Specific Chromosomal Aberrations Research Articles

EPEN-28. NOVEL TARGET IDENTIFICATION IN RECURRENT POSTERIOR FOSSA EPENDYMOMA GROUP A

Abstract BACKGROUND Posterior fossa ependymoma group A (PF-EPN-A) are an aggressive subgroup of ependymal tumors which occur predominantly in infants and possess a poor prognosis. Treatment is limited to surgical resection and adjuvant radiation with no approved chemotherapies or targeted therapies available. Despite treatment efforts, nearly half of all PF-EPN-A cases will experience tumor recurrence within a decade of initial diagnosis. Our current understanding of relapse is limited to clinical risk stratification based on subtotal resection of the primary tumor, male sex and specific chromosomal copy number aberrations. In an effort to overcome the shortcomings of current literature on PF-EPN-A recurrence, we present a concerted effort to investigate the biology of both recurrent human tissue specimens and patient-derived cell models to identify novel drivers of recurrence. METHODS Utilizing a multi-omics approach, we have conducted transcriptomic (bulk RNA-Seq), proteomic and phosphoproteomic (LC-MS/MS) analyses in order to identify molecular pathways that are enriched in recurrent disease as compared to primaries. We have also performed genome-wide CRISPR essentiality screens to further pinpoint essential genes and pathways with therapeutic potential. RESULTS Through the comparison of primary and recurrent PF-EPN-A samples, we have identified both previously implicated and novel genes and pathways in recurrent disease. Our multi-omic analyses identified the upregulation of genes involved in DNA repair, DNA damage response and cell cycle pathways in recurrent PF-EPN-A. CONCLUSIONS Our combined approach has enabled the identification of novel targets with therapeutic potential in the treatment of recurrent PF-EPN-A. The identified targets will be validated using a combination of drug screening and knockdown approaches in vitro. Top candidates will be further validated in vivo with established recurrent xenograft mouse models. PF-EPN-A is an aggressive disease with limited treatment options and the identification of novel targets with potential therapeutic utility will allow us to better treat this patient population.

Gain-Type Aneuploidies Influence the Burden of Selective Long Non-Coding Transcripts in Colorectal Cancer.

Chromosomal instability is a hallmark of colorectal carcinogenesis and produces an accumulation of different forms of aneuploidies or broad copy number aberrations. Colorectal cancer is characterized by gain-type broad copy number aberrations, specifically in Chr20, Chr8q, Chr13 and Chr7, but their roles and mechanisms in cancer progression are not fully understood. It has been suggested that broad copy number gains might contribute to tumor development through the so-called caricature transcriptomic effect. We intend to investigate the impact of broad copy number gains on long non-coding RNAs' expression in colorectal cancer, given their well-known role in oncogenesis. The influence of such chromosomal aberrations on lncRNAs' transcriptome profile was investigated by SNP and transcriptome arrays in our series of colorectal cancer samples and cell lines. The correlation between aneuploidies and transcriptomic profiles led us to obtain a class of Over-UpT lncRNAs, which are transcripts upregulated in CRC and further overexpressed in colon tumors bearing specific chromosomal aberrations. The identified lncRNAs can contribute to a wide interaction network to establish the cancer driving effect of gain-type aneuploidies.

Measuring cancer driving force of chromosomal aberrations through multi-layer Boolean implication networks.

Multi-layer Complex networks are commonly used for modeling and analysing biological entities. This paper presents the advantage of using COMBO (Combining Multi Bio Omics) to suggest a new role of the chromosomal aberration as a cancer driver factor. Exploiting the heterogeneous multi-layer networks, COMBO integrates gene expression and DNA-methylation data in order to identify complex bilateral relationships between transcriptome and epigenome. We evaluated the multi-layer networks generated by COMBO on different TCGA cancer datasets (COAD, BLCA, BRCA, CESC, STAD) focusing on the effect of a specific chromosomal numerical aberration, broad gain in chromosome 20, on different cancer histotypes. In addition, the effect of chromosome 8q amplification was tested in the same TCGA cancer dataset. The results demonstrate the ability of COMBO to identify the chromosome 20 amplification cancer driver force in the different TCGA Pan Cancer project datasets.

