Multiple Chromosomal Aberrations Research Articles

NTRK-fused central nervous system tumours: clinicopathological and genetic insights and response to TRK inhibitors

Background Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are found in 1% of gliomas across children and adults. TRK inhibitors are promising therapeutic agents for NTRK-fused gliomas because they are tissue agnostic and cross the blood–brain barrier (BBB). Methods We investigated twelve NGS-verified NTRK-fused gliomas from a single institute, Seoul National University Hospital. Results The patient cohort included six children (aged 1–15 years) and six adults (aged 27–72 years). NTRK2 fusions were found in ten cerebral diffuse low-grade and high-grade gliomas (DLGGs and DHGGs, respectively), and NTRK1 fusions were found in one cerebral desmoplastic infantile ganglioglioma and one spinal DHGG. In this series, the fusion partners of NTRK2 were HOOK3, KIF5A, GKAP1, LHFPL3, SLMAP, ZBTB43, SPECC1L, FKBP15, KANK1, and BCR, while the NTRK1 fusion partners were TPR and TPM3. DLGGs tended to harbour only an NTRK fusion, while DHGGs exhibited further genetic alterations, such as TERT promoter/TP53/PTEN mutation, CDKN2A/2B homozygous deletion, PDGFRA/KIT/MDM4/AKT3 amplification, or multiple chromosomal copy number aberrations. Four patients received adjuvant TRK inhibitor therapy (larotrectinib, repotrectinib, or entrectinib), among which three also received chemotherapy (n = 2) or proton therapy (n = 1). The treatment outcomes for patients receiving TRK inhibitors varied: one child who received larotrectinib for residual DLGG maintained stable disease. In contrast, another child with DHGG in the spinal cord experienced multiple instances of tumour recurrence. Despite treatment with larotrectinib, ultimately, the child died as a result of tumour progression. An adult patient with glioblastoma (GBM) treated with entrectinib also experienced tumour progression and eventually died. However, there was a successful outcome for a paediatric patient with DHGG who, after a second gross total tumour removal followed by repotrectinib treatment, showed no evidence of disease. This patient had previously experienced relapse after the initial surgery and underwent autologous peripheral blood stem cell therapy with carboplatin/thiotepa and proton therapy. Conclusions Our study clarifies the distinct differences in the pathology and TRK inhibitor response between LGG and HGG with NTRK fusions.

Meningioma

Meningioma is considered as a single type by the World Health Organization 2021 classification, encompassing 15 subtypes. These classifications are primarily based on histological attributes. However, recent advancements in understanding driver gene mutations and molecular prognostic markers are of particular importance for comprehending the clinical behavior of these tumors. Meningiomas are stratified molecularly, predominantly based on the presence of NF2 mutation. Over 50% are associated with mutations in the NF2 gene and/or monosomy of chromosome 22, whereas the remaining are associated with gene mutations in either of AKT1, SMO, KLF4, TRAF7, or the other less common ones. Importantly, these driver gene mutations are mutually exclusive and significantly associated with tumor location, histological subtype, and grading. The majority of higher-grade meningiomas is associated with multiple chromosomal aberrations, including the loss of 1p, 6q, and 14q, along with NF2 mutations. Homozygous deletion of CDKN2A/CDKN2B genes or TERT promoter mutations serves as a predictor of poor prognosis. Notably, these molecular markers are now incorporated into the criteria for anaplastic meningioma. The amalgamation of molecular insights with clinical and histological parameters, alongside patient prognosis, holds significant utility in the management of patients with meningiomas. It is anticipated to pave the way for treatments founded on molecular-clinical associations.

Lung Ultrasound Role in Diagnosis of Neonatal Respiratory Disorders: A Prospective Cross-Sectional Study.

Lung ultrasound (LUS) has become one of the most exciting applications in neonatal point-of-care ultrasound (POCUS), yet still lacks routine clinical use. This study assesses the utility of LUS for neonatal respiratory disorders (NRDs) diagnosis and follow-up compared to chest X-ray (CXR). A prospective cross-sectional study was conducted on 100 neonates having NRDs with a gestational age ≥28 weeks, excluding those having multiple congenital anomalies, chromosomal aberrations, hydrops fetalis and/or heart failure. CXR and LUS were done on admission for diagnosis and were repeated after 7 days, or if needed earlier within the 7 days. The diagnosis of NRDs by CXR and LUS on admission and after 7 days was comparable (p > 0.05). LUS diagnosis sensitivity and specificity for respiratory distress syndrome, pneumonia, meconium aspiration syndrome, pneumothorax and pulmonary atelectasis were 94.7/100%, 97.5/95%, 92.3/100%, 90.9/98.9% and 100/97.8%, respectively. The total agreement between LUS and CXR was 98.5% with 95% CI (0.88 to 0.92). LUS and CXR had considerable agreement in the diagnosis of NRDs. Being a reliable bedside modality of diagnosis and safer than CXR, LUS may be considered an alternative method for the diagnosis of neonates with NRDs.

