P53 Loss Of Heterozygosity Research Articles

Chromosomal Instability (CIN) Phenotype, CIN High or CIN Low, Predicts Survival for Colorectal Cancer

To examine whether chromosomal instability (CIN) phenotype, determined by the severity of CIN, can predict survival for stages II and III colorectal cancer (CRC). We determined microsatellite instability (MSI) and loss of heterozygosity (LOH) status in 1,103 patients (training [n = 845] and validation [n = 258] sets with stages II and III CRC). The LOH ratio was defined as the frequency of LOH in chromosomes 2p, 5q, 17p, and 18q. According to the LOH ratio, non-MSI high tumors were classified as CIN high (LOH ratio ≥ 33%) or CIN low (LOH ratio < 33%). CIN-high tumors were subclassified as CIN high (mild type; LOH ratio < 75%) or CIN high (severe type; LOH ratio ≥ 75%). We used microarrays to identify a gene signature that could classify the CIN phenotype and evaluated its ability to predict prognosis. CIN high showed the worst survival (P < .001), whereas there was no significant difference between CIN low and MSI high. CIN high (severe type) showed poorer survival than CIN high (mild type; P < .001). Multivariate analysis revealed that CIN phenotype was an independent risk factor for disease-free and overall survival, respectively, in both the training (P < .001 and P = .0155) and validation sets (P < .001 and P = .0076). Microarray analysis also revealed that survival was significantly poorer in those with the CIN-high than in the CIN-low gene signature (P = .0203). In a validation of 290 independent CRCs (GSE14333), the CIN-high gene signature showed significantly poorer survival than the CIN-low signature (P = .0047). The CIN phenotype is a predictive marker for survival and may be used to select high-risk patients with stages II and III CRC.

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.

Clinical and Genomic Characterization of Chromosome 7 Lesions in Myeloid Malignancies,

Abstract 3549Acute myeloid leukemia and myelodysplastic syndrome cases with monosomy 7 or del(7q) comprise a heterogeneous group. Complex karyotypes with multiple aberrations such as del(5q) are more frequent, and there is evidence that the overall survival is significantly lower in this group, compared with patients who have monosomy 7 or del(7q) as a sole abnormality. In this study, our purpose was to gain insights into these heterogeneous subsets among myeloid disorders with lesions of chromosome 7, taking advantage of the better definition of chromosomal aberrations which provides SNP-A karyotyping.We studied a large cohort of patients (N=1,153) with myeloid disorders using SNP-A karyotyping. Loss of heterozygosity (LOH) in 7q was identified in 9.7% (112/1153) of patients. It included monosomy 7 (n=38, 3.3%), del(7q) (n=55, 4.8%) and UPD7 (n=19, 1.6%). The LOH 7 cohort included men (70%) and women (30%) with a mean age of 57 years (S.D. 22.2 years). The presence of chromosome 7 material in 35% of our cases with apparent monosomy 7 by conventional MC serves as an illustration for SNP array-based mapping allowing for a more precise definition of the breakpoints. Clinical and chromosomal lesions association made possible to distinguish between three subsets: UPD 7: with 60% of the patients included in a myeloproliferative or myeloproliferative/myelodysplastic disease and 50% of presence of EZH2 mutations; del(7q): with 85% of patients included in the high risk group (RAEB and AML) and frequently associated with complex karyotypes (including 5q and 17p LOH); and monosomy 7: frequently (59%) as a sole abnormality and, in the case of MDS patients, associated with hypoplastic features. The existence of those three subsets is supported by the difference survival among the MDS cohort: median overall survival of 1250, 512 and 209 days for UPD 7, monosomy 7 and del(7q) patients, respectively (p=0.03). Three SNP-A defined commonly deleted regions were described in bands 7q22 (100634238–101658775), 7q34 (137841484–139319208), and between bands 7q35 and 7q35q36.1 (144338001–148545983) but among the candidate tumor suppressor genes (TSG), only EZH2 showed to be recurrently mutated in UPD7. The lack of a TSG mutation in monosomy and del(7q) cases led us to determine that the expression of majority of genes included in the CDRs was significantly reduced in MDS CD34+ cells from 9 cases with monosomy or partial deletion of chromosome 7 (Multiple testing by Benjamin Hochberg correction, FDR<10%). These genes included LUC7L2, ZNHIT1, TTC26, RABL5, TRIM24, EZH2, ZC3HAV1L, CNTNAP2, TRIM24, CUX1, FIS1, RABL5, ZC3HAV1 and TBXAS. The mean decrease in gene expression was 42–33 % supporting haploinsufficiency as a probable cause of disease.In summary, the present study shows how SNP-A karyotyping enables us to refine our knowledge about lesions of chromosome 7. The secondary nature of del(7q), accompanied almost invariably by “founder” 5q aberrations, the proliferative phenotype and presence of EZH2 mutations of UPD7q, and the description of monosomy 7 as isolated lesion and associated with hypoplastic disease phenotype, are the main correlations found herein, which prompt to investigate for different underlying pathogenic origin for each subset. Disclosures:No relevant conflicts of interest to declare.

