Non-invasive prenatal test identifies circulating cell-free DNA chromosomal abnormalities derived from clonal hematopoiesis in aggressive hematological malignancies.

Liquid biopsy is a minimally invasive diagnostic tool for identification of tumor-related mutations in circulating cell-free DNA (cfDNA). The aim of this study was to investigate feasibility, sensitivity, and specificity of non-invasive prenatal test (NIPT) for identification of chromosomal abnormalities in cfDNA from a total of 77 consecutive patients with non-Hodgkin B-cell lymphomas, Hodgkin lymphoma (HL), or plasma cell dyscrasia. In this case series, half of patients had at least one alteration, more frequently in chromosome 6 (23.1%), chromosome 9 (20.5%), and chromosomes 3 and 18 (16.7%), with losses of chromosome 6 and gains of chromosome 7 negatively impacting on overall survival (OS), with a 5-year OS of 26.9% and a median OS of 14.6 months, respectively (P = 0.0009 and P = 0.0004). Moreover, B-cell lymphomas had the highest NIPT positivity, especially those with aggressive lymphomas, while patients with plasma cell dyscrasia with extramedullary disease had a higher NIPT positivity compared to conventional cytogenetics analysis and a worse outcome. Therefore, we proposed a NIPT-based liquid biopsy a complementary minimally invasive tool for chromosomal abnormality detection in hematological malignancies. However, prospective studies on larger cohorts are needed to validate clinical utility of NIPT-based liquid biopsy in routinely clinical practice.

Identification of Tissue miRNA Signatures for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), a neoplasm of the gastrointestinal tract, is the most common pancreatic malignancy (90%) and the fourth highest cause of cancer mortality worldwide. Surgery intervention is currently the only strategy able to offer an advantage in terms of overall survival, but prognosis remains poor even for operated patients. Therefore, the development of robust biomarkers for early diagnosis and prognostic stratification in clinical practice is urgently needed. In this work, we investigated deregulated microRNAs (miRNAs) in tissues from PDAC patients with high (G3) or low (G2) histological grade and with (N+) or without (N-) lymph node metastases. miRNA expression profiling was performed by a comprehensive PCR array and subsequent validation by RT-qPCR. The results showed a significant increase in miR-1-3p, miR-31-5p, and miR-205-5p expression in G3 compared to G2 patients (** p < 0.01; *** p < 0.001; *** p < 0.001). miR-518d-3p upregulation and miR-215-5p downregulation were observed in N+ compared to N- patients. A statistical analysis performed using OncomiR program showed the significant involvement (p < 0.05) of two miRNAs (miR-31 and miR-205) in the histological grade of PDAC patients. Also, an expression analysis in PDAC patients showed that miR-31 and miR-205 had the highest expression at grade 3 compared with normal and other tumor grades. Overall, survival plots confirmed that the overexpression of miR-31 and miR-205 was significantly correlated with decreased survival in TCGA PDAC clinical samples. A KEGG pathway analysis showed that all three miRNAs are involved in the regulation of multiple pathways, including the Hippo signaling, adherens junction and microRNAs in cancer, along with several target genes. Based on in silico analysis and experimental validation, our study suggests the potential role of miR-1-3p, miR-31-5p, and miR-205-5p as useful clinical biomarkers and putative therapeutic targets in PDAC, which should be further investigated to determine the specific molecular processes affected by their aberrant expression.

Clinical Experience with Genome-Wide Noninvasive Prenatal Screening in a Large Cohort of Twin Pregnancies.

Non-invasive prenatal screening (NIPS) in twin gestations has been shown to have high detection rates and low false-positive rates for trisomy 21, as seen in singleton pregnancies, although there have been few large cohort twin studies, genome-wide studies in particular, to date. In this study, we looked at the performance of genome-wide NIPT in a large cohort consisting of 1244 twin pregnancy samples collected over a two-year period in a single laboratory in Italy. All samples underwent an NIPS for common trisomies, with 61.5% of study participants choosing to undergo genome-wide NIPS for additional fetal anomalies (namely, rare autosomal aneuploidies and CNVs). There were nine initial no-call results, all of which were resolved upon retest. Based on our NIPS results, 17 samples were at high risk for trisomy 21, one for trisomy 18, six for a rare autosomal aneuploidy, and four for a CNV. Clinical follow-up was available for 27 out of 29 high-risk cases; a sensitivity of 100%, a specificity of 99.9%, and a PPV of 94.4% were noted for trisomy 21. Clinical follow-up was also available for 1110 (96.6%) of the low-risk cases, all of which were true negatives. In conclusion, we found that NIPS was a reliable screening approach for trisomy 21 in twin pregnancies.

