Presence Of Loss Of Heterozygosity Research Articles

Predictive and prognostic significance of loss of heterozygosity in ABC transporter genes in breast cancer

ABC-transporter family genes have been well studied and their involvement in the development of drug resistance has been assessed. The presence of aberrant conditions in these genes can affect the treatment and prognosis of the disease. Loss of heterozygosity (LOH) is one of these conditions; it is a common event in cancer development. therefore, The aim of this study was to investigate the relationship between LOH in ABC transporter genes in breast cancer and response to chemotherapy and disease prognosis. Material and methods. a total of 130 breast cancer patients were included in the study. microarray analysis was performed on Affymetrix Cytoscantm HD Array high-density DNA chips to assess LOH status. Chromosome Analysis Suite 4.1 software (Affymetrix, USA) was used to process microarray results. Results. Forty-nine ABC transporter genes were evaluated for LOH. the frequency of LOH ranged from 6.9 % to 90 %. an association analysis identified two genes: ABCG5 and ABCG8, in which the presence of LOH was associated with a lack of objective response to neoadjuvant chemotherapy. the presence of LOH in the ABCA5, ABCA6, ABCA8, ABCA9, ABCA10 and ABCC3 genes was associated with high rates of metastasis-free survival (log-rank test, p<0.04). Conclusion. The presence of loss of heterozygosity in the ABC transporter genes was found to have no significant effect on the response to chemotherapy. However, a high prognostic potential of ABCA family genes was found.

Abstract 2452: Evidence of decreased X chromosome inactivation in primary ovarian tumors

Abstract Copy number alterations and loss of heterozygosity (LOH) are common in ovarian tumors, including those on the X chromosome. In females, one of the two copies of the X chromosome is transcriptionally silenced during embryonic development in the epigenetic process of X chromosome inactivation. Specifically, the long non-coding RNA XIST is expressed from the inactive X (Xi) and triggers chromosome-wide DNA methylation. In breast and ovarian cancer cell line studies, Xi deletion with concomitant duplication of the active X (Xa) has been observed. We sought to examine which copy of the X chromosome, Xi or Xa, was gained or lost in a set of primary ovarian tumors, using levels of XIST expression as a marker of X chromosome inactivation. To determine which copy of the X chromosome harbors the alterations, we compared local copy number and LOH in Xq13.2 to the levels of XIST expression in a set of N=134 primary tumors from Mayo Clinic ovarian cancer patients. Associations with XIST expression were examined using ANOVA, with copy number of two and absence of LOH as the reference level. Associations between expression of XIST and expression of other genes were examined with a mixed effects model, to account for the repeated observations across patients, after adjusting for local copy number and LOH. Associations with XIST expression and overall survival were performed with Cox regression. Additional analyses were performed in a subset of 24 patient tumors with X chromosome-wide alterations to simplify interpretation, including a visual examination of chromosome-wide DNA methylation patterns. Analyses were replicated in N=106 ovarian tumors from TCGA with X chromosome-wide alterations. Across N=134 ovarian tumors from Mayo Clinic, abnormal copy number (not equal to two) was associated with reduced XIST expression (p=0.0016). Levels of XIST expression were similar for tumors with one copy and tumors with three or more copies. Similarly, levels of XIST expression were also lower for tumors with LOH (p=5.2E-5). Reduced XIST expression was also associated with poorer survival, after adjusting for age (p=0.02). Results were consistent in the subset of tumors with whole chromosome alterations (pCN=0.0013, pLOH=0.0019). For these tumors, when LOH was present, XIST expression was low and the DNA methylation distribution is bi-modal, suggesting that the inactive X was lost and the active X was gained. Furthermore, after adjusting for copy number and LOH, XIST expression was weakly positively correlated with the median expression of other genes (R2=0.04, p=0.026). In N=106 TCGA tumors with whole X chromosome alterations, XIST expression was also reduced in tumors with abnormal copy number (p=0.006) and with LOH (p=1.5E-10). Taken together, because XIST is expressed from Xi and not Xa, associations of reduced XIST expression with abnormal copy number and presence of LOH suggest that Xi is frequently lost and Xa is gained in ovarian tumors, consistent with cell line studies. These results should be further validated in larger sample sets and examined for association with clinical outcome. Citation Format: Yilin Song, Bryan M. McCauley, Melissa C. Larson, Sebastian M. Armasu, Kate Lawrenson, Ellen L. Goode, Nicholas B. Larson, Stacey J. Winham. Evidence of decreased X chromosome inactivation in primary ovarian tumors [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 2452.

