Loss Of Heterozygosity For Chromosome Research Articles

Abstract 4806: Strategies to overcome the heterogeneity of tumor cells in breast cancer therapy

Abstract Breast carcinogenesis is a multistage process that involves mutations and alterations attributed to exposure to exogenous environmental substances and endogenous agents as female hormones. To overcome the heterogeneity of tumor cells in breast cancer therapy several strategies must be considered from the bench to clinical settings. It is proposed four strategies: Analysis to determine 1) Apoptosis, 2) Epithelial-mesenchymal transition (EMT), 3) CD44/CD24 gene and protein expression in cells derived from mammospheres and 4) MicroRNAs as miR34a and others by the effects of chemotherapeutic drugs (as pamidronate (Pam), 5-Fluorouracil (5-FU) and antioxidants as Curcumin (Cur) (diferuloylmethane) derived from Curcuma longa. Pam, a bisphosphonate is used in the treatment of breast cancer. 5-FU is a chemotherapeutic agent for the treatment of a variety of solid cancers that arrest cell cycle and induce apoptosis in cancer cells. We evaluated genes and proteins targeted by these drugs and antioxidants in a triple positive cell line, as MCF7 and a negative, MDA-MB-231 for hormonal receptors, respectively and in an in vitro breast cancer model induced by radiation and estrogen that was developed with a normal immortalized breast epithelial cell line, MCF-10F exposed to low doses of high LET (linear energy transfer) alpha particles (150 keV/μm) of radiation, and cultured in presence of 17β-estradiol. We used: i) MCF-10F, ii) Alpha3, a malignant non-tumorigenic, iii) Alpha5, a tumorigenic one and iv) Tumor2 cell line derived from Alpha5 injected into the nude mice. Previous results showed increased cell proliferation, anchorage independency, invasive capabilities and tumor formation in nude mice, microsatellite instability and loss of heterozygosity in chromosomes 6, 8, 11 and 17 and mutations of c-Ha-ras and Rho-A in Alpha5 and Tumor2 compared to the control MCF-10F and their counterparts. Pam, 5-FU and Cur inhibited migration and invasion in both cell lines, and also decreased c-Ha-ras, Rho-A, p53, and Cav-1 gene expression by RT-qPCR. These compounds also induced 1) apoptotic effect on Bcl-xL and Bax gene and protein expression and by flow cytometry; 2) changes in EMT markers such as Snail, Slug, Axl, 3) CD44/CD24 alterations and 4) target MicroRNAs as miR34a on different process. It can be concluded that strategies to overcome the heterogeneity of tumor cells can be used to interfere with genes involved in critical steps in breast carcinogenesis. Grant: Tarapacá University, Arica, Chile (GMC). Short tittle: Strategies in breast cancer therapy Citation Format: Gloria M. Calaf, Marcela Gallardo, Debasish Roy, Richard Ponce-Cusi. Strategies to overcome the heterogeneity of tumor cells in breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4806. doi:10.1158/1538-7445.AM2017-4806

Abstract 1448: Unraveling the oncogenic pathway of serrated polyposis syndrome driven by RNF43 germline mutation

Abstract Serrated polyps can arise in a sporadic or familial polyposis setting and predispose to colorectal cancer (CRC). Recently, we have identified RNF43 germline mutation in a family with Serrated Polyposis Syndrome (Yan et al, GUT 2016). The presence of second somatic hit in all serrated polyps examined from members of this family further confirmed the pathogenicity of RNF43 germline mutation. In an attempt to further delineate the global genomic alterations in these serrated polyps, so as to understand the RNF43 driven serrated neoplasia pathway for the development of colorectal cancer, we performed whole exome sequencing (WES) on two sessile serrated adenomas (SSAs) resected from an RNF43 germline mutation carrier, together with the paired blood DNA. Consistent with the results from our previous Sanger sequencing study, WES detected complete inactivation of RNF43 through a 2nd hit somatic mutation, c.461 C>T P154L, or loss of heterozygosity (LOH) in the two SSAs, respectively. We also identified 51 and 58 somatic mutations in the two SSAs, respectively. BRAFV600E mutation was the only shared mutation between the two polyps. Interestingly, each of the SSAs carried a truncating mutation in a histone-methyltransferase gene, either PRDM9 or SETD1B, respectively. Other truncating or deleterious mutations included chromatin modifiers (CHD2, CHD4, TSPYL2) or other genes with methyltransferase activity (TRMT2B, METTL12, METTL14, ECE2). In addition, we found few instances of chromosomal aberration or LOH, apart from a region of LOH at chromosome 17 encompassing RNF43 in one of the SSAs. Overall, we have revealed the genomic landscape of two sessile serrated adenomas resected from a germline RNF43 mutation carrier. The results confirmed somatic RNF43 2nd hit and BRAFV600E mutation as the key events, along with putative roles for histone methyltransferase, as well as other chromatin modifiers. These findings highlight the potentially important role of an altered chromatin in the oncogenic pathway of serrated neoplasia. Citation Format: Helen Hoi Ning Yan, Jeffrey C W Lai, Siu Lun Ho, Anthony K W Chan, Wai Yin Tsui, Annie S Y Chan, Siu Tsan Yuen, Suet Yi Leung. Unraveling the oncogenic pathway of serrated polyposis syndrome driven by RNF43 germline mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1448. doi:10.1158/1538-7445.AM2017-1448

