Spectral Karyotyping Analysis Research Articles

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication.

Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

Molecular Cytogenetics Reveals Mosaicism in Human Umbilical Vein Endothelial Cells

Primary human umbilical vein endothelial cells (HUVECs) are consistently the most reliable in vitro model system for studying the inner lining of blood and lymphatic vessels or the endothelium. Primary human cells originate from freshly isolated tissues without genetic manipulation and generally show a modal number of 46 chromosomes with no structural alterations, at least during early passages. We investigated the cytogenetic integrity of HUVECs with conventional (G-banding) and molecular cytogenetic methods (spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH)). Our G-band data shows two X-chromosomes, confirming these HUVECs originate from a female donor. Notably, some cells consistently exhibit an unfamiliar banding pattern on one X chromosome toward the distal end of the long arm (Xq). Our FISH analysis confirms that approximately 50% of these HUVECs have a deletion of the Xq terminal region. SKY analysis indicates that the deleted region is apparently not integrated into any other chromosome. Finally, we demonstrated the presence of a similar Xq deletion in the daughter cell line, EA.hy926, which was generated by fusing HUVECs with A549 (a thioguanine-resistant clone of adenocarcinomic human alveolar basal epithelial cells). These findings will advance comprehension of HUVECs biology and will augment future endothelial studies.

Abstract 5821: The role of SMARCAL1 loss-of-function mutations in glioblastoma telomere maintenance

Abstract Malignant tumor cells overcome the restriction of progressive telomere shortening by employing telomere maintenance mechanisms (TMMs) to achieve replicative immortality. Over 80% of human cancers maintain telomere length using the enzyme telomerase, which uses an RNA template subunit. However, other cancers maintain their telomeres in a telomerase-independent manner, also known as Alternative Lengthening of Telomeres (ALT). Recently, we identified loss-of-function mutations in the SMARCAL1 gene as novel genetic alterations associated with the ALT phenotype in a subset of GBM that is wildtype for IDH1 and TERT promoter mutations. SMARCAL1 encodes an annealing helicase that localizes to sites of DNA damage and/or replication stress and resolves stalled replication fork structures to facilitate fork progression within difficult-to-replicate DNA sequences, such as telomeres. However, limited models are available to study the underlying mechanisms associated with SMARCAL1 deficiency and ALT induction, and tumorigenic patient-derived xenograft (PDX) models of SMARCAL1 deficient GBM have yet to be established. In this study, we sought to generate an ALT-positive, SMARCAL1-deficient PDX by subcutaneously transplanting the D06MG cell line, which harbors a native SMARCAL1 loss-of-function mutation, into immune-compromised mice. Transplanted cells formed palpable subcutaneous tumors in nude mice within approximately 6 months. Tissue from these tumors was harvested, disaggregated, and intracranially transplanted into nude mice. Immune compromised mice bearing intracranial xenografts showed neurological symptoms due to tumor growth within three months. Cell lines were derived from PDX tissue for further characterization of telomere-related endpoints. PDX-derived D06MG cell lines maintained ALT status, as evidence by a lack of telomerase activity, presence of APBs (ALT-associated promyelocytic leukemia (PML) bodies), and extrachromosomal C-circles. PDX-derived D06MG cell lines were characterized relative to the parental cell line using SKY (spectral karyotyping) and RNA-seq analyses. SKY analysis revealed that, during the in vivo selection, a unique homogeneous subpopulation emerged, which remained genetically stable even after intracranial injection. The majority of PDX-derived cells are diploid and share similar chromosome aberrations. RNA-sequencing and pathway enrichment analyses revealed that several signal transduction pathways were upregulated in the D06MG PDX lines, while cytosolic DNA sensing pathways were partially suppressed. In summary, we have generated and characterized a PDX model of SMARCAL1-deficient GBM that maintains telomeres via the ALT pathway. Citation Format: Heng Liu, Cheng Xu, Lee H. Chen, Bill H. Diplas, Jinjie Ling, Paula K. Greer, Martin Roskoski, Stephen T. Keir, David M. Ashley, Hai Yan, Matthew S. Waitkus. The role of SMARCAL1 loss-of-function mutations in glioblastoma telomere maintenance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5821.