105. Clinical whole-genome sequencing identifies NSD3 as the correct fusion partner of NUP98 in a patient with acute myeloid

<h3>Background</h3> Only rare cases of acute myeloid leukemia (AML) have been shown to harbor a t(8;11)(p11.2;p15.4). This translocation is believed to involve the fusion of NSD3 or FGFR1 with NUP98; however, apart from targeted mRNA quantitative PCR analysis, no molecular approaches have been utilized to define the chimeric fusions present in these rare cases. <h3>Case Presentation</h3> Here we present the case of a 51-year-old female with AML with myelodysplastic-related morphologic changes, 13q deletion and t(8;11), where initial fluorescence in situ hybridization (FISH) assays were consistent with the presence of NUP98 and FGFR1 rearrangements, and suggestive of NUP98/FGFR1 fusion. Using a streamlined clinical whole-genome sequencing approach, we resolved the breakpoints of this translocation to intron 4 of NSD3 and intron 12 of NUP98, indicating NUP98/NSD3 rearrangement as the underlying aberration. Furthermore, our approach identified small variants in WT1 and STAG2, as well as an interstitial deletion on the short arm of chromosome 12, which were cryptic in G-banded chromosomes. <h3>Conclusions</h3> NUP98 fusions in acute leukemia are predictive of poor prognosis. The associated fusion partner and the presence of co-occurring mutations, such as WT1, further refine this prognosis with potential clinical implications. Using a clinical whole-genome sequencing analysis, we resolved t(8;11) breakpoints to NSD3 and NUP98, ruling out the involvement of FGFR1 suggested by FISH while also identifying multiple chromosomal and sequence level aberrations.

Noninvasive Prenatal Test Results Indicative of Maternal Malignancies: A Nationwide Genetic and Clinical Follow-Up Study.

Noninvasive prenatal testing (NIPT) for fetal aneuploidy screening using cell-free DNA derived from maternal plasma can incidentally raise suspicion for cancer. Diagnostic routing after malignancy suspicious-NIPT faces many challenges. Here, we detail malignancy suspicious-NIPT cases, and describe the clinical characteristics, chromosomal aberrations, and diagnostic routing of the patients with a confirmed malignancy. Clinical lessons can be learned from our experience. Patients with NIPT results indicative of a malignancy referred for tumor screening between April 2017 and April 2020 were retrospectively included from a Dutch nationwide NIPT implementation study, TRIDENT-2. NIPT profiles from patients with confirmed malignancies were reviewed, and the pattern of chromosomal aberrations related to tumor type was analyzed. We evaluated the diagnostic contribution of clinical and genetic examinations. Malignancy suspicious-NIPT results were reported in 0.03% after genome-wide NIPT, and malignancies confirmed in 16 patients (16/48, 33.3%). Multiple chromosomal aberrations were seen in 23 of 48 patients with genome-wide NIPT, and a malignancy was confirmed in 16 patients (16/23, 69.6%). After targeted NIPT, 0.005% malignancy suspicious-NIPT results were reported, in 2/3 patients a malignancy was confirmed. Different tumor types and stages were diagnosed, predominantly hematologic malignancies (12/18). NIPT data showed recurrent gains and losses in primary mediastinal B-cell lymphomas and classic Hodgkin lymphomas. Magnetic resonance imaging and computed tomography were most informative in diagnosing the malignancy. In 231,896 pregnant women, a low percentage (0.02%) of NIPT results were assessed as indicative of a maternal malignancy. However, when multiple chromosomal aberrations were found, the risk of a confirmed malignancy was considerably high. Referral for extensive oncologic examination is recommended, and may be guided by tumor-specific hallmarks in the NIPT profile.

Histopathologic and genetic findings in atypical spindle cell/pleomorphic lipomatous tumors and atypical pleomorphic fibromas.