A Case Report of a Feto-Placental Mosaicism Involving a Segmental Aneuploidy: A Challenge for Genome Wide Screening by Non-Invasive Prenatal Testing of Cell-Free DNA in Maternal Plasma.

Non-invasive prenatal testing (NIPT) using cell-free DNA can detect fetal chromosomal anomalies with high clinical sensitivity and specificity. In approximately 0.1% of clinical cases, the NIPT result and a subsequent diagnostic karyotype are discordant. Here we report a case of a 32-year-old pregnant patient with a 44.1 Mb duplication on the short arm of chromosome 4 detected by NIPT at 12 weeks' gestation. Amniocentesis was carried out at 18 weeks' gestation, followed by conventional and molecular cytogenetic analysis on cells from the amniotic fluid. SNP array analysis found a de novo deletion of 1.2 Mb at chromosome 4, and this deletion was found to be near the critical region of the Wolf-Hirschhorn syndrome. A normal 46,XY karyotype was identified by G-banding analysis. The patient underwent an elective termination and molecular investigations on tissues from the fetus, and the placenta confirmed the presence of type VI true fetal mosaicism. It is important that a patient receives counselling following a high-risk call on NIPT, with appropriate diagnostic analysis advised before any decisions regarding the pregnancy are taken. This case highlights the importance of genetic counselling following a high-risk call on NIPT, especially in light of the increasing capabilities of NIPT detection of sub-chromosomal deletions and duplications.

Non-Invasive Prenatal Screening: The First Report of Pentasomy X Detected by Plasma Cell-Free DNA and Karyotype Analysis.

Pentasomy X is a sex chromosome anomaly caused by the presence of three extra X chromosomes in females (49,XXXXX instead of 46,XX) and is probably due to a nondisjunction during the meiosis. So far, only five cases prenatally diagnosed were described. The main features in 49,XXXXX karyotype include severe intellectual disability with delayed speech development, short stature, facial dysmorphisms, osseous and articular abnormalities, congenital heart malformations, and skeletal and limb abnormalities. Prenatal diagnosis is often difficult due to the lack of a clear echographic sign like nuchal translucency (NT), and mostly cases were postnatally described. We report the first case of a 49,XXXXX female that was detected by non-invasive prenatal screening (NIPS), quantitative fluorescence polymerase chain reaction (QF-PCR) and a fetal karyotype.

PD-L1 Overexpression in the Lungs of Subjects Who Died from COVID-19: Are We on the Way to Understanding the Immune System Exhaustion Induced by SARS-CoV-2?

Knowledge of the pathogenic mechanisms of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. The presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. PD-L1 is overexpressed in the pulmonary tissue of subjects died from COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Detection of 46, XY Disorder of Sex Development (DSD) Based on Plasma Cell-Free DNA and Targeted Next-Generation Sequencing.

Mutations in the HSD17B3 gene cause HSD17B3 deficiency and result in 46, XY Disorders of Sex Development (46, XY DSD). The diagnosis of 46, XY DSD is very challenging and not rarely is confirmed only at older ages, when an affected XY female presents with primary amenorrhea or develops progressive virilization. The patient described in this paper represents a case of discrepancies between non-invasive prenatal testing (NIPT) and ultrasound based fetal sex determination detected during prenatal screening. Exome sequencing was performed on the cell free fetal DNA (cffDNA), amniotic fluid, and the parents. Libraries were generated according to the manufacturer's protocols using TruSight One Kits (Illumina Inc., San Diego, CA, USA). Sequencing was carried out on NEXT Seq 500 (Illumina) to mean sequencing depth of at least 100×. A panel of sexual disease genes was used in order to search for a causative variant. The finding of a mutation (c.645 A>T, p.Glu215Asp) in HSD17B3 gene in amniotic fluid as well as in cffDNA and both parents supported the hypothesis of the HSD17B3 deficiency. In conclusion, we used clinical exome sequencing and non-invasive prenatal detection, providing a solution for NIPT of a single-gene disorder. Early genetic diagnoses are useful for patients and clinicians, contribute to clinical knowledge of DSD, and are invaluable for genetic counseling of couples contemplating future pregnancies.

MED12 Mutation in Two Families with X-Linked Ohdo Syndrome.