Molecular prognostic factors of anaplastic oligodendroglial tumors and its relationship: a single institutional review of 77 patients from China

The increased chemosensitivity of oligodendroglial tumors has been associated with loss of heterozygosity (LOH) on chromosomes 1p and 19q. Other clinical and molecular factors have also been identified as being prognostic and predictive for treatment outcome. Seventy-seven patients with anaplastic oligodendroglioma (AO) or anaplastic oligoastrocytoma (AOA), treated in Beijing Tiantan Hospital from 2006 through 2008, were reviewed. LOH 1p, LOH 19q, IDH1 mutation, O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation, and protein expression level of MGMT, P53, EGFR, and Ki-67 were evaluated. Age at diagnosis, LOH 1p and 19q, IDH1 mutation, P53 expression level, reoperation when progression, and adjuvant chemotherapy were statistically significant factors for overall survival (OS) in univariate analysis. Further multivariate analysis showed that age at diagnosis (P = .010), LOH 1p and 19q (P = .016), IDH1 mutation (P = .011), and reoperation after progression (P = .048) were independent predictors for longer survival in these patients. Nonrandom associations were found between LOH 1p and LOH 19q, MGMT promoter methylation and LOH 1p or 19q, IDH1 mutation and LOH 1p and 19q, IDH1 mutation and MGMT promoter methylation, whereas mutual exclusion was found between MGMT promoter methylation and MGMT expression level. The present study confirmed that age at diagnosis, LOH 1p and 19q, IDH1 mutation, and reoperation after progression were independent significant prognostic factors for patients with anaplastic oligodendroglial tumors. Inter-relationship between LOH 1p, LOH 19q, IDH1 mutation, MGMT promoter methylation, and MGMT expression level were also revealed. Future clinical trials for AO and AOA should consider the molecular alterations of patients.

Correlation between loss of heterozygosity on chromosome 1p and 19q and expression of MGMT, p53 and Ki-67 proteins in gliomas

To study the correlation of loss of heterozygosity (LOH) on chromosome 1p and 19q with the expression of MGMT, p53 and Ki-67 proteins in gliomas. One hundred and forty six cases of gliomas (45 oligodendrogliomas, 42 oligodendroastrocytomas, and 59 astrocytomas) were included in this study. Their tissue and blood samples were retrospectively analyzed by PCR-denaturing high-performance liquid chromatography (DHPLC) for 1p and 19q status and by immunohistochemistry for MGMT, p53 and Ki-67 expression patterns. The correlation among them and with clinicopathological characteristics were analyzed by chi-square test and t-test. In the oligodendrogliomas, the positive rate of 1p LOH was 59.8%, significantly higher than 33.9% in astrocytomas (P = 0.002), and 1p and 19q LOH was 42.5%, significantly higher than 16.9% in astrocytomas (P = 0.001). Combined with LOH on 1p and 19q, low MGMT expression (65.5%), and high Ki-67 expression (54%) were more frequent in oligodendrogliomas, whereas high p53 expression was more frequent in astrocytomas and mixed tumors (75.2%). 1p LOH (72.5%) and low MGMT (87.5%) expressions were more frequent in grade II oligodendrogliomas, whereas high expressions of p53 (83.0%) and Ki-67 (76.6%) were more frequent in grade III oligodendrogliomas. In addition, high Ki-67 expression was more frequent in grade III astrocytomas. LOH on 1p and 19q LOH was more frequent in nontemporal oligodendrogliomas (55.6%) than that in temporal ones (22.2%, P = 0.002). Non-random associations were found between LOH 1p and 19q LOH, MGMT and p53 protein expressions, and MGMT and Ki-67 protein expressions (all P < 0.05), whereas mutual exclusions were found between LOH on 1p and 19q and p53 expression, and LOH 1p and Ki-67 expression. There is a significant interrelationship of the investigated molecular markers and clinicopathological features of gliomas, which support a promising role of molecular markers in guiding diagnostic assessment and clinical management of gliomas.