Spindle cell lipomas, pleomorphic lipomas (SCL/PLs), and pleomorphic fibromas (PF) are tumors with loss of retinoblastoma (RB). The latest World Health Organization classification includes a category of atypical spindle cell/pleomorphic lipomatous tumors (ASPLT), which encompasses tumors in this spectrum that show atypical histopathologic features. We have observed PFs that show similar atypical features. Cases of SCL/PL and PF with atypical features were collected from tissue archives between 2010 and 2019. Genetic alterations were investigated using array comparative genomic hybridization (aCGH). Of 15 cases found, most tumors were dermal based with fibrocytic or fibroadipocytic appearance and occasional lipoblasts. All cases had a high proliferation index with atypical mitotic figures in 71% of cases. Chromosome 13q loss was present in all cases with CGH data. Additional recurrent chromosomal losses included 17p, 16q, 17q, 20p, 4, and 10. No recurrence was found in limited follow-up. ASPLTs are characterized by loss of RB, prominent nuclear pleomorphism, mitotic activity including atypical mitotic figures, and genomic instability with multiple chromosomal aberrations. A similar group of tumors with these histopathologic features lacks lipomatous differentiation, and we propose the diagnosis of atypical PF as a fibromatous variant of ASPLT. Limited clinical follow-up appears benign.

Investigation of Association with First-Trimester Free Human Chorionic Gonadotropin - ß, Pregnancy - Associated Plasma Protein-A, and Adverse Pregnancy Outcomes

OBJECTIVES: This study aimed to investigate the association between first-trimester screening maternal serum markers (free human chorionic gonadotropin-beta (β-hCG), pregnancy-associated plasma protein-A, and adverse pregnancy outcomes (gestational hypertension, preeclampsia, preterm delivery, intrauterine growth restriction, oligohydramnios, intrauterine ex-fetus, abruptio placentae, and gestational diabetes mellitus). STUDY DESIGN: This was a retrospective cohort study including 1516 women who delivered a singleton pregnancy at GATA Haydarpasa Education Hospital from 2006 to 2009. Patients with multiple pregnancies, chromosome aberrations, or fetal anomalies were excluded. Extreme values of corrected- pregnancy-associated plasma protein-A and free β-hCG, and their association with adverse pregnancy outcomes were analyzed. RESULTS: Adverse pregnancy outcomes at the cutoff level of ≤0.25 c-pregnancy-associated plasma protein-A MOM values positive likelihood ratio (+LR) was 7.5 (95%CI 2.426-19.836) and had a significant correlation (r=0.108, p&lt;0.01). It was also a significant correlation with adverse pregnancy outcomes (r=0.189, p&lt;0.01) at the cut-off level of ≤0.50 corrected-pregnancy-associated plasma protein-A MOM values and the +LR was 2.388 (95%CI 1.889-2.991). Association between low corrected-pregnancy-associated plasma protein-A MOM values and adverse pregnancy outcomes were statistically significant and had a poor association (AUC, 0.630 95%CI 0.596-0.663, p&lt;0.01). Preeclampsia was statistically significant, however had a fair association (AUC, 0.74 95% CI 0.690-0.802, p&lt;0.01). Preterm birth was statistically significant but had a poor association (AUC, 0.568 95% CI 0.512-0.624, p&lt;0.05). CONCLUSION: First-trimester maternal serum low pregnancy-associated plasma protein-A values are significantly associated with adverse pregnancy outcomes. It may be a useful tool to predictive not only chromosome anomalies but also adverse pregnancy outcomes.

Dissection and physical mapping of wheat chromosome 7B by inducing meiotic recombination with its homoeologues in Aegilops speltoides and Thinopyrum elongatum.

