<p>Detection and precise genomic mapping of balanced chromosomal abnormalities in patients with impaired fertility or a clinical phenotype represent a challenge for current cytogenomics owing to difficulties with precise breakpoint localization in the regions enriched for DNA repeats and high genomic variation in such regions. Here, we present a comprehensive cytogenomic approach to breakpoint mapping in a rare paracentric inversion on 10q (in a patient with oligoasthenoteratozoospermia and necrozoospermia) that does not affect other phenotype traits. Multicolor banding, chromosomal microarray analysis, chromosome microdissection with reverse painting, and single-copy sequencing of the rearranged chromosome were performed to determine the length and position of the inverted region as well as to rule out a genetic imbalance at the breakpoints. As a result, a paracentric 19.251 Mbp inversion at 10q22.2q23.3 was described. The most probable location of the breakpoints was predicted using the hg38 assembly. The problems of genetic counseling associated with enrichment for repeats and high DNA variability of usual breakpoint regions were discussed. Possible approaches for cytogenomic assessment of couples with balanced chromosome rearrangements and problems like reproductive failures were considered and suggested as useful part of effective genetic counseling.</p>

Digital object identifier (DOI): 10.3390/biomedicines10123255

<p>Protontherapy is a rapidly expanding radiotherapy modality where accelerated proton beams are used to precisely deliver the dose to the tumor target but is generally considered ineffective against radioresistant tumors. Proton-Boron Capture Therapy (PBCT) is a novel approach aimed at enhancing proton biological effectiveness. PBCT exploits a nuclear fusion reaction between low-energy protons and B atoms, i.e. p+ B→ 3α (p-B), which is supposed to produce highly-DNA damaging α-particles exclusively across the tumor-conformed Spread-Out Bragg Peak (SOBP), without harming healthy tissues in the beam entrance channel. To confirm previous work on PBCT, here we report new in-vitro data obtained at the 62-MeV ocular melanoma-dedicated proton beamline of the INFN-Laboratori Nazionali del Sud (LNS), Catania, Italy. For the first time, we also tested PBCT at the 250-MeV proton beamline used for deep-seated cancers at the Centro Nazionale di Adroterapia Oncologica (CNAO), Pavia, Italy. We used Sodium Mercaptododecaborate (BSH) as B carrier, DU145 prostate cancer cells to assess cell killing and non-cancer epithelial breast MCF-10A cells for quantifying chromosome aberrations (CAs) by FISH painting and DNA repair pathway protein expression by western blotting. Cells were exposed at various depths along the two clinical SOBPs. Compared to exposure in the absence of boron, proton irradiation in the presence of BSH significantly reduced DU145 clonogenic survival and increased both frequency and complexity of CAs in MCF-10A cells at the mid- and distal SOBP positions, but not at the beam entrance. BSH-mediated enhancement of DNA damage response was also found at mid-SOBP. These results corroborate PBCT as a strategy to render protontherapy amenable towards radiotherapy-resilient tumor. If coupled with emerging proton FLASH radiotherapy modalities, PBCT could thus widen the protontherapy therapeutic index.</p>

Digital object identifier (DOI): 10.3389/fonc.2021.682647

Chromosome analysis of prenatal samples and products of conception (POC) has conventionally been done by karyotyping (KT). Shortcomings of KT like high turnaround time and culture failure led to technology innovations, such as the bacterial artificial chromosomes (BAC)s-on-Beads (BoBs)-based tests, Prenatal BoBs (prenatal samples) and KaryoLite BoBs (POC samples). In the present study, we validated and evaluated the utility of each test on prenatal, POC and blood samples. Study A (n = 305; 259 prenatal + 46 blood/POC) and Study B (n = 176; 146 POC/chorionic vill + 30 blood/amniotic fluid) samples were analyzed using Prenatal and KaryoLite BoBs kits, respectively. KT, array-based Comparative Genomic Hybridization (arrayCGH) and fluorescence in situ hybridization (FISH) were used for comparison of results. Ability of KaryoLite BoBs to identify ring chromosomes was tested. Prenatal BoBs had zero test failure rate and results of all samples were concordant with KT results. Totally four microdeletions were identified by Prenatal BoBs but not by KT. In Study B, all but two POC samples (one triploid and one tetraploid) were concordant with KT and arrayCGH. Partial chromosomal imbalance detection rate was ~64% and KaryoLite BoBs indicated the presence of a ring chromosome in all four cases. The failure rate of KaryoLite BoBs was 3%. We conclude that Prenatal BoBs (common aneuploidies and nine microdeletions) together with KT constitutes more comprehensive prenatal testing compared to FISH and KT. KaryoLite BoBs for aneuploidies of all chromosomes is highly successful in POC analysis and the ability to indicate presence of ring chromosomes improves its clinical sensitivity. Both tests are robust and could also be used for different specimens.