O-196 Evidence-based management of mosaic embryos - a single Centre experience from prospective transfer and birth outcomes of 565 mosaic embryos

Abstract Study question What are the pregnancy and neonatal outcomes of prospective transfer of 565 mosaic embryos and what are the key determinants influencing the outcome? Summary answer Mosaic (diploid/aneuploid) embryos have a significant developmental potential with good neonatal outcomes allowing personalized management and evidence based prioritization of embryos for transfer. What is known already Since the first report of healthy babies born after mosaic transfer, the gradual accumulation of knowledge on mosaic embryo transfers in the International Registry of Mosaic Embryo Transfers provided the necessary reassurance for clinical management of mosaic embryos. Large and comprehensive data from single centres with consistent clinical genetic counselling and laboratory practices for preimplantation and prenatal testing offer valuable contribution to the current practices. The aim of this study was to evaluate the pregnancy and neonatal outcomes after mosaic embryo transfers and to evaluate for associations between embryo genetic aberrations, static morphology and specific outcomes. Study design, size, duration This is a retrospective cohort study that took place in a single academic IVF centre (The CReATe Fertility Centre, Toronto, Canada), where we analysed the results from high resolution PGT-A, embryo morphologic features and pregnancy outcomes of 3529 frozen embryo transfers from 2016-2022. Associated patient demographic data, transfer outcome and neonatal outcome of 2964 euploid and 565 mosaic embryo transfers were analyzed. Participants/materials, setting, methods All patients received genetic counselling prior to frozen embryo transfer. High resolution NGS PGT-A was performed using the Illumina platform and BluGnome and NxClinical for data analysis. Clinically relevant findings were aberrations >10Mb and mosaicism from 25%-75%. Comparison of clinical and morphological characteristics of euploid, low-level mosaic (25%-<50% aneuploid cells in trophectoderm biopsy), and high-level mosaics (50% -75%) was performed with R-statistical package, Mathlab and SSPS software. p < 0.05 with CI 95% was considered significant. Main results and the role of chance Of the total 565 mosaic embryos transferred 77.1% had mosaicism levels ≥25%-<50% (Group1-n=436): 54.8% had segmental chromosome mosaic (SCM)gain/loss, 45.2% had whole chromosome mosaicism(WCM)(trisomy/monosomy); and 22.9% had mosaic levels of ≥ 50-<70% (Group2-n=129)(50.4% with SCM and 49.6% with WCM). The overall ongoing pregnancy rate(OPR) for mosaic embryos was 33% and the miscarriage rate was 6%. Compared with euploid embryos, mosaic embryos have significantly lower implantation (p = 0,OD0.62-CI[0.5-0.7]) and OPR(p = 0,OD0.5-CI[0.4-0.6]). Pregnancy outcomes are determined by level of mosaicism: Group1 have better implantation(p = 0.005,OD1.8-CI[1.18-2.78]) and OPR compared to Group2. High level of mosaicism independently affected the OPR of SCM(p = 0.003) and WCM(p = 0.02) embryos. WCM had lower implantation rates compared to SCM (p = 0.005,OD1.6-CI[1.15-2.27]). Mosaic monosomies had the lowest impanation and OPR of all mosaic embryos (14% and 8%,respectively). Within Group1 and Group2 good static morphology was associated with higher OPR for SCM and WCM embryos. Birth outcomes were available for 118 babies from mosaic-FET and from 802 euploid-FET. There is no difference in duration of pregnancy and birth weight between mosaic and euploid-FET. No gross-fetal anomalies were reported in the mosaic group at birth. There was one termination<20GW due to fetal anomalies and one stillbirth. For euploid transfers, 11 had fetal anomalies, there were 7-terminations and 2-stillbirths. Limitations, reasons for caution Although this is the largest single centre study to date that evaluated genomic, morphology and outcome data of mosaic transfers, the numbers are still low to analyze the impact of specific chromosomal aberrations on pregnancy outcome. Wider implications of the findings Mosaic embryos should be considered for transfer and prioritized based on the level of mosaicism, type of chromosomal aberration and static morphology. The implantation potential is lower than euploid embryos however established pregnancies result in healthy babies born at term and normal birth weight. Trial registration number CReATe Fertility Centre, Toronto, Canada

Genetic alterations of TP53 and OTX2 indicate increased risk of relapse in WNT medulloblastomas