Utilization of Whole-Exome Next-Generation Sequencing Variant Read Frequency for Detection of Lesion-Specific, Somatic Loss of Heterozygosity in a Neurofibromatosis Type 1 Cohort with Tibial Pseudarthrosis

A subset of neurofibromatosis type 1 patients develop tibial dysplasia, which can lead to pseudarthrosis. The tissue from the tibial pseudarthrosis region commonly has a somatic second hit in NF1: single-nucleotide variants, small deletions, or loss of heterozygosity (LOH). We used exome next-generation sequencing (NGS) variant frequency data (allelic imbalance analysis) to detect somatic LOH in pseudarthrosis tissue from three individuals with clinically and diagnostically confirmed neurofibromatosis type 1, and verified the results with microarray. The variant files were parsed and plotted using python scripts, and the NGS variant frequencies between the affected tissue and blood sample were compared. Individuals without somatic single-nucleotide variants or small insertions/deletions were tested for somatic LOH using the NGS variant allele frequencies. One individual's NGS data indicated no LOH in chromosome 17. The other two individuals demonstrated somatic LOH inclusive of NF1: one had an LOH region of approximately one million bases and Contra (NGS copy number program) indicated a somatic deletion and the other individual had LOH for most of chromosome 17q and Contra indicated no copy number change (microarray data verified this sample as copy neutral somatic LOH). Both LOH and copy number variation detected by NGS data correlated with microarray data, demonstrating the somatic LOH second hit can be detected directly from the NGS data.

The correlation of clinical and chromosomal alterations of benign meningiomas and their recurrences

Meningiomas (MGs) are the frequent benign intracranial tumors. Their complete removal does not always guarantee relapse-free survival. Recurrence-associated chromosomal anomalies in MGs haves been proposed as prognostic factors in addition to the World Health Organisation (WHO) grading, tumor size and resection rate. The aim of this study was to evaluate the frequency of deletions on chromosomes in sporadic MGs and to correlate them with the clinical findings and tumor behaviour. Along with survival, the tumor recurrence was the main endpoint. Chromosomal loss of heterozygosity (LOH) was studied. 46 benign MGs were subjected to the analysis, complete tumor resection was intended and no early mortalities were observed. Incomplete removal was related to parasagittal location and psammomatous hisptopathology (p<0.01). Chromosomal alterations were present in 82.6% of cases; LOH at 22q (67.4%) and 1p (34.8%) were the most frequent and associated with male sex (p=0.04). Molecular findings were not specific for any of the histopathologic grade. Tumor recurrence (14 of 46) correlated with tumor size (≥35mm), LOH at 1p, 14q, coexistence of LOH at 1p/14q, 10q/14q, ‘complex karyotype’ status (≥2 LOHs excluding 22q), patient age (younger <35), and Simpson grading of resection rate (≥3 of worse prognosis). The last 3 variables were independent significant prognostic factors in multivariate analysis and of the same importance in recurrence prediction (Receiver Operating Characteristic curves comparison p>0.05). Among the cases of recurrence, tumor progression was observed in 3 of 14. In 2 cases, LOH on 1p and/or coexistence of LOH 1p/14q correlated with anaplastic transformation.

