Gain Of Chromosome Research Articles

A phase II study of the selective MET kinase inhibitor INC280 in advanced papillary renal cell cancer.

5075 Background: There are currently no approved systemic agents for patients with advanced type 1 papillary renal cell carcinoma (pRCC). Type 1 pRCC can occur in a hereditary form with germline alterations of MET (hereditary papillary RCC: HPRC) or in a sporadic form. Both hereditary and sporadic forms have similar histologic features and somatic MET alterations have been found in 15-20% of patients with sporadic pRCC. Furthermore, gain of chromosome 7, where MET and the gene for its ligand HGF are located, has been reported in a majority of patients with type 1 pRCC and may represent an alternative means of activating the MET pathway. This is a phase II study evaluating the activity of INC280, a highly selective and potent MET inhibitor, in patients with pRCC. Methods: Patients with advanced type 1 pRCC were treated with INC280 at a starting dose of 600mg PO twice daily (capsules). Later in the study course, the capsules were replaced with exposure-equivalent tablets (400mg PO twice daily). Eligible patients had an ECOG PS 0-2, no active brain metastases, and less than 4 prior lines of therapy. The primary endpoint was overall response rate (ORR) assessed by RECIST 1.1. Secondary endpoints included progression-free survival, disease control rate, and tolerability. Exploratory endpoints included the correlation between MET alteration/copy number gain and response to INC280. Results: Twenty subjects were enrolled from January 2014 to October 2019. Median age was 62 years, 60% of patients were classified as IMDC good risk and 40% as IMDC intermediate risk. Three patients (15%) achieved a confirmed partial response and seven (35%) had stable disease, including 4 (20%) who maintained stable disease for over 6 months. The most common treatment-related adverse events (AEs) were of grade 1-2 including nausea (85%), increased creatinine(70%) diarrhea(60%), fatigue(55%), limb edema(40%), increase in amylase(20%), triglycerides(20%), LFTs(20%), lipase(10%) and leukopenia(10%). INC280-related Grade 3-4 AEs included asymptomatic increased lipase (20%), increased LFTs (10%), increased amylase(5%), leukopenia (5%), lymphopenia (5%) and syncope (5%). Conclusions: INC280 demonstrated clinical activity in patients with advanced type 1 pRCC with an acceptable toxicity profile. The correlation between the presence of MET alteration and/or copy number gain and response to INC280 is under evaluation.

Molecular profiling and clinical characteristic of malignant melanoma in younger patients.

e22087 Background: Combination of clinical and molecular data has not yet been well established in adolescent and younger adult patients with melanoma Methods: We performed a retrospective analysis of the molecular profiles and clinical outcome of patients diagnosed at the age younger than 40 treated at UCLA during 2010 to 2019. Patient’s molecular profile, characteristics, and treatment outcome were described using descriptive statistic. Kaplan-Meier curve was used for disease-free survival (DFS) and overall survival (OS). Results: 150 patients with a median age of 29 year (12-39) were analyzed. 101 (67.3%) patients had cutaneous melanoma, 49 (32.7%) had localized uveal melanoma. Of those 101, 23.8% had pre-existing benign or congenital nevus. 70/101 (69.3%) was stage I-II, 26/101 (25.7%) stage III and 5/101 (4.9%) stage IV. Of those 37 patients with molecularly characterized tissue, 23 (62%) had BRAF mutation including 17 V600E, 4 V600 unknown codon, 2 G469A, 2 NRAS and 1 NF-1 mutation. 11/37 were BRAF negative. 79.6% of patients who were not tested were stage I. For patients with stage III, 16/26 (61%) received adjuvant immunotherapy, none of adjuvant targeted therapy. At 35.1 months follow-up time, 27/96 (28%) localized cutaneous melanoma experienced relapse. 1-year DFS was 73% in adjuvant vs. 90% in no adjuvant immunotherapy group. Among 49 uveal melanoma patients, the median age was 27 years old. 23/49 (49%) had a T1 tumor, 18/49 (36.7%) T2, 6/49(12.2%) T3, 1/49(2%) T4. At 45 months follow-up time, three (6.1%) patients developed metastasis. 5-year DFS and OS was 90% and 100%, respectively. 45 patients had tissue for gene expression profiling and/or FISH testing. 15/45 (33%) had class IA, 13/45 (28%) had class IB, and 8/45 (17.7%) had class 2. 24 samples were analyzed by FISH: 14 with disomy 3, 5 with complex disomy 3 with gain of chromosome 6 or 8, and 5 with monosomy 3. Conclusions: Similarly to general population, most of younger patients with melanoma are diagnosed with stage I disease. Among patients with stage III melanoma treated with adjuvant immunotherapy 1-year DFS was comparable to the general population. Patients with stage III who did not receive adjuvant therapy had an excellent DFS which confirms that the decision on selecting patients for treatment was appropriate. The prognosis of younger patients with uveal melanoma was much better than predicted by molecular testing, and much better than expected regardless of gene expression profile or cytogenetic testing.

