Incidence Of Loss Of Heterozygosity Research Articles

Ionizing radiation induces cells with past caspase activity that contribute to the adult organ in Drosophila and show reduced Loss of Heterozygosity

There is increasing recognition that cells may activate apoptotic caspases but not die, instead displaying various physiologically relevant consequences. Mechanisms that underlie the life-or-death decision in a cell that has activated apoptotic caspases, however, are incompletely understood. By optimizing a published reporter for past caspase activity, we were able to visualize cells that survived caspase activation specifically after exposure to ionizing radiation in Drosophila larval wing discs. We found that cells with X-ray-induced past active caspases (XPAC) did not arise at random but were born at specific locations within the developing wing imaginal discs of Drosophila larvae. Inhibiting key components of the apoptotic pathway decreased XPAC number, suggesting that apoptotic signaling is needed to induce XPAC cells. Yet, XPAC cells appeared in stereotypical patterns that did not follow the pattern of IR-induced apoptosis, suggesting additional controls at play. Functional testing identified the contribution of wingless (Drosophila Wnt1) and Ras signaling to the prevalence of XPAC cells. Furthermore, by following irradiated larvae into adulthood, we found that XPAC cells contribute to the adult wing. To address the relationship between XPAC and genome stability, we combined a reporter for past caspase activity with mwh, an adult marker for Loss of Heterozygosity (LOH). We found a lower incidence of LOH among XPAC compared to cells that did not activate the reporter for past caspase activity. In addition, at time points when wing disc cells are finishing DNA repair, XPAC cells show an anti-correlation with cells with unrepaired IR-induced double-stranded breaks. Our data suggest that non-lethal caspase activity safeguards the genome by facilitating DNA repair and reducing LOH after transient exposure to X-rays. These results identify a physiological role for non-lethal caspase activity during recovery from radiation damage.

E2F1, DIAP1, and the presence of a homologous chromosome promote while JNK inhibits radiation-induced loss of heterozygosity in Drosophila melanogaster.

Loss of heterozygosity (LOH) can occur when a heterozygous mutant cell loses the remaining wild-type allele to become a homozygous mutant. LOH can have physiological consequences if, for example, the affected gene encodes a tumor suppressor. We used fluorescent reporters to study the mechanisms of LOH induction by X-rays, a type of ionizing radiation (IR), in Drosophila melanogaster larval wing discs. IR is used to treat more than half of patients with cancer, so understanding its effects is of biomedical relevance. Quantitative analysis of IR-induced LOH at different positions between the telomere and the centromere on the X chromosome showed a strong sex dependence and the need for a recombination-proficient homologous chromosome, whereas, paradoxically, position along the chromosome made little difference in LOH incidence. We propose that published data documenting high recombination frequency within centromeric heterochromatin on the X chromosome can explain these data. Using a focused screen, we identified E2F1 as a key promotor of LOH and further testing suggests a mechanism involving its role in cell-cycle regulation. We leveraged the loss of a transcriptional repressor through LOH to express transgenes specifically in cells that have already acquired LOH. This approach identified JNK signaling and apoptosis as key determinants of LOH maintenance. These studies reveal previously unknown mechanisms for the generation and elimination of cells with chromosome aberrations after exposure to IR.

Genome-scale patterns in the loss of heterozygosity incidence in Saccharomyces cerevisiae.

Former studies have established that loss of heterozygosity can be a key driver of sequence evolution in unicellular eukaryotes and tissues of metazoans. However, little is known about whether the distribution of loss of heterozygosity events is largely random or forms discernible patterns across genomes. To initiate our experiments, we introduced selectable markers to both arms of all chromosomes of the budding yeast. Subsequent extensive assays, repeated over several genetic backgrounds and environments, provided a wealth of information on the genetic and environmental determinants of loss of heterozygosity. Three findings stand out. First, the number of loss of heterozygosity events per unit time was more than 25 times higher for growing than starving cells. Second, loss of heterozygosity was most frequent when regions of homology around a recombination site were identical, about a half-% sequence divergence was sufficient to reduce its incidence. Finally, the density of loss of heterozygosity events was highly dependent on the genome’s physical architecture. It was several-fold higher on short chromosomal arms than on long ones. Comparably large differences were seen within a single arm where regions close to a centromere were visibly less affected than regions close, though usually not strictly adjacent, to a telomere. We suggest that the observed uneven distribution of loss of heterozygosity events could have been caused not only by an uneven density of initial DNA damages. Location-depended differences in the mode of DNA repair, or its effect on fitness, were likely to operate as well.

