Personalized health risk assessment based on single-cell RNA sequencing analysis of a male with 45, X/48, XYYY karyotype.

Numeric sex chromosome abnormalities are commonly associated with an increased cancer risk. Here, we report a 14-year-old boy with a rare mosaic 45, X/48, XYYY karyotype presenting with subtle dysmorphic features and relative height deficiency, requiring growth hormone therapy. As only 12 postnatal cases have been described so far with very limited follow-up data, to assess the proband's long-term prognosis, including cancer risk, we performed high-throughput single-cell RNA sequencing (scRNA-seq) analysis. Although comprehensive cytogenetic analysis showed seemingly near perfect balance between 45, X and 48, XYYY cell populations, scRNA-seq revealed widespread differences in genotype distribution among immune cell fractions, specifically in monocytes, B- and T-cells. These results were confirmed at DNA level by digital-droplet PCR on flow-sorted immune cell types. Furthermore, deregulation of predominantly autosomal genes was observed, including TCL1A overexpression in 45, X B-lymphocytes and other known genes associated with hematological malignancies. Together with the standard hematological results, showing increased fractions of monocytes and CD4+/CD8+T lymphocytes ratio, long-term personalized hemato-oncological surveillance was recommended in the reported patient.

Bilateral Ovarian Germ Cell Tumor in a 46,XX Female with Nijmegen Breakage Syndrome and Hypergonadotropic Hypogonadism

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disease, affecting mainly patients of Slavic origin. It is caused by a defect in the NBN gene, resulting in defective nibrin protein formation. This leads to chromosomal instability, which predisposes to cancer, with lymphoid malignancies predominating. Nibrin is also involved in gonadal development and its disfunction in females with NBS frequently results in a pure gonadal dysgenesis (PGD) causing hypergonadotropic hypogonadism. However, only a few ovarian tumors in NBS patients have been reported to date. We describe the first case of a girl with NBS with PGD, who developed metachronous bilateral ovarian germ cell tumors (dysgerminoma and gonadoblastoma). Pathogenesis of PGD, neoplastic transformation and therapeutic approach in females with NBS are discussed.

Expression of BARD1 ß Isoform in Selected Pediatric Tumors.

Currently, many new possible biomarkers and mechanisms are being searched and tested to analyse pathobiology of pediatric tumours for the development of new treatments. One such candidate molecular factor is BARD1 (BRCA1 Associated RING Domain 1)-a tumour-suppressing gene involved in cell cycle control and genome stability, engaged in several types of adult-type tumours. The data on BARD1 significance in childhood cancer is limited. This study determines the expression level of BARD1 and its isoform beta (ß) in three different histogenetic groups of pediatric cancer-neuroblastic tumours, and for the first time in chosen germ cell tumours (GCT), and rhabdomyosarcoma (RMS), using the qPCR method. We found higher expression of beta isoform in tumour compared to healthy tissue with no such changes concerning BARD1 full-length. Additionally, differences in expression of BARD1 ß between histological types of neuroblastic tumours were observed, with higher levels in ganglioneuroblastoma and ganglioneuroma. Furthermore, a higher expression of BARD1 ß characterized yolk sac tumours (GCT type) and RMS when comparing with non-neoplastic tissue. These tumours also showed a high expression of the TERT (Telomerase Reverse Transcriptase) gene. In two RMS cases we found deep decrease of BARD1 ß in post-chemotherapy samples. This work supports the oncogenicity of the beta isoform in pediatric tumours, as well as demonstrates the differences in its expression depending on the histological type of neoplasm, and the level of maturation in neuroblastic tumours.

ALK alterations in salivary gland carcinomas.

Salivary gland carcinomas represent a heterogeneous group of poorly characterized head and neck tumors. The purpose of this study was to evaluate ALK gene and protein aberrations in a large, well-characterized cohort of these tumors. A total of 182 salivary gland carcinomas were tested for anaplastic lymphoma kinase (ALK) positivity by immunohistochemistry (IHC) using the cut-off of 10% positive cells. ALK positive tumors were subjected to FISH analysis and followed by hybrid capture-based next generation sequencing (NGS). Of the 182 tumors, 8 were ALK positive by IHC. Further analysis using hybrid capture NGS analysis revealed a novel MYO18A (Exon1-40)-ALK (exon 20-29) gene fusion in one case of intraductal carcinoma. Additional genomic analyses resulted in the detection of inactivating mutations in BRAF and TP53, as well as amplifications of ERBB2 and ALK. ALK rearrangements are a rare entity in salivary gland carcinomas. We identified a potentially targetable novel ALK fusion in an intraductal carcinoma of minor salivary glands.