MON-917 Carney Complex Due to a Contiguous Gene Deletion Syndrome (17q24.2-17q24.3)

BackgroundWhile genomic rearrangements of chromosome 17 are not uncommon, deletions of chromosome band 17q24.2-q24.3 are rare, and associated features include cardiac abnormalities, characteristic facial appearance, short stature, obesity, syndactyly, intellectual disability, seizures, delayed dentition, and features of Carney Complex. It has been suggested that the involvement of KCNJ2, PRKCA, CACNG gene cluster (CACNG1, CACNG4, CACNG5), and PRKAR1A genes contribute to this phenotype. A case of a child with a 3.7 Mb deletion at chromosome band 17q24.2-q24.3, as well as a 2.1 Mb gain at chromosome 17q22, is described.Clinical CaseA now 6 year old female was born at 34 weeks gestational age with prenatal course complicated by oligohydramnios and intrauterine growth restriction. Birth weight was at the 9th percentile, and birth length was at the 92nd percentile. She was noted to have a patent ductus arteriosus (PDA), poor suck and swallow, and dysmorphic features. Chromosome microarray revealed a 3.7 Mb deletion at Chromosome 17q24.2-q24.3, involving KCNJ2, PRKCA, CACNG gene cluster (CACNG1, CACNG4, CACNG5), and PRKAR1A, as well as a 2.1 Mb gain at Chromosome 17q22, involving C17orf112 and KIP2B.At 6 years old, she continues to be small for weight (-4.5 SDs), BMI (-4.22 SDs), and height (-2.5 SDs), though with appropriate pre-pubertal linear growth velocity. She is minimally verbal and continues to receive physical, occupational and speech therapies. Examination showed dysmorphic facial features, including triangular face with pointed chin, prominent forehead with low-set ears, retro-micrognathia, almond-shaped eyes with up-slanting palpebral fissures, bulbous nose, thin lips, and irregularly-shaped teeth. She had bilateral 5th digit clinodactyly, tapering of the distal aspects of bilateral first digits of the hands, and syndactyly of bilateral 2nd/3rd digits of the feet. She had scant freckling over the nasal bridge and cheeks, as well as freckles of the left arm, left groin, and back. She had no clinical stigmata of hypercortisolism. Echocardiogram continues to show a PDA with no cardiac myxomas. Thyroid ultrasound was normal. However, she does have mild hypercalcemia, most recently 2.61 mmol/L (2.15-2.55), and mildly elevated alkaline phosphatase of 341 U/L (96-297).ConclusionThis case highlights a child with many of the previously reported findings associated with 17q24.2-q24.3 deletions. However, she also was noted to have a 2.1 Mb gain at chromosome 17q22 involving C17orf112 and KIP2B genes, which have not yet been associated with a clinical phenotype. It is therefore unclear if her phenotype is partially explained by the chromosomal gain. Clinicians should suspect a contiguous gene deletion syndrome in a patient with Carney Complex and atypical features. Patients with this condition have also been described as “Carney Complex-plus”, a term that we do not recommend be used.

MON-LB48 The Genomic Landscape of Sporadic Thyrotrophinomas

Background: Thyrotrophinoma (TSHoma) is rare and knowledge on the genomic landscape of this tumour type is very limited. Aim: To perform whole-exome sequencing (WES) in a population of TSHomas to identify recurrent somatic genetic events Method: WES was performed on paired tumour and germline DNA of 7 patients with TSHomas. Three tissue samples were formalin-fixed paraffin-embedded and 4 fresh frozen tumour samples. Fresh blood samples were also collected from each patient. The average of mean depth of coverage amongst all samples was 129X, and 97% of target bases were covered ≥20X. Results:Four (57%) of the seven patients were male and median age at diagnosis was 52 years. (IQR 46, 60) Six patients (86%) had macroadenomas. Four patients (57%) had central thyrotoxicosis at diagnosis and three patients’ tumour stained positive for TSH on histology examination. Two patients (29%) had growth hormone co-secreting tumours. In total, 69 somatic variants were identified to be of potential interest, averaging 1.4 variants per million base-pair of DNA read. No variants were observed in more than one individual. According to the GTEx database, 9 of 69 genes (DRC3, HDAC5, KDM1A, POLR21, TCF25, THAP7, TTC13, UNC5D, UNC13A) were highly expressed in the pituitary (top 10%). Four of these genes appear to contribute to tumour development via epigenetic pathway. Specifically, three of these genes (HDAC5, KDM1A, THAP7) either interact with or form part of histone deacetylases whilst POLR21 encodes a subunit of RNA polymerase II which is responsible for mRNA synthesis. On the other hand, TCF25 gene is thought to act as transcriptional repressor and UNC5D plays a role in cell-cell adhesion.Large scale copy number variations involving gain or loss of whole chromosome or chromosome (chr) arm were observed in six (86%) tumour samples. Chr 5, 9, 13 and 19 were most commonly affected by chromosomal gains. Deletion of chr 1p was seen in two cases and mutations in KDM1A (p.Glu161fs/c.482_491delAGGAAGAAAA) and ADGRB2 gene (p.Leu1565Gln/c.4694T>A) were found in each of the remaining single copy of chr 1p. ADGRB2 gene is thought to be involved in cell adhesion and angiogenesis inhibition. Copy neutral loss-of-heterozygosity were present in two (29%) of the tumour samples (chr 2 and 12q). However, no somatic mutation was found in these regions. Gene level copy number analysis identified a potential deletion in TTI2 gene which encodes for a regulator in DNA damaging response as well as telomere length regulation. ConclusionOverall, the rate of somatic variant mutations in TSHomas is low, consistent with the relative benign nature of this tumour type. No classical driver mutations were identified by this study however, chromosomal anomalies and epigenetics may play an important part in TSHoma development.