Abstract LB-086: Genes targeted by drugs and curcumin in a breast carcinogenesis model

Abstract Breast carcinogenesis is a multistage process that involves mutations and cellular phenotypic alterations attributed to exposure to exogenous environmental substances as well as endogenous agents as female hormones. Pamidronate (Pam), one of the nitrogen-containing bisphosphonates, is used in the treatment of bone metastases of breast cancer. It has recently been reported that it is also related to cell proliferation and apoptosis. 5-Fluorouracil (5-FU) is a chemotherapeutic agent for the treatment of a variety of solid cancers that arrest cell cycle and induce apoptosis in cancer cells. Curcumin (Cur) is an antioxidant known as a dietary natural yellow pigment derived from the rhizome of the herb Curcuma longa. The aim of this study was to evaluate genes that could be targeted by these drugs and curcumin in a breast carcinogenesis in vitro model induced by radiation and estrogen. Such model was developed with a normal immortalized breast epithelial cell line, MCF-10F that was exposed to low doses of high LET (linear energy transfer) alpha particles (150 keV/μm) of radiation, and cultured in presence of 17β-estradiol. This model consisted of the following cell lines: i) MCF-10F, ii) Alpha3, a malignant non-tumorigenic, iii) Alpha5, a tumorigenic one and iv) Tumor2, derived from Alpha5 injected into the nude mice. Previous results showed that Alpha5 and Tumor2 increased cell proliferation, presented anchorage independency, invasive capabilities and tumor formation in nude mice, as well as microsatellite instability and loss of heterozygosity in chromosomes 6, 8, 11 and 17 and mutations of c-Ha-ras and Rho-A among others. Pam, 5-FU and Cur were analyzed with MCF-10F by MTT indicating that the mean LD50 was 10, 2 and 30 μM after 48 hrs, respectively. Such substances inhibited migration and invasion in both Alpha5 and Tumor2 cell lines compared to the control MCF-10F and their counterparts, and also decreased c-Ha-ras, Rho-A, p53, Serpin-1 and Cav-1 gene expression in Alpha5 and significantly in Tumor2 cell lines by RT-qPCR. A significant apoptotic activity was observed by flow cytometry in Alpha5 and Tumor2 cell lines in comparison to control MCF-10F and their counterparts. These compounds had a direct antitumor and apoptotic effect on Bcl-xL and Bax by down-regulation of a transcription factor as NFĸB gene expression in Alpha5 and Tumor2 cell lines. It can be concluded that c-Ha-ras, RhoA, p53, Serpin-1 and Cav-1 genes were targeted by drugs and curcumin in a model transformed by low doses of alpha particles and estrogen. Such genes are involved in critical steps in breast carcinogenesis. Supported by FONDECYT #1120006 (GMC) and Ministry of Education (MINEDUC), Universidad de Tarapacá, Arica, Chile Citation Format: Gloria M. Calaf, Debasish Roy, Richard Ponce-Cusi. Genes targeted by drugs and curcumin in a breast carcinogenesis 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 LB-086. doi:10.1158/1538-7445.AM2015-LB-086

Chemotherapy induced microsatellite instability and loss of heterozygosity in chromosomes 2, 5, 10, and 17 in solid tumor patients.

BackgroundThe inevitable side effects of the currently used chemotherapy are associated with serious syndromes. Genotoxic effects and consequent genetic instability may play an important role in these syndromes. The aim of the study was to evaluate chemotherapy-related microsatellite instability (MSI), loss of heterozygosity (LOH), and loss of mismatch repair (MMR) expression in solid tumor patients.MethodsSamples were collected from 117 de novo patients with solid tumors of different origins. Specimens, taken pre- and post-treatment, were screened for MSI and LOH in 10 microsatellite sequences in blood, and expression of five MMR proteins were analyzed in cancer tissues using immunohistochemistry. Statistical analysis included the use of; Fisher’s exact test, Chi Square, and an inter-rater reliability test using Cohen’s kappa coefficient.ResultsMicrosatellite analysis showed that 66.7% of the patients had MSI, including 23.1% high-positive MSI and 43.6% low-positive MSI. A large portion (41%) of the patients exhibited LOH in addition to MSI. MSI and LOH were detected in seven loci in which incidence rates ranged from 3.8% positive for Bat-26 to 34.6% positive for Tp53-Alu. Immunohistochemistry revealed that human mutL homolog 1 (hMLH1) expression was deficient in 29.1% of the patients, whereas 18.8%, 23.9%, 13.4%, and 9.7% were deficient for human mutS homolog 2 (hMSH2), P53, human mutS homolog 6 (hMSH6) and human post-meiotic segregation increased 2 (hPMS2), respectively. There was a significant correlation between MSI and LOH incidence in Tp53-Alu, Mfd41, and APC with low or deficient expression of hMLH1, hMSH2, and P53. A significant association between MSI and LOH, and incidence of secondary tumors was also evident.ConclusionsThe negative correlation between MMR expression, MSI, and LOH and increased resistance to anti-cancer drugs and development of secondary cancers demonstrates a useful aid in early detection of potential chemotherapy-related side-effects. The diagnostic value demonstrated in our earlier study on breast cancer patients was confirmed for other solid tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-014-0118-4) contains supplementary material, which is available to authorized users.