Digital object identifier (DOI): 10.1111/jog.13920

In the WHO classification, double or triple-hit lymphoma depicts rare and aggressive lymphomas displaying BCL2 and/or MYC and/or BCL6 gene rearrangements that are categorized as B-cell lymphomas unclassified, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma. Bacher et al.2 described an interesting series of 10 cases of such neoplasms. In addition, they reported the two first cases displaying four different lymphoma-specific events (quadruple hit) involving the genes MYC, BCL2, BCL6 and CCND1. We describe here a third case occurring in a 79-year-old male patient suffering from paraesthesias for 4 months who was referred for polyneuritis in a context of poor general condition. Clinical examination showed the presence of numerous axillary, supraclavicular, mediastinal and inguinal lymphadenopathies, neuro-meningeal invasion and skin infiltration. The biopsy of a left arm skin nodule revealed large proliferating cells (Ki-67 80%) stained by anti-CD20, BCL2 and BCL6 antibodies, CD10 and CD23 remaining negative, consistent with the diagnosis of diffuse large B-cell lymphoma (DLBCL), not otherwise specified. Blood cell counts showed 8.1 × 109/l leukocytes, 13.2 g/dl hemoglobin, 166 × 109/l platelets. LDH and β-2 microglobulin were elevated (989 U/I and 9.14 mg/l, respectively). Blood cell film examination showed the presence of 28% abnormal lymphocytes (medium sized, with intense basophilia, irregular nuclear contours, slightly clumped chromatin and frequent prominent nucleoli) suggestive of a high grade lymphoma. Flow cytometry revealed a lambda immunoglobulin light chain restriction. These cells expressed pan-B markers such as CD19, CD20, FMC7, CD22, with weak CD5 and CD43 positivity. CD10 and 23 were negative. Both the morphology and immunophenotype of the blood cells favored a pleomorphic mantle cell lymphoma (MCL) aggressive variant diagnosis. Cytogenetic study performed in the WBCs found a complex hyperdiploid karyotype (47 chromosomes, Figure 1) with a t(3;22) translocation involving the BCL6 and IGL genes, a structural abnormality of chromosome 8 resulting in juxtaposition of 5′ MYC and BCL2 in fluorescence in situ hybridization (with break of the MYC probe), a derivative chromosome 18 from a t(14;18) translocation with fusion of 5′IGH and BCL2, and a t(11;14) complex translocation involving IGH and CCND1 (Figure 2). Other numeral (trisomy 12) and structural abnormalities (involving the 1, 7, 14 and 21 chromosomes) were also detected (Figure 1). Overexpression of cyclin D1 was detected in the WBCs by real-time quantitative PCR, as well as in the skin lesion using immunochemistry. Anti-SOX11 antibody staining was found to be negative. Chemotherapy combining rituximab, ifosfamide, cytosine arabinoside and intrathecal methotrexate was initiated, but the patient died 4 months after the diagnosis. This third case of quadruple-hit lymphoma underlines the complexity of the classification of such aggressive malignancies. Initial rearrangement of the CCND1 gene characterizes MCL that may harbor in very rare cases additional rearrangements of MYC or BCL6, but histological transformation to typical large cell lymphoma is not retained in the WHO classification. In addition, cyclin D1 overexpression is considered to be a rare feature in DLBCL. Recently, Ok et al.3 proposed to reclassify DLBCL with expression of cyclin D1, CCND1 chromosomal rearrangement and CD5 positivity as an aggressive pleomorphic MCL variant. However, no observation of multiple lymphoma-specific gene rearrangements was described in that study. Juskevicius et al.4 suggest the existence of a ‘gray zone’ in which morphologic, clinical and genetic features are insufficient to segregate lymphomas with overexpression of cyclin D1/translocations involving CCND1 between blastoid MCL and cyclin D1-positive DLBCL. Regarding the immunophenotyping and molecular data, our case is possibly a genetically unstable aggressive pleomorphic MCL variant, which acquired three additional genetic hits.

Digital object identifier (DOI): 10.1038/bcj.2015.99

Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.

<p>Myelodysplastic syndrome (MDS) represents a group of clonal hematological disorders characterized by progressive cytopenia, and reflects to defects in erythroid, myeloid and megakaryocytic maturation. MDS is more frequently observed in older aged patients with cytogenetic abnormalities like monosomy of chromosome(s) 5 and/or 7. In 50% of de novo MDS cases, chromosomal aberrations are found and rearrangements involving the retinoblastoma (RB1) gene in 13q14 are found. Here, we are presenting a case report of a rare biclonal MDS with a karyotype of 45, XY,-4, der(6)t(4;6)(p15.1;p21.3), der(8)t(4;8)(q31.2;q22), t(13;16)(q21.3;p11.2)11/45, XY, der(7)t(7;13)(p22.2~22.3;q21.3),-13 9. The patient was diagnosed according to WHO classification as refractory anemia with excess of blasts (RAEB-II).Immunophenotyping was positive for CD11b, CD11c, CD10, CD13, CD15, CD16 and CD33. We report, a novel and cytogenetically rare case of a biclonal MDS with complex chromosomal aberrations and deletion of RB1-gene in both clones. These findings are associated with a poor prognosis as the patient died 3 months after diagnosis.</p>

Chromosome aberrations are important prognostic markers in multiple myeloma (MM), but their identification may be hampered by complexity of the karyotypes. Using multicolor fluorescence in situ hybridization (mFISH), we found cryptic aberrations in 7 of 10 patients with a complex karyotype. Moreover, in addition to typical aberrations involving 1q, 13q, 14q and 17p and structural aberrations in chromosomes 1, 6, 9 and 19, (iso)dicentric chromosomes and whole-arm translocations were detected. These chromosome aberrations were generated by breaks in heterochromatic regions indicating an increased breakage of these regions, which may predispose to the generation of chromosome aberrations in multiple myeloma.