Clinicopathological and molecular features of hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinomas

AimsHereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder caused by germline mutations in fumarate hydratase (FH). Affected families have an increased risk of renal cell carcinoma...

From the Editor’s Desk…

From the Editor’s Desk…

Abstract P1-05-13: Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer

Abstract Background and Aims: Triple-negative breast cancer (TNBC) is the most aggressive subtype, and is often associated with mutations in the breast cancer susceptibility protein (BRCA) genes, which are known tumor-suppressor genes involved in DNA damage repair. Chromosomal instability (CIN) has been added to the hallmarks of cancer and is characterized by a frequent gain or loss of chromosomes during mitosis (i.e. chromosomal aberrations). BRCA1 and BRCA2 breast tumors develop by specific and distinct evolutionary paths, as their gene profiles and genome aberration spectra differ from each other and from those in sporadic BC. The lncRNA Non-Coding RNA Activated by DNA Damage (NORAD) has been demonstrated to contribute to the progression of several cancer entities by regulating genomic stability. Therefore, we wanted to explore a potential additive anti-cancerous effect by treating TNBC cells with a combination of NORAD silencing and PARP inhibition. Material and Methods: To clarify the potential clinical significance of NORAD in human TNBC, patient data from publicly available databases were analyzed. In order to further characterize NORAD, we performed phenotypic experiments after establishing an siRNA-mediated knock-down approach. Expression levels of NORAD as well as DNA-damage repair proteins were analyzed by qRT-PCR and Western Blot after combined cell treatment with NORAD silencing and PARP inhibition. To further unravel the molecular mechanism behind, we performed immunofluorescent experiments against markers for DNA-double strand breaks and homologous recombination efficiency (i.e. Rad51 and yH2AX) after TNBC treatment with NORAD siRNAs and PARP inhibitors. Results: High NORAD levels were identified to be a negative prognostic factor for disease-free survival of TNBC patients (HR with 95% CI: 2.4 (1.35-4.25); n=161; p=0.002). Furthermore, the NORAD gene is amplified in up to 3% of BC cases and mRNA levels are higher in tumor tissue when compared to healthy tissue. A qPCR based screening of several breast cancer cell lines showed an at least 4-times increased expression of NORAD when compared to normal mammary cells (i.e. MCF 12A). Knock-down of NORAD in TNBC cells resulted in a slightly reduced proliferation as well as to a decreased expression of several DNA repair proteins. Immunofluorescent experiments of TNBC cells treated with the PARP inhibitor olaparib and/or after NORAD silencing revealed increased yH2AX-dependent nuclear foci formation when compared to control conditions, indicating augmented DNA double-strand breaks after treatment. Conclusion: Our first data let us hypothesize that a combination of NORAD knock-down and PARPi treatment increases inhibition of DNA repair mechanisms and leads to a more pronounced “anti-tumorigenic” phenotype in TNBC cells. This approach could lead to the optimization of current treatment concepts. Furthermore, as increased NORAD expression is associated with an unfavorable patient survival, this lncRNA could serve as potential prognostic biomarker. Citation Format: Christiane Klec, Anita Kapeller, Felix Prinz, Christoph Trenk, Herbert Stöger, Martin Pichler. Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-13.

Single-Chromosomal Gains Can Function as Metastasis Suppressors and Promoters in Colon Cancer.

High levels of cancer aneuploidy are frequently associated with poor prognosis. To examine the relationship between aneuploidy and cancer progression, we analyzed a series of congenic cell lines that harbor single extra chromosomes. We found that across 13 different trisomic cell lines, 12 trisomies suppressed invasiveness or were largely neutral, while a single trisomy increased metastatic behavior by triggering a partial epithelial-mesenchymal transition. In contrast, we discovered that chromosomal instability activates cGAS/STING signaling but strongly suppresses invasiveness. By analyzing patient copy-number data, we demonstrate that specific aneuploidies are associated with distinct outcomes, and the acquisition of certain aneuploidies is in fact linked with a favorable prognosis. Thus, aneuploidy is not a uniform driver of malignancy, and different aneuploidies can uniquely influence tumor progression. At the same time, the gain of a single chromosome is capable of inducing a profound cell state transition, thereby linking genomic plasticity, phenotypic plasticity, and metastasis.

