Minimal residual disease detection by multicolor flow cytometry in cryopreserved ovarian tissue from leukemia patients.

BACKGROUND: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. RESULTS: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. CONCLUSIONS: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients.

Complete lung agenesis caused by complex genomic rearrangements with neo-TAD formation at the SHH locus.

During human organogenesis, lung development is a timely and tightly regulated developmental process under the control of a large number of signaling molecules. Understanding how genetic variants can disturb normal lung development causing different lung malformations is a major goal for dissecting molecular mechanisms during embryogenesis. Here, through exome sequencing (ES), array CGH, genome sequencing (GS) and Hi-C, we aimed at elucidating the molecular basis of bilateral isolated lung agenesis in three fetuses born to a non-consanguineous family. We detected a complex genomic rearrangement containing duplicated, triplicated and deleted fragments involving the SHH locus in fetuses presenting complete agenesis of both lungs and near-complete agenesis of the trachea, diagnosed by ultrasound screening and confirmed at autopsy following termination. The rearrangement did not include SHH itself, but several regulatory elements for lung development, such as MACS1, a major SHH lung enhancer, and the neighboring genes MNX1 and NOM1. The rearrangement incorporated parts of two topologically associating domains (TADs) including their boundaries. Hi-C of cells from one of the affected fetuses showed the formation of two novel TADs each containing SHH enhancers and the MNX1 and NOM1 genes. Hi-C together with GS indicate that the new 3D conformation is likely causative for this condition by an inappropriate activation of MNX1 included in the neo-TADs by MACS1 enhancer, further highlighting the importance of the 3D chromatin conformation in human disease.

Genotype-first in a cohort of 95 fetuses with multiple congenital abnormalities: when exome sequencing reveals unexpected fetal phenotype-genotype correlations.

PURPOSE: Molecular diagnosis based on singleton exome sequencing (sES) is particularly challenging in fetuses with multiple congenital abnormalities (MCA). Indeed, some studies reveal a diagnostic yield of about 20%, far lower than in live birth individuals showing developmental abnormalities (30%), suggesting that standard analyses, based on the correlation between clinical hallmarks described in postnatal syndromic presentations and genotype, may underestimate the impact of the genetic variants identified in fetal analyses. METHODS: We performed sES in 95 fetuses with MCA. Blind to phenotype, we applied a genotype-first approach consisting of combined analyses based on variants annotation and bioinformatics predictions followed by reverse phenotyping. Initially applied to OMIM-morbid genes, analyses were then extended to all genes. We complemented our approach by using reverse phenotyping, variant segregation analysis, bibliographic search and data sharing in order to establish the clinical significance of the prioritised variants. RESULTS: sES rapidly identified causal variant in 24/95 fetuses (25%), variants of unknown significance in OMIM genes in 8/95 fetuses (8%) and six novel candidate genes in 6/95 fetuses (6%). CONCLUSIONS: This method, based on a genotype-first approach followed by reverse phenotyping, shed light on unexpected fetal phenotype-genotype correlations, emphasising the relevance of prenatal studies to reveal extreme clinical presentations associated with well-known Mendelian disorders.

Autopsy findings of ectodermal dysplasia and sex development disorder in a fetus with 19q12q13 microdeletion.

A 5,6 Mb de novo 19q12-q13.12 interstitial deletion was diagnosed prenatally by array-comparative genomic hybridization in a 26 weeks male fetus presenting with intra-uterine growth retardation, left clubfoot, atypical genitalia and dysmorphic features. Autopsic examination following termination of pregnancy identified a severe disorder of sex development (DSD) including hypospadias, micropenis, bifid scrotum and right cryptorchidism associated with signs of ectodermal dysplasia: scalp hypopigmentation, thick and frizzy hair, absence of eyelashes, poorly developed nails and a thin skin with prominent superficial veins. Other findings were abnormal lung lobation and facial dysmorphism. This new case of DSD with a 19q12q13 deletion expands the phenotypic spectrum associated with this chromosomal rearrangment and suggests that WTIP is a strong candidate gene involved in male sex differentiation.

How to Explore Fetal Sacral Agenesis Without Open Dysraphism: Key Prenatal Imaging and Clinical Implications.

The estimated prevalence of fetal caudal dysgenesis is 1 per 100,000 births. The functional prognosis of sacral agenesis is dominated by the large spectrum of associated caudal malformations. Except for cases associated with hydrocephalus secondary to open spinal dysraphism or chromosomal anomalies, association with mental deficiency is rare. We propose a systematic prenatal approach to cases of fetal sacral agenesis based on 9 etiologic items: clinical context, type of sacral dysgenesis, associated spinal cord malformations, mobility of lower limbs, investigation of the presacral region, analysis of the gastrointestinal tract, analysis of the genitourinary tract, associated vertebral defects, and cytogenetic analysis.