Preliminary whole-exome sequencing reveals mutations that imply common tumorigenicity pathways in multiple endocrine neoplasia type 1 patients

Whole-exome sequencing studies have not established definitive somatic mutation patterns among patients with sporadic hyperparathyroidism (HPT). No sequencing has evaluated multiple endocrine neoplasia type 1 (MEN1)-related HPT. We sought to perform whole-exome sequencing in HPT patients to identify somatic mutations and associated biological pathways and tumorigenic networks. Whole-exome sequencing was performed on blood and tissue from HPT patients (MEN1 and sporadic) and somatic single nucleotide variants (SNVs) were identified. Stop-gain and stop-loss SNVs were analyzed with Ingenuity Pathways Analysis (IPA). Loss of heterozygosity (LOH) was also assessed. Sequencing was performed on 4 MEN1 and 10 sporadic cases. Eighteen stop-gain/stop-loss SNV mutations were identified in 3 MEN1 patients. One complex network was identified on IPA: Cellular function and maintenance, tumor morphology, and cardiovascular disease (IPA score = 49). A nonsynonymous SNV of TP53 (lysine-to-glutamic acid change at codon 81) identified in a MEN1 patient was suggested to be a driver mutation (Cancer-specific High-throughput Annotation of Somatic Mutations; P = .002). All MEN1 and 3/10 sporadic specimens demonstrated LOH of chromosome 11. Whole-exome sequencing revealed somatic mutations in MEN1 associated with a single tumorigenic network, whereas sporadic pathogenesis seemed to be more diverse. A somatic TP53 mutation was also identified. LOH of chromosome 11 was seen in all MEN1 and 3 of 10 sporadic patients.

Utilization of Magnetic-Activated Cell Sorting and High-Density Single Nucleotide Polymorphism Microarrays Improves Diagnostic Yield and Prognostic Value in Clinical Testing for Patients with Multiple Myeloma and Normal Routine Chromosome Study

Utilization of Magnetic-Activated Cell Sorting and High-Density Single Nucleotide Polymorphism Microarrays Improves Diagnostic Yield and Prognostic Value in Clinical Testing for Patients with Multiple Myeloma and Normal Routine Chromosome Study

Dysplastic Nodules with Glypican-3 Positive Immunostaining: A Risk for Early Hepatocellular Carcinoma

Glypican-3 (GPC3) has been reported to be a novel serum and histochemical marker for HCC. The positivity or negativity for GPC3 in hepatic precancerous lesions, such as dysplastic nodules (DN), has also been described. Moreover, our previous studies have demonstrated that some DN in liver cirrhosis represent monoclonal hyperplasia, and confirmed their neoplastic nature. However, additional studies must be performed to investigate further the relationship between DN with GPC3 positivity and HCC. Thus, we first investigated the expression of GPC3 in 136 HCC and 103 small DN (less than 1 cm in diameter) by immunohistochemical staining and determined the clonality of 81 DN from female patients using X-chromosome inactivation mosaicism and polymorphism of androgen receptor (AR) gene. Then we examined these samples for chromosomal loss of heterozygosity (LOH) at 11 microsatellite polymorphism sites. The results demonstrated that GPC3 immunoreactivity was detected in 103 of 136 HCC (75.7%) and 19 of 103 DN (18.4%), and the positive ratio correlated with HBsAg positivity. Clonality assays showed that 15 GPC3-positive DN from female patients, including 12 high-grade DN (HGDN), and 28 (42.4%) of 66 GPC3-negative DN, were monoclonal. In addition, among 19 GPC3-positive DN, chromosomal LOH was found at loci D6S1008 (100%, 19/19), D8S262 (52.6%, 10/19) and D11S1301 (57.9%, 11/19). However, the LOH frequency in GPC3-negative DN was 5.95% (5/84), 23.8% (20/84), and 4.76% (4/84) in three loci, respectively. Thus, we concluded that GPC3-positive DN, especially GPC3-positive HGDN, was really a late premalignant lesion of HCC.

Cytogenetic analysis of choroidal melanoma

PurposeTo investigate the presence of known cytogenetic alterations of choroidal melanoma in a series of patients diagnosed and treated in our Ocular Oncology Service. A review of the present literature on this topic is also presented. MethodsMicrosatellite analysis (MSA) studies on loss of heterozygosity (LOH) of chromosome 3, as well as multiplex ligation probe amplification (MLPA) on chromosomes 1, 3, 6 and 8, were performed on enucleation or local resection samples obtained from a total of 27 patients, over a 2-year period. ResultsTwenty patients showed at least one of the cytogenetic alterations looked for. A total of 11 cases were found that showed LOH of chromosome 3 (44%), 8 gains of chromosome 8 (30%), 8 gains of chromosome 6 p (30%), and 7 partial or total losses of chromosome 1 (26%). ConclusionsThis is the first study on the cytogenetics of choroidal melanoma performed in our country.The results are similar to that published in the literature.Cytogenetic analysis provides more accurate knowledge on a vital individual prognosis. It also may become a valuable tool for establishing the most adequate follow-up regimes, and the need for adjuvant therapies.