X-linked intellectual deficiency (XLID) is a widely heterogeneous group of genetic disorders that involves more than 100 genes. The mediator of RNA polymerase II subunit 12 (MED12) is involved in the regulation of the majority of RNA polymerase II-dependent genes and has been shown to cause several forms of XLID, including Opitz-Kaveggia syndrome also known as FG syndrome (MIM #305450), Lujan-Fryns syndrome (MIM #309520) and the X-linked Ohdo syndrome (MIM #300895). Here, we report on two first cousins with X-linked Ohdo syndrome with a missense mutation in MED12 gene, identified through whole exome sequencing. The probands had facial features typical of X-linked Ohdo syndrome, including blepharophimosis, ptosis, a round face with a characteristic nose and a narrow mouth. Nextera DNA Exome kit (Illumina Inc., San Diego, CA, USA) was used for exome capture. The variant identified was a c.887G > A substitution in exon 7 of the MED12 gene leading to the substitution of a glutamine for a highly conserved arginine (p. Arg296Gln). Although the variant described has been previously reported in the literature, our study contributes to the expanding phenotypic spectrum of MED12-related disorders and above all, it demonstrates the phenotypic variability among different affected patients despite harboring identical mutations.

Performance of cell-free DNA sequencing-based non-invasive prenatal testing: experience on 36,456 singleton and multiple pregnancies.

BACKGROUND: This paper describes the clinical practice and performance of cell-free DNA sequencing-based non-invasive prenatal testing (NIPT) as a screening method for fetal trisomy 21, 18, and 13 (T21, T18, and T13) and sex chromosome aneuploidies (SCA) in a general Italian pregnancy population. METHODS: The AMES-accredited laboratory offers NIPT in maternal blood as a screening test for fetal T21, T18, T13 and SCA. Samples were sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. RESULTS: A retrospective analysis was performed on 36,456 consecutive maternal blood samples, including 35,650 singleton pregnancies, 800 twin pregnancies, and 6 triplet pregnancies. Samples were tested between April 2017 and September 2019. The cohort included 46% elevated-risk and 54% low-risk patients. A result indicative of a classic trisomy was found in 356 (1%) of singleton or twin samples: 254 T21, 69 T18, and 33 T13. In addition, 145 results (0.4%) were indicative of a SCA. Of the combined 501 screen-positive cases, 484 had confirmatory diagnostic testing. NIPT results were confirmed in 99.2% (247/249) of T21 cases, 91.2% (62/68) of T18 cases, 84.4% (27/32) of T13 cases, and 86.7% (117/135) of SCA cases. In the 35,955 cases reported as unaffected by a classic trisomy or SCA, no false negative cases were reported. Assuming that false negative results would be reported, the sensitivity of NIPT was 100.00% for T21 (95% Cl 98.47-100.0), T18 (95% Cl 94.17-100.0), and T13 (95% Cl 87.54-100.0). The specificities were 99.99% (95% Cl 99.98-100.0), 99.98% (95% Cl 99.96-100.0), 99.99% (95% Cl 99.97-100.0), and 99.95% (95% Cl 99.92-99.97) for T21, T18, T13, and SCA, respectively. CONCLUSION: This retrospective analysis of a large cohort of consecutive patients who had whole-genome sequencing-based NIPT for classic trisomies and SCA shows excellent detection rates and low false positive rates.

Detection of SRY-positive46,XX male syndrome by the analysis of cell-free fetal DNA via non-invasive prenatal testing.

We report a new case of 46,XX male syndrome that was detected following an anomalous result by non-invasive prenatal testing (NIPT) and a discrepancy between the fetal karyotype and the ultrasonographic investigation. With the increasing use of NIPT, more gender discordances can be identified prenatally and be amenable to early therapy.

In-Site Monocyte Implantation in Bone Grafting for Maxillary Atrophy Reconstruction: A Preliminary Observational Proof of Concept Study.

PURPOSE: The main objective of this study, involving 11 patients, is to share our experience on an integrated treatment modality, namely, the use of cellular therapy simultaneously with surgical reconstruction. Published works show that the implantation of monocytes in ischemic tissue enhances healing by providing neo-angiogenesis, a key mechanism in tissue regenerative processes. MATERIALS AND METHODS: Our approach included the utilization of autologous monocytes and endothelial precursor cells in the bone graft itself to improve the success rate of the integration of the bone graft and its long-term viability/survival by promoting angiogenesis. We compared the standard regenerative procedures, namely sinus lift grafting performed with xenogeneic particle bone graft and posterior mandible grafting performed with on-lay or in-lay autologous cortical/medullary bone-block graft harvested from the iliac crest, with and without the use of cellular implementation. We evaluated results by both radiological and histological assessment. RESULTS: Autologous cortical/medullary bone-block graft had a different response to implementation with monocytes, showing a better osteointegration than expected conversely to the xenogeneic particle bone graft. CONCLUSIONS: Monocytes seem to improve autologous bone-block graft according to the "Therapeutic Angiogenesis" concept. Implementation with monocytes does not always improve xenogeneic particle bone graft.