This study aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in WNT-activated medulloblastoma (WNT-MB). In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by DNA sequencing. Chromosomal copy-number aberrations were assessed by molecular inversion probe technology (MIP), SNP6, or 850k methylation array hybridization. Prognostic impact was evaluated in 120 patients with follow-up data from the HIT2000 medulloblastoma trial or HIT registries. CTNNB1 mutations were present in 92.2%, and APC mutations in 6.8% of samples. One CTNNB1 wild-type tumor gained WNT activation due to homozygous FBXW7 deletion. Monosomy 6 was present in 78.6%, and more frequent in children than adults. 16.1% of tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. Loss of heterozygosity at the TP53 locus (chromosome 17p13.1) was found in 40.7% (11/27) of TP53 mutant tumor samples and in 12.6% of TP53 wild-type cases (13/103). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; 5-year-PFS 68% versus 93%, p = 0.001), and were enriched for chromosomes 17p (p = 0.001), 10, and 13 losses. Gains of OTX2 (14q22.3) occurred in 38.9% of samples and were associated with poor PFS and OS (5-year-PFS 72% versus 93%, p = 0.017 resp. 5-year-OS 83% versus 97%, p = 0.006). Multivariable Cox regression analysis for PFS/OS identified both genetic alterations as independent prognostic markers. Our data suggest that patients with WNT-MB carrying TP53 mutations or OTX2 gains (58.1%) are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

JS04.4.A Beyond β-catenin: Genetic alterations ofTP53 andOTX2 and older age indicate increased risk of relapse in WNT medulloblastomas

Abstract Background This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of age, TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers. Material and Methods In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Association with prognosis was evaluated in 133 patients with follow-up data from the HIT2000 medulloblastoma trial, HIT registries, and the NOA-07 trial. Results CTNNB1 mutations were present in 92.2% of the samples. APC mutations were found in 6.8% (13 samples). One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, and more frequent in children compared to adolescents/adults (≥16 years). Adolescents/adults showed worse overall survival (OS; p=0.009) compared to children, but not worse progression-free survival (PFS; p=0.106). With an age cut-off at 18 years, no survival difference was found. Also adolescents alone (16-20 years) had worse OS (p=0.003) compared to children, whereas in patients ≥21 (n=12 adults with PFS/OS data) no tumor progression/relapse occurred. Only one adult died due to therapy-related complications. WNT-MB patients with tumors harboring TP53 mutations (24/133, 18.1%) showed significant worse PFS (p=0.001), which was also found in children and adolescents individually (p=0.004, resp. p=0.017). Gains of the OTX2 locus on chromosome 14q found in 40.2% (35/87) of samples were independent of TP53 mutations and also associated with poor PFS and OS (p=0.034, resp. p=0.016). Individual analyses of OTX2 gains within age groups showed only worse OS in children (p=0.012). Multivariate Cox regression analysis for PFS identified both genetic alterations, but not age, as independent prognostic markers. For OS, multivariate analysis found OTX2 gains and older age as independent prognostic markers. Conclusion Our data suggest that adolescent patients with WNT-MB and those patients carrying TP53 mutations or OTX2 gains - independent of age - are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

Systemic ALK-negative anaplastic large cell lymphoma with distinctive myxoid change and DUSP22 rearrangement.

Systemic anaplastic lymphoma kinase-negative (ALK-) anaplastic large cell lymphoma (ALCL) comprises a genomically heterogeneous disease that is considered a distinct entity by the 2016 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. Other than lymph nodes, systemic ALK- ALCL may affect extranodal tissues, sites where the inflammatory background may be especially prominent. In this scenario, myxoid change is exceptional in systemic ALK- ALCL. We describe a rare case of systemic ALK- ALCL with distinctive myxoid changes, carrying specific chromosomal aberrations that affect the clinical outcome. Careful morphological, immunohistochemical, and molecular workup is mandatory because a myxoid background should not be a reason to ignore the possibility of a lymphoma. Finally, extensive correlation with staging and the detection of prognostic biomarkers such as DUSP22 and TP63 rearrangements are essential for the diagnosis and prediction of clinical outcome in ALK- ALCL.

O-076 Genomic and morphokinetic blueprint of mosaic embryos is evidence supporting their distinct clinical category