Pediatric genitourinary tumors

To review the 2009/2010 literature on pediatric genitourinary tumors and highlight the most significant publications. New techniques such as gene expression profiling, PET, nephron-sparing surgery, and stem cell transplantation are being incorporated into contemporary treatments for pediatric patients with genitourinary tumors. Biologic markers are increasingly being used to help with risk stratification of patients. WT1 mutation and 11p15 loss of heterozygosity have been associated with relapse in very low-risk Wilms tumors treated with surgery alone and may help reduce the use of chemotherapy in some children. Discussion continues on the use of fusion gene status to risk stratify alveolar rhabdomyosarcoma. Meta-analysis of the use of high-dose chemotherapy with autologous hematopoetic stem cell rescue in patients with relapsed Wilms tumor and rhabdomyosarcoma suggests that some patients may benefit more from conventional salvage chemotherapy. New agents are needed for patients with high-risk and relapsed disease to improve outcomes. In general, the prognosis for patients with pediatric genitourinary tumors is favorable. The elucidation of the molecular abnormalities in these tumors is determining risk stratification, treatment strategies, and candidates for new drug development.

Abstract 4876: Proteomic analysis of pelvic tissues at the time of risk-reducing surgery in BRCA mutation carriers

Abstract Objective: To develop a workflow for collecting specimens for proteomic analysis from the peritoneum, fallopian tubes (FT), and ovarian surface epithelium (OSE) at the time of risk-reducing surgery in women at increased risk for developing malignancies due to a germline BRCA mutation. Methods: A pilot set of 6 subjects with BRCA2 mutations were consented for an IRB-approved protocol for surgical specimen collection. During laparoscopic removal of the FT and ovaries, peritoneal washings, OSE brushings, and FT lavage specimens were collected without interfering with standard pathological analysis of the tissues. After immunodepletion, samples were loaded in a stacking gel and digested in-gel with trypsin. Digests were analyzed in duplicate by nanoflow reversed-phase liquid chromatography coupled online to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database from the European Bioinformatics Institute using SEQUEST. Differences in protein abundance between samples were derived by spectral counting. Immunohistochemical (IHC) staining for p53 and loss of heterozygosity (LOH) testing for p53 at the D17S-1844, D17S-516, and D17S-786 loci were performed on all paraffin-embedded FT specimens. Results: A total of 265, 328, and 241 different proteins were identified by 2 or more unique peptides each from the any of the OSE, FT and peritoneal samples, respectively. The number of proteins identified on both the right and left side in individual subjects ranged from 16-33% for OSE and 31-36% for FT. 37 common proteins involved in cell signaling, cell growth, cellular assembly, acute phase response, and DNA replication and repair were found in at least 1 specimen from all 6 subjects. 23 of the common proteins are involved in cancer pathways. All surgical pathology was negative both for malignancy and possible precursor lesions such as tubal intraepithelial carcinoma. MUC16 (CA-125) was elevated by proteomic analysis in the right FT of 1 subject; the left FT lavage in this subject could not be collected due to tubal scarring. Although IHC for p53 was normal in both of these FT, LOH for p53 was present in both. IHC was also negative for all remaining FT specimens. LOH was absent in both FT in 3 subjects, present in 1 FT but not the other in 2 subjects, and present bilaterally in 1 subject mentioned above. Conclusions: This workflow allows analysis of the proteomes of peritoneal, FT, and OSE specimens collected at the time of laparoscopic risk-reducing surgery, a technique which has not been described previously. It presents a unique opportunity for closer examination of the tissues at risk for malignant transformation in women with BRCA mutations. Harvesting proteins from the environment where ovarian and related cancers arise will inform current attempts to use the “p53 signature” to evaluate the FT as the potential source of many pelvic serous tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2011-4876