Análisis citogenético del melanoma de coroides

PurposeTo investigate the presence of known cytogenetic alterations of choroidal melanoma in a series of patients diagnosed and treated in our Ocular Oncology Service. A review of the present literature on this topic is also presented. MethodsMicrosatellite analysis (MSA) studies on loss of heterozygosity (LOH) of chromosome 3, as well as multiplex ligation prove amplification (MLPA) on chromosomes 1, 3, 6 and 8, were performed on enucleation or local resection samples obtained from a total of 27 patients, over a 2 year period. ResultsTwenty patients showed at least one of the cytogenetic alterations looked for. A total of 11 cases were found that showed LOH of chromosome 3 (44%), 8 gains of chromosome 8 (30%), 8 gains of chromosome 6p (30%), and 7 partial or total losses of chromosome 1 (26%). ConclusionsThis is the first study on the cytogenetics of choroidal melanoma performed in our country. The results are similar to that published in the literature.Cytogenetic analysis provides more accurate knowledge on a vital individual prognosis. It also may become a valuable tool for establishing the most adequate follow-up regimes, and the need for adjuvant therapies.

Immune escape of cancer cells with beta2‐microglobulin loss over the course of metastatic melanoma

Cancer cells escape T-cell-mediated destruction by losing human leukocyte antigen (HLA) class I expression via various mechanisms, including loss of beta2-microglobulin (β2m). Our study illustrates the immune escape of HLA class I-negative tumor cells and chronological sequence of appearance of tumor β2m gene mutation in successive lesions obtained from a patient with metastatic melanoma. We observed a gradual decrease in HLA expression in consecutive lesions with few HLA-negative nodules in the primary tumor and the emergence of a totally negative lesion at later stages of the disease. We detected loss of β2m in β2m-negative nests of the primary tumor caused by a combination of two alterations: (i) a mutation (G to T substitution) in codon 67 in exon 2 of β2m gene, producing a stop codon and (ii) loss of the second gene copy by loss of heterozygosity (LOH) in chromosome 15. The same β2m mutation was found in a homogeneously β2m-negative metastasis 10 months later and in a cell line established from a biopsy of a postvaccination lymph node. Microsatellite analysis revealed the presence of LOH in chromosomes 6 and 15 in tumor samples, showing an accumulation of chromosomal loss at specific short tandem repeats in successive metastases during disease progression. HLA loss correlated with decreased tumor CD8+ T-cell infiltration. Early incidence of β2m defects can cause an immune selection and expansion of highly aggressive melanoma clones with irreversible genetic defects causing total loss of HLA class I expression and should be taken into consideration as a therapeutic target in the development of cancer immunotherapy protocols.

Abstract 3813: Uniparental disomy is associated with embryonal rhabdomyosarcoma in Costello Syndrome and nonsyndromic patients.

Abstract Embryonal rhabdomyosarcoma (ERMS) is the most prevalent pediatric soft tissue tumor and is characterized at the molecular level by loss of heterozygosity (LOH) at 11p15.5. We aimed to study ERMS in the context of Costello syndrome (CS), a rare syndromic predisposition to ERMS. Because CS arises from a typically paternal germline mutation in the proto-oncogene HRAS, it is considered a rasopathy. CS results in failure to thrive, intellectual disabilities, short stature, coarse facial features, skeletal abnormalities, congenital heart disease, and a predisposition for cancer, most commonly ERMS. We molecularly characterized seven ERMS tumors from six unrelated CS patients, carrying paternally derived HRAS p.G12S (5) or p.G12A (2) mutations. LOH was evaluated in all CS ERMS tumors by microarray and/or using short tandem repeat (STR) markers along both arms of chromosome 11. One patient developed a second primary ERMS tumor showing the ERMS hallmark LOH limited to 11p15.5. The six other CS ERMS tumors displayed paternal uniparental disomy (UPD) of the entire chromosome 11. Paternal UPD was also observed in a pure ERMS cell line, established using fresh tumor sample from one of the patients with a p.G12A germline mutation. This represents, to our knowledge, the first CS ERMS cell line. Additional cell lines derived from non CS patients were used in this study and included two ERMS (RD and RH36), one Alveolar RMS (RH30), and one mixed A/ERMS (RH18). RH36 cells carried an HRAS mutation at position 61 (p.Q61K). Unexpectedly, both ERMS cell lines, RD and RH36, displayed a complete LOH for chromosome 11. The ARMS RH30 cells carried no mutation in HRAS or LOH. RH18 cells derived from an A/ERMS had no HRAS mutation and showed LOH on both, the p and q arm of chromosome 11, except at locus D11S1338. Cytogenetic analysis with fluorescence in situ hybridization (FISH) on three CS tumors revealed multiple copies of chromosome 11 leading to trisomies and tetrasomies in the tumor and cultured cells. Gene expression revealed loss of imprinting in the growth regulating genes H19, IGF2, and CDKN1C on chromosome 11 not only in CS tumors, but also in nonsyndromic ERMS. Citation Format: Katherine M. Robbins, Deborah L. Stabley, Katrina Conard, David J. Wu, Hunter Stitik, Edward A. Kolb, Andrew Napper, Robert W. Mason, Karen W. Gripp, Katia Sol-Church. Uniparental disomy is associated with embryonal rhabdomyosarcoma in Costello Syndrome and nonsyndromic patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3813. doi:10.1158/1538-7445.AM2013-3813