Abstract Study question Is there a correlation between blastocyst mosaicism, morphokinetic and morphologic characteristics and can it inform the clinical outcome of mosaic embryo transfers? Summary answer Mosaic (diploid/aneuploid) embryos have a distinct genetic, morphokinetic and morphologic signature that distinguishes them from euploid and abnormal embryos and supports their unique clinical identity. What is known already Advances in technologies for preimplantation aneuploidy testing (PGT-A) as the ultimate test for embryo selection has increased the resolution and sensitivity to detect intermediate chromosomal copy number as low as 20%. This has brought to light a biological phenomenon that has created clinical dilemma. Current recommendations support mosaic embryo transfer but with conflicting standpoints ranging from complete dismissal of mosaic embryos to arguing their existence. Time-lapse imaging technologies allow collection of morphokinetic data as a non-invasive assessment tool for embryo development. The aim of this study was to correlate genomic and morphokinetic data with clinical outcomes of mosaic embryos. Study design, size, duration This is a retrospective cohort study that took place in a single academic IVF centre, where we analysed the results from high resolution PGT-A, morphokinetic, morphologic and cell division features of 4113 embryos cultured in the time lapse imaging incubator (Embryoscope™) from 2016-2021. Associated patient data and transfer outcome of 770 euploid and 147 mosaic embryo transfers were analyzed. Participants/materials, setting, methods High resolution NGS PGT-A was performed using the Illumina platform and BluGnome and NxClinical for data analysis. Clinically relevant findings were aberrations >10Mb and mosaicism from 25%-75%. Time-lapse imaging was performed with an Embryoscope™. Comparison of clinical, morphological, morphokinetic characteristics of euploid, aneuploid, low-level mosaic (25%-<50% aneuploid cells in trophectoderm biopsy), and high-level mosaics (50% -75%) was done with R-statistical package, Mathlab and SSPS software were used, and p < 0.05 with CI 95% was considered significant. Main results and the role of chance Aneuploidy was detected in 26.2%, euploidy in 56.7% and diploid-aneuploid mosaicism in 17.1% of embryos included in this study. Compared with euploid embryos, mosaic embryos had significant delays in t2 (27.04±0.35vs.26.4±0.17 h-post-ICSI(hpi), p = 0.0029), which was not correlated with transfer outcome. Embryos with whole chromosome mosaicism (WCM) had longer t2 compared to those with segmental chromosome mosaicism (SCM) (27.29±0.59vs.26.4±0.3hpi,p=0.013). The time to first mitosis was also longer for mosaic vs. euploid embryos (4.57± 0.59vs.3.4±0.25hpi,p=0.000056) and for mosaic embryos with WCM vs SCM embryos (4.64± 0.9 vs 3.5 ±0.5hpi,p=0.04). Mosaic embryos had longer tB &tSB than euploid embryos (110.12±0.69vs.109.13hpi±0.4,p=0.01). Embryos with WCM had longer tB &tSB than those with SCM (111.07±1.09vs.108.7±0.8hpi,p=0.0006). Time to blast was longer for mosaic vs euploid embryos (13.69±0.52vs.12.7±0.4hpi,p=0.0035) and for mosaic WCM vs. SCM embryos (14.3±0.7vs.12.8±0.5hpi,p=0.00084). Both euploid and mosaic embryos that implanted had shorter time to tB&tSB than mosaic embryos that didn’t (p = 0.029; p = 0.007 and p = 0.002; p = 0.006 and p = 0.029 respectively); and mosaic embryos that implanted had shorter time to first mitosis than euploid embryos that didn’t (p = 0.00228). High level mosaic embryos had significantly more multinucleation at 2-cell stage (MN2) vs. euploid (p = 0.04,OD1.58-95%CI[1.01-2.45]),aneuploid (p = 0.02,OD1.7-95%CI[1.08-2.8]), as well as MN at 4-cell stage (euploid-p=0.0007,OD2.6-95CI1.4-4.7] and aneuploid-p=0.039,OD1.8-95CI1.02-3.4]). Limitations, reasons for caution Although this is the largest study to date that evaluated genomic, morphokinetic and outcome data of mosaic transfers, the numbers are still low to analyze the impact of specific chromosomal aberrations on morphokinetics and pregnancy outcome. The retrospective nature and cohort design limited the generalizability of our results. Wider implications of the findings Mosaic embryos have significant developmental potential and result in birth of healthy babies. Our study reveals the genomic interconnection of genomic and morphokinetic features of mosaic embryos and provides further evidence that mosaic embryos are a distinct category that requires specific clinical attention. Trial registration number Not applicable

MEDB-61. Genetic alterations ofTP53 andOTX2 indicate increased risk of relapse in WNT medulloblastomas