Detection of Allelic Status of 1p and 19q by Microsatellite-based PCR Versus FISH

Combined loss of chromosome arms 1p and 19q in oligodendroglial tumors has become a powerful predictor of prognosis and treatment response, and hence clinical testing for their detection is widely used nowadays. Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) analysis and fluorescence in-situ hybridization (FISH) are the 2 important relatively common clinical molecular diagnostic techniques used for this purpose and they have their unique advantages and limitations. The preference of methodology used depends on local expertise and existing laboratory capabilities. However, there is no consensus on which methodologic approach has a better potential. The objective of the study was to compare the accuracy, reliability, and feasibility of FISH and PCR in detecting the 1p and 19q LOH status. Using the PCR-based method, a LOH analysis was performed on 30 oligodendrogliomas and 10 glioblastomas using fresh-frozen tissue and peripheral blood as control. A FISH assay using paraffin blocks was performed on all the cases. Concordance for 1p and 19q was found in 92.5% (37 of 40) and 82.5% (33 of 40) cases, respectively. The relative advantages and limitations of both the techniques are analyzed and discussed. The main issue pertains to the use of the best technique in large clinical trials whose results are translated to patient care services. Unless the technique used is correct, the results of clinical trials and their correlations may be somewhat questionable.

CASZ1, a candidate tumor-suppressor gene, suppresses neuroblastoma tumor growth through reprogramming gene expression

Neuroblastoma (NB) is a common childhood malignant tumor of the neural crest-derived sympathetic nervous system. In NB the frequent loss of heterozygosity (LOH) on chromosome 1p raises the possibility that this region contains tumor-suppressor genes whose inactivation contributes to tumorigenesis. The human homolog of the Drosophila neural fate determination gene CASZ1, a zinc-finger transcription factor, maps to chromosome 1p36.22, a region implicated in NB tumorigenesis. Quantitative real-time PCR analysis showed that low-CASZ1 expression is significantly correlated with increased age (≥18 months), Children's Oncology Group high-risk classification, 1p LOH and MYCN amplification (all P<0.0002) and decreased survival probability (P=0.0009). CASZ1 was more highly expressed in NB with a differentiated histopathology (P<0.0001). Retinoids and epigenetic modification agents associated with regulation of differentiation induced CASZ1 expression. Expression profiling analysis revealed that CASZ1 regulates the expression of genes involved in regulation of cell growth and developmental processes. Specific restoration of CASZ1 in NB cells induced cell differentiation, enhanced cell adhesion, inhibited migration and suppressed tumorigenicity. These data are consistent with CASZ1 being a critical modulator of neural cell development, and that somatically acquired disruption of normal CASZ1 expression contributes to the malignant phenotype of human NB.

Molecular analysis of TP53, D9S1747, D9S162 and RPS6 in oral squamous cell carcinoma