[Authorization of pathologists for the estimation of the tumor cell percentage on tissue sample for molecular analysis purpose]. / Habilitation des pathologistes pour l'évaluation du pourcentage de cellules tumorales lors du contrôle morphologique avant extraction d'acides nucléiques.

Before molecular analysis is performed, morphological control with an estimation of the tumour cell percentage (%TC) could have a major impact on mutation detection. Accreditation according to NF EN ISO 15189 commands an authorization through evaluation of skills. The objective of this work was to validate the empowerment of pathologists to estimate %TC in tissue sample prior to molecular analysis. The accreditation technical guidance methods in Medical biology and histopathology were taken as references. %TC was the ratio of tumour cell nuclei on all nuclei within the area selected for the DNA extraction. External evaluations quality scores were used for accuracy. In order to assess the intermediate precision, 35 %TC estimation were performed 15 days apart in 4 samples (biopsies, transparietal biopsies or surgical specimen, either fixed or frozen) by 7 pathologists. Three other cases with interference (inflammation, mucus, necrosis) were evaluated. A result was satisfactory if %TC were within ±20 % of expected percentage obtained by the average of 35 estimates. The performances were satisfactory since no estimate was made more than 20 % of the expected percentage. Low interpathologists reproducibility has been reported in the literature and can have a consequence on molecular analysis in samples with low %TC, where the value reach the analytical sensitivity thresholds of molecular techniques. The current report is an example of a step of the accreditation process, which is a challenge for pathologists' activity in the future.

Primary Linitis Plastica of the Rectum: Focus on Magnetic Resonance Imaging Patterns and Treatment Options.

BACKGROUND: Rectal linitis plastica (RLP) is a rare disease with poor outcome. It is often accompanied by a delayed histopathological diagnosis, primarily due to submucosal disease. A concentric ring pattern or "target sign" on T2-weighted magnetic resonance imaging (MRI) has been proposed as being characteristic for early suspicion. Even though RLP is more aggressive and has poorer survival than other rectal adenocarcinomas, no specific treatment is recommended. In this case report of 3 patients, we challenge the sensitivity of the characteristic radiological pattern, and we review the existing data for a treatment strategy. CASE REPORT: One patient presented classic clinical characteristics of RLP with young age and advanced stage at diagnosis, with chemo-refractory disease and rapid fatal evolution. Biopsies confirmed the RLP with the presence of signet-ring cells (SRC) in a strong desmoplastic stromal reaction. However, the characteristic concentric ring pattern was absent. Instead, he had a large vegetative lesion with important tumor infiltration in mesorectum and pelvic organs, with major lymph node involvement. The 2 other patients presented resectable locally advanced disease with characteristic concentric ring pattern. No clinical and radiological responses were observed to neo-adjuvant chemoradiotherapy (CRT), including 1 patient with non-resectable disease at surgery and another with upstaged disease at pathological specimen after resection. However, data suggest 2 types of RLP: about half of patients are extremely sensitive to CRT with pathological complete response, and the other half are highly resistant with no response to CRT. Current data are insufficient to distinguish between these 2 populations. CONCLUSIONS: The absence of a concentric ring pattern should not eliminate the suspicion of RLP, especially in young patients with aggressive clinical presentation. There are probably 2 types of RLP in terms of chemoradiosensitivity, and neoadjuvant CRT could delay the curative-intent surgery in refractory patients. Future molecular analysis of the tumor and its environment are required to characterize the 2 different forms of RLP to develop more personalized treatment strategies.

Echinococcus ortleppi infections in humans and cattle, France.

In 2011 and 2012, liver infections caused by Echinococcus ortleppi tapeworms were diagnosed in 2 humans in France. In 2012, a nationwide slaughterhouse survey identified 7 E. ortleppi infections in cattle. The foci for these infections were spatially distinct. The prevalence of E. ortleppi infections in France may be underestimated.

Severe sex differentiation disorder in a boy with a 3.8 Mb 10q25.3-q26.12 microdeletion encompassing EMX2.

The molecular basis of male disorders of sex development (DSD) remains unexplained in a large number of cases. EMX2 has been proposed to play a role in the masculinization process for the past two decades, but formal evidence for this causal role is scarce. The aim of this study is to yield additional support to this hypothesis by reporting on a male patient who presented with 46,XY DSD, a single kidney, intellectual disability, and the smallest microdeletion including EMX2 reported to date. EMX2 haploinsufficiency is likely to explain the masculinization defect observed in our patient, similar to what has been described in the mouse. In the case of cytogenetically diagnosed cases, deletions of EMX2 have been associated with a wide range of DSD, ranging from hypospadias to complete sex reversal.