LOSS OF HETEROZYGOSITY IN THE BRCA1 AND BRCA2 LOCUS IN BREAST CANCER

One of the factors of variability of malignant neoplasms is the loss of heterozygosity (LOH). The biological meaning of LOH, in relation to carcinogenesis, is associated with the inactivation of heterozygous loci of pathogenetically significant genes. Thus, the aim of this work was to study BRCA1/2 LOH in breast tumors.Material and Methods. The study included 122 patients with stage IIAIIIC breast cancer. DNA was isolated from 122 biopsy samples of tumor tissue using the QIAamp DNA mini Kit (Qiagen, Germany). To assess the status of LOH, microarray analysis was performed on high-density DNA chips from Affymetrix CytoScanTM HD Array. To process the results of microchipping, we used the Chromosome Analysis Suite 3.3 program (Affymetrix, USA).Results. The loss of heterozygosity in the BRCA1 gene was found to be associated with response to NAC. It was shown that in 59 patients LOH in the BRCA1gene was associated with an objective response to treatment (p=0.005). The presence of LOH in the studied genes was associated with a favorable prognosis. The 5-year non-metastatic survival rates were 75 % and 100 % in patients with LOH in the BRCA1 and BRCA2 genes, respectively (log-rank test: p=0.003 and p=0.05, respectively).Conclusion. The phenomenon of LOH in the BRCA1/2 genes was shown to be associated with response to NACT. BRCA1/2. Further studies are needed to evaluate the frequency of BRCA1/2 LOH after NAC for choosing and changing treatment tactics.

Abstract 435: Cell-free circulating tumor DNA (ctDNA) detects somatic copy number loss in homologous recombination repair genes

Abstract Background: In addition to BRCA1/2 germline and somatic inactivating mutations, loss of heterozygosity (LOH) and biallellic somatic copy number loss are associated with BRCA1/2 loss of function. Patients with cancers harboring these somatic features may benefit from treatment with PARP inhibitors. ctDNA analysis has proven a viable alternative to tumor tissue genotyping, especially in tissue- or time-limited clinical scenarios. However, ctDNA assessment of LOH and biallelic copy number loss is challenging given that ctDNA is diluted by cell-free leukocytic DNA making it difficult to confidently call these genomic events. We developed a method to identify somatic biallelic copy number loss in ctDNA using targeted sequencing of cell-free (cfDNA) across a wide range of cancer types. Methods: A novel statistical model was developed using coverage profiles and single nucleotide polymorphism (SNP) allelic frequencies estimated from plasma samples to determine the presence of LOH and biallellic copy number loss in BRCA1 or BRCA2. For each gene of interest, the model uses observed coverage and fragment size distribution, together with allele frequency of germline SNPs. The model was applied to plasma samples from 28,000 patients with advanced solid tumors sequenced using a 73-gene next generation sequencing ctDNA panel (Guardant360®, Guardant Health, Redwood City, CA). Results: The model was analytically validated using in-silico simulations in order to assess both the limit of blank and limit of detection. This method shows 95% sensitivity in detecting LOH and bi-allellic copy number loss for samples with a maximum somatic variant allele frequency as low as 9%. Sensitivity is mainly driven by the panel size and the depth of coverage of the gene of interest. The model was then applied to the 28,199 patient cohort. BRCA1 and BRCA2 LOH was observed in 2.4% (134/5568) and 7.4% (415/5568) of classic homologous recombination deficient (HRD) cancers including breast, ovarian, prostate, and pancreas. BRCA1 and BRCA2biallelic copy number loss was observed in 0.3% (19/5568) and 0.6% (31/5568) of this same group of HRD cancers. Discussion: In this cohort of 5,568 patients with classic HRD associated cancers, somatic LOH and biallelic copy number loss was detected in BRCA1 in 2.7% of samples and in BRCA2 in 8.0% of samples, which is aligned with previously reported tissue prevalence. BRCA1/2 somatic LOH and biallelic copy number loss can accurately be detected in ctDNA utilizing likelihood-based scores based on coverage and germline SNPs allele frequencies. The ability to identify this therapeutically targetable genomic alteration through a non-invasive ctDNA assessment has significant clinical implications. Citation Format: Catalin Barbacioru, Victoria M. Raymond, Marcin Sikora, Elena Helman, Sante Gnerre, Stephen Fairclough, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Cell-free circulating tumor DNA (ctDNA) detects somatic copy number loss in homologous recombination repair genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 435.