To investigate the correlation between the frequency of molecular abnormalities of 4 loci at chromosomal 9p21 (D9S1747, D9S162, RPS6) and 17p13 (TP53) and the clinical characteristics and prognosis. The oral squamous cell carcinoma (OSCC) lesions in 71 patients were manually microdessected. Genomic DNA from these lesions and normal lymphnode tissu or peripheral blood of the same patients were extracted using the Watson's tissue kit. The loss of heterozygosity (LOH) and microsatellite instability (MI) of 17p13 and 9p21 were analyzed by PCR-page electrophoresis after DNA extraction. LOH and MI were detected in the OSCC of 48 patients (68%). The LOH and MI frequency at chromosomes 17p13 and 9P21 were 56% (35/63) and 59% (40/68) respectively. The LOH and MI frequency at 9p21 was significantly associated with WHO grading (P < 0.01) and lymphonode metastasis (P < 0.01). The LOH and MI frequency at 17p13 was significantly associated with clinical stage (P < 0.05). TP53 genetic aberration and 9p21 genetic aberration were significant prognostic factors for OSCC. The prognosis was poor in the LOH and MI positive group of chromosome 17p13 and 9p21. The frequency of LOH and MI at TP53 was the only independent factor for overall survival (P < 0.05). The LOH and MI of 17p13 and 9p21 were related to clinical stage and lymphonode metastasis. LOH of TP53 was an independent prognostic factor for OSCC.

Genomic and genetic alterations influence the progression of gastric cancer

Gastric cancer is one of the leading causes of cancer-related deaths worldwide, although the incidence has gradually decreased in many Western countries. Two main gastric cancer histotypes, intestinal and diffuse, are recognised. Although most of the described genetic alterations have been observed in both types, different genetic pathways have been hypothesized. Genetic and epigenetic events, including 1q loss of heterozygosity (LOH), microsatellite instability and hypermethylation, have mostly been reported in intestinal-type gastric carcinoma and its precursor lesions, whereas 17p LOH, mutation or loss of E-cadherin are more often implicated in the development of diffuse-type gastric cancer. In this review, we summarize the sometimes contradictory findings regarding those markers which influence the progression of gastric adenocarcinoma.

WT1 Mutation and 11P15 Loss of Heterozygosity Predict Relapse in Very Low-Risk Wilms Tumors Treated With Surgery Alone: A Children's Oncology Group Study

Children's Oncology Group defines very low-risk Wilms tumors (VLRWT) as stage I favorable histology Wilms tumors weighing less than 550 g in children younger than 24 months of age. VLRWTs may be treated with nephrectomy alone. However, 10% to 15% of VLRWTs relapse without chemotherapy. Previous studies suggest that VLRWTs with low WT1 expression and/or 11p15 loss of heterozygosity (LOH) may have increased risk of relapse. The current study validates these findings within prospectively identified children with VLRWT who did not receive adjuvant chemotherapy. Fifty-six VLRWTs (10 relapses) were analyzed for mutation of WT1, CTNNB1, and WTX; for 11p15 LOH using microsatellite analysis; and for H19DMR and KvDMR1 methylation. 11p15 LOH was identified in 19 (41%) of 46 evaluable VLRWTs and was significantly associated with relapse (P < .001); 16 of 19 were isodisomic for 11p15. WT1 mutation was identified in nine (20%) of 45 evaluable VLRWTs and was significantly associated with relapse (P = .004); all nine cases also had 11p15 LOH. All evaluable tumors showing LOH by microsatellite analysis also showed LOH by methylation analysis. Retention of the normal imprinting pattern was identified in 24 of 42 evaluable tumors, and none relapsed. Loss of imprinting at 11p15 was identified in one of 42 tumors. WT1 mutation and 11p15 LOH are associated with relapse in patients with VLRWTs who do not receive chemotherapy. These may provide meaningful biomarkers to stratify patients for reduced chemotherapy in the future. VLRWTs show a different incidence of WT1 mutation and 11p15 imprinting patterns than has been reported in Wilms tumors of all ages.