PURPOSE: This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in a retrospective cohort of patients treated according to the protocols of the HIT medulloblastoma studies. PATIENTS AND METHODS: In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations (CNAs) were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Complete clinical data were available from 120 patients. RESULTS: Patients with WNT-MBs had a female predominance (1.4:1) and a median age of 13 years (range 3-69 years). CTNNB1 mutations were present in 92.2% of the samples, APC mutations in 6.8%. One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, but more frequent in children compared to adults. 16.1% of the tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. A loss of heterozygosity at the TP53 locus on chromosome 17p13.1 was found in 40.7% (11/27) of TP53 mutant tumor samples and in 18.5% of the whole cohort (24/130 cases). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; p=0.001), but not overall survival (OS) and were enriched for chromosomes 17p (p=0.001), 10, and 13 losses. Gains of the OTX2 locus on chromosome 14q were also associated with poor PFS and OS (p=0.017 resp. p=0.006). Multivariate Cox regression analysis identified both genetic alterations as independent prognostic markers for PFS and OS. CONCLUSION: For ongoing and future de-escalation trials for patients with WNT medulloblastomas, we recommend to exclude patients with tumors carrying TP53 mutations or OTX2 gains.

Chromosomal Analysis of Myelodysplastic Syndromes Among Atomic Bomb Survivors in Nagasaki

[Background] Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders that develop de novo and also secondary to chemotherapy and / or radiation therapy. We previously demonstrated that the risk of MDS is increased among atomic bomb survivors in Nagasaki, Japan with significant correlation to radiation dose (Iwanaga et al. JCO 2011). The latest our study showed that MDS among proximally radiated survivors to the hypocenter had higher frequency of chromosomal aberrations, especially those related to poor prognosis such as complex karyotypes, however, survival and leukemia transformation did not show statistical difference by the distance from hypocenter (Matsuo et al. Caner Sci 2016).[Aim] In this study, we investigated chromosomal abnormalities of MDS among survivors to address (1) whether specific chromosomal aberration exists, and (2) why karyotype did not have strong impact on the survival and leukemia transformation.[Methods] We collected clinical information of MDS patients diagnosed from 1985 to 2013 registered in the Nagasaki-City MDS database. This database includes information from five hospitals in Nagasaki city. Patients were diagnosed according to the French-American-British (FAB) classification criteria. Patient risk was evaluated using the International Prognostic Scoring System (IPSS), and the revised IPSS (IPSS-R).[Results and Discussion] There were 133 MDS patients among A-bomb survivors, and 269 were unexposed. Among survivors, the number of cases according to distance from the hypocenter was 29 in the <1.5km group (Group I), 35 in the 1.5-2.99 km group (Group II) and 69 in the ≥3 km group (Group III). Abnormal karyotype frequency was significantly higher with more very poor karyotypes, according to the revised International Prognostic Scoring System, among those exposed close to the hypocenter compared with unexposed cases. However, there was no difference in prognosis between exposed and unexposed cases. In the Group I, chromosomal translocations and inversions were more frequent, and the frequency of structural alterations in chromosomes 3, 8, and 11 was significantly increased compared with unexposed cases (P=0.007, 0.01, and 0.001, respectively). These results suggest that chromosomal alterations in MDS among survivors have different features compared with those in de novo or therapy-related MDS. Detailed molecular study is warranted. DisclosuresMiyazaki:Nippon Shinyaku: Honoraria. Haase:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Study of the Age Dynamics of Morphological Subtypes of Acute Myeloid Leukemia in Adults

Aim: to determine the age dynamics of morphological subtypes of acute myeloid leukemia in adult patients. Materials and methods. The study group consisted of 132 patients (including 34 aged 15 to 45 years, 46 aged 45-60 years, 52 aged over 60 years) with newly diagnosed AML. Patients with acute promyelocytic leukemia were not included in the study. The diagnosis was made in accordance with WHO recommendations and FAB classification criteria. In all cases, morphological verification was performed, including cytological, cytochemical studies and immunophenotyping. Detection of chromosomal abnormalities was performed using standard cytogenetic and real-time polymerase chain reaction methods. Point mutations were screened in 7 genes: c-KIT, DNMT3A, FLT3, NRAS, NPM1, TP53 and WT1 by direct sequencing method. Results. The distribution of morphological variants of AML according to the FAB classification was as follows: M2 — 47.7%, M4 — 26.5%, M1 — 7.6%, M0 — 4.5%, M6 — 4.5%, M5 — 3.0%, M4eo — 2.3%, blastic plasmacytoid dendritic cell neoplasm — 1.5%, acute myelofibrosis — 0.8%, acute hybrid leukemia — 0.8%. Complex genetic abnormalities were detected in 18.2%, specific chromosome abnormalities associated with a favorable prognosis — 7.6%, specific chromosome abnormalities associated with an unfavorable prognosis — 5.3%, normal cytogenetics — 28.8%. Mutations in the FLT3 gene were detected in 15.0%, NPM1 — 14.3%, DNMT3A — 12.5%, NRAS — 12.2%, TP53 — 9.6%, c-KIT — 5.7%, WT1 — 4.2%. It was revealed that the frequency of acute myeloid leukemias with monocytic differentiation (M4, M4eo and M5 according to FAB) had a statistically significant tendency to decrease with increasing age: in the subgroup of patients younger than 45 years, it was 44.1%, in the age of 45-60 years — 32.6%, in the age of 60 years and older — 23.1%. These differences correlated with the age-related dynamics of the frequency of genetic abnormalities in AML, including a decrease in the frequency of NPM1 gene mutations and specific chromosomal aberrations, as well as an increase in the frequency of TP53 abnormalities in older age groups.