Loss of heterozygosity (LOH) Analysis in individuals with developmental delay/congenital anomalies previously investigated for 22q11.2 Deletion Syndrome

LLoss of heterozygosity (LOH) also called absence of heterozygosity (AOH) or regions of homozygosity (ROH) can be detected by SNP-array analysis. It is observed in regions identical by descent or occurs because of errors in the mechanisms of recombination between homologous chromosomes, DNA replications or DNA repair. The 22q11.2 deletion syndrome (22q11.2DS) has a prevalence of 1/4000 live births and an extensive clinical variability. About 30% of individuals with 22q11.2DS clinical suspicion have been diagnosed with the typical 22q11.2 deletion and other genomic imbalances are often reported related to similar phenotypes. However, most of patients remain without an etiological diagnosis. Currently, there are few studies about LOH in individuals with developmental delay/congenital anomalies. The aim of this study is to describe the preliminary results of LOH analysis in individuals with clinical suspicion of 22q11.2DS. Inclusion criteria was individuals with clinical suspicion without the typical 22q11.2 deletions, previously tested by Fluorescent in situ Hybridization (FISH) or Multiplex Ligation Probe-dependent Amplification (MLPA). Exclusion criteria was parental consanguinity. A total of 124 individuals were investigated by chromosomal microarray analysis (CMA) using the CytoScan HD or 750K Array (Affymetrix®). Data were compared with a database composed by 114 control individuals from Brazilian general population. We considered only LOH biggest than 3Mb and not frequent (<1%) in control individuals. The presence of LOH was more often in the patients than in the control group (12,1% against 3,5%). In the patient group the LOH size were between 3,3Mb and 46,2Mb (average size 22,7Mb) and in the control group it was between 3,1Mb and 8,5Mb (average size 6,0Mb). It was observed only one large LOH for almost all patients, and for almost all the control group it was observed two small LOHs. Among patients with LOH, six also carried VOUS (variants of uncertain significance) and one had a pathogenic imbalance. Laboratorial confirmations and genotype-phenotype correlation are in progress. Our results clearly show a higher proportion and bigger size of LOHs in individuals with developmental delay/congenital anomalies and a possible applicability of LOH analysis as a diagnostic approach in individuals with the same characteristics of the present sample.Support: Fapesp and CNPq

Abstract S2-02: The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers

Abstract Introduction: Women carrying BRCA1 or BRCA2 germline mutations have a 45-80% lifetime risk of developing breast cancer (BC). BRCA1 and BRCA2 are perceived as bona fide tumor suppressor genes, whereby bi-allelic inactivation in tumor cells is required for tumorigenesis. Recent studies have indicated that loss of heterozygosity (LOH) of the wild-type allele of BRCA1 may be heterogeneous and constitute a late event. Therefore, additional somatic events prior to full BRCA1/2 inactivation may be required for tumorigenesis. Given that the somatic events that result in the development of BRCA1/2-BCs and their chronology are not understood, here we sought to define the genomic landscape of BRCA1/2-BCs and whether LOH of BRCA1/2 wild-type allele and/or mutations affecting additional tumor suppressor genes would be clonal or subclonal in these cancers. Methods: We retrieved 29 BRCA1-BCs and 10 BRCA2-BCs from the Pathology Departments of the authors' institutions. DNA extracted from microdissected tumor and normal breast samples was subjected to targeted capture massively parallel sequencing using either the MSK-IMPACT assay or an assay targeting all exons of 254 genes recurrently mutated in BC or related to DNA repair. Somatic single nucleotide variants, small insertions and deletions and copy number alterations affecting genes present in both sequencing assays (111 genes) were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to define clonal (i.e. present virtually in 100% of the cancer cells of a given case) and subclonal mutations and the presence of LOH of the BRCA1 and BRCA2 wild-type alleles. Results: Our analysis revealed bi-allelic inactivation of BRCA1 in 28 of 29 BRCA1-BCs (93% harbored LOH of the BRCA1 wild-type allele and 3% harbored a second somatic BRCA1 pathogenic mutation). The only BRCA1-BC lacking bi-allelic inactivation of BRCA1 was an estrogen receptor-positive lobular carcinoma, lacking genomic features consistent with homologous recombination DNA repair defects, diagnosed at 62 years of age. Bi-allelic inactivation of BRCA2 was found in all cases (100% of harbored LOH of the BRCA2 wild-type allele). A clonal somatic 'second hit' resulting in bi-allelic inactivation of BRCA1 or BRCA2 was detected in 76% and 100% of BRCA1-BCs and BRCA2-BCs, respectively. In BRCA1-BCs, TP53 mutations were detected in 76% of cases, and these mutations were found to be clonal in 58% of cases. The repertoire of somatic mutations affecting BRCA1-BCs included clonal somatic mutations or homozygous deletions of known tumor suppressor genes, such as PTEN, RB1, CDKN2A and NF1. In contrast, only 10% of the BRCA2-BCs harbored TP53 somatic mutations. Though clonal somatic mutations in several cancer genes were detected, 40% of BRCA2-BCs had no mutations affecting the cancer genes analyzed. Conclusions: Bi-allelic inactivation of BRCA1 and BRCA2 are frequent events in BRCA1-BCs and BRCA2-BCs, respectively. In a subset of BRCA1-BCs, however, the second 'hit' appeared to be subclonal, whereas mutations affecting TP53 and other tumor suppressor genes were clonal, supporting the notion that at least in a subset of these tumors, loss of the wild-type allele of BRCA1 may be preceded by inactivation of another tumor suppressor gene. Citation Format: Geyer FC, Burke KA, Macedo GS, Piscuoglio S, Ng CK, Martelotto LG, Papanastatiou AD, De Filippo MR, Schultheis AM, Brogi E, Robson M, Wen YH, Weigelt B, Schnitt SJ, Tung N, Reis-Filho JS. The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S2-02.