We constructed a homoeologous recombination-based bin map of wheat chromosome 7B, providing a unique physical framework for further study of chromosome 7B and its homoeologues in wheat and its relatives. Homoeologous recombination leads to the dissection and diversification of the wheat genome. Advances in genome sequencing and genotyping have dramatically improved the efficacy and throughput of homoeologous recombination-based genome studies and alien introgression in wheat and its relatives. In this study, we aimed to physically dissect and map wheat chromosome 7B by inducing meiotic recombination of chromosome 7B with its homoeologues 7E in Thinopyrum elongatum and 7S in Aegilops speltoides. The special genotypes, which were double monosomic for chromosomes 7B' + 7E' or 7B' + 7S' and homozygous for the ph1b mutant, were produced to enhance 7B - 7E and 7B - 7S recombination. Chromosome-specific DNA markers were developed and used to pre-screen the large recombination populations for 7B - 7E and 7B - 7S recombinants. The DNA marker-mediated preselections were verified by fluorescent genomic in situ hybridization (GISH). In total, 29 7B - 7E and 61 7B - 7S recombinants and multiple chromosome aberrations were recovered and delineated by GISH and the wheat 90K SNP assay. Integrated GISH and SNP analysis of the recombinants physically mapped the recombination breakpoints and partitioned wheat chromosome 7B into 44 bins with 523 SNPs assigned within. A composite bin map was constructed for chromosome 7B, showing the bin size and physical distribution of SNPs. This provides a unique physical framework for further study of chromosome 7B and its homoeologues. In addition, the 7B - 7E and 7B - 7S recombinants extend the genetic variability of wheat chromosome 7B and represent useful germplasm for wheat breeding. Thereby, this genomics-enabled chromosome engineering approach facilitates wheat genome study and enriches the gene pool of wheat improvement.

Neuropathologic Changes in Sudden Unexplained Death in Childhood.

Sudden unexplained death in childhood (SUDC) affects children >1-year-old whose cause of death remains unexplained following comprehensive case investigation and is often associated with hippocampal abnormalities. We prospectively performed systematic neuropathologic investigation in 20 SUDC cases, including (i) autopsy data and comprehensive ancillary testing, including molecular studies, (ii) ex vivo 3T MRI and extensive histologic brain samples, and (iii) blinded neuropathology review by 2 board-certified neuropathologists. There were 12 girls and 8 boys; median age at death was 33.3 months. Twelve had a history of febrile seizures, 85% died during apparent sleep and 80% in prone position. Molecular testing possibly explained 3 deaths and identified genetic mutations in TNNI3, RYR2, and multiple chromosomal aberrations. Hippocampal abnormalities most often affected the dentate gyrus (altered thickness, irregular configuration, and focal lack of granule cells), and had highest concordance between reviewers. Findings were identified with similar frequencies in cases with and without molecular findings. Number of seizures did not correlate with hippocampal findings. Hippocampal alterations were the most common finding on histological review but were also found in possibly explained deaths. The significance and specificity of hippocampal findings is unclear as they may result from seizures, contribute to seizure pathogenesis, or be an unrelated phenomenon.

Whole Exome Sequencing Reveals Multiple Driver Events in Chronic Lymphocytic Leukemia Patients with Acquired Ibrutinib Resistance

Disease progression in chronic lymphocytic leukemia (CLL) patients receiving single-agent ibrutinib has been associated with either histological transformation or acquired mutations in BTK and PLCG2 genes which are found in up to 85% of patients with progressive CLL (PD-CLL), less commonly in patients with histologic transformation. Variant allele frequencies of these mutations are highly variable and in some patients remain low and thus cannot fully explain the emergence of drug-resistant clones. Cooperation between mutant and "wild-type" clones has been proposed to explain expansion of clones that have no BTK or PLCG2 mutation. Alternatively, additional drivers have been suggested, including 8p deletion. Here we performed a comprehensive analysis of resistant samples' clonal composition using droplet digital polymerase chain reaction (ddPCR) and whole exome sequencing across multiple time points and compartments to reveal additional hits responsible for drug resistance. Eighty-four CLL patients have been enrolled to a phase II study for ibrutinib monotherapy. Eligible patients had either TP53 aberration or age ≥65 years regardless of prior treatment status. All patients received single-agent ibrutinib 420 mg once daily until disease progression or intolerable side effects occurred. At a median follow-up of 5.5 years, 23 (27.4%) patients developed progressive disease (PD) including 17 patients with PD-CLL, five with Richter's transformation and one with prolymphocytic leukemia. Majority of PD-CLL patients had TP53 aberration (88.2%) and relapsed or refractory CLL (58.8%). The median time-to-progression was 38.8 months for all PD patients and was shorter for those with histologic transformations compared to those with PD-CLL (7.0 vs 44.2 months). We tested for BTK/PLCG2 mutations in 17 PD-CLL patients using archival samples collected at baseline, during response to ibrutinib and at progression using variant-specific ddPCR assays. Fifteen PD-CLL patients (88%) had detectable BTK and/or PLCG2 variants at the time of progression. Notably, only 5 PD-CLL patients (29.4%) demonstrated a strong BTK/PLCG2-driven resistance with cumulative clonal fraction (cCF) above 50% at PD and a significant tumor burden in the peripheral blood (PB). Seven patients (41.2%) demonstrated partial BTK/PLCG2-associated resistance (cCF ranging 4-40%) with additional sub-clonal variants (TP53, NFKBIE, SF3B1, BIRC3, KRAS) and chromosomal aberrations (del8p, del6q, amp2p) as putative drivers in concurrently evolving subclones at PD. Finally, 29.4% of patients had very low (&lt;4%, 3 patients) or undetectable (2 patients) frequencies of BTK/PLCG2 variants but often carried multiple chromosomal aberrations (del17p, del8p, del3p, del2p, del13q) and known CLL driver mutations (NOTCH1, SF3B1) in the resistant clone(s). In all PD-CLL patients, the resistant clones were detected in at least one of the 3 compartments: PB, lymph node (LN) or bone marrow (BM). Expanding clones were present in all 3 sites in three patients (18%), in PB and either LN or BM in 10 (59%) patients, and in LN and/or BM but not in PB in 4 patients (23%). In summary, BTK/PLCG2 mutations are present in most patients progressing on ibrutinib but in many cases these mutations cannot completely account for drug resistance. Exome sequencing followed by clonal decomposition analysis revealed additional genetic alterations in ibrutinib-resistant clones including acquisition of multiple copy number alterations and/or additional known CLL driver mutations. In most patients resistant clones are equally detected in blood and tissue sites. Disclosures Wiestner: Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding; Pharmayclics: Research Funding.