Role of cytogenetic abnormalities detected by fluorescence in situ hybridization as a prognostic marker: Pathogenesis & clinical course in patients with B-chronic lymphocytic leukaemia.

Background & objectives:B-cell chronic lymphocytic leukaemia (B-CLL) is one of the most common forms of adult leukaemia, with a highly variable clinical course. Specific chromosomal and genetic aberrations are used clinically to predict prognosis, independent from conventional clinical markers. Molecular cytogenetic methods such as fluorescence in situ hybridization (FISH) detect aberrations in up to 80 per cent B-CLL patients. This study was conducted to score the frequencies of recurrent aberrations, i.e., del(13q14), trisomy 12, del(11q22), del(17p13), del(6q21) and IgH (immunoglobulin heavy chain) translocations and to understand their role in prognostication and risk stratification.Methods:FISH studies were performed on bone marrow aspirate or peripheral blood of 280 patients using commercially available disease-specific probe set. The data were correlated with clinical and haematological parameters such as low haemoglobin, splenomegaly and lymphadenopathy.Results:Chromosomal aberrations were detected in 79 per cent of patients, with del(13q14) (57%) as the most common cytogenetic aberration, followed by trisomy 12 (27%), del(11q22) (22%), t(14q32) (19%), del(17p13) (18%) and del(6q21) (9%). Single or in coexistence with other aberration del(13q14) had a favourable outcome in comparison to del(11q22), t(14q32), del(17p13) and del(6q21) which were associated with advanced stages of the disease. Trisomy 12 had a variable clinical course.Interpretation & conclusions:FISH was found to be a sensitive and efficient technique in detecting the prevalence of recurrent cytogenetic abnormalities. Each of these aberrations is an important independent predictor of disease progression and survival which aids in designing risk-adapted treatment strategies for better disease management.

Stratified treatment of fit and unfit acute myeloid leukemia

The etiology and pathogenesis of acute myeloid leukemia (AML) have been elucidated at chromosomal and genetic levels. The classification and prognosis for its treatment has clearly involved specific chromosomal aberrations and genetic mutations. The recent comprehensive genomic analysis represented by next-generation sequencers has led to discovering new genetic mutations in AML. These findings have not only been applied clinically as prognostic factors and MRD markers but also contributed to the development of new molecular-targeting drugs. Many new drugs have already been approved in the USA and Europe, and new stratified treatments have tried to incorporate them. With the advent of venetoclax, treatment strategies, especially for patients with poor prognosis and who are unfit, have been substantially revised, and the maintenance therapy for AML is also being reevaluated in accordance to the National Comprehensive Cancer Network guidelines. This article will review the current status of AML treatment in Japan and according to Western guidelines.

Frequent activating PIK3CA mutations in sporadic angiolipoma.

Angiolipoma (AL) is considered as a lipoma variant that is characterized by the combination of mature adipocytes and capillary blood vessels diffusely distributed within the tumor. With the exception of recurrent PRKD2 mutations of uncertain pathogenetic significance, the genetic abnormalities of ALs are unknown, in the absence of any of the specific chromosomal aberrations described in other lipoma variants. Formalin-fixed and paraffin-embedded blocks of 13 conventional ALs and 5 cellular ALs from 17 individuals were retrieved and analyzed for mutations in exons 9 and 20 of PIK3CA by polymerase chain reaction and Sanger sequencing. Activating PIK3CA mutations were identified in 14 tumors (78%). All PIK3CA-mutated samples carried the same exon 9 mutation, c.1634A>C (p.E545A). No mutation was detected in exon 20 of PIK3CA. No significant difference between PIK3CA-mutated and wild-type samples appeared to exist based on age, gender, and location of the tumor. All 5 cellular ALs carried the p.E545A PIK3CA mutation. The high frequency of the p.E545A PIK3CA mutation in both conventional and cellular ALs suggests that activation of the PI3K/AKT pathway plays a key role in AL pathogenesis and reinforces the concept that cellular AL should be regarded as a variant of AL.