Loss of Heterozygosity of Tumor Suppressor Genes (p16, Rb, E‐cadherin, p53) in Hypopharynx Squamous Cell Carcinoma

Microsatellite alterations, especially those that cause loss of heterozygosity (LOH), have recently been postulated as a novel mechanism of carcinogenesis and a useful prognostic factor in many kinds of malignant tumors. However, few studies have focused on a specific site, hypopharynx. The aim of this study was to evaluate the relationship between LOH and hypopharyngeal squamous cell carcinoma (HPSCC). Laboratory-based study. Integrated health care system. Matched normal and cancerous tissues from 30 patients with HPSCC were examined for LOH in 4 tumor suppressor genes (TSGs) (p16, Rb, E-cadherin, and p53) at loci 9p21, 13q21, 6q22, and 17p13, respectively, using microsatellite markers amplified by polymerase chain reaction. The results for each loci were compared with clinicopathological features. Among the 30 cases, 26 (86.7%) exhibited LOH, with the most common alteration being LOH at p53 (52.6%). Significantly higher rates of LOH detection were seen in Rb, p53, and the LOH-high group (cases where 2 or more loci with LOH were found) in cases of lymph node metastasis. Compared with stage I and II carcinoma, tumors of stages III and IV had significantly higher frequencies of LOH in Rb, p53, and the LOH-high group. However, the presence of LOH was not significantly correlated with survival. These results suggest that LOH in TSGs such as Rb and p53 may contribute to the development and progression of HPSCC. The presence of LOH in the primary tumor may also be predictive of lymph node metastasis.

Concomitant loss of heterozygosity at the BRCA1 and FHIT genes as a prognostic factor in sporadic breast cancer

Previous studies have shown loss of heterozygosity (LOH) at the BRCA1 and FHIT genes in sporadic primary breast cancer. The aim of this study was to evaluate concomitant LOH at the BRCA1 and FHIT genes in sporadic breast cancer and investigate its influence on patient survival. Loss of heterozygosity was determined using microsatellite markers. The analysis on the informative cases ( n = 72) indicated LOH at both the BRCA1 and FHIT loci in 25 cases (35%), the absence of LOH at both loci in 23 cases (32%), and the presence of LOH at one of the loci in 24 cases (33%). The concomitant LOH was associated with poor prognostic factors, such as large tumors ( P = 0.01), axillary nodal involvement ( P < 0.01), histologic grade III ( P < 0.01), vascular invasion ( P = 0.01), and negative hormone receptor ( P = 0.02). After a median follow-up period of 48 months, the concomitant LOH group had the shortest survival ( P < 0.02 by log-rank test; P < 0.05 by Cox model; hazard ratio of 4.87), compared with patients without LOH. These data suggest that concomitant allelic losses of the BRCA1 and FHIT genes are associated with more aggressive breast tumors.