ABCL-261: STR Profiling Reveals Tumor Genome Instability in Primary Mediastinal B-Cell Lymphoma

<h3>Context</h3> Molecular factors to determine the prognosis for primary mediastinal B-cell lymphoma (PMBCL) are not described yet. In this regard, the loss of heterozygosity (LOH) in STR loci and elevated microsatellite alteration at selected tetranucleotides (EMAST) in the tumor genome are of interest. <h3>Objective</h3> To analyze the frequency of LOH and EMAST in tumor cells of PMBCL patients at diagnosis and to check possible association with disease features. <h3>Design</h3> STR profiles of the DNA of tumor cells were analyzed in a cohort of 72 PMBCL patients undergoing EPOCH regimen treatment at the National Research Center for Hematology (Moscow, Russia). <h3>Setting</h3> DNA was isolated from tumor biopsy samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission or from the buccal epithelium. STR profiles for LOH and EMAST analysis were assessed by PCR with the COrDIS Plus multiplex kit for amplification of 19 STR markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on an ABI3130 Genetic Analyzer. <h3>Patients</h3> Inclusion criterion: <i>de novo</i>-diagnosed PMBCL patients. Exclusion criterion: pre-treatment. <h3>Main Outcomes Measures</h3> Progression-free survival (PFS) and overall survival (OS) at 24 months from diagnosis were used to assess the independent impact of genetic instability as a risk factor. <h3>Results</h3> LOH was detected in 37 of 72 patients (51.4%). EMAST was found in 40 patients (55.5%); 24 had a combination of EMAST with LOH. For all EMAST-positive patients, the number of involved loci was no more than 4 out of 19 (EMAST-low, less than one-third of the panel of microsatellite markers). It was shown that OS and PFS were the highest in patients with a stable STR tumor profile. <h3>Conclusions</h3> STR profiling of PMBCL tumor genome revealed LOHs associated with deletions or uniparental disomy and detected EMAST attributed to failure of DNA mismatch repair. The high incidence of LOH and EMAST in PMBCL can be associated with standard therapy failure in a significant number of patients. <h3>Funding</h3> This study was supported by Rakfond grant 2/2020.

Cells with loss-of-heterozygosity after exposure to ionizing radiation in Drosophila are culled by p53-dependent and p53-independent mechanisms.

Loss of Heterozygosity (LOH) typically refers to a phenomenon in which diploid cells that are heterozygous for a mutant allele lose their wild type allele through mutations. LOH is implicated in oncogenesis when it affects the remaining wild type copy of a tumor suppressor. Drosophila has been a useful model to identify genes that regulate the incidence of LOH, but most of these studies use adult phenotypic markers such as multiple wing hair (mwh). Here, we describe a cell-autonomous fluorescence-based system that relies on the QF/QS transcriptional module to detect LOH, which may be used in larval, pupal and adult stages and in conjunction with the GAL4/UAS system. Using the QF/QS system, we were able to detect the induction of cells with LOH by X-rays in a dose-dependent manner in the larval wing discs, and to monitor their fate through subsequent development in pupa and adult stages. We tested the genetic requirement for changes in LOH, using both classical mutants and GAL4/UAS-mediated RNAi. Our results identify two distinct culling phases that eliminate cells with LOH, one in late larval stages and another in the pupa. The two culling phases are genetically separable, showing differential requirement for pro-apoptotic genes of the H99 locus and transcription factor Srp. A direct comparison of mwh LOH and QF/QS LOH suggests that cells with different LOH events are distinguished from each other in a p53-dependent manner and are retained to different degrees in the final adult structure. These studies reveal previously unknown mechanisms for the elimination of cells with chromosome aberrations.

ABCL-349: Genetic Instability of Microsatellite Loci in Primary Mediastinal B-cell Lymphoma