Shallow whole genome sequencing of adenoid cystic carcinomas of the salivary glands identifies specific chromosomal aberrations related to tumor progression.

Adenoid cystic carcinomas (ACC) are characterized by high rate of local recurrence and late distant metastasis. Chromosomal changes in the evolution from primary tumors to metastatic disease of ACC have not been appointed. Here we investigated the chromosomal alterations of 53 primary tumors from ACC patients with different progressive states by shallow whole genome sequencing to identify potential new markers for metastatic spread. Illumina paired-end libraries were generated using DNA from the primary tumor of 53 ACC patients. Fragmented DNA was end-repaired, A-tailed and multiplex sequencing adapters were ligated. Sequence data were mapped to HG19 and a copy-number analysis was conducted using the QDNAseq R package (version 1.10.0). Outliers were removed and data was smoothed by applying the circular binary segmentation algorithm implemented in the R package copynumber version 1.22.0. A modified chromosomal instability (CNI) score was used to analyze deletions and amplifications. Cluster analysis of the whole genome sequencing revealed that the frequency of chromosomal aberrations were increased in ACC with local recurrence and distant metastases in comparison to ACC patients with no metastatic spread. Specifically, chromosome 6 and 12 and exclusively the entire chromosome 4 showed an increased frequency of chromosomal alterations with tumor progression. Our data show a molecular evolution from primary tumors to local recurrences and distant metastases and pinpoint the critical chromosomal regions involved in this process. These regions should be in the focus of the search for therapeutic targets of progressive ACC.

Paraoxonase 1 (PON1) Q192R and L55M Polymorphisms as Potential Predisposition Factors for Chronic Lymphocytic Leukemia.

PON1 gene has an executive role in antioxidant defense, protecting cells from genotoxic factors. Q192R and L55M PON1 polymorphisms reduce catalytic activity of the encoded protein. These polymorphisms were studied in 300 chronic lymphocytic leukemia (CLL) patients and 106 healthy donors. They were also associated with patients' cytogenetic findings, to investigate their possible implication in CLL pathogenesis. SNP genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Karyotypic analysis was also performed by chromosome G-banding analysis and fluorescence in situ hybridization. Genotypic and allelic distribution of Q192R polymorphism showed a statistically significant higher frequency of mutant genotypes and mutant alleles in patients compared to controls. The same observation was noted in patients with abnormal karyotypes and those carrying abn14q32 and del(6q). A statistically increased frequency for the mutant allele was also revealed in patients with del(11q). On the contrary, L55M polymorphism showed a similar distribution between patients and controls. Q192R polymorphism plays a role in CLL predisposition and the formation of specific chromosomal aberrations.

Specific Chromosomal Aberrations in Primary Amenorrhoea: Study on 3776 Cases from Indian Population.

To verify the prevalence of chromosomal abnormalities in women with primary amenorrhoea in India aiming at appropriate genetic counselling. In a 16-year retrospective (2001-2016) study, 3776 women with primary amenorrhoea were evaluated. Chromosomal analysis of all the cases was done by GTG banding. Clinical history and other laboratory findings were taken into consideration to determine the diagnosis. The karyotype results revealed 31.2% cases with chromosomal abnormalities (n = 1177/3776). In patients with abnormal chromosome complement, 31.2% exhibited numerical aberrations (n = 367) and 34.9% with structural aberrations (n = 411). About 33.9% of cases were with XY male karyotype (n = 399). As per the literature till date, this study is the largest with high incidence of chromosomal abnormalities; early detection of abnormalities is necessary for guidance to reproductive management and genetic counselling.