Hsp27 (HSPB1): a possible surrogate molecular marker for loss of heterozygosity (LOH) of chromosome 1p in oligodendrogliomas but not in astrocytomas

In oligodendrogliomas, 1p loss of heterozygosity (LOH) is a predictor of good prognosis and treatment response. In contrast, in uveal melanomas, LOH of chromosome 3 has been linked to poor prognosis and downregulation of Hsp27. In the present study, we have analyzed the expression of heat-shock proteins (Hsps) to characterize subtypes of gliomas and their histopathologic features and to correlate with other molecular markers including LOH of 1p. Biopsies from patients with primary gliomas (n = 65) were analyzed by immunohistochemistry, chromogenic in situ hybridization and fluorescent in situ hybridization and methylation-specific PCR (MSP). Elevated Hsp27 and total Hsp70 expression levels were associated with high-grade astrocytomas (p = 0.0001 and p = 0.01, respectively). In grade III oligodendrogliomas, the Hsp27 levels were significantly higher (p = 0.03). Low O6-methylguanine-DNA methyltransferase (MGMT) expression was associated with grade II astrocytomas. Elevated β-catenin expression was associated with grade III/IV astrocytomas (p = 0.003); p53 (+) tumors were more frequently found in grade III/IV astrocytomas (p = 0,001). LOH on 1p was associated with oligodendroglial tumours. In addition, a higher Hsp27 expression correlated with LOH of 1p (p = 0.017); this was also tested in two glioma cell lines. MSP was successful in only six samples. No significant correlations were found for the other markers. In conclusion, in oligodendroglial tumors, Hsp27 appeared as a surrogate marker of LOH of 1p which could also help to predict the disease prognosis. In gliomas, p53, Hsp27, Hsp70, MGMT, and β-catenin correlated with histopathological characteristics, suggesting that these markers could predict the disease outcome and the response to treatments.

OC-100 The clonal origins of gastric adenocarcinoma

IntroductionWe have previously shown that entire fields of dysplasia in the human stomach are derived from a single mutated, metaplastic gland.1 This suggests that intestinal metaplasia (IM) can be considered...

SNP Array Profiling of Childhood Adrenocortical Tumors Reveals Distinct Pathways of Tumorigenesis and Highlights Candidate Driver Genes

Childhood adrenocortical tumors (ACT) are rare malignancies, except in southern Brazil, where a higher incidence rate is associated to a high frequency of the founder R337H TP53 mutation. To date, copy number alterations in these tumors have only been analyzed by low-resolution comparative genomic hybridization. We analyzed an international series of 25 childhood ACT using high-resolution single nucleotide polymorphism arrays to: 1) detect focal copy number alterations highlighting candidate driver genes; and 2) compare genetic alterations between Brazilian patients carrying the R337H TP53 mutation and non-Brazilian patients. We identified 16 significantly recurrent chromosomal alterations (q-value < 0.05), the most frequent being -4q34, +9q33-q34, +19p, loss of heterozygosity (LOH) of chromosome 17 and 11p15. Focal amplifications and homozygous deletions comprising well-known oncogenes (MYC, MDM2, PDGFRA, KIT, MCL1, BCL2L1) and tumor suppressors (TP53, RB1, RPH3AL) were identified. In addition, eight focal deletions were detected at 4q34, defining a sharp peak region around the noncoding RNA LINC00290 gene. Although non-Brazilian tumors with a mutated TP53 were similar to Brazilian tumors, those with a wild-type TP53 displayed distinct genomic profiles, with significantly fewer rearrangements (P = 0.019). In particular, three alterations (LOH of chromosome 17, +9q33-q34, and -4q34) were significantly more frequent in TP53-mutated samples. Finally, two of four TP53 wild-type tumors displayed as sole rearrangement a copy-neutral LOH of the imprinted region at 11p15, supporting a major role for this region in ACT development. Our findings highlight potential driver genes and cellular pathways implicated in childhood ACT and demonstrate the existence of different oncogenic routes in this pathology.