Identification of novel breakpoints for locus- and region-specific translocations in 293 cells by molecular cytogenetics before and after irradiation

The human kidney embryonic 293 cell line (293 cells) is extensively used in biomedical and pharmaceutical research. These cells exhibit a number of numerical and structural chromosomal anomalies. However, the breakpoints responsible for these structural chromosomal rearrangements have not been comprehensively characterized. In addition, it is not known whether chromosomes with structural rearrangement are more sensitive to external toxic agents, such as ionizing radiation. We used G-banding, spectral karyotyping (SKY), and locus- and region-specific fluorescence in situ hybridization (FISH) probes designed in our lab or obtained from commercial vendor to address this gap. Our G-banding analysis revealed that the chromosome number varies from 66 to 71, with multiple rearrangements and partial additions and deletions. SKY analysis confirmed 3 consistent rearrangements, two simple and one complex in nature. Multicolor FISH analysis identified an array of breakpoints responsible for locus- and region-specific translocations. Finally, SKY analysis revealed that radio-sensitivity of structurally rearranged chromosomes is dependent on radiation dose. These findings will advance our knowledge in 293 cell biology and will enrich the understanding of radiation biology studies.

Our Unique Cell Lines Derived Serially After Injection With Human T24 HRAS Transformed NIH/3T3 Mouse Cells (GhrasT‐NIH/3T3) Showed The Highly Tumorigenic T1‐A Cells Cultured From The First Primary Tumor Had A Six‐Fold Higher LD50 For Resveratrol Killing Than The LD50 For Highly Metastatic Lung T4‐PA Cells Derived After Three Subsequent In Vivo / In Vitro Serial Manipulations

Our new cell lines demonstrating progressively increasingly metastatic behavior as they were derived in a series of in vivo/in vitro manipulations (Figure 1) provide the opportunity to evaluate in vivo and in vitro compounds and methods to kill primary tumor cells and various forms of increasingly metastatic cells derived from the same primary tumor (Figure 2) (Ray, D.B., et. al., Experimental Cell Research, 2016 Jan; 340 (1): pp. 1–11). We determined the dose of the anti‐cancer natural phytoalexin resveratrol to kill 50% (LD50s) of our various cells that represent progressive cancer stages. Cell counts were recorded daily, over the course of 3 to 6 days, using gridded plates and photomicrographs of six pre‐determined 2 mm square sections of each plate. An average percent change in cell number from each individual grid was then calculated. P‐values were determined between each treatment time and dose and significance was determined. The NIH Swiss and NIH/3T3 cells were not significantly affected by 3–300 μM after 3 days. After 3 or 4 days the LD50s for the tumorigenic cells were GhrasT‐NIH/3T3 cells: LD50 = 3.5+/− 1.0 μM; T1‐A cells: LD50 = 45 +/− 5.0 μM; T4‐PA cells: LD50 = 7.0 +/− 1.5 μM. Thus, unexpectantly, the highly tumorigenic T1‐A cells were significantly more difficult to kill than the highly metastatic T4‐PA cells. This pattern may mimic the epithelial to mesenchymal transition (EMT) seen in breast and other cancer progression. EMT enhances mobility, invasion, and resistance to apoptotic stimuli as tumor cells acquire stem cell properties and exhibit marked therapeutic resistance. RNA‐Seq, genome sequencing (WGS) and spectral karyotyping (SKY) analysis of these cells demonstrating increasingly aggressive metastatic cancer tumorigenesis that progressed from a single nuclear genotype and mitochondrial haplotype should help contribute to the understanding of cancer progression. These cells are available by contacting our laboratory.Support or Funding InformationSupported in part by the National Science Foundation grant # 8602787, the Grove City College Research Fund, the Grove City College Swezey Research Fund and the Grove City College Janicki Foundation Research Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The genomic landscape of UM-SCC oral cavity squamous cell carcinoma cell lines

ObjectivesWe sought to describe the genetic complexity of 14 UM-SCC oral cavity cancer cell lines that have remained uncharacterized despite being used as model systems for decades. Materials and MethodsWe performed exome sequencing on 14 oral cavity UM-SCC cell lines and denote the mutational profile of each line. We used a SNP array to profile the multiple copy number variations of each cell line and use immunoblotting to compare alterations to protein expression of commonly amplified genes (EGFR, PIK3CA, etc.). RNA sequencing was performed to characterize the expression of genes with copy number alterations. ResultsThe cell lines displayed a highly complex network of genetic aberrations that was consistent with alterations identified in the HNSCC TCGA project including PIK3CA amplification, CDKN2A deletion, as well as TP53 and CASP8 mutations, enabling genetic stratification of each cell line in the panel. Copy number FISH and spectral karyotyping analysis demonstrate that cell lines retain chromosomal heterogeneity. ConclusionsCollectively, we developed an important resource for future oral cavity HNSCC cell line studies and highlight the complexity of genomic aberrations in cell lines.