Genetic pathways of multiple esophageal squamous cell carcinomas

Whether multiple esophageal squamous cell carcinomas (SCCs) in a patient develop through an identical genetic pathway is still unclear. We examined multiple esophageal SCCs for alterations of p53, p16, IRF and mitochondrial DNA (mtDNA) and microsatellite instability (MSI). Thirty patients with multiple superficial esophageal SCCs, 23 with double lesions and 7 with triple lesions, were enrolled. Loss of heterozygosity (LOH) of p53 (TP53), p16 (D9S171), IRF (IRF) and other microsatellite loci including D1S191, D17S858, D18S58 and D18S61 of the tumors was examined by microsatellite assay. Mutations of p16 and mtDNA were examined with PCR single-strand conformation polymorphism (SSCP) analysis. LOH of p53, p16 and IRF were detected in 16 of 50 (32%), 5 of 38 (13%) and 5 of 48 (10%) tumors, respectively. Mutations of p16 were detected in 4 of 67 (6%) tumors. Six of 67 (9%) tumors had mtDNA alterations and none of the tumors showed high-frequency MSI. All 30 patients showed one or more gene alterations in one or more genetic loci. Discordant genetic patterns among individual lesions within a patient were observed in 28 of the 30 (93%) patients. The most discordant locus was TP53, present in 11 of 29 (38%) informative cases, followed by D18S61, present in 11 of 30 (37%) informative cases. These results suggest that the genetic pathways of multiple esophageal SCCs may differ even within the same patient.

Convergent Mechanisms of Somatic Mutations In Polycythemia Vera

AbstractAbstract 1979Polycythemia vera (PV) is an acquired clonal blood disorder characterized by variable increased number of clonal myeloid cells (erythrocytes, granulocytes and platelets) and mostly normal polyclonal T lymphocytes. Most patients carry a gain-of function JAK2 V617F mutation in 2 copies acquired by a uniparenteral disomy; i.e. a copy-neutral loss of heterozygosity (LOH) in chromosome 9p in PV pathogenesis. Yet, JAK2 V617F mutation is not a PV-initiating event and the family clustering of PV suggests a contribution of inherited genetic events to its pathogenesis. Interestingly, majority of PV subjects acquire the JAK2 V617F mutation on a distinct a germline SNP 9p24.1 haplotype. Using whole-genome SNP arrays, we assayed 34 T-cells and 66 granulocytes from PV patients (including 32 pairs from the same patient). Comparison to a control population identified multiple germline SNPs around JAK2 as being associated with PV susceptibility (rs11999802, P=1.8×10-8, OR=4.4). Analysis of somatic genomic changes in granulocytes revealed strong genetic heterogeneity: we detected 9p LOH in 36% of the patients, 9p chromosomal gain in 1 patient, as well as “fractional LOH” in 14% of the patients where a small fraction of granulocytes have LOH. Interestingly, the magnitude of somatic LOH in 9p was strongly associated with somatic V617F allelic dosage (r=0.74, P=4.8×10-12), suggesting that LOH preferentially increase V617 PV subclone without altering genomic copy number. Adjusting for the fractional LOH, we did not have sufficient power to detect association between rs11999802 germline genotypes and increased dosage of somatic V617F mutations. Analysis of granulocytes with heterozygous rs11999802 genotypes demonstrated that LOH increases the relative fraction of germline risk alleles versus non-risk alleles. In summary, germline risk variants surrounding JAK2 predispose to somatic SNVs within JAK2, whose allelic dosage can be further increased by a serial subclonal expansion of allele-specific copy-neutral LOH or chromosomal duplications. Our study illustrates how convergent mechanisms of somatic mutations increase the genetic load of gain-of-function mutations contributing to the clonal expansion and disease pathogenesis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Study of loss of heterozygosity at 9p21 and P16 expression in gastrointestinal stromal tumors

To evaluate the impact of loss of heterozygosity (LOH) at chromosome 9p21 and P16(INK4A)(CDKN2A) expression on the prognosis of gastrointestinal stromal tumor (GIST). A total of 51 cases with GISTs were characterized by immunohistochemistry and evaluated for LOH at 9p21 by microsatellite analysis in 4 markers(D9S1751, D9S1846, D9S942 and D9S1748). Associations of LOH at 9p21 and P16(INK4A) expression encoded by CDKN2A with clinicopathological parameters and prognosis in GISTs were analyzed. The frequency of 9p21 LOH was 37.0% (10/27) at D9S1751, 37.5%(12/32) at D9S1846, 42.1%(16/38) at D9S942 and 24.2%(8/33) at D9S1748. The overall frequency of LOH at 9p21 was 63.3%(31/49). In 21 samples of 51 GISTs(41.2%), P16 expression was not detected. Loss of P16 expression was 60%(12/20) in high risk group and 23.5%(4/17) in very low and low risk groups(P<0.05). The 5-year overall survival rate of p16-negative patients was 70.8%, while in P16-positive patients it was 92.0%(P<0.05). LOH at 9p21 is a frequent event in GIST. Loss of CDKN2A gene at 9p21 may contribute to the progression and malignant transformation of GIST. P16 expression in GIST is associated with prognosis.