Is it forever? Genotoxicological impact of marine contaminants on Arabian/Persian Gulf bivalves: An experimental approach

A novel approach of combining manipulative field experiments and cytogenetic endpoints was used to assess the genotoxic impact of chemical contamination on a local model bivalve species, the pearl oyster -Pinctada imbricata radiata. Baseline levels of Polycyclic Aromatic Hydrocarbons (PAHs), Total Petroleum Hydrocarbons (TPHs) and trace metals were determined from surface sediments and pearl oyster tissue collected from 3 coastal locations in Qatar (Umm Bab, Dukhan and Al-Wakra). Initial aneuploidy –numerical chromosomal abnormality-level in oysters was also assessed during the baseline survey. The oysters’ ability to adapt to new sites, with distinct signatures in terms of chemical contamination, was assessed using a reciprocal transplantation experiment among the same 3 coastal locations, from February to April 2016. Significant differences in sediment contamination levels were detected among the 3 sites. TPHs were in higher concentrations at Umm Bab (western coast), while higher concentrations of metals and PAHs were found at Dukhan (western coast) and Al-Wakra (eastern coast), respectively. Oysters transplanted between locations showed lower levels of aneuploidy, relative to those kept in the original location (controls). Transplanted oysters, regardless of the contamination levels at the origin and destination, showed recovery from initial aneuploidy levels. Moreover, there was a larger percentage of aneuploid cells with chromosomal gain than with chromosomal loss in all treatments, which contrasts with the pattern usually observed in bivalves.

Immunohistochemical expression of p63 in oral premalignant disorders and its correlation with oral squamous cell carcinoma

Introduction: Oral squamous cell carcinoma (OSCC) represents 95% of all head and neck cancers, and its incidence has increased by 50% in the past decade. Accumulation of genetic alterations (mutations, loss of heterozygosity, loss or gain of chromosomes) is the basis for the progression from a normal cell to a cancer cell. OSCCs are frequently preceded by oral potentially malignant disorders (PMDs). It is difficult to predict the malignant transformation of these PMDs. Thus, early detection of genetic changes in PMDs can facilitate detection of those lesions, which may have potential to progress to malignancy. p63 is a protein coded by p63 gene which is a homolog of p53 gene. It has a critical role in cell cycle regulation and is associated with epithelial tumorigenesis. The present study aims to evaluate the role of p63 in carcinogenesis in PMDs (Leukoplakia, Oral submucous fibrosis and Lichen planus) and OSCCs using immunohistochemistry. Aims and Objectives: The aim is to evaluate the expression of the p63 IHC marker in oral premalignant disorders and to compare its expression in OSCC. Materials and Methods: Tissue sections of PMDs (105 cases) and OSCCs (35 cases) were stained for IHC marker p63. The percentage of positive cells and staining patterns were assessed for all the lesions. Results and Conclusions: Progressive accumulation of p63-immunopositive cells from PMDs to OSCC reflects its major role in the development of OSCC according to a multiple-stage model of carcinogenesis and can be a useful indicator of dysplastic change, thus serving as a biomarker for cancer progression.

Meningeal Melanocytoma - Focus on Molecular Aspects with 3 New Molecular Alterations: A Literature Review and Report of Two Cases

Background: Meningeal melanocytoma (MM) is a locally aggressive, low-grade primary melanocytic tumor of the central nervous system (PMN-CNS). According to the literature, GNAQ and GNA11 mutations are relatively frequent. In contrast, BRAF and NRAS mutations are very rare. Other series have reported BAP1 mutation, monosomy 3 and gain of chromosome 6. In this paper, we discussed two cases of MM for whom a FoundationOne CDxTM assay was performed.

Cytogenetic and Molecular Study of an Adult Sclerosing Rhabdomyosarcoma of the Extremity: MYOD1-mutation and Clonal Evolution

Spindle cell/sclerosing rhabdomyosarcoma is a genomically heterogeneous, uncommon subtype of rhabdomyosarcoma, particularly rare in adults. Its MYOD1-mutant variant is aggressive irrespective of age. Cytogenetic data on spindle cell/sclerosing rhabdomyosarcoma are sparse and disparate. Cytogenetic and molecular analyses were performed on an adult sclerosing rhabdomyosarcoma. The karyotype of the sclerosing rhabdomyosarcoma displayed clonal evolution corresponding to two hyperdiploid clones: 48,XY,+i(19)(p10),+22/48,idem,der(9)t(2;9)(q21~22;p21). The changes were gain of chromosome 19 with the overrepresentation of 19p arm, gain of chromosome 22, gain of the 2q arm, and loss of 9p21. Mutation analysis revealed a homozygous c.T365G (p.L122R) mutation of the MYOD1 gene, but none of PIK3CA. To our knowledge, this is the first adult MYOD1-mutant sclerosing rhabdomyosarcoma studied cytogenetically. The only other reported sclerosing rhabdomyosarcoma with MYOD1 mutation and abnormal karyotype was pediatric. Since these tumors are highly aggressive, further studies unravelling their cytogenetic and molecular characteristics are warranted.