Signature patterns of human papillomavirus type 16 in invasive anal carcinoma.

Many studies have reported that most invasive anal carcinomas contain high-risk human papillomaviruses (HPVs), HPV16 being the most prevalent type. This study aimed to investigate HPV status and cellular biomarkers in invasive anal cancers. HPV genotype distribution was determined in 76 anal cancers by the INNO-LiPA assay (Innogenetics, Gent, Belgium). HPV16-positive samples were then tested for viral load and physical state with type-specific real-time polymerase chain reaction targeting E6, E2, and albumin genes. Samples were also subjected to immunohistochemical analysis of p16, Ki-67, p53, and p21. Of the analyzable tumors, 98.6% were positive for α-HPV DNA. HPV16 was the most prevalent genotype (89.0%), followed by HPV39 (4.1%) and HPV33 (2.7%). HPV16 viral load was high, ranging from 2.1 × 10(3) to 1.5 × 10(7) copies/10(3) cells. Integration of HPV16 estimated by the E2/E6 ratio was detected in 77.8% of cases, among which 70.4% were mixed integrated and episomal DNA cases and 7.4% were fully integrated DNA cases. The latter cases were associated with a low HPV16 load compared with cases containing either episomes or mixed integrated and episomal DNA. As expected, most HPV16-positive tumors expressed p16 (92.6%) with a high proliferative index, whereas a minority of them overexpressed p53 (10.3%). p21 expression did not appear to correlate with p53 expression. Although HPV16 was almost exclusively detected, high viral load and differences in DNA integration have been identified in the present series of anal cancers. HPV features assessed in conjunction with expression of cell-cycle regulators could be helpful, as joint biomarkers, in predicting clinical outcome.

Coronal craniosynostosis and radial ray hypoplasia: a third report of Twist mutation in a 33 weeks fetus with diaphragmatic hernia.

We describe a multiple malformation syndrome comprising coronal craniosynostosis, unilateral radial ray hypoplasia and diaphragmatic hernia in a 33w female fetus born to a 46 y-old male with an alleged personal and family history of Crouzon syndrome. By identifying an already described c.445C>T TWIST missense mutation, we were able to reassign the diagnosis of the family condition to Saethre-Chötzen syndrome. The present report illustrates clinical variability of a dominantly inherited TWIST mutation and provides a third example of Baller-Gerold/Saethre-Chötzen overlapping phenotype. We also add diaphragmatic hernia in the spectrum of TWIST-related malformations, although we couldn't prove the co-occurrence is not coincidental.

Prognostic value of phosphorylated STAT3 in advanced rectal cancer: a study from 104 French patients included in the EORTC 22921 trial.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) has been implicated as an oncogene in several neoplastic diseases. However, the biological effects of STAT3 have not been extensively studied in rectal carcinogenesis. AIMS: To evaluate STAT3 activation in advanced rectal cancers and its association with clinicopathological variables and prognosis. METHODS: Nuclear immunohistochemical expression of phosphorylated STAT3 (p-STAT3) was studied in 104 advanced rectal cancers (T3-T4). All patients were participating in the EORTC 22921 trial to assess whether preoperative chemoradiotherapy followed by postoperative chemotherapy improved overall and progression-free survival. RESULTS: Nuclear p-STAT3 expression was detected in 37.5% of rectal cancer patients. No correlation was observed between p-STAT3 and any clinicopathological variables tested. However, patients with tumours positive for p-STAT3 had significantly improved overall survival. CONCLUSION: These results highlight an unexpected role for nuclear p-STAT3 expression in advanced rectal cancers and need further investigation to clarify this finding.

Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer.

Hepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/beta-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. beta-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy.

The uterosacral complex: ligament or neurovascular pathway? Anatomical and histological study of fetuses and adults.

The aim of this study was to define the anatomical relationships of the uterosacral ligament complex (USLC) and to analyze histologically its content. Three fetal and four adult cadavers were used. Anatomical dissections were carried out. Eight fresh biopsies (four fetal and four adult) of the USLC were analyzed histologically and immunohistochemically. Specimens were stained with hematoxylin eosin safran coloration, with anti-nervous cell antibodies (PS 100) and with anti-smooth muscle antibodies (to visualize vessel walls). By removing the visceral pelvic fascia, nervous fibers were found within the USLC forming the hypogastric plexus. Histologically, the USLC contained connective tissue, nervous fibers, sympathetic nodes, vessels, and fatty tissue. No structured ligamentous organization was identified. The uterosacral "ligament" is a "complex" integrating connective tissue as well as nervous and vascular elements. Radical excisions and USLC suspension during pelvic floor reconstructive surgery should be performed with caution in order to preserve pelvic innervation.