Abstract 3104: Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient

Abstract (Objective) The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. (Materials and methods) Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109.(Results) UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays and mouse xenografts supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers.(Conclusion) We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers. Citation Format: Steven J. Wang, Saurabh Asthana, Annemieke van Zante, Adam B. Olshen, Frank McCormick, Osamu Tetsu. Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3104.

Abstract 3388: Molecular characterization of HPV type 18 cervical cancer: Upregulation of telomerase expression and induced chromosomal instability by E6 and E7 oncoproteins

Abstract Human papillomavirus (HPV), high-risk type 18, is directly associated with approximately 98% incidence of invasive cervical cancer. Epithelial cells infected with HPV-18 become transformed and exhibit overexpression of telomerase activity and chromosomal instability. As a result of this transformation, E6 and E7 oncoproteins are perpetually expressed: E6 degrades the tumor suppressor p53; E7 inhibits the tumor suppressor pRB, leading to uncontrollable growth and the extension of telomere length. While a few studies explored the chromosomal instability induced by HPV E6 and E7 oncoproteins, the full scope of the problem has not been clearly characterized. In this research we investigated HPV-18 acquired genomic instability by E6 and E7 oncoproteins. HPV-positive cervical cancer cells, HeLa, were studied through spectral karyotyping (SKY), Giemsa banding (G-banding), telomere length, and telomerase activity using PCR ELISA assay and the TRAPeze XL Kit, which uses fluorescence energy transfer. With the use of SKY, G-banding, and chromosomal instability analysis, HeLa cells exhibited many aneuploidies. In our results, there were consistent translocations on chromosomes 4 and 11, with deletions on chromosomes 11 and 20. There were also other translocations on chromosomes 9 and 20, with deletions on chromosome 10. Additionally, copies of chromosome 5 and unidentifiable marker chromosomes were noted. Moreover, telomerase data suggest that upregulated expression of telomerase activity correlates with the increase in chromosomal instability. The aforementioned aneuploidy is demonstrative of the induced chromosomal instability from HPV-18 infection. In addition to the tumor-suppressor disruption acquired by E6 and E7 oncoproteins, HeLa cells showed gene deletion on chromosome 11. The ATM gene, which is specifically located between distal regions 11q22.3 and 11q23 of chromosome 11, is known to help identify breaks in DNA and plays a crucial role in DNA repair. Damage to the ATM gene may further play an important role in increasing cancer progression. Citation Format: Mai Do, Nichelle Cox, Naomi Long, Liliana Zarate, Chinelo Ezechukwu, Jenna Cormier, Hyun Chung, Meidrah Tyler, Judith Okoro, Diondra Harris, Victoria Vidal, Jerica Watson, Ellie Canty, Shyam Arya, Benjamin Liu, Roland Pattillo, Billy Ballard, Jay Vadgama, Eva McGhee. Molecular characterization of HPV type 18 cervical cancer: Upregulation of telomerase expression and induced chromosomal instability by E6 and E7 oncoproteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3388.

Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells

Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as “Limited” or “Significant”. Previously reported stepwise acquisition of a spontaneous neoplastic phenotype during three-year continuous culture of telomerized cells (hBMSC-TERT20) didn't alter a diploid karyotype measured by spectral karyotype analysis (SKY). Such screening may not adequately monitor abnormal and potentially tumorigenic hBMSC in clinical scenarios. We here used array comparative genomic hybridization (aCGH) to more stringently compare non-tumorigenic parental hBMSC-TERT strains with their tumorigenic subcloned populations. Confirmation of a known chromosome 9p21 microdeletion at locus CDKN2A/B, showed it also impinged upon the adjacent MTAP gene. Compared to reference diploid human fibroblast genomic DNA, the non-tumorigenic hBMSC-TERT4 cells had a copy number variation (CNV) in at least 14 independent loci. The pre-tumorigenic hBMSC-TERT20 cell strain had further CNV including 1q44 gain enhancing SMYD3 expression and 11q13.1 loss downregulating MUS81 expression. Bioinformatic analysis of gene products reflecting 11p15.5 CNV gain in tumorigenic hBMSC-TERT20 cells highlighted networks implicated in tumorigenic progression involving cell cycle control and mis-match repair. We provide novel biomarkers for prospective risk assessment of expanded stem cell cultures.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 21q11.2-q21.1 and a literature review