BAP1 mutations define a homogeneous subgroup of hepatocellular carcinoma with fibrolamellar-like features and activated PKA

DNAJB1-PRKACA fusion is a specific driver event in fibrolamellar carcinoma (FLC), a rare subtype of hepatocellular carcinoma (HCC) that occurs in adolescents and young adults. In older patients, molecular determinants of HCC with mixed histological features of HCC and FLC (mixed-FLC/HCC) remain to be discovered. A series of 151 liver tumors including 126 HCC, 15 FLC, and 10 mixed-FLC/HCC were analyzed by RNAseq and whole-genome- or whole-exome sequencing. Western blots were performed to validate genomic discoveries. Results were validated using the TCGA database. Most of the mixed-FLC/HCC RNAseq clustered in a robust subgroup of 17 tumors, which all had mutations or translocations inactivating BAP1, the gene encoding BRCA1-associated protein-1. Like FLC, BAP1-HCC were significantly enriched in females, patients with a lack of chronic liver disease, and fibrotic tumors compared to non-BAP1 HCC. However, patients were older and had a poorer prognosis than those with FLC. BAP1 tumors were immune hot, showed progenitor features and did not show DNAJB1-PRKACA fusion, while almost none of these tumors had mutations in CTNNB1, TP53 and TERT promoter. In contrast, 80% of the BAP1 tumors showed a chromosome gain of PRKACA at 19p13, combined with a loss of PRKAR2A (coding for the inhibitory regulatory subunit of PKA) at 3p21, leading to a high PRKACA/PRKAR2A ratio at the mRNA and protein levels. We have characterized a subgroup of BAP1-driven HCC with fibrolamellar-like features and a dysregulation of the PKA pathway, which could be at the root of the clinical and histological similarities between BAP1 tumors and DNAJB1-PRKACA FLCs. Herein, we have defined a homogeneous subgroup of hepatocellular carcinomas in which the BAP1 gene is inactivated. This leads to the development of cancers with features similar to those of fibrolamellar carcinoma. These tumors more frequently develop in females without chronic liver disease or cirrhosis. The presence of PKA activation and T cell infiltrates suggest that these tumors could be treated with PKA inhibitors or immunomodulators.

MPC-14 BRAF V600E MUTANT OLIGODENDROGLIOMA-LIKE TUMORS WITH CHROMOSOMAL INSTABILITY IN ADOLESCENTS AND YOUNG ADULTS

We performed genome-wide methylation analysis on 136 pediatric low grade gliomas, identifying a unique cluster consisting of oligodendroglioma-like BRAF V600E mutant tumors with Recurrent gain of Chromosome 7 and loss of Chromosome 10 (OLIVER). Hierarchical clustering and t-stochastic neighbor embedding analyses cluster them with previously described pediatric-type low grade gliomas, separate from adult gliomas. OLIVERS exhibit distinct clinical behavior as temporal lobe lesions in adolescents and young adults, prolonged history of seizures and all are alive with no recurrence (follow-up 3.2 to 13.2 years). Morphogically, all showed oligodendroglioma-like features, including round nuclei with perinuclear halos, a chicken-wire pattern of branching capillaries and microcalcification. None showed astrocytic features or characteristics suggestive of high-grade tumors including necrosis or mitotic figures. All tumors harbored multiple chromosomal copy number abnormalities (more than 10 chromosomes per OLIVER), but none showed 1p/19q co-deletion or IDH1 mutation. Interestingly, one tumor showed a TERT promoter mutation. Although the series is small, OLIVER may represent a new category of IDH wild-type low grade gliomas which may be confused with molecular GBM. Further, they highlight the heterogeneity of IDH wild-type gliomas and the relatively indolent behavior of pediatric-type gliomas.

Individual Assignment of Adult Diffuse Gliomas into the EM/PM Molecular Subtypes Using a TaqMan Low-Density Array.

We aimed to develop a diagnostic platform to capture the transcriptomic resemblance of individual adult diffuse gliomas of WHO grades II to IV to neural development and the genomic signature associated with glioma progression. Based on the EM/PM classification scheme, we designed a RT-PCR-based TaqMan low-density array (TLDA) containing 44 classifier and 4 reference genes. Samples of a training dataset (GSE48865), characterized by RNA-sequencing, were utilized to optimize the TLDA design and to develop a support vector machine (SVM)-based prediction model. Complemented with Sanger sequencing for IDH1/2, and low coverage whole genome sequencing (WGS), the TLDA and SVM prediction model were tested in a validation (31 gliomas) and a test (121 gliomas) dataset. Independent of morphologically defined subtypes and grades, gliomas can be individually assigned into the EM and PM glioma subtypes with the respective areas under ROC curves at 0.86 and 0.85 in the validation dataset. The EM gliomas showed a medium overall survival (OS) of 15.6 months, whereas the medium OS for PM gliomas was not reached (HR = 3.55; 95% confidence interval, 1.96-6.45). The EM and PM gliomas showed distinct patterns of genomic alterations, with IDH mutation and 1p19q codeletion in the PM gliomas and gain of chromosome 7/loss of chromosome 10 in the EM gliomas. Extensive chromosomal abnormalities marked the progression of PM gliomas. The integration of EM/PM subtyping, IDH sequencing, and low coverage WGS may improve the risk stratification and selection of treatment regimens for patients with glioma.