MAML2 rearrangement as a useful diagnostic marker discriminating between Warthin tumour and Warthin-like mucoepidermoid carcinoma.

Warthin tumour is the second most common benign neoplasm of salivary glands. Despite its relatively characteristic histology, it may sometimes mimic other lesions. Here, we report two female non-smoker patients diagnosed with low-grade mucoepidermoid carcinoma with oncocytic epithelium and prominent lymphoid (Warthin-like) stroma and with molecularly confirmed MAML2 rearrangement. In addition, we screened a consecutive series of 114 Warthin tumour cases by means of MAML2 break apart fluorescence in situ hybridization to assess its value in differential diagnosis. MAML2 rearrangement was detected in both mucoepidermoid carcinoma cases, while all Warthin tumours were negative. Taking into account the literature data, Warthin-like mucoepidermoid carcinomas are more frequently observed in women, while a slight male predominance and smoking history are typical for Warthin tumour. In addition, the patients with Warthin-like mucoepidermoid carcinoma were significantly younger than those with Warthin tumour. To conclude, Warthin-like mucoepidermoid carcinoma may usually be suspected based on histology, while the diagnosis can be confirmed by means of molecular assays such as FISH. The investigation of MAML2 status is particularly advised when Warthin tumour is considered in a young, non-smoking, female patient.

Expression of Female Sex Hormone Receptors, Connective Tissue Growth Factor and HER2 in Gallbladder Cancer.

Gallbladder cancer (GBC) is a highly malignant tumor with poorly understood etiology. An insight into phenotypic features of this malignancy may add to the knowledge of its carcinogenesis and pave the way to new therapeutic approaches. We assessed the expression of female sex hormone receptors (ERα, ERß, PR), connective tissue growth factor (CTGF) and HER2 in GBC, and adjacent normal tissue (NT), and determined their prognostic impact. Immunohistochemical (IHC) expression of all biomarkers was performed in formalin-fixed, paraffin-embedded specimens in 60 Caucasian GBC patients (51 women and 9 men). ERß, cytoPR and CTGF expression were found in 89%, 27%, 91% of GBC, and in 63%, 87%, 100% of NT, respectively. No ERα expression was found in GBC and NT. Strong (3+) HER2 expression by IHC or HER2 amplification was seen in five GBC (10.4%). A positive correlation was found between HER2 and CTGF and ERß expression in GBC and matched NT. In the multivariate analysis, patient age >70 years, tumor size and ERß expression in GBC was highly predictive for OS (p = 0.003). The correlation between HER2, CTGF and ERß expression in GBC and NT may indicate the interaction of these pathways in physiological processes and gallbladder pathology.

Clinical and Biological Significance of ESR1 Gene Alteration and Estrogen Receptors Isoforms Expression in Breast Cancer Patients.

The amplification of estrogen receptor alpha (ERα) encoded by the ESR1 gene has been described as having a prognostic role in breast cancer patients. However, increased dosage of the ESR1 gene (tested by real-time PCR) is also observed in ER-negative breast cancers, which might suggest the expression of alternative isoforms of ERα (other than classical ERα of 66 kDa). In the current work, we have investigated the ESR1 gene dosage in 402 primary breast cancer patients as well as the expression of ERα isoforms-ERα66 and ERα36-on mRNA and protein levels. The obtained results were correlated with clinicopathological data of the patients. Results showed that increased ESR1 gene dosage is not related to ESR1 gene amplification measured by fluorescent in situ hybridization (FISH), but it correlates with the decreased expression of ERα66 isoform (p = 0.01). Interestingly, the short ER isoform ERα36 was expressed in samples with increased ESR1 gene dosage, suggesting that genomic aberration might influence the expression of that particular isoform. Similarly to ESR1 increased gene dosage, high ERα36 expression was linked with the decreased disease-free survival of the patients (p = 0.05), which was independent of the status of the classical ERα66 level in breast tumors.

Application of high-resolution genomic profiling in the differential diagnosis of liposarcoma.