Microsatellite Alterations of Major Tumor Suppressor Genes in Hepatocellular Carcinoma

Purpose : Hepatocellular carcinoma (HCC) shows various molecular and genetic alterations in its development and progression. The major tumor suppressor genes (TSGs) such as p16, PTEN, E-cadherin and p53 on chromosomes 9p21, 10q23, 16q22, 17p13 may play important roles in various regulation pathways as well as tumor suppression. Microsatellite alteration such as loss of heterozygosity (LOH) has been reported is a predominant mechanism of inactivation of TSGs. Also, LOH is known to be related with allelic loss of various TSGs. This study evaluated LOH of 4 TSGs in HCCs and correlated these results with the clinicopathological factors. Methods : LOH analysis was carried out using a polymerase chain reaction with 16 polymorphic microsatellite markers of 4 chromosomes having TSGs in 50 surgically resected tumors and their non-tumorous counterparts. Results : There was no detectable LOH in normal tissue. LOH was detected in 82% of 50 cases of HCCs. LOH of 9p21, 10q23, 16q22, 17p13 was detected in 36%, 54%, 38% and 66%, respectively. Advanced HCCs showed significant LOH results of 9p21, 10q23 & 17p13. According to tumor grade, those of 10q23, 16q22, 17p13 correlated with higher grade III. Vascular invasion & lymphatic emboli showed correlation with those of 9p21, 10q23, 17p13. Cancer recurrence & metastasis significantly correlated with 9p21 LOH. Conclusion : These results suggest that LOH of various chromosomes may be predominantly involved in hepatocarcinogenesis and tumor invasion. Also, those LOH may be useful clinical indicator in determining the poorer prognosis among patients with HCC.

Acute Lymphoblastic Leukemia Natural History in Neurofibromatosis Type 1 Monozygotic Twins