Chromosomal aberrations in ascetic fluid of metastatic gastric cancer patients: A clustering analysis.

To date, no specific pattern of chromosomal abnormalities has been established in gastric cancer (GC). Cytogenetic analysis was performed using G-banding and fluorescence in situ hybridization (FISH) in 9 ascetic fluids from GC patients, and the clustering patterns of chromosomal abnormalities were studied. Twenty-six different types of chromosomal abnormalities were identified. In contrast to structural abnormalities, the gain or loss of chromosomes was infrequent. Moreover, five main clusters of chromosomal abnormalities were identified by clustering analysis. Extensive cytogenetic complexity, specific chromosomal abnormalities and karyotype heterogeneity are the main characterizations of GC. Some of the recurrent and novel chromosomal abnormalities with distinct clustering patterns identified in this study may play important roles for GC initiation and progression and could serve as promising diagnostic and prognostic markers in GC patients.

Evolutionary analysis of genus Channa based on karyological and 16S rRNA sequence data

A wide range of diploid number of chromosomes and the body size of Channa congeners are useful combination of characters for studying the factors controlling the body size. In this study, the karyological information was superimposed on the evolutionary tree generated by 16S rRNA mitochondrial gene sequences. Here, the metaphase chromosome complements stained with Giemsa, AgNO3 and CMA3 were prepared from six snakehead murrel fish species collected from northeast India. The diploid chromosome numbers and the fundamental arms of C. aurantimaculata (2n = 52, NF = 98), C. gachua (2n = 56, NF = 84), C. marulius (2n = 44, NF = 58), C. orientalis (2n = 52, NF = 74), C. punctata (2n = 32, NF = 60) and C. striata (2n = 40, NF = 48) were calculated by the analysis of metaphase chromosome complements. Both methods of nucleolar organizer region (NOR) localization, silver nitrate and chromomycin A3, revealed NOR pairs of 1, 2, 3, 1, 4 and 3 in C. aurantimaculata, C. gachua, C. marulius, C. orientalis, C. punctata and C. striata, respectively. The subject species showed primitive type of asymmetrical chromosomes, except the C. punctata. The variation in 2n for C. orientalis (2n = 52, 78) and C. gachua (2n = 52, 78, 104) of a complete haploid set indicates the possibility of either ploidy change in C. orientalis and C. gachua, if we consider 2n = 52 or the Robertsonian rearrangements in different populations of these two species. The chromosome evolution tree was constructed on 16S rRNA ML-phylogenetic tree using ChromEvol 1.3. The analysis of chromosome evolution explained the loss or gain of chromosome, duplications or semiduplications mechanism. For time scaling the chromosomeevolution, the node age of available 16S rRNA gene of Channa species were estimated, which was also used for estimating the time when chromosomal changes occurred in context of geological time-scale.