Comparative assessment of conventional chromosomal analysis and fluorescence in situ hybridization in the evaluation of suspected myelodysplastic syndromes: a single institution experience.

Background:Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic neoplasms, roughly half of which harbor cytogenetic abnormalities with diagnostic, prognostic, and therapeutic significance. Fluorescence in situ hybridization (FISH) for the most commonly seen abnormalities (5/5q, –7/7q, +8, and –20/20q–) is routinely performed alongside conventional cytogenetics (CC) in the evaluation of suspected MDS despite conflicting reports of its relative contribution compared to CC alone.Objectives:To assess the additional diagnostic and prognostic value of performing concurrent FISH versus CC alone in cases of suspected MDS.Materials and Methods:A total of 127 bone marrow samples submitted to our cytogenetic laboratory with a presumptive diagnosis of MDS were evaluated by concurrent CC and an MDS FISH panel.Results:CC was used as the gold standard method with 100% sensitivity in detecting suspected MDS-associated cytogenetic abnormalities. FISH alone had a sensitivity of 76%, whereas CC alone achieved a sensitivity of 97%. The addition of FISH did not change the diagnosis nor change the Revised International Prognostic Scoring System score in any patient. Moreover, in 12 cases identified as positive by both CC and FISH, CC identified multiple chromosomal aberrations of clinical significance not interrogated by the FISH probe panel.Conclusion:CC alone is sufficiently sensitive in detecting suspected MDS-associated cytogenetic abnormalities that influence clinical decision-making. Routine FISH testing does not provide a significant increase in test sensitivity when an adequate karyotype is obtained. Therefore, FISH testing is best reserved for suspected MDS cases lacking sufficient metaphases.

Widely metastatic IDH1-mutant glioblastoma with oligodendroglial features and atypical molecular findings: a case report and review of current challenges in molecular diagnostics

BackgroundGliomas with 1p/19q-codeletion as well as mutation of isocitrate dehydrogenase (IDH) 1 are typically characterized as oligodendrogliomas with comparatively good response to treatment with radiation and chemotherapy.Case presentationWe present the case of a 28-year-old man with an IDH1 and TP53 mutant high grade glioma with abnormalities in chromosomes 1 and 19 suggestive of anaplastic oligodendroglioma that rapidly progressed to widespread metastatic disease. Biopsy of a liver lesion confirmed metastasis of the patient’s known brain primary and chemotherapy with temozolomide was initiated. The patient’s rapidly growing tumor burden with fulminant liver failure and tumor lysis led to multisystem failure of which the patient died. Further molecular testing illustrated features more consistent with glioblastoma: multiple large chromosomal aberrations including loss of whole chromosome 1 and 2q; gain/amplification of MYCN, MET, and CDK4; loss of CDKN2A/B; and an ATRX mutation.ConclusionThis case illustrates the importance of higher level molecular diagnostic testing for patients with particularly aggressive disease progression that is not concordant with standard prognoses. Additional data on cases with atypical alterations of 1p and 19q are needed to better understand the distinct biology of these cancers so that appropriate therapies can be developed.