ObjectiveWe present prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 21q11.2-q21.1, and we review the literature of an sSMC(21) with a duplication of 21q11.2-q21.1. Case reportA 40-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XX,+mar [18]/46,XX [4]. The parental karyotypes were normal. Prenatal ultrasound findings were unremarkable. aCGH analysis of cultured amniocytes revealed a 2.855-Mb duplication of 21q11.2-q21.1 encompassing the genes of LIPI, ABCC13 and NRIP1. Metaphase fluorescence in situ hybridization analysis on cultured amniocytes revealed a result of 47,XX,+mar .ish der(13/21) (D13/21Z1+) [10]. Spectral karyotyping analysis determined the origin of chromosome 21 in the sSMC. A female fetus was delivered with no phenotypic features of Down syndrome and no structural abnormalities. We discuss the genotype–phenotype correlation of LIPI, ABCC13 and NRIP1, and review the literature of an sSMC(21) associated with dup(21)(q11.2q21.1). ConclusionaCGH is useful for identification of the nature and genetic component of a prenatally detected sSMC.

Establishment and characterization of an oral tongue squamous cell carcinoma cell line from a never-smoking patient

ObjectiveThe rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. Materials and methodsFresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109. ResultsUCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers. ConclusionWe have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 2

ObjectiveWe present prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 2. Case ReportA 42-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XY,+mar[10]/46,XY[12]. The parental karyotypes were normal. Array comparative genomic hybridization analysis of the DNA extracted from cultured amniocytes revealed no genomic imbalance. Spectral karyotyping analysis failed to identify the sSMC. Metaphase fluorescence in situ hybridization analysis using the satellite probes CEP1/5/19, CEP2, CEP3, CEP4, CEP6, CEP7, CEP8, CEP9, CEP10, CEP12, CEP13/21, CEP14/22, CEP15, CEP16, and CEP20 revealed a result of 47,XY,+mar .ish der(2)(D2Z+)[10]. The sSMC was derived from the α satellite of chromosome 2. Polymorphic DNA marker analysis using the markers specific for chromosome 2 on the DNAs extracted from cultured amniocytes and parental bloods excluded uniparental disomy 2. At 39 weeks of gestation, a healthy 3394-g male baby was delivered with no phenotypic abnormality. The cord blood had a karyotype of 47,XY,+mar[21]/46,XY[19]. ConclusionArray comparative genomic hybridization and spectral karyotyping may fail to detect an sSMC derived from α satellite, which needs satellite probes for confirmation.

First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation

BackgroundNeurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined.Case presentationHere, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansionConclusionsThis is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma. Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may lead to elucidation of their molecular implications in meningioma pathogenesis.

Abstract 4072: Molecular cytogenetic characterization of HPV types 16 and 18 cervical cancers: Acquired genomic instability by E6 and E7 oncoproteins