Cold Agglutinin Disease Shows Highly Recurrent Gains of Chromosome 3, 12 and 18

Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia, caused by a distinct type of B-cell lymphoproliferative disease of the bone marrow. Anemia is mediated by binding of monoclonal cold agglutinin antibodies to the erythrocyte surface I antigen at temperatures below central body temperature, followed by agglutination and complement activation. These antibodies are almost exclusively encoded by IGHV4-34. We recently found recurrent KMT2D and CARD11 gene mutations in CAD (Malecka, et al 2017). We analyzed 12 CAD samples from the CAD-5 study (NCT02689986) (Berentsen, et al 2017) using Affymetrix OncoScan CNV Plus Assay and exome sequencing data in order to detect chromosomal aberrations. All samples were sorted using fluorescence activated cell sorting prior to analysis. Complete or partial gain of chromosome 3 (+3 or +3q) was detected in all samples (Table 1). Additionally, most cases showed either gain of chromosome 12 or 18 (9/12) (Table 1). Additional small regions of recurrent gain or loss were also detected in other chromosomes. The recurrent changes in least 4 samples are: +1p36.31-p36.13, -8p21.3-p21.2, +9q34.2-q34.3, +11q13.1-q13.2, +17q25.3, +21q22.3, +22q13.31-q13.33. Gains and losses of large parts of chromosomes are clearly visible in both Affymetrix OncoScan CNV Plus Assay and exome sequencing data (Figure 1). In contrast smaller changes are often not visible in exome sequencing data and therefore could not be fully validated. Further validation will be required for smaller chromosomal changes. Gain of chromosome 3/3q was characteristic for all CAD cases analyzed. Gain of chromosome 3 in CAD has previously been reported in 9 of 26 patients (Gordon, et al 1990, Michaux, et al 1995). Gain of chromosome 3 is not a highly recurrent finding in most B-cell lymphomas with the exception of marginal zone lymphoma (MZL). Of interest, gain of chromosome 12 and 18, demonstrated in our study, is also a feature of MZL. These findings, together with our previous findings (Malecka, et al 2017), suggest that the CAD-associated lymphoproliferative disease, although exclusively present in the bone marrow, might be related to MZL. The CAD-5 study assessed the efficacy of bendamustine plus rituximab in CAD patients. We explored whether a correlation existed between presence or absence of trisomy 12 and 18 with response to therapy. Although the series is small, we found a trend towards poorer response in patients with trisomy 18 compared to patients with trisomy 12. Furthermore, both patients without trisomy 12 or 18 had the best responses (Table 1). Despite the limited number of cases the Pearson correlation was statistically significant (p=0.02). However, more samples need to be analyzed to confirm these results. A correlation between response to therapy and presence of trisomy 12 or 18 has previously been demonstrated for other B-cell lymphomas. Trisomy 12 is frequent in patients with small lymphocytic lymphoma (28-36%) and in chronic lymphocytic leukemia (10-25%) and in the latter +12 is associated with intermediate prognosis (Autore, et al 2018). Trisomy 3 and 18 are found to be correlated with advanced stage extranodal marginal zone lymphoma (Krugmann, et al 2005). There is evidence from previous studies that trisomy 18 may be associated with upregulation of BCL2, an anti-apoptotic gene located on chromosome 18. Whether BCL2 is upregulated in CAD with trisomy 18 and whether it might be the cause of therapy resistance, needs to be investigated. Such a study is of importance since BCL2 inhibitors might be considered to improve treatment responses. We conclude that gain of chromosome 3 is a highly recurrent finding in CAD-associated lymphoproliferative disease. Further gain of chromosome 12 and 18 might be predictors of therapy outcome. These genetic findings are similar to what has previously been demonstrated in nodal and extranodal MZL, suggesting that CAD-associated lymphoproliferative disease, a disease of the bone marrow, might be related to MZL. Disclosures Berentsen: Mundipharma: Research Funding; Apellis, Bioverativ (a Sanofi company), Momenta Pharmaceuticals, and True North Therapeutics: Consultancy; Alexion, Apellis, and Janssen-Cilag: Honoraria. Tjønnfjord:Mundipharma: Research Funding.

Characterization of a Novel JAK1 Pseudokinase Mutation in the First Case of Trisomy 21-Independent GATA1-Mutated Transient Abnormal Myelopoiesis

Clonal proliferation of megakaryoblasts, called transient abnormal myelopoiesis (TAM), is a rare disease of newborns triggered by trisomy 21 (constitutional or somatic) together with acquired mutations of GATA1 resulting in the exclusive production of its short variant - GATA1s. No other TAM drivers have been described so far. We have diagnosed a unique TAM case with a typical clinical and laboratory manifestation but without the gain (or any other aberration) of chromosome 21. Thorough genomic profiling revealed 4 somatic mutations: GATA1 D65_C228del, JAK1 F636del, FN1 R2420C and SPIRE2 R471W. With respect to the generally accepted 2-hit theory, we hypothesized that this TAM arose from a collaboration of the atypical GATA1 mutation (not inducing GATA1s) with (at least) one of the other identified mutations. Unlike SPIRE2 and FN1 aberrations, various mutations of the JAK1 kinase have been previously described as leukemia drivers, suggesting JAK1 F636del as a top candidate for the second hit. Moreover, JAK1 mutations have been associated with the transformation of TAM into acute megakaryoblastic leukemia (Nikolaev et al., Blood, 2013). The aim of our project was to functionally characterize this novel JAK1 mutation. Phenylalanine 636 belongs to a phylogenetically conserved triad of amino acids suggested to control catalytic activity of JAK1 via mediating a switch between the supposedly active and inactive conformations (Toms et al., Nat Struct Mol Biol, 2013). Hence, F636 seems to be essential for JAK1 function. Surprisingly, homology modeling showed that loss of F636 is compatible with both functionally opposite conformations. Indeed, Western blot analysis of JAK/STAT signaling in transiently transformed HEK293T cells showed that catalytic activity is preserved in JAK1 F636del. However, we observed lower levels of auto- and STATs- phosphorylation compared to wild-type (wt) JAK1 suggesting decreased kinase activity of JAK1 F636del. Subsequently, we tested the oncogenic potential of JAK1 F636del in the Ba/F3 cell assay; unlike the known oncogenic JAK1 variant (JAK1 V658I), JAK1 F636del did not induce IL3-independent growth. To further assess phenotypic impact of F636del, we introduced JAK1 F636del into murine bone marrow and fetal liver hematopoietic stem and progenitor cells (HSPCs) using lentiviruses and performed colony forming assays. The number and morphology of colonies did not differ in JAK1 F636del compared to wt JAK1. Furthermore, we assessed the impact of JAK1 F636del in the context of mutated GATA1. We utilized the in-vitro model recently described by Labuhn et al. (Cancer Cell, 2019), in which the CRISPR/Cas9-mediated induction of Gata1s expression leads to the expansion and sustained proliferation of fetal liver HSPCs from embryonic day 13.5 ROSA26:Cas9-EGFPki/wt mice. Similar to wt JAK1, lentivirally introduced JAK1 F636del had no impact on the proliferation and maturation status of such Gata1-edited HPSCs irrespective of the timing of its introduction (simultaneously with Gata1 editing versus into fully established Gata1-edited culture) or of culturing conditions (fully cytokine-supplemented growth-supportive versus cytokine-depleted growth-restrictive medium). Altogether, we show that unlike known oncogenic variants, F636del identified in the first case of trisomy 21-independent TAM attenuates JAK1 kinase activity. The results of our phenotypic studies question the potential contribution of this mutation to TAM development. Interestingly, Labuhn et al. (2019) recently showed that non-activating JAK mutations occur at higher than random frequency in trisomy 21-dependent TAM. This tempts us to speculate that JAK1 mutations may still play a role in TAM. Yet, this role may significantly differ from that of known oncogenic mutations; it may result from attenuation/modulation instead of activation of downstream signaling and it may remain unrevealed utilizing the currently available sophisticated, yet still imperfect experimental models. Support: GAUK 86218, EHA Research Mobility Grant Disclosures No relevant conflicts of interest to declare.