Prognostic effect of whole chromosomal aberration signatures in standard-risk, non-WNT/non-SHH medulloblastoma: a retrospective, molecular analysis of the HIT-SIOP PNET 4 trial.

SummaryBackgroundMost children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features (metastatic disease, large-cell/anaplastic histology, and MYC amplification), which includes 50–60% of patients and has a 5-year event-free survival of 75–85%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies.MethodsThe HIT-SIOP PNET 4 trial recruited 338 patients aged 4–21 years with medulloblastoma between Jan 1, 2001, and Dec 31, 2006, in 120 treatment institutions in seven European countries to investigate hyperfractionated radiotherapy versus standard radiotherapy. In this retrospective analysis, we assessed the remaining tumour samples from patients in the HIT-SIOP PNET 4 trial (n=136). We assessed the clinical behaviour of the molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a molecular inversion probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies (maximal surgical resection followed by adjuvant craniospinal irradiation [all patients] and chemotherapy [65 of 70 patients], at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with ClinicalTrials.gov, number NCT01351870.FindingsWe analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH (we considered Group3 and Group4 medulloblastoma together in our analysis because of their similar molecular and clinical features). Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation (TP53mut) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associated with this whole chromosomal aberration signature (at least two of chromosome 7 gain, chromosome 8 loss, and chromosome 11 loss) predicted favourable prognosis. Patients with non-WNT/non-SHH medulloblastoma could be reclassified by these markers as having favourable-risk or high-risk disease. In patients in the HIT-SIOP PNET4 cohort with non-WNT/non-SHH medulloblastoma, with a median follow-up of 6·7 years (IQR 5·8–8·2), 5-year event-free survival was 100% in the favourable-risk group and 68% (95% CI 57·5–82·7; p=0·00014) in the high-risk group. In the validation cohort, with a median follow-up of 5·6 years (IQR 3·1–8·1), 5-year event-free survival was 94·7% (95% CI 85·2–100) in the favourable-risk group and 58·6% (95% CI 45·1–76·1) in the high-risk group (hazard ratio 9·41, 95% CI 1·25–70·57; p=0·029). Our comprehensive molecular investigation identified subgroup-specific risk models which allowed 69 (51%) of 134 accessible patients from the standard-risk medulloblastoma HIT-SIOP PNET 4 cohort to be assigned to a favourable-risk group.InterpretationWe define a whole chromosomal signature that allows the assignment of non-WNT/non-SHH medulloblastoma patients normally classified as standard-risk into favourable-risk and high-risk categories. In addition to patients younger than 16 years with WNT tumours, patients with non-WNT/non-SHH tumours with our defined whole chromosomal aberration signature and patients with SHH-TP53wild-type tumours should be considered for therapy de-escalation in future biomarker-driven, risk-adapted clinical trials. The remaining subgroups of patients with high-risk medulloblastoma might benefit from more intensive therapies.FundingCancer Research UK, Swedish Childhood Cancer Foundation, French Ministry of Health/French National Cancer Institute, and the German Children's Cancer Foundation.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Łomianki/Kiełpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Complex Karyotype in Hematological Diseases: A 6-Year Single Centre Study from Pakistan.

Background Most of the hematological disorders are heterogenous with regard to morphology, immunophenotype, and genetic rearrangements. Multiple recurrent chromosomal aberrations have been identified by conventional cytogenetic analysis, which is now widely recognized as one of the most important diagnostic and prognostic determinants in these patients. Though rarer, complex karyotype has been associated with worst prognosis. Materials and Methods A total of 1185 bone marrow or peripheral blood cytogenetics samples were taken with different hematological diseases. They included both benign and malignant disease entities. In each case, cells were cultured and conventional cytogenetic analysis was performed. Results Among 1185 subjects, 41 (3.4%) patients possessed complex cytogenetic abnormalities. Out of these 41, 33 (80%) were males. The mean age was 37 years (median age 39 years). Myelodysplastic syndromes had the most numbers of complex karyotypes (8%), followed by acute myeloid leukemia (7%) and acute lymphoblastic leukemia (4%). Also we found few patients with acute promyelocytic leukemia, aplastic anemia , chronic myeloid leukemia, and diffuse large B cell Lymphoma possessing complex karyotype. Frequencies of different cytogenetic abnormalities were assessed with respect to disease as well as independently. Trisomy 21 was the most common chromosomal abnormality found in 28% of patients. Conclusion Complex karyotype was most frequently associated with myelodysplastic syndromes and acute myeloid leukemia. Trisomy 21 and deletion 5q were the commonest cytogenetic abnormalities found. We also assessed complex karyotype in benign diseases and detected one patient of aplastic anemia with complex karyotype. This is the first study highlighting the presence of complex karyotypes in hematological disorders in our region.