<p>BACKGROUND: The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. RESULTS: Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. CONCLUSION: These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.</p>

A complex low-repetitive human DNA probe (BAC RP11-35B4) together with two microdissection-derived region-specific probes of the multicolor banding (MCB) probe-set for chromosome 1 were used to re-analyze the evolution of human chromosome 1 in comparison to four ape species. BAC RP11-35B4 derives from 1q21 and contains 143 kb of non-repetitive DNA; however, it produces three specific FISH signals in 1q21, 1p12 and 1p36.1 of Homo sapiens (HSA). Human chromosome 1 was studied in comparison to its homologues in Hylobates lar (HLA), Pongo pygmaeus (PPY), Gorilla gorilla (GGO) and Pan troglodytes (PTR). A duplication of sequences homologous to human 1p36.1 could be detected in PPY plus an additional signal on PPY 16q. The region homologous to HSA 1p36.1 is also duplicated in HLA, and split onto chromosomes 7q and 9p; the region homologous to HSA 1q21/1p12 is present as one region on 5q. Additionally, the breakpoint of a small pericentric inversion in the evolution of human chromosome 1 compared to other great ape species could be refined. In summary, the results obtained here are in concordance with previous reports; however, there is evidence for a deletion of regions homologous to human 1p34.2-->p34.1 during evolution in the Pongidae branch after separation of PPY.

Small supernumerary marker chromosomes (SMCs) in human can be defined as additional centric chromosome fragments smaller than chromosome 20. For most small or minute SMCs a correlation with clinical symptoms is lacking, mostly due to problems in visualizing their euchromatic content. Recently we described two new molecular cytogenetic approaches for the comprehensive characterization of small SMCs, excluding those few cases with neo-centromeres. Minute SMCs, consisting preferentially of alpha-satellite DNA, are characterizable in one step by the centromere-specific multicolor FISH (cenM-FISH) approach. For further characterization of minute SMCs and eventually present euchromatic content, the recently developed centromere-near-specific multicolor FISH (subcenM-FISH) technique can be applied. These two approaches are highly informative and easy to perform, as demonstrated in the present report on the example of a prenatal case with a minute SMC derived from chromosome 3 cytogenetically described as min(3)(:p12.1→q11.2:).

Multicolor fluorescence in situ hybridization (FISH) assays are nowadays indispensable for a precise description of complex chromosomal rearrangements. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole chromosome painting probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Since then different approaches for chromosomal differentiation based on multicolor-FISH (mFISH) assays have been described. Predominantly, they have been established to characterize marker chromosomes identified in conventional banding analysis. Their characterization is of high clinical impact and is the requisite condition for further molecular investigations aimed at the identification of disease-related genes. Here we present a review on the available mFISH methods including their advantages, limitations and possible applications.

Using comparative genomic hybridization (CGH), we present a genome-wide screening of a mixed mesenchymal-epithelial hepatoblastoma, its recurrence and 2 novel hepatoblastoma cell lines raised from the ascites, 18 (HepU1) and 23 (HepU2) months after diagnosis of a hepatoblastoma in a 35-month-old boy. Both cell lines were also characterized by GTG-banding, multicolor-fluorescence in situ hybridization (M-FISH) and multicolor banding (M-Band). On the basis of CGH, we compared the cytogenetics of histologically different tumor areas of the parental tumor and its recurrence with the hepatoblastoma cell lines. We found different CGH profiles in the parental tumor rev ish enh(1q31-q32,8p,12,17,20,X), dim(4q34-q35,18q23)[cp] and its recurrence rev ish enh(8q24,17,Xq26-q28), dim(7q11.2-q21,13q34)[cp]. Although both epithelial cell lines were obtained at different times and the clonal ancestor of HepU2 had been exposed to a higher cumulative dose of chemotherapy, HepU1 and HepU2 have an identical karyotype: 48-56,XY,+Y,dup(2)(q32-q34),t(3;4)(q21;q34),+8,+12,+13, +17,+t(18;19)(q21;q?),+20[cp] and identical CGH profiles: rev ish enh(2q24-q33,8,12,13q,17,20), dim(4q34-q35,18q22-q23). In common with previously described hepatoblastoma cell lines, HepU1 and HepU2 demonstrate a gain of chromosome 20. The in situ aberrations most closely resembling that of HepU1 and HepU2 were found in areas of fetal-embryonal differentiation of the primary tumor. Interestingly, both cell lines mimic this histology in their three-dimensional growth pattern in vitro. HepU1 and HepU2 are thus cytogenetically and phenotypically highly characteristic of fetal-embryonal hepatoblastoma.