, this new clinico-pathological entity, even with lack of documented evidence of the ETV6-NTRK3 translocation, should be included in the differential diagnosis of congenital bowel perforation or obstruction and may represent an intestinal counterpart of IFS.

Germ-line and somatic DICER1 mutations in pineoblastoma.

Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours.

Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations.

Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5' arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 "hits" occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.

Diagnostic utility of molecular and cytogenetic analysis in lipoblastoma: a study of two cases and review of the literature.

AIMS: Lipoblastoma is a benign neoplasm of embryonic white fat tissue that results from the proliferation of primitive adipocytes, in which histological features can be ambiguous. In order to discriminate between lipoblastoma and other lipogenic and lipomatous tumours, we studied chromosomal alterations and protein expression in two cases of lipoblastoma in infants. METHODS AND RESULTS: Standard cytogenetic analysis, fluorescence in-situ hybridization, array comparative genomic hybridization and Western blotting allowed us to demonstrate the presence of chromosome abnormalities involving the 8q11-13 region containing the pleomorphic adenoma gene 1 (PLAG1), which are classically reported in lipoblastoma, and aberrant expression of PLAG1. CONCLUSIONS: This report illustrates two different tumorigenic pathways implicating PLAG1 in lipoblastoma: amplification through multiple copies of a small marker chromosome derived from chromosome 8, and a paracentric inversion of the long arm of chromosome 8. Both these anomalies induced aberrant expression of PLAG1, emphasizing the role of PLAG1 in tumorigenesis. The aberrant expression of PLAG1 protein has been hypothesized, but this is the first report to demonstrate its occurrence in lipoblastoma.

Identification of divergent placental profiles in clinically distinct pregnancy complications revealed by the transcriptome.

INTRODUCTION: Pregnancy complications, including preeclampsia (PE), preterm birth (PTB), and intra-uterine growth restriction (IUGR) have individually been associated with inflammation but the combined comparative analysis of their placental profiles at the transcriptomic and histological levels is lacking. METHODS: Bulk RNA-sequencing of human placental biopsies from uncomplicated term pregnancies (CTL) and pregnancies complicated with early-onset (EO), and late-onset (LO) PE, as well as PTB and term IUGR were used to characterize individual molecular profiles. We also applied immune-cell-specific cellular deconvolution to address local immune cell compositions and analyzed placental lesions by histology to further characterize these complications. RESULTS: Transcriptome analysis revealed that clinically distinct complications differentiated themselves in unique ways compared to CTLs. Only TMEM136 was commonly modulated. Compared to CTLs, we found that PTB and IUGR were the most distinct, with LOPE being the least distinct. PTB and IUGR revealed differently enhanced inflammatory pathways, where PTB had general inflammatory responses and IUGR had immune cell activation. This inflammation was reflected in the histological profile for PTB only, whereas structural lesions were elevated in all complications. Placental lesions additionally had corresponding enhancement in inflammatory and structural biological processes. We observed that having co-complications, particularly for PTB with or without IUGR, impacted placental transcriptomes. Lastly, cellular deconvolution uncovered shared immune features among the complications. DISCUSSION: Overall, we provide evidence that these pregnancy complications are not only distinct in their clinical manifestations but also in their placental profiles, which could be leveraged to understand their underlying mechanisms and could offer therapeutic targets.