Establishment of cholangiocarcinoma cell lines from patients in the endemic area of liver fluke infection in Thailand.

Cholangiocarcinoma is a rare type of cancer which is an increasingly discernible health threat. The disease is usually very difficult in diagnosis and various treatment modalities are typically not effective. Cholangiocarcinoma is a complex and very heterogeneous malignancy characterized by tumor location, different risk factors, molecular profiling, and prognosis. Cancer cell lines represent an important tool for investigation in various aspects of tumor biology and molecular therapeutics. We established two cell lines, KKU-452 and KKU-023, which were derived from patients residing in the endemic area of liver fluke infection in Thailand. Both of tumor tissues have gross pathology of perihilar and intrahepatic mass-forming cholangiocarcinoma. Two cell lines were characterized for their biological, molecular and genetic properties. KKU-452 and KKU-023 cells are both adherent cells with epithelium morphology, but have some differences in their growth pattern (a doubling time of 17.9 vs 34.8 h, respectively) and the expression of epithelial bile duct markers, CK7 and CK19. Cytogenetic analysis of KKU-452 and KKU-023 cells revealed their highly complex karyotypes; hypertriploid and hypotetraploid, respectively, with multiple chromosomal aberrations. Both cell lines showed mutations in p53 but not in KRAS. KKU-452 showed a very rapid migration and invasion properties in concert with low expression of E-cadherin and high expression of N-cadherin, whereas KKU-023 showed opposite characters. KKU-023, but not KKU-452, showed in vivo tumorigenicity in xenografted nude mice. Those two established cholangiocarcinoma cell lines with unique characters may be valuable for better understanding the process of carcinogenesis and developing new therapeutics for the patients.

High number of chromosomal copy number aberrations inversely relates to t(11;19)(q21;p13) translocation status in mucoepidermoid carcinoma of the salivary glands.

Although rare, mucoepidermoid carcinoma (MEC) is one of the most common malignant salivary gland tumors. The presence of the t(11;19)(q21;p13) translocation in a subset of MECs has raised interest in genomic aberrations in MEC. In the present study we conducted genome-wide copy-number-aberration analysis by micro-array comparative-genomic-hybridization on 27 MEC samples.Low/intermediate-grade MECs had significantly fewer copy-number-aberrations compared to high-grade MECs (low vs high: 3.48 vs 30; p = 0.0025; intermediate vs high: 5.7 vs 34.5; p = 0.036). The translocation-negative MECs contained more copy-number-aberrations than translocation-positive MECs (average amount of aberrations 15.9 vs 2.41; p =0.04).Within all 27 MEC samples, 16p11.2 and several regions on 8q were the most frequently gained regions , while 1q23.3 was the most frequently detected loss.Low/intermediate-grade MEC samples had copy-number-aberrations in chromosomes 1, 12 and 16, while high-grade MECs had a copy-number-aberration in 8p. The most commonly observed copy-number-aberration was the deletion of 3p14.1, which was observed in 4 of the translocation-negative MEC samples. No recurrent copy-number-aberrations were found in translocation-positive MEC samples.Based on these results, we conclude that MECs may be classified as follows: (i) t(11;19)(q21;p13) translocation-positive tumors with no or few chromosomal aberrations and (ii) translocation-negative tumors with multiple chromosomal aberrations.