RNA-Seq Can Help Identify IGH Disease Clones for MRD Monitoring in Childhood B-Lymphoblastic Leukemia

Background As a single platform, RNA sequencing (RNA-Seq) can accurately identify majority of oncogenic driver fusion genes and aneuploidy. This provides refined molecular subtyping and risk stratification; RNA-Seq is increasing used upfront for genetic subtype assignment in on-going contemporary ALL trials. The IGH gene is highly expressed in our diagnostic ALL samples using RNA-Seq. We investigated the usefulness of RNA-Seq in identifying IGH disease sequences for MRD monitoring in 258 childhood B-lymphoblastic leukemia (B-LL) samples. Methods RNA-seq was performed on a total of 259 diagnostic bone marrow or peripheral blood samples from children with B-LL treated in the Ma-Spore ALL 2003 and ALL 2010 studies. The reads were aligned to germline IGH gene V, D and J segments, and reads covered IGH VHDJH or DJH junctional regions are clustered into clones. Overall, the distribution of read count of the IGH background clones follows Zipf's law, i.e. the read count of the IGH background clones is negative linearly correlated with the number of IGH clones with that read count in the log-log scale (Figure 1A). IGH disease clones are outliers with exceedingly high read count. For each sample, a linear model is fitted between log transformed read count (using only read count ≤10) and log transformed number of IGH clones. The expected number of IGH clones (E-value) of a read count can be predicted by the fitted linear model. A clone with E-value <0.01 is defined as a disease clone (Figure 1B). Disease clones identified by RNA-Seq are compared with conventional Sanger sequencing. Results In 90.3% of the patients, 497 IGH disease clones (median 2, range 0-7 clones/patient) are identified. The number of IGH disease clones significantly correlated with genetic subtype (P=1.9×10-8; Table 1). Specifically, high hyperdiploid patients have the most IGH disease clones (median 3) with 67% (22/33) have >2 IGH disease clones, due to the gain of chromosome 14. In contrast, DUX4 high expressers have the least (median 1) IGH disease clones with 19% (8/43) have no IGH disease clone, due to the rearrangements involving one of the IGH locus. Of the 348 IGH disease clones identified by Sanger sequencing, 90.8% are also identified by RNA-Seq; and in the 199 Sanger sequences that yielded sensitive markers, 191 (96.0%) are also defined as IGH disease clones by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets by Sanger sequencing, the disease clones identified by RNA-Seq were used collectively for MRD monitoring by targeted deep sequencing of IGH (IGH-Seq) and showed high correlation with conventional RQ-PCR MRD using non-IGH targets (R=0.93; P=1.3x10-14;Figure 1C). In addition, IGH-Seq showed improved sensitivity with quantifiable MRD in 20% more samples compared to RQ-PCR (78% vs 58%; Figure 1C). In the samples that are positive below sensitivity or negative by RQ-PCR, IGH-Seq obtained quantifiable MRD for 60% (9/15) or 25% (5/20) of the samples (Figure 1C). Conclusion In conclusion, IGH disease clone sequences are highly expressed and can be distinctly identified from whole transcriptome RNA-Seq in >90% of children with B-LL. These can be used for molecular MRD monitoring using RQ-PCR or NGS-based IGH-Seq. In addition to identifying the constellation of oncogene fusions and chromosomal aneuploidies that drive B-LL and RNA-Seq can identify IGH MRD sequences for molecular MRD monitoring. Disclosures No relevant conflicts of interest to declare.