BACKGROUND: Rarity and heterogeneity of liposarcomas (LPS) make their diagnosis difficult even for sarcoma-experts pathologists. The molecular mechanism underlying the development and progression of liposarcomas (LPS) remains only partially known. In order to identify and compare the genomic profiles, we analyzed array-based comparative genomic hybridization (array-CGH) profiles of 66 liposarcomas, including well-differentiated (WDLPS), dedifferentiated (DDLPS) and myxoid (MLPS) subtypes. RESULTS: Copy number aberrations (CNAs) were identified in 98% of WDLPS and DDLPS and in 95% of MLPS cases. The minimal common region of amplification at 12q14.1q21.1 was observed in 96% of WDLPS and DDLPS cases. Four regions of CNAs, including losses of chromosome 6, 11 and 13 and gains of chromosome 14 were classified as recurrent in DDLPS; at least one was identified in 74% of DDLPS tumors. The DDLPS-associated losses were much more common in tumors with increased genomic complexity. In MLPS, the most frequent CNAs were losses of chromosome 6 (40%) and gains of chromosome 1 (30%), with the minimal overlapping regions 6q14.1q22.31 and 1q25.1q32.2, respectively. CONCLUSIONS: Our findings show that the application of array-CGH allows to delineate clearly the genomic profiles of WDLPS, DDLPS and MLPS that reflect biological differences between these tumors. Although CNAs varied widely, the subtypes of tumors have characteristic genomic profiles that could facilitate the differential diagnosis of LPS subtypes, especially between WDLPS and DDLPS.

Concomitance of monosomal karyotype with at least 5 chromosomal abnormalities is associated with dismal treatment outcome of AML patients with complex karyotype - retrospective analysis of Polish Adult Leukemia Group (PALG).

Monosomal karyotype (MK) and complex karyotype (CK) are poor prognostic factors in acute myeloid leukemia (AML). A comprehensive analysis of cytogenetic and clinical factors influencing an outcome of AML-CK+ was performed. The impact of cladribine containing induction on treatment results was also evaluated. We analyzed 125 patients with AML-CK+ treated within PALG protocols. MK was found in 75 (60%) individuals. The overall complete remission (CR) rate of 66 intensively treated patients was 62% vs. 28% in CK+ MK- and CK+ MK+ group (p = .01). No difference in CR rate was observed between DA and DAC arms. The overall survival (OS) in intensively treated patients was negatively influenced by MK, karyotype complexity (≥5 abnormalities), and WBC >20 G/L in multivariate analysis. The addition of cladribine to DA regimen improved OS only in MK- but not in MK+ group. In conclusion, concomitance of MK with ≥5 chromosomal abnormalities is associated with dismal treatment outcome in AMK-CK+.

Genomic findings in patients with clinical suspicion of 22q11.2 deletion syndrome.

Chromosome 22q11.2 deletion syndrome, one of the most common human genomic syndromes, has highly heterogeneous clinical presentation. Patients usually harbor a 1.5 to 3 Mb hemizygous deletion at chromosome 22q11.2, resulting in pathognomic TBX1, CRKL and/or MAPK1 haploinsufficiency. However, there are some individuals with clinical features resembling the syndrome who are eventually diagnosed with genomic disorders affecting other chromosomal regions. The objective of this study was to evaluate the additive value of high-resolution array-CGH testing in the cohort of 41 patients with clinical features of 22q11.2 deletion syndrome and negative results of standard cytogenetic diagnostic testing (karyotype and FISH for 22q11.2 locus). Array-CGH analysis revealed no aberrations at chromosomes 22 or 10 allegedly related to the syndrome. Five (12.2 %) patients were found to have other genomic imbalances, namely 17q21.31 microdeletion syndrome (MIM#610443), 1p36 deletion syndrome (MIM#607872), NF1 microduplication syndrome (MIM#613675), chromosome 6pter-p24 deletion syndrome (MIM#612582) and a novel interstitial deletion at 3q26.31 of 0.65 Mb encompassing a dosage-dependent gene NAALADL2. Our study demonstrates that the implementation of array-CGH into the panel of classic diagnostic procedures adds significantly to their efficacy. It allows for detection of constitutional genomic imbalances in 12 % of subjects with negative result of karyotype and FISH targeted for 22q11.2 region. Moreover, if used as first-tier genetic test, the method would provide immediate diagnosis in ∼40 % phenotypic 22q11.2 deletion subjects.

On the significance of germline cytogenetic rearrangements at MYCN locus in neuroblastoma.

BACKGROUND: MYCN oncogene amplification is the most important prognostic factor in neuroblastoma. 25% neuroblastoma tumors have somatic amplifications at this locus but little is known about its constitutional aberrations and their potential role in carcinogenesis. Here, we have performed an array-CGH and qPCR characterization of two patients with constitutional partial 2p trisomy including MYCN genomic region. RESULTS: One of the patients had congenital neuroblastoma and showed presence of minute areas of gains and losses within the common fragile site FRA2C at 2p24 encompassing MYCN. The link between 2p24 germline rearrangements and neuroblastoma development was reassessed by reviewing similar cases in the literature. CONCLUSIONS: It appears that constitutional rearrangements involving chromosome 2p24 may play role in NB development.