The Prognostic Value of Ki-67, p53, Epidermal Growth Factor Receptor, 1p36, 9p21, 10q23, and 17p13 in Skull Base Chordomas

Skull base chordomas are rare, locally aggressive, notochord-derived neoplasms for which prognostically relevant biomarkers are not well established. To evaluate whether newly discovered molecular alterations in chordomas have prognostic significance similar to what has been described regarding Ki-67 proliferation index. We conducted a retrospective study of 28 cases of primary clival chordomas. Ki-67 proliferation index 5% or more, p53 accumulation, and epidermal growth factor receptor expression were seen in 32%, 44%, and 8% of chordomas, respectively. 1p loss of heterozygosity (LOH) and/or 1p36 hemizygous deletion was seen in 30% of tumors, while 9p LOH and/or 9p21 homozygous deletion was seen in 21% of cases. Loss of heterozygosity at 10q23 and 17p13 were identified in 57% and 52% of cases, respectively. Ki-67 proliferation index 5% or more and 9p LOH were significantly associated with a shorter overall survival, while homozygous deletion at 9p21 via fluorescence in situ hybridization approached significance. No correlation with survival was found for p53 or epidermal growth factor receptor expression, 1p36 hemizygous deletion, or LOH at 1p, 10q23, or 17p13. Chordomas with elevated Ki-67 proliferation index or deletion at 9p21 may be at risk for a more aggressive clinical course and shorter survival. These biomarkers may thus be used to improve therapeutic stratification.

Characteristics of combining loss of heterozygosity of 1p/19q in glioma

To find possible factors correlated with combined loss of heterozygosity (LOH) of 1p and 19q. The status of 1p and 19q of 138 glioma specimen from January 2009 to December 2009 was evaluated by Fluorescence in situ hybridization (FISH) method, and the frequencies of combining LOH of 1p/19q were compared between different pathologies, brain sub-regions, genders and ages. The frequencies of combined LOH of 1p and 19q of oligodendroglial (81.3%) and oligo astrocytic tumors (55.8%) were significantly higher than that of astrocytic tumor (22.2%) (P < 0.01), and the frequency of oligodendroglial tumor was significantly higher than that of oligo astrocytic tumor (P < 0.05). The frequency of combining LOH of 1p and 19q in frontal lobe (61.8%) was higher than that in temporal (31.8%) and insular lobes (34.6%) (P < 0.05). Combining LOH of 1p and 19q has significant correlation with the pathologies and brain sub-regions.

902 Investigating Clonal Competition in Barrett's Associated Tumorigenesis Using Spatial Maps of Genetic Heterogeneity

Introduction Genetic heterogeneity within Barrett9s mucosa is significantly associated with the development of oesophageal adenocarcinoma (OA). Why genetic diversity promotes tumorigenesis is unclear. Pro-tumorigenic interaction between clones is a possible explanation. To look for evidence of clonal interaction within Barrett9s lesions, we have undertaken a high-resolution clonal-ordering analysis to produce phylogenetic trees and spatial maps that illustrate the spread of genetically distinct clones in endoscopic mucosal resection specimens (EMRs). Methods Entire EMR specimens were serially sectioned. Each crypt was classified according to histological grade. Aneuploidy was assessed using image cytometry. More than 50 individual crypts were then laser-capture microdissected from each EMR. The genetic mutation burden of individual crypts was assessed (K-ras, p53 and p16 point mutations and microsatellite loss of heterozygosity (LOH) data for up to16 loci on chromosomes 3p (FHIT), 5q (APC), 9p (p16), 17p (p53), 17q and 18q (SMAD4)). In each EMR, a point mutation in at least one gene was identified. The relative location of each analysed crypt was recorded and used to reconstruct a genetic and histological map of each EMR. Results EMRs were rarely genetically homogeneous. Typically, dysplastic crypts within individual lesions were clonal for a founding p53 or p16 point-mutated clone, whereas the adjacent metaplastic (non-dysplastic) crypts analysed did not contain the point mutation. Mosaic subclones with distinct patterns of LOH (often 18q and/or 3p) were present. In one EMR, the same p53 and p16 point mutations and 5q and 17p LOH were detected across the entire EMR. Conclusion Clonal expansion of mutated cells is frequently restricted to small numbers of crypts. Interactions between genetically distinct clones appears to be predominantly competitive: a crypt acquiring an additional pro-tumorigenic mutation can outcompete, and so clonally expand to the detriment of, the original crypts. Further investigation will reveal whether this progression is driven by mutation and selection or by additional mutations altering the behaviour of neighbouring pre-existing clones.