Diagnostic utility of fluorescence in situ hybridization testing on cytology cell blocks for the definitive classification of salivary gland neoplasms

Fine needle aspiration biopsy (FNAB) is a minimally invasive modality to evaluate salivary gland neoplasms and help guide clinical management. However, significant overlap in the cytomorphology findings among salivary gland neoplasms often renders the definitive diagnosis challenging. Recently, a number of benign and malignant salivary gland tumors have been characterized by specific chromosomal aberrations detectable using fluorescence in situ hybridization (FISH) testing. In the present study, we evaluated the role of FISH testing performed on cytology cell blocks in the diagnosis of salivary gland neoplasms by FNAB. The data from 57 cases of primary salivary gland tumors diagnosed using FNAB at our institution and sent for ancillary FISH testing between 2012 and 2017 were retrospectively reviewed. The FISH studies were performed on cytology cell blocks, and break-apart probes were used to detect characteristic gene rearrangements for PLAG1, MYB, MAML2, and ETV6 for pleomorphic adenoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, and secretory carcinoma (mammary analogue secretory carcinoma), respectively. Of the 57 cases sent for FISH testing, 6 were excluded because of FISH analysis failure (insufficient cell block cellularity). Of the 51 cases included in the analysis, 15 samples were successfully subclassified after FISH testing, and 10 of these 15 FISH-positive cases were diagnostically confirmed by the surgical pathology review of excision material. Forty cases overall had undergone subsequent excision with the histopathologic follow-up diagnosis available, and all subclassified cases had concordant FNAB, FISH, and excision diagnoses. FISH testing performed on cytology cell blocks is a useful adjunct in establishing the diagnosis of salivary gland neoplasms by FNAB.

14. A novel cytogenomic approach to diagnosis of plasma cell neoplasms

Plasma cell neoplasm (PCN) is characterized by specific chromosomal aberrations with diagnostic and prognostic significance. Low percentage of neoplastic cells is a drawback in application of karyotyping and fluorescence in situ hybridization (FISH) for evaluation of bone marrow aspirates. Enrichment techniques to isolate the CD138+ cells have improved the detection rates. However, a complete FISH analysis for various abnormalities may not be achieved due to limited number of isolated cells. In 2017, we validated and implemented a novel approach for a whole genome profiling of plasma cells in PCN. The protocol consisted of isolation of CD138+ cells, whole genome DNA amplification and microarray comparative genomic hybridization. Separate FISH analysis was performed for detection of balanced IGH rearrangements. Of 412 clinical cases to date, 234 (56.8%) had morphologic and immunohistochemical studies indicative of PCN with 0.5–90% plasma cells in a bone marrow aspirates. Among PCN patients, chromosomal abnormalities were identified in 223 (95.2%) demonstrated by both microarray and FISH (n = 76, 32.9%), microarray (n = 116, 50.2%) or FISH only (n = 31, 13.9%). The most common findings included IGH rearrangement (49%), 13q deletion (45%), hyperdiploidy (41%), and 1q duplication (38%). Tetraploidy and MYC rearrangements were observed in 2.1% (5/234) and 1.7% (4/234) of patients, respectively. Interestingly, microarray revealed characteristic deletions involving the 6q21q25 region in 4/7 patients with Waldenström Macroglobulinemia. Our diagnostic approach to PCN is a cost-efficient replacement of multiple FISH assays that provides a superior detection rate and additional information for disease classification, prognosis, treatment and differentiation from other B cell malignancies.

Strong associations between chromosomal aberrations in blood lymphocytes and the risk of urothelial and squamous cell carcinoma of the bladder

Chromosomal aberrations (CAs) in blood lymphocytes have been shown to be associated with overall cancer risk and aging. However, their relationship to bladder cancer risk remains to be elucidated. In a case-control study of bladder cancer in Egypt, we examined the relationship between the increased frequency of CAs in blood lymphocytes and bladder cancer risk. High frequency of CAs was significantly associated with an increased risk of bladder cancer [adjusted odds ratios (OR) = 3.90, 95% confidence interval (CI) = 2.65–5.73]. The associations were somewhat stronger in squamous cell carcinomas (SCC, OR = 4.90) than in urothelial carcinomas (UC, OR = 3.62). We also identified chromosome specific CAs for chromosomes 3, 4, 5, 8, 9, 10, 11, 12, 17, 19 that were significantly associated with an increased risk of bladder cancer. We observed particularly strong associations between aberrations of chromosomes 12, 13, 17 and risk of SCC (OR = 7.06, 6.91 and 6.23, respectively). Conclusion: increased frequency of chromosomal aberrations in blood lymphocytes was significantly associated with bladder cancer risk. Overall and chromosome specific aberrations in blood lymphocytes may be a unique set of biomarkers for risk assessments of SCC and UC.