Downregulation of SERPINB13 expression in head and neck squamous cell carcinomas associates with poor clinical outcome

Tumorigenesis of head and neck squamous cell carcinomas (HNSCC) is associated with various genetic changes such as loss of heterozygosity (LOH) on human chromosome 18q21. This chromosomal region maps a gene cluster coding for a family of intracellular serine protease inhibitors (serpins), including SERPINB13. As SERPINB13 expression in HNSCC has recently been shown to be downregulated both at the mRNA and protein levels, here we investigated if such a low SERPINB13 expression is associated with histopathological and clinical parameters of HNSCC tumors and patient survival. By generating specific antibodies followed by immunohistochemistry on a well-defined cohort of 99 HNSCC of the oral cavity and oropharynx, SERPINB13 expression was found to be partially or totally downregulated in 75% of the HNSCC as compared with endogenous expression in non-neoplastic epithelial cells. Downregulation of SERPINB13 protein expression in HNSCC was significantly associated with the presence of LOH at the SERPINB13 gene in the tumors (p = 0.006), a poor differentiation grade of the tumors (p = 0.001), the presence of a lymph node metastasis (p = 0.012), and a decreased disease-free (p = 0.033) as well as overall (p = 0.018) survival of the patients. This is the first report demonstrating that downregulation of SERPINB13 protein expression in HNSCC is positively associated with poor clinical outcome. Therefore, SERPINB13 seems to act as an important protease inhibitor involved in the progression of HNSCC.

Aurora-A Expression Is Independently Associated with Chromosomal Instability in Colorectal Cancer

AURKA (the official symbol for Aurora-A, STK15, or BTAK) regulates the function of centrosomes, spindles, and kinetochores for proper mitotic progression. AURKA overexpression is observed in various cancers including colon cancer, and a link between AURKA and chromosomal instability (CIN) has been proposed. However, no study has comprehensively examined AURKA expression in relation to CIN or prognosis using a large number of tumors. Using 517 colorectal cancers in two prospective cohort studies, we detected AURKA overexpression (by immunohistochemistry) in 98 tumors (19%). We assessed other molecular events including loss of heterozygosity (LOH) in 2p, 5q, 17q, and 18q, the CpG island methylation phenotype (CIMP), and microsatellite instability (MSI). Prognostic significance of AURKA was evaluated by Cox regression and Kaplan-Meier method. In both univariate and multivariate logistic regressions, AURKA overexpression was significantly associated with CIN (defined as the presence of LOH in any of the chromosomal segments; multivariate odds ratio, 2.97; 95% confidence interval, 1.40–6.29; P = .0045). In multivariate analysis, AURKA was associated with cyclin D1 expression (P = .010) and inversely with PIK3CA mutation (P=.014), fatty acid synthase expression (P=.028), and family history of colorectal cancer (P = .050), but not with sex, age, body mass index, tumor location, stage, CIMP, MSI, KRAS, BRAF, BMI, LINE-1 hypomethylation, p53, p21, β-catenin, or cyclooxygenase 2. AURKA was not significantly associated with clinical outcome or survival. In conclusion, AURKA overexpression is independently associated with CIN in colorectal cancer, supporting a potential role of Aurora kinase-A in colorectal carcinogenesis through genomic instability (rather than epigenomic instability).

Loss of heterozygosity on chromosome 10q23 and mutation of the phosphatase and tensin homolog deleted from chromosome 10 tumor suppressor gene in Korean hepatocellular carcinoma patients

Loss of heterozygosity (LOH) in the 10q23 chromosomal region was analyzed in 18 tissue samples from Korean hepatocellular carcinoma (HCC) patients. LOH at the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) region (D10S215, AFMa086wg9 and D10S541) was found in 8 of the 18 (44.4%) HCCs. LOH (20%) and microsatellite instability (26.7%) were also frequently found at the D10S2177 locus, which is located on the telomere side of the PTEN region. LOH was found in other loci, such as AFM280we1 and D10S2281. The presence of LOH in regions other than the PTEN region on chromosome 10q23 suggested the presence of additional tumor suppressor gene(s). PTEN mutation was found in only a subset of HCCs: A single base insertion at the end of the 5'-end splice signal (AG-GUAAGUU) in intron 5 and a silent mutation in exon 6 (codon 188, CTG-Val to CTA). Our data collectively suggest that the genetic alterations of chromosome 10q23, including the PTEN gene, could be important in hepatocarcinogenesis in the Korean population.