Establishment and Characterization of a Novel Cell Line, ASAN-PaCa, Derived From Human Adenocarcinoma Arising in Intraductal Papillary Mucinous Neoplasm of the Pancreas

Our aim was to establish and characterize a novel pancreatic ductal adenocarcinoma cell line from a patient in whom the origin of the invasive carcinoma could be traced back to the intraductal papillary mucinous neoplasm (IPMN) precursor lesion. The primary patient-derived tumor was propagated in immunocompromised mice for 2 generations and used to establish a continuous in vitro culture termed ASAN-PaCa. Transplantation to fertilized chicken eggs confirmed the tumorigenic potential in vivo. Molecular analyses included karyotyping, next-generation genomic sequencing, expression analysis of marker proteins, and mucin-profiling. The analysis of marker proteins confirmed the epithelial nature of the established cell line, and revealed that the expression of the mucin MUC1 was higher than that of MUC2 and MUC5AC. ASAN-PaCa cells showed rapid in vitro and in vivo growth and multiple chromosomal aberrations. They harbored mutations in KRAS (Q61H), TP53 (Y220C), and RNF43 (I47V and L418M) but lacked either IPMN-specific GNAS or presumed pancreatic ductal adenocarcinoma-driving mutations in KRAS (codons 12/13), SMAD, and CDKN2A genes. ASAN-PaCa cell line represents a novel preclinical model of pancreatic adenocarcinoma arising in the background of IPMN, and offers an opportunity to study how further introduction of known driver mutations might contribute to pancreatic carcinogenesis.

Biphasic Squamoid Alveolar Renal Cell Carcinoma: A Distinctive Subtype of Papillary Renal Cell Carcinoma?

Biphasic squamoid alveolar renal cell carcinoma (BSARCC) has been recently described as a distinct neoplasm. Twenty-one cases from 12 institutions were analyzed using routine histology, immunohistochemistry, array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization. Tumors were removed from 11 male and 10 female patients, whose age ranged from 53 to 79 years. The size of tumors ranged from 1.5 to 16 cm. Follow-up information was available for 14 patients (range, 1 to 96 mo), and metastatic spread was found in 5 cases. All tumors comprised 2 cell populations arranged in organoid structures: small, low-grade neoplastic cells with scant cytoplasm usually lining the inside of alveolar structures, and larger squamoid cells with more prominent cytoplasm and larger vesicular nuclei arranged in compact nests. In 9/21 tumors there was a visible transition from such solid and alveolar areas into papillary components. Areas composed of large squamoid cells comprised 10% to 80% of total tumor volume. Emperipolesis was present in all (21/21) tumors. Immunohistochemically, all cases were positive for cytokeratin 7, EMA, vimentin, and cyclin D1. aCGH (confirmed by fluorescence in situ hybridization) in 5 analyzable cases revealed multiple numerical chromosomal changes including gains of chromosomes 7 and 17 in all cases. These changes were further disclosed in 6 additional cases, which were unsuitable for aCGH. We conclude that tumors show a morphologic spectrum ranging from RCC with papillary architecture and large squamoid cells to fully developed BSARCC. Emperipolesis in squamoid cells was a constant finding. All BSARCCs expressed CK7, EMA, vimentin, and cyclin D1. Antibody to cyclin D1 showed a unique and previously not recognized pattern of immunohistochemical staining. Multiple chromosomal aberrations were identified in all analyzable cases including gains of chromosomes 7 and 17, indicating that they are akin to papillary RCC. Some BSARCCs were clinically aggressive, but their prognosis could not be predicted from currently available data. Present microscopic, immunohistochemical, and molecular genetic data strongly support the view that BSARCC is a distinctive and peculiar morphologic variant of papillary RCC.

Lymphocytes with multiple chromosomal damages in a large cohort of West Siberia residents: Results of long-term monitoring

Cells with specific multiple chromosome aberrations, defined as rogue cells (RC) have been described in different populations, predominantly those exposed to radiation. The frequency, etiology and related health risks have still not been elucidated due to their low frequency of occurrences and rarely performed studies. This study reports RC frequency using chromosome aberration (CA) assay in peripheral lymphocytes in the group of 3242 subjects, during a 30-year long follow-up study in a general rural and urban population, children environmentally exposed to radon, occupationally exposed population and lung cancer patients from the Kemerovo region (Siberia, Russian Federation). Results show that the highest RC frequency was present in children environmentally exposed to radon and the lowest in the general urban population. Total frequency of CA did not correlate with frequency of RC. Genotoxic analysis of air and water samples excluded anthropogenic pollution as a possible cause of genome damage and RC frequency. In 85% of RCs, double minutes, observed in a large number of human tumors, were present. Results of CA analysis suggested that radon and its decay products (alpha-emitters) were the leading factors causing RC in subjects exposed to high LET radiation. Thus, RC may be a candidate biomarker for exposure to this type of radiation.