Different prognosis of acute myeloid leukemia harboring monosomal karyotype with total or partial monosomies determined by FISH: retrospective PALG study.

A monosomal karyotype (MK) was identified by banding techniques (BT) in acute myeloid leukemia (AML). However, BT may be insufficient to determine the actual loss of a complete chromosome, especially in complex karyotypes. We have investigated the effect of monosomy type, total (MK-t) and partial (MK-p), reevaluated by FISH, on prognosis. We have found that complete remission rate and probability of overall survival at 1 year was higher in MK-p (n=27) than MK-t (n=15) group (40% vs. 15.4%, P=0.19 and 30% vs. 9%, P=0.046, respectively). Our results indicate for the first time that monosomy type influences the prognosis of MK-AML.

[Probability rate of unbalanced offspring at birth and risk of unfavorable pregnancy outcomes in families of carriers of chromosomal reciprocal translocations involving chromosome 7]. / Ocena prawdopodobienstwa urodzenia dziecka z niezrównowafonym kariotypem oraz ryzyka wystqpienia róznych patologii ciazy w rodzinach nosicieli translokacji chromosomowych wzajemnych angazujacych chromosom 7.

INTRODUCTION: Carriership of reciprocal chromosomal translocation (RCT) may be the reason the occurrence of congenital malformations in the offspring, early neonatal death, stillbirth, and recurrent miscarriages due to unbalanced karyotype of gametes. The probability rate for individual categories of unfavorable outcomes depends on the kind of chromosome involved and is individually variable. OBJECTIVES: The aim of study was to estimate the probability rates for unbalanced offspring and to evaluate the risk for different categories of unfavorable pregnancy outcomes, depending on the size of chromosomal segment with differentiation between maternal/paternal origin of the reciprocal chromosomal translocations involving chromosome 7p (RCT-7p) and 7q (RCT-7q). In addition, the use of the obtained results has been illustrated by the example of a family with unique RCT t(7;9)(p21.3,p23). MATERIAL AND METHODS: Empirical and cytogenetic data on 341 pregnancies and offspring of 133 carriers were collected from 69 pedigrees of carriers of RCT-7p and RCT-7q at risk for a single 7 segment imbalance. The probability rates of particular form of pregnancy pathology have been calculated according to the method of Stengel-Rutkowski and Stene, including all forms of meiotic segregation and their survival rates after fertilization to term childbirth. RESULTS: The probability rates for unbalanced offspring for carriers of RCT-7p after 2:2 disjunction and adjacent-1 segregation were calculated as 5.5% +/- 2.2% (6/108); for maternal (MAT) and paternal (PAT) carriers were about < 1% (0/56) and 13.6 +/- 5.2% (6/44) (p = 0.04) respectively. Considering different segment lengths of 7p, the following values for shorter and longer segments were obtained: 23.0 +/- 11.7% (3/13) for 7p21-->pter; 3.3 +/- 3.3% (1/30) for 7p 14-->pter and 3.1 +/- 2.1% (2/65) for 7p 1-->pter The risk figures for stillbirth/earl neonatal death were estimated at 2.8 +/- 1.6% (3/108), but for miscarriage were calculated at 25.9 +/- 4.2% (28/108) for carriers RCT-7p. The probability rates for unbalanced offspring at birth for carriers of RCT-7q were calculated as 2.7 +/- 1.5% (3/111); for MAT and PAT carriers were 3.5 +/- 2.0% (3/86) and < 2.6% (0/19) respectively. Considering different segment lengths of 7q, the following values for shorter and longer segments were obtained: 6.2 +/- 6.1% (1/16) for 7q33-->qter; 5.3 +/- 3.6% (2/38) for 7q32-->qter and < 0.82% (0/57) for 7q11-->qter. The risk figures for stillbirth/early neonatal death were estimated at 9.9 +/- 2.8% (11/111), but for miscarriage were calculated at 34.2 +/-4.5% (38/111) for carriers RCT-7q. The probability estimated values for unbalanced fetuses, evaluated prenatally in the second trimester of pregnancy for carriers of RCT-7p and RCT-7q were similar i.e. 41.7 +/- 14.2% (5/12) and 46.7 +/-12.9% (7/15), respectively. CONCLUSIONS: 1. The probability rates for unbalanced offspring and the risk values for individual categories of unfavorable outcomes for carriers of RCT-7 are different and depend on the size of chromosome 7 segment involved in RCT 2. The probability rate for unbalanced offspring for paternal carriers of RCT-7p is higher than for maternal carriers (p = 0.04). 3. It is suggested that the probability rate for unbalanced offspring for maternal carriers of RCT-7q may be higher than for paternal carriers.