Altered structure and deregulated expression of the tumor suppressor gene retinoblastoma (RB1) in human brain tumors

A total of 40 human brain tumor samples were analyzed for tumor-specific alterations at the RB1 gene locus. Gliomas were more prevalent in younger males and meningiomas in older females. Southern blot analysis revealed loss of heterozygosity (LOH) at the intron 1 locus of RB1 gene in 19.4% of informative cases and this is the first report showing LOH at this locus in human brain tumors. Levels of RB1 mRNA and protein, pRb, and the percentage of hyperphosphorylated form of pRb were also analyzed in these tumors. Normal human fibroblast cell line WI38 was used as control in northern and western analysis. Normal sized RB1 mRNA and protein were present in all the tumor samples. Majority of the gliomas had 2.0-fold or higher levels of RB1 mRNA and most meningiomas had less than 2.0-fold of RB1 mRNA compared to control WI38 cells. The total pRb levels were 2.0-fold or higher in all the tumor samples compared to control. More than 50% of pRb existed in hyperphosphorylated form in all gliomas except two. However, six out of 13 meningiomas had less than 50% of total pRb in the hyperphosphorylated form. These results indicate that the increased percentage of hyperphosphorylated form of pRb in gliomas could provide growth advantage to these tumors. Presence of LOH at the RB1 gene locus and the increased levels of RB1 RNA and protein and increased percentage of hyperphosphorylated form of pRb are indicative of an overall deregulation of pRb pathway in human brain tumors.

Microsatellite Instability Is Uncommon in Lymphoepithelioma-like Carcinoma of the Lung

Primary lymphoepithelioma-like carcinoma of the lung (LELC) shares some morphologic and clinical characteristics with malignancies associated with microsatellite instability (MSI). The aims of our study were to determine the MSI status in LELC and compare these findings with stage I non-small cell lung carcinoma (NSCLC) with marked lymphocytic host response (MLHR). We assessed MSI by a DNA-based polymerase chain reaction assay using mononucleotide (BAT25 and BAT26) and dinucleotide (D2S123, D5S346, and D17S250) repeats. MSI was detected in 2 (29%) of 7 LELC cases with only 1 marker (D17S250), and in 3 (19%) of 16 NSCLC cases with MLHR with only 2 markers (1D2S123 and 2 D17S250). Loss of heterozygosity (LOH) was detected at 1 or 2 of 3 dinucleotide repeats in 11 NSCLC cases (69%) with MLHR and 3 LELC cases (43%) (P = .36). The overall frequencies of LOH in NSCLC with MLHR were 29% and 19% in LELC (P = .55). MSI is very uncommon in LELC, indicating that MSI is not an important event in carcinogenesis for this tumor subtype. The presence of LOH suggests a probable role of tumor suppressor genes in LELC carcinogenesis.

Relationship Between the Loss of Heterozygosity and Tobacco Smoking in Pulmonary Adenocarcinoma

A loss of heterozygosity (LOH) is a major cause of lung carcinogenesis, and it is considered to be related to tobacco smoking in central type lung cancer. We investigated the relationship between LOH in lung adenocarcinoma and tobacco smoking. In a consecutive series of 50 patients with lung adenocarcinoma who underwent a surgical resection, cancer tissue specimens and corresponding normal peripheral lung and central bronchial tissue specimens were analyzed for LOH at the regions of D3S1234 (FHIT), D3S1300 (FHIT), D9S171 (CDKN2), and D17S796 (p53) by polymerase chain reaction using four fluorescence-labeled dinucleotide markers. To examine how cells are influenced by smoking, the A549 cell line was exposed to benzo[a]pyrene (B[a]P) for 24 weeks and then was subjected to the above analysis. The LOH in cancer tissue was thus detected in four (17%) patients at D3S1234, six (14%) at D3S1300, and seven (18%) at D17S796, but no LOH was detected in any normal tissue specimens. The incidence of LOHs in cancer tissue specimens from active smokers was 21% at D3S1234, 11% at D3S1300, and 19% at D17S796, whereas that of LOHs from nonactive smokers was 0% at D3S1234, 19% at D3S1300, and 14% at D17S796. Analyzing the relationship between the pack-year index and the presence of LOH, a significant difference was found among the active smokers. Besides, in the A549 cell line exposed to B[a]P, LOH was de novo detected in one (D2S123) of the nine regions examined. The incidence of LOH could be influenced by tobacco smoking in lung adenocarcinoma, thus suggesting the presence of an important event in the carcinogenesis of this disease.