Abstract Human Papillomavirus (HPV) high-risk types 16 and 18 are directly associated with approximately 90% incidence of invasive cervical carcinoma. Epithelial cells infected with HPV become transformed and acquire genomic instability. As a result of this transformation, E6 and E7 oncogenes are perpetually expressed. E6 degrades the tumor suppressor p53; E7 inhibits tumor suppressor pRB, leading to disproportionate target cell growth and proliferation. While there have been studies exploring genomic instability induced by HPV oncoproteins E6/E7, the full scope of the genomic damage has not been clearly characterized. Three HPV positive cervical cancer cell lines, HeLa (HPV18), SiHa (HPV16) and CaSki (HPV16), were studied using Spectral Karyotyping (SKY) and Giemsa banding (G-banding). Our results using SKY and G-banding analysis showed HeLa cells exhibited frequent translocations on chromosomes 4 and 11, and deletions on chromosomes 11 and 20. The SiHa cells exhibited translocations primarily on chromosomes 9 and 20, with deletions on chromosome 10. CaSki revealed translocations on chromosomes 10 and 4, with deletions on chromosome 21. All three cell lines gained copies on chromosome 5. The aforementioned aneuploidy is demonstrative of the induced genomic instability acquired from HPV infection, which include tumor suppressor genes; p53 and pRB disruptions caused by E6 and E7 oncoproteins. HeLa cells showed extensive genomic instability on chromosome 11q22-23, where the ATM gene is located. Acquired genomic instability to the ATM chromosome region may further increase the rate of cervical cancer. Citation Format: Karen Tate, Lucy Tran, Melanie Baker, Kamilah Evans, Mechelle Rouse, Seyung Chung, Duy Nguyen, Juri Kim, Enijah Smith-Joe, Jay Vadgama, Mengtao Li, Yu-Ling Lin, Roland Patillo, Eva McGhee. Molecular cytogenetic characterization of HPV types 16 and 18 cervical cancers: Acquired genomic instability by E6 and E7 oncoproteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4072.

A der(11)t(4;11)(q21;p15) in a T-ALL/LBL patient

Translocation t(4;11)(q21;p15) is a rare recurrent change associated to T-cell acute leukemia. In most cases, this alteration appears as the only abnormality or as part of a simple karyotype. In this report, we present the first case of T acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) with the unbalanced translocation der(11)t(4;11)(q21;p15) as part of a very complex karyotype with multiple chromosome abnormalities, most of them not previously described in the literature. FISH (fluorescence in situ hybridization) and spectral karyotype (HiSKY) analysis confirmed the presence of complex alterations. The patient, a 16-year-old male, showed poor response to treatment and short survival (11 months). A detailed review of previously reported cases with t(4;11)(q21;p15) is also provided. The description of this type of alterations may contribute to the identification of new molecular mechanism associated to neoplastic development.

Abstract A19: A novel cell line model for chromophobe renal cell carcinoma, UOK276, derived from an aggressive sarcomatoid differentiated tumor

Abstract Background: Renal cell carcinoma (RCC) represents a heterogeneous group of cancers that arise from the nephron and are subtyped by histopathological features. The most common subtypes are clear cell RCC (~75%) and papillary RCC (~15%); whereas chromophobe renal cell carcinoma (ChRCC) represents only 5% of RCC cases. ChRCC typically demonstrate a well-known karyotype of multiple chromosomal losses and a relatively indolent pattern of local growth, but can present with aggressive features and demonstrate resistance to treatment in a metastatic setting. Some ChRCC cases demonstrate regions of sarcomatoid RCC and the exact cause of this differentiation has yet to be elucidated. Cell line models are an important tool for both the investigation of tumor biology and therapeutic drug efficacy. Currently, numerous cell lines models exist that have been derived from sporadic clear cell or papillary RCCs, but there are few cell lines derived from chromophobe RCCs and none are well characterized. This study produced a novel ChRCC-derived cell line model and provides an initial genetic and metabolic characterization. Materials and Methods: A patient presented with a 20 cm ChRCC with regions of sarcomatoid differentiation that was surgically excised and a section of this tumor was used to establish a spontaneously immortal cell line model, UOK276. This line was grown for over 20 passages and cytogenetically assessed by spectral karyotyping (SKY). Mutation analysis was performed using a cancer gene specific chip, OncoVar V3, which analyses 232 genes. Identified mutations were confirmed in both UOK276 and the original tumor tissue and further investigated for their effects of mRNA and protein expression. UOK276 cells were injected into nude mice to assess the production of xenograph tumors. The metabolic, bioenergetic profile was assessed using a Seahorse XF96 Extracellular Flux Analyzer. Results: The chromosomal SKY analysis did not demonstrate the classic pattern of chromophobe chromosomal losses, but demonstrated hyper-aneuploidy, with a modal number of 49 chromosomes per cell, and identified a balanced translocation t(X;8)(q10;q24). The break on chromosome 8q occurred near the MYC gene, but break-apart FISH analysis demonstrated no alterations to MYC although amplification of this derivative chromosome was observed and increased MYC mRNA expression was demonstrated. Mutation analysis identified a missense mutation (p.H193Y) of TP53, commonly mutated in ChRCC, which was only present in the sarcomatoid region of the tumor. Mutation of TP53 has previously been associated with sarcomatoid differentiation. Protein expression analysis demonstrated the presence of the mutant TP53 protein in UOK276. A heterozygous germline mutation in TRAF7 was identified resulting in an in-frame loss of 4 amino acids (del T22-P25) that was homozygous in the sarcomatoid tumor region and UOK276. Xenograph tumors were successfully grown in nude mice and provide an in vivo animal model for the investigation of potential therapeutic regimes. The recent TCGA study of ChRCC demonstrated increased expression of the electron transport chain (ETC) genes suggesting increased oxidative phosphorylation within these tumors. Metabolic analysis of UOK276 demonstrated a relatively low level of oxygen consumption (OCR) in comparison to a normal kidney cell line and this was supported by mRNA expression data showing normal or reduced levels of expression for several ETC-related genes. Conclusions: Our study has produced a novel ChRCC cell line model that exhibits a TP53 mutation, commonly seen in ChRCC, and represents a sarcomatoid differentiated region of the tumor. UOK276 should provide a unique in vitro and in vivo preclinical model system for studying the deregulated pathways and testing therapeutic strategies in sarcomatoid differentiated ChRCC. Citation Format: Youfeng Yang, Cathy D. Vocke, Christopher J. Ricketts, Darmood Wei, Hesed M. Padilla-Nash, Shawna L. Boyle, Robert Worrell, Thomas Ried, Maria J. Merino, W. Marston Linehan. A novel cell line model for chromophobe renal cell carcinoma, UOK276, derived from an aggressive sarcomatoid differentiated tumor. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A19.