Bidirectionality and transcriptional activity of the EWSR1 promoter region.

EWSR1 is involved in chimeric proteins which play crucial roles in the development of a variety of bone and soft tissue tumors. Many of the chimeric genes involving EWSR1 have been extensively studied, whereas less is known about the wild-type (wt) gene and its regulation. As the expression of the chimeric gene is driven by the EWSR1 promoter, it is of importance to study the mechanisms regulating wt EWSR1 expression. We estimated the transcriptional activity of the EWSR1 promoter through deletion fragments driving reporter gene expression. This assay identified the 100-bp region immediately downstream of the EWSR1 transcriptional start site (+1) and the downstream region from +100 to +300 as important regions for transcriptional regulation. We also found that EWSR1 and RHBDD3, a gene located directly upstream of EWSR1 that is likely to share regulatory elements with EWSR1, were co-expressed in the tissue panels, Ewing tumor biopsies and cell lines. Thus, our results show that the EWSR1 promoter functions in a bidirectional manner, thereby regulating also RHBDD3, and identifies specific regions that strongly influence promoter activity.

Prenatal diagnosis of an atrioventricular canal in a foetus with deletion of chromosome 8 (pter-->p21).

Congenital heart malformations, detected during a pregnancy, are associated in 20-48% of cases with a chromosomal aberration. In the following study we have reported the deletion of chromosome 8 (pter-->p21), diagnosed prenatally at 22 weeks of gestation, because of a visible defect in the upper part of the interventricular septum and a partial defect of the atrial septum. The atria and the ventricles were joined with a common central valve. The cordocentesis was performed and karyotype: 46, XX ish del(8)(wcp8x2) was detected. Because of the persistent bradycardia of the foetus, indicating a danger of intrauterine asphyxia of the foetus, as well as features of premature placental detachment, the caesarean section was performed at 27 weeks of gestation. The patient gave birth to a daughter weighing 960 g. The child died in the 4th hour of her life. On the basis of the present observation it is safe to say that when an AV-canal defect is diagnosed prenatally, special attention must be paid to the detection of chromosomal abnormalities and amniocentesis or cordocentesis should be performed to assess the state of affairs.

Periventricular heterotopia in a boy with interstitial deletion of chromosome 4p.

We report on a 4-year-old boy with a proximal interstitial deletion in the short arm of chromosome 4p with the karyotype 46,XY,del(4)(p14p15.32),inv(9)(p13q13). For a precise delineation of the deleted region, an array-based comparative genomic hybridization (a-CGH) analysis was performed. The proband's phenotype and cytogenetic findings are compared with previously reported cases with proximal 4p deletion syndrome. The syndrome is associated with normal growth, varying degrees of mental retardation, characteristic facial appearance and minor dysmorphic features. Additionally, our patient developed a seizure disorder due to abnormal neuronal migration, i.e., periventricular heterotopia.

A girl with duplication 9q34 syndrome.

We report on a 17-year-old female with numerous developmental abnormalities associated with 46,XX,dup(9)(q33.3q34.1), where the duplication occurred de novo. The patient presented with dysmorphic features and notable psychomotor delays, manifestations similar to those described in other reported cases of duplication of 9q34-qter. Due to late presentation and diagnosis, our patient was not evaluated and characterized until adolescence, when particular attention was paid to the development of secondary sexual characteristics, secondary amenorrhea and obesity.

Karyotypic characterization of 64 nonmalignant thyroid goiters.

Cytogenetic analyses were performed on 64 nonmalignant thyroid goiters (11 common and 53 multinodular goiters) after short-term culture. The majority of goiters (67%) were characterized by a normal karyotype, but in 5 common (45%) and 16 nodular (30%) goiters, small clones with various numerical and/or structural aberrations were found, in addition to many normal cells. Trisomy or tetrasomy 7 was the most frequent numerical aberration, seen in five cases. Deletion of 18p11 was found in four cases, and in three of them as the sole change. Selection and clonal evolution of aneuploid cells present in nonmalignant goiters could underlie progression into adenoma formation.