Functional Loss of the γ-Catenin Gene through Epigenetic and Genetic Pathways in Human Prostate Cancer

Gamma-catenin is a cell adhesion molecule and a candidate mediator of Wnt signal transduction. We hypothesized that impaired regulation of gamma-catenin through genetic and epigenetic pathways is associated with the pathogenesis of prostate cancer. To test this hypothesis, cytosine-phosphate-guanine methylation, loss of heterozygosity (LOH), and mutation status of the gamma-catenin gene were analyzed in cultured prostate cancer cell lines, 180 localized prostate cancers, 69 benign prostatic hyperplasias, and 11 hormone refractory prostate cancers (HRPC). In prostate cancer cell lines (DuPro, LNCaP, ND-1, and PC3), gamma-catenin mRNA transcripts were increased after 5-aza-2'-deoxycytidine treatment. In localized prostate cancer, gamma-catenin expression was lower but prevalence of gamma-catenin methylation was higher compared with benign prostatic hyperplasia. However, gamma-catenin methylation did not correlate with Gleason sum, pT category, or capsular penetration. Among localized prostate cancers with positive gamma-catenin methylation, the presence of LOH at chromosome 17q21 was closely related to down-regulation of gamma-catenin mRNA expression. The gamma-catenin mutations were not found in localized prostate cancers, whereas six mutations were found in five HRPCs within or close to the GSK-3beta consensus motif phosphorylation site, among which four HRPCs showed strong nuclear gamma-catenin accumulation. In these four HRPCs, Bcl-2 expression was increased, whereas the target of the Wnt signal, c-myc, was only expressed in one HRPC. Therefore, although epigenetic gamma-catenin methylation is an early event in the development of prostate cancer, simultaneous events of epigenetic cytosine-phosphate-guanine methylation and genetic LOH may be responsible for functional loss of gamma-catenin. The gamma-catenin mutation related to Bcl-2 overexpression has a significant effect on the pathogenesis of HRPC. This is the first report to characterize the epigenetic and genetic regulation of gamma-catenin in human prostate cancer.

High frequency of allelic losses in high-grade prostate cancer is associated with biochemical progression after radical prostatectomy

Loss of heterozygosity (LOH) is the most consistent genetic change in prostate cancer (CaP). We aimed, to correlate specific LOH and the overall LOH frequency, to disease progression after radical prostatectomy (RP) in high-grade CaP. Between January 1990 through December 1998, 126 patients who underwent RP (cT1-T2), Gleason 8–10, were pT3, or pN1, or SM(+) (surgical margins). Nine were lost of follow-up, 39/117 (33%) had no biochemical progression (mean follow-up: 45 months). After exclusion for preoperative PSA >50 ng/mL, a case-control study was designed by matching 26 of these cases with 26 similar patients without biochemical progression (criteria: pT, pN, year of surgery). Using microsatellite markers, LOH were assessed on six chromosomal regions (7q31, 8p22, 12p13, 13q14, 16q23.2, 18q21). No prognostic value was associated with LOH at any one specific locus. However, the overall LOH frequency (five classes, cutoff of 60%), was significantly higher if progression ( P = 0.02; P = 0.03) in SM(+) patients, and was near statistical significance ( P = 0.08; P = 0.07) for the overall case-control population. In multivariate analysis (overall population), the overall LOH rate ≥60% was independently associated with progression [ P = 0.035; Odds Ratio (OR) = 5.54]. An overall LOH rate ≥60% predicted poor outcome in 85% of SM(+) patients and 69% of the whole population. Our results suggest that the overall rate of LOH at chromosomal “hot spots” is more likely to be predictive of recurrence than the presence of LOH at any one particular locus. Moreover, the identification of a threshold of LOH could help in predicting patients with poor outcome who may be candidates for local or systemic adjuvant therapies.

Loss of heterozygosity (LOH) in non-small cell lung cancer: difference between adenocarcinoma and squamous cell carcinoma

In non-small cell lung cancer, a loss of heterozygosity (LOH) is frequently observed; however, few studies have investigated the differences in the LOH status between adenocarcinoma and squamous cell carcinoma. In a consecutive series of 49 patients with adenocarcinomas and 22 patients with squamous cell carcinomas, the LOH in tumors was analyzed using polymerase chain reaction employing 5 fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, D13S175) and an autosequencer. LOH was more frequently observed in squamous cell carcinoma (20 of 22, 90%) than in adenocarcinomas (33 of 49, 67%) (P=0.0348), and the number of LOH per patient was also higher in the patients with squamous cell carcinoma (2.2+/-1.4) than in those with adenocarcinoma (1.5+/-1.2, P=0.037). In adenocarcinomas, the number of LOH per patients correlated significantly with the pack-year index, whereas the pathological stage significantly affected the number of LOH in squamous cell carcinomas. The presence of LOH is relatively uncommon in adenocarcinoma of the lung; however, the incidence of LOH tends to be associated with the smoking status.