Abstract 2414 Introduction:Neurofibromatosis 1 (NF1) is an autosomal dominant disease, caused by mutation of neurofibromin (NF1) gene, located in the 17q11.2 chromosomal region. Children affected by NF1 have an increased risk to develop tumors, including leukemia. It has been suggested that NF1 aberrancy is a tumor predisposing factor, and that secondary events in somatic cells gives rise to the formation of neoplasm. However, the clonal evolution from NF1 mutation to tumor development has never been elucidated. Monozygotic twins constitute an ideal tool to understand the clonal evolution of leukemia in the context of a common genetic background. Patients:We performed the genomic characterization of a pair of monozygotic twins with diagnosis of NF1, who developed concordant B-cell precursor ALL at the age of 6 (Twin 1, T1) and 6.5 years (T2). The diagnostic and relapse samples showed common and unique lesions, allowing us to hypothesize a model for leukemia clonal evolution. At diagnosis, T1 had common ALL with 45,XX,-7,del(9)(p12),del(10)(q23)[8]/46,XX[12] whereas T2 had common ALL with 45,XX,-7,del(10)(q22)[14]. Both were treated according to AIEOP-BFM ALL 2000 protocol; T1 was classified as MRD high risk (HR), and T2 as MRD Intermediate risk (IR). T1 underwent BMT 8 months after diagnosis, and achieved clinical remission, still persisting, whilst T2 relapsed 13 months after diagnosis with 47,XX,del(9)(p21),+21[11]/46,XX[5]; she entered AIEOP REC 2003 protocol and, after achieving second complete remission and negativity for MRD, underwent allogeneic HLA-compatible BMT; she is in clinical remission 3.5 years after BMT. Results:Genome-wide copy number alteration (CNA) analysis by Cytogenetics Whole Genome 2.7M Arrays (Affymetrix) indicated that T1 and T2 shared a copy number neutral Loss Of Heterozygosity (LOH) of 17q arm, were the NF1 gene is located. At diagnosis, both had chromosome 7 monosomy but showed completely different Ig/TCR rearrangements. Three additional clonal rearrangements were found in T2 relapse sample. By backtracking the rearrangements, the relapse clone was detected in about 1% of T2 diagnostic cells, but not in T1 diagnosis sample.Several twin-specific abnormalities were detected: both had a deletion involving the q arm of chromosome 10, with different extent: del(10)(q23.33) in T1 and del(10)(q23.1) in T2. Moreover, T1 carried hemizygous deletions on chromosome 9 and a focal loss of 6q15 locus, containing the B-cell specific transcriptional regulator BACH2. Genomic analysis of T2 revealed a gain in the telomeric region of chromosome 2 and heterozygous losses in 14q32.13 and 15q21.3 regions, involving BX247990 (human full-length cDNA clone of B cells) and TCF12 (transcription factor 12) genes, respectively. The T2 relapse showed LOH 17q and del(15)(q21.3), as found in her diagnostic sample, and several additional CNAs, including the heterozygous deletions of IKZF1 (7p12.2), ETV6 (12p13.2) and C20orf94 (20p12.2) genes, trisomy of chromosome 21 and LOH of chromosome 20. Discussion:This is the first study that describes the natural history of events in NF1 patients (with 17q LOH) who developed ALL.The unique feature common to both twins in diagnosis and relapse samples was 17q LOH, that probably arose prenatally in a common hematopoietic progenitor (before somatic recombination) of one twin and spread to the other twin through intraplacental circulation. After birth, an independent clonal evolution is sustained by different Ig/TCR rearrangements as well as specific and different oncogenetic lesions. This model support a preleukemic state followed by at least-two-step mechanism for progression to leukemia. The absence of monosomy 7 at relapse indicates an independent and postnatal acquisition of this lesion; and that relapse in T2 occurs from a pre-leukemic clone before monosomy 7, which further evolved through acquisition of additional abnormailities, commom in BCP-ALL (i.e. ETV6 and Ikaros deletions).The role of BACH2 and TCF12 gene deletions in NF1 and ALL must be further explored. BACH2 is a human B-cell specific transcriptional repressor already described as tumor suppressor gene, involved in IgH@ translocations. TCF12 (also named HEB) is a basic helix-loop-helix transcription factor involved in B and T cell commitment. The altered expression of these genes could contribute to the differentiation arrest and the uncontrolled proliferation of leukemic blasts. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Molecular and clinical analysis of glioblastoma with an oligodendroglial component (GBMO)

The genetic and clinical features of glioblastoma with an oligodendroglial component (GBMO), pathologically defined as anaplastic oligo-astrocytoma with necrosis, remain unclear. We investigated the correlation between genetic alterations and clinical outcomes in 19 GBMO patients we have encountered since 1997. Using single nucleotide polymorphism oligonucleotide genomic (SNP) microarrays, we analyzed gene amplification, loss of heterozygosity (LOH), and homozygous deletions in their whole genome. We also analyzed their overall survival (OS). Pathological studies revealed the presence of calcification in 11 and of a cyst in 9 of the 19 patients. Whole-genome analysis using SNP microarrays revealed LOH of chromosome 10 in 11, EGFR amplification in 8, 9p21 (INK4 locus) deletion in 12, PDGFR amplification in 2, and LOH of 1p19q in 2 patients. Median OS was 14months (average 22.8months). The pattern of genetic alterations was similar in GBMO and glioblastoma multiforme (GBM) patients, and the clinical outcomes were similar in GBMO and GBM patients.

Rad52 function prevents chromosome loss and truncation in Candida albicans

RAD52 is required for almost all recombination events in Saccharomyces cerevisiae. We took advantage of the heterozygosity of HIS4 in the Candida albicans SC5314 lineage to study the role of Rad52 in the genomic stability of this important fungal pathogen. The rate of loss of heterozygosity (LOH) at HIS4 in rad52-ΔΔ strains was ∼10(-3) , at least 100-fold higher than in Rad52(+) strains. LOH of whole chromosome 4 or truncation of the homologue that carries the functional HIS4 allele was detected in all 80 rad52-ΔΔ His auxotrophs (GLH -GL lab His(-)) obtained from six independent experiments. Isolates that had undergone whole chromosome LOH, presumably due to loss of chromosome, carried two copies of the remaining homologue. Isolates with truncations carried centric fragments of broken chromosomes healed by de novo telomere addition. GLH strains exhibited variable degrees of LOH across the genome, including two strains that became homozygous for all the heterozygous markers tested. In addition, GLH strains exhibited increased chromosomal instability (CIN), which was abolished by reintroduction of RAD52. CIN of GLH isolates is reminiscent of genomic alterations leading to cancer in human cells, and support the mutator hypothesis in which a mutator mutation or CIN phenotype facilitate more mutations/aneuploidies.