T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA

Cancer cells often arise progressively from “normal” to “pre-cancer” to “transformed” to “local metastasis” to “metastatic disease” to “aggressive metastatic disease”. Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic “punctuated evolution” (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6–10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7–10 days. T1-A, T-2A, T3-HA and T4-PA cells when injected i.v. into the tail produced local metastasis in non-nude or nude NIH/Swiss mice. T4-PA cells were more widely metastatic than T3-HA cells when injected i.v. into nude mice. Evaluation of the injected mice indicated a general increase in metastatic potential of each cell line in the progression as compared to the GhrasT-NIH/3T3 transformed cells. A new photomicrographic technique to follow growth rates within six preselected 2×2mm2 grids per plate is described. Average doubling times of the transformed cells GhrasT-NIH/3T3 (17h), T1A (17.5h), T2A (15.5h), T3-HA (17.5h) and T4-PA (18.5h) (average 17.2h) were significantly faster (P=0.006) than NIH Swiss primary embryonic cells and NIH/3T3 cells (22 h each). This cell series is currently used in this lab for studies of cancer cell inhibitors, mitochondrial biogenesis and gene expression and is available for further study by other investigators for intra- and inter-laboratory comparisons of WGS, transcriptome sequencing, SKY and other analyses. The genome rearrangements in these cells together with their phenotypic properties may help provide more insights into how one tumorigenic progression occurred to produce the various cell lines that led to the highly metastatic T4-PA cell line.

Abstract 2029: Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model

Abstract MCPH1, also known as BRIT1, has recently been identified as a novel key regulatory gene of the DNA damage response pathway. MCPH1 is located on human chromosome 8p23.1, where human cancers frequently show loss of heterozygosity. As such, MCPH1 is aberrantly expressed in many malignancies, including breast and ovarian cancers, and the function of MCPH1 has been implicated in tumor suppression. However, it remains poorly understood whether MCPH1 deficiency leads to tumorigenesis. Here, we generated and studied both Mcph1−/− and Mcph1−/−p53−/− mice; we showed that Mcph1−/− mice developed tumors with long latency, and that primary lymphoma developed significantly earlier in Mcph1−/−p53−/− mice than in Mcph11+/+p53−/− and Mcph1+/−p53−/- mice. The Mcph1−/−p53−/− lymphomas and derived murine embryonic fibroblasts (MEFs) were both more sensitive to irradiation. Mcph1 deficiency resulted in remarkably increased chromosome and chromatid breaks in Mcph1−/−p53−/− lymphomas and MEFs, as determined by metaphase spread assay and spectral karyotyping analysis. Additionally, Mcph1 deficiency significantly enhanced aneuploidy as well as abnormal centrosome multiplication in Mcph1−/−p53−/− cells. Meanwhile, Mcph1 deficiency impaired double strand break (DSB) repair in Mcph1−/−p53−/− MEFs as demonstrated by neutral Comet assay. Compared with Mcph1+/+p53−/− MEFs, homologous recombination and non-homologous end joining activities were significantly decreased in Mcph1−/−p53−/− MEFs. Notably, reconstituted MCPH1 rescued the defects of DSB repair and alleviated chromosomal aberrations in Mcph1−/−p53−/- MEFs. Taken together, our data demonstrate MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. Our study using knockout mouse models provides convincing genetic evidence that MCPH1 is a bona fide tumor suppressor gene. Its deficiency leading to defective DNA repair in tumors can be utilized to develop novel targeted cancer therapies in the future. Citation Format: Yulong Liang, Hong Gao, Shiaw-Yih Lin, John A. Goss, Chunying Du, Kaiyi Li. Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2015-2029