Clinical value of alterations in p73 gene, related to p53 at 1p36, in human hepatocellular carcinoma

A novel gene, p73, encoding a protein with significant homology to p53 and showing functional similarities to p53, was identified at chromosome 1p36, at which tumor suppressor gene of hepatocellular carcinoma (HCC) is supposed to be. Involvement of p73 in hepatocarcinogenesis is controversial and clinical value of p73 alterations remains obscure. We investigated allelic status of p73 in 63 patients with HCC. Loss of heterozygosity (LOH) in p73 was analyzed by PCR-RFLP analysis. The results were compared with LOH on chromosome 1p surrounding p73 locus, mutations of p53 and p73, and clinicopathologic characteristics. LOH on p73 was observed in 33% of informative tumors. LOH in p73 was not always observed between the regions with LOH on chromosome 1p examined despite the significant association of LOH in p73 with LOH on chromosome 1p. No mutations were detected in p73. Tumors with LOH in p73 were more frequently detected in liver without cirrhosis than that with cirrhosis. There was no significant statistic association between the presence of LOH in p73 and six different clinicopathologic characteristics such as age, sex, histological type, T stage, tumor diameter, and virus status. Disease-free survival rates of the patients with LOH in p73 were significantly poorer than those without LOH in p73. Multivariate analysis indicated that presence of LOH in p73 was independent prognostic factor in patients with HCC. These findings suggested that p73 might play some role in tumor progression of HCC even though p73 should not be considered a candidate gene on chromosome 1p of HCC and does not function as a tumor suppressor gene like p53. Identifying the patients with LOH of p73 in tumors could be useful to predict early recurrence and to stratify the patients who need adjuvant therapy after operation.

Circulating DNA microsatellites: molecular determinants of response to biochemotherapy in patients with metastatic melanoma.

Although biochemotherapy appears to be a promising treatment for metastatic melanoma, its impact remains unpredictable. Microsatellite markers for loss of heterozygosity (LOH) appear to have prognostic significance when identified in primary tumors and serum and/or plasma from cancer patients. However, their association with response to systemic therapy has yet to be assessed. To determine whether microsatellite markers are associated with response to therapy, serum from 41 patients with metastatic melanoma, drawn before the initiation of biochemotherapy, was analyzed for LOH with nine microsatellite markers. During a median follow-up of 13 months, the overall response rate for these 41 patients was 56%, including 13 (32%) complete responses and 10 (24%) partial responses. LOH was detected in sera from 12 (29%) of the 41 patients. The response rate of these 12 patients was 17% (95% confidence interval [CI] = 5% to 45%), whereas that of the 29 patients without LOH was 72% (95% CI = 54% to 85%) (P =.001). All statistical tests were two-sided. The presence of LOH was statistically significant and independently associated with disease progression (multivariable analysis, P =.003). Circulating tumor DNA markers may be useful in assessing prognosis for advanced melanoma patients and their response to biochemotherapy.

Microsatellite instability at chromosome 8p in non-small cell lung cancer is associated with lymph node metastasis and squamous differentiation

Genetic alterations at chromosome arm 8p are associated with advanced disease and poor patient outcome in several types of malignant tumors. We studied the frequency of microsatellite instability (MSI) and loss of heterozygosity (LOH) at chromosome 8p in early stage non-small cell lung cancer (NSCLC) of 47 patients with stage I or II disease (25 squamous cell carcinomas and 22 adenocarcinomas). Microsatellite analysis was performed after laser microdissection using 5 polymorphic tetranucleotide microsatellite markers and 4 dinucleotide markers at chromosome 8p. A pentanucleotide repeat marker at the chromosomal locus 17p13.1 (TP53.Alu) was also analyzed. Expression of the mismatch repair (MMR) proteins hMSH2, hMSH6 and hMLH1 was evaluated by immunohistochemistry. Microsatellite instability (MSI) in at least 2 markers was detected in 9 of 47 patients (19.1%) and was predominantly found at tetranucleotide repeats. Sixteen of 47 (34.0%) NSCLC demonstrated LOH at chromosome 8p. All MSI-positive tumors showed normal expression of the MMR proteins. The presence of MSI at chromosome 8p was associated with lymph node metastasis (p=0.02), squamous differentiation (8/25; 32%-p=0.03), and the presence of LOH at the p53 locus (p=0.06). None of the other investigated clinical, pathologic or molecular factors correlated with MSI. Our study showed that an elevated MSI at selected tetranucleotide sequences (EMAST) on chromosome 8p is frequent in early stage squamous cell carcinomas of the lung with lymphatic spread. The tetranucleotide marker panel used in this study was able to indicate lymph node metastasis and high risk disease in patients with resectable squamous cell lung cancer.