Heterozygosity status of 1p and 19q and its correlation with p53 protein expression and EGFR amplification in patients with astrocytic tumors: novel series from India

There are few reports of loss of heterozygosity (LOH) of 1p and 19q in astrocytic tumors, especially glioblastoma multiforme (GBM). We evaluated 1p and 19q (either or both) heterozygosity status in 71 astrocytomas, including 6 pediatric cases: 20 diffuse astrocytomas (DA), 9 anaplastic astrocytomas (AA), and 42 GBM. In the GBMs, p53 protein expression was assessed by immunohistochemistry and epidermal growth factor receptor ( EGFR) gene amplification by fluorescence in situ hybridization; TP53 sequencing was done in 15 of the GBMs. In adults, LOH of 1p or 19q was detected in 16% of DAs and 50% of GBMs; none of the AAs showed this alteration. In GBMs, LOH of 19q was most common (26%), followed by combined 1p and 19q LOH (13%) and 1p LOH (10%). Pediatric GBMs also harbored isolated 1p and 19q LOH (50%). Notably, LOH of 1p or 19q LOH was more frequent in p53 immunopositive secondary GBMs (61%) than in primary GBMs (17%). This suggests that LOH of 1p and 19q may be acquired during progression to secondary GBMs. Thus, 1p and 19q LOH can occur in astrocytic tumors, most commonly in secondary GBMs without morphological correlation with an oligodendroglial histology. The clinical significance of recognition of this subset of GBMs is based on several recent reports of association with better prognosis, although long-term follow-up studies are required.

Serum DNA can define tumor-specific genetic and epigenetic markers in gliomas of various grades

We evaluated whether cell-free circulating DNA can be used as a noninvasive approach for detection of genetic/epigenetic alterations in brain tumors during the course of the disease. Paired tumor-serum samples from 70 patients with either high-grade astrocytomas (n = 41) or oligodendrogliomas of various grades were analyzed. The median interval between surgery and serum sampling was 1 month (range 0.5-168 months). DNA was extracted from whole blood, serum, and paraffin-embedded tumor sections. Loss of heterozygosity (LOH) in chromosomes 1p, 19q, and 10q was assessed by polymerase chain reaction (PCR)-based microsatellite analysis. The methylation status of O(6)-methyl guanine methyltransferase (MGMT) and phosphatase and tensin homolog promoters was studied by methylation-specific PCR. LOH and/or methylation that could identify DNA as tumor-specific was found in 80.5% of astrocytic tumors and in all oligodendrogliomas. The rate of serum detection of these biomarkers was 51% and 55%, respectively, with specificity around 100%. The rate of serum detection did not differ between low- and high-grade oligodendrogliomas. Statistically significant tumor-serum concordance was found for MGMT methylation in both astrocytic tumors (83%; P < .001) and oligodendroglial tumors (72%; P < .003) and for LOH of 10q (79%; P < .002) and 1p (62%; P < .03) in oligodendrogliomas. We conclude that serum DNA in glial tumors is informative for both LOH and aberrant gene promoter methylation analysis during the course of the disease. The sensitivity is moderate and specificity is high for both low- and high-grade tumors. Future studies should identify a panel of biomarkers that bear the highest potential for clinical application.