Assessment of Cancer Cell Line Representativeness Using Microarrays for Merkel Cell Carcinoma

When using cell lines to study cancer, phenotypic similarity to the original tumor is paramount. Yet, little has been done to characterize how closely Merkel cell carcinoma (MCC) cell lines model native tumors. To determine their similarity to MCC tumor samples, we characterized MCC cell lines via gene expression microarrays. Using whole transcriptome gene expression signatures and a computational bioinformatic approach, we identified significant differences between variant cell lines (UISO, MCC13, and MCC26) and fresh frozen MCC tumors. Conversely, the classic WaGa and Mkl-1 cell lines more closely represented the global transcriptome of MCC tumors. When compared to publicly available cancer lines, WaGa and Mkl-1 cells were similar to other neuroendocrine tumors, but the variant cell lines were not. WaGa and Mkl-1 cells grown as xenografts in mice had histological and immunophenotypical features consistent with MCC, while UISO xenograft tumors were atypical for MCC. Spectral karyotyping and short tandem repeat analysis of the UISO cells matched the original cell line’s description, ruling out contamination. Our results validate the use of transcriptome analysis to assess the cancer cell line representativeness and indicate that UISO, MCC13, and MCC26 cell lines are not representative of MCC tumors, whereas WaGa and Mkl-1 more closely model MCC.

Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model.

MCPH1, also known as BRIT1, has recently been identified as a novel key regulatory gene of the DNA damage response pathway. MCPH1 is located on human chromosome 8p23.1, where human cancers frequently show loss of heterozygosity. As such, MCPH1 is aberrantly expressed in many malignancies, including breast and ovarian cancers, and the function of MCPH1 has been implicated in tumor suppression. However, it remains poorly understood whether MCPH1 deficiency leads to tumorigenesis. Here, we generated and studied both Mcph1−/− and Mcph1−/−p53−/− mice; we showed that Mcph1−/− mice developed tumors with long latency, and that primary lymphoma developed significantly earlier in Mcph1−/−p53−/− mice than in Mcph11+/+p53−/− and Mcph1+/−p53−/− mice. The Mcph1−/−p53−/− lymphomas and derived murine embryonic fibroblasts (MEFs) were both more sensitive to irradiation. Mcph1 deficiency resulted in remarkably increased chromosome and chromatid breaks in Mcph1−/− p53−/− lymphomas and MEFs, as determined by metaphase spread assay and spectral karyotyping analysis. Additionally, Mcph1 deficiency significantly enhanced aneuploidy as well as abnormal centrosome multiplication in Mcph1−/−p53−/− cells. Meanwhile, Mcph1 deficiency impaired double strand break (DSB) repair in Mcph1−/−p53−/− MEFs as demonstrated by neutral Comet assay. Compared with Mcph1+/+p53−/− MEFs, homologous recombination and non-homologous end joining activities were significantly decreased in Mcph1−/−p53−/− MEFs. Notably, reconstituted MCPH1 rescued the defects of DSB repair and alleviated chromosomal aberrations in Mcph1−/−p53−/− MEFs. Taken together, our data demonstrate MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. Our study using knockout mouse models provides convincing genetic evidence that MCPH1 is a bona fide tumor suppressor gene. Its deficiency leading to defective DNA repair in tumors can be utilized to develop novel targeted cancer therapies in the future.