Context: Loss of heterozygosity (LOH) in STR loci, elevated microsatellite alteration at selected tetranucleotides (EMAST) and instability of mononucleotide tandem repeats (MSI - microsatellite instability) are intrinsic features of diverse tumor cells. For primary mediastinal B-cell lymphoma (PMBCL) these molecular factors to determine the prognosis of the disease have not been described yet. Objective: To analyze the frequency of LOH, EMAST, and MSI in tumor cells of PMBCL patients at diagnosis and to check possible association with disease features. Design: STR profiles of the DNA of tumor cells were analyzed on a cohort of 36 PMBCL patients undergoing treatment at the National Research Center for Hematology (Moscow, Russia). Setting: DNA was isolated from tumor biopsy samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission or from the buccal epithelium. STR-profiles for LOH and EMAST analysis were assessed by PCR with COrDIS Plus multiplex kit for amplification of 19 STR-markers and amelogenin loci (Gordiz Ltd, Russia). COrDIS MSI (BAT-25, BAT-26, NR-21, NR-24 and NR-27) kit was used for MSI testing. The fragment analysis was performed on ABI3130 Genetic Analyzer. Patients: Inclusion criteria: de novo diagnosed PMBCL patients. There were 10 males and 26 females, the median age at presentation was 30 years (range 21–61). Main outcome measures: Progression-free survival (PFS) and overall survival (OS) at 24 months from diagnosis were used to assess the independent impact of the genetic instability as a risk factor. Results: LOH was revealed in 21 out of 36 patients (58%). EMAST was found in 18 patients (50%); in 12 of them EMAST was accompanied by LOH, and in one by MSI. MSI was found in three patients only. OS and PFS were highest in the group with stable STR profile of a tumor, but not formally significant because of the small sample size. Conclusions: The high incidence of LOH and EMAST in PMBCL can cause standard therapy failure in a significant number of patients. EMAST as a variant of microsatellite instability might be useful for further consideration as a predictor of response to checkpoint inhibitor therapy.

Loss of heterozygosity detected at three short tandem repeat locus commonly used for human DNA identification in a case of paternity testing.

Short tandem repeat (STR) is widely used for DNA profiling in forensic sciences for its stable inheritance. Genomic variations in STR loci may affect the results of the genotyping. In this study, using STR profiling and genome-wide chromosomal microarray assay, we detected the incidence of uniparental disomy or copy-neutral loss of heterozygosity (LOH) in a case of a parental testing, which altered the genotype of three commonly used STR markers including D2S1338, D2S441 and D2S1776. To the best of our knowledge, this is the first time found that LOH affect the genotyping of STR markers commonly used for paternity testing. Our findings demonstrated that the incidence of LOH in the genome may dramatically alter the results of DNA identification, and suggested that genomic structure variation need to be taking into consideration in the DNA identification using STR markers.

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.

A preliminary study of the relationship between breast cancer metastasis and loss of heterozygosity by using exome sequencing

We explored the feasibility of studying loss of heterozygosity (LOH) by using exome sequencing and compared the differences in genetic LOH between primary breast tumors and metastatic lesions. Exome sequencing was conducted to investigate the genetic LOH in the peripheral blood, a primary tumor, and a metastatic lesion from the same patient. LOH was observed in 30 and 48 chromosomal loci of the primary tumor and metastatic lesion, respectively. The incidence of LOH was the highest on chromosome 19, followed by chromosomes 14, 3, and 11 in the metastatic lesion. Among these ‘hot' regions, LOH was observed for multiple genes of the CECAM, MMP and ZNF families. Therefore, the use of exome sequencing for studying LOH is feasible. More members of gene families appeared with LOH in ‘hot' regions, suggesting that these gene families had synergistic effects in tumorigenesis.

Brca2 and tp53 Collaborate in Tumorigenesis in Zebrafish

Germline mutations in the tumor suppressor genes BRCA2 and TP53 significantly influence human cancer risk, and cancers from humans who inherit one mutant allele for BRCA2 or TP53 often display loss of the wildtype allele. In addition, BRCA2-associated cancers often exhibit mutations in TP53. To determine the relationship between germline heterozygous mutation (haploinsufficiency) and somatic loss of heterozygosity (LOH) for BRCA2 and TP53 in carcinogenesis, we analyzed zebrafish with heritable mutations in these two genes. Tumor-bearing zebrafish were examined by histology, and normal and neoplastic tissues were collected by laser-capture microdissection for LOH analyses. Zebrafish on a heterozygous tp53M214K background had a high incidence of malignant tumors. The brca2Q658X mutation status determined both the incidence of LOH and the malignant tumor phenotype. LOH for tp53 occurred in the majority of malignant tumors from brca2 wildtype and heterozygous mutant zebrafish, and most of these were malignant peripheral nerve sheath tumors. Malignant tumors in zebrafish with heterozygous mutations in both brca2 and tp53 frequently displayed LOH for both genes. In contrast, LOH for tp53 was uncommon in malignant tumors from brca2 homozygotes, and these tumors were primarily undifferentiated sarcomas. Thus, carcinogenesis in zebrafish with combined mutations in tp53 and brca2 typically requires biallelic mutation or loss of at least one of these genes, and the specific combination of inherited mutations influences the development of LOH and the tumor phenotype. These results provide insight into cancer development associated with heritable BRCA2 and TP53 mutations.

Abstract P2-08-04: Subtype specific chromosomal aberrations in breast cancer

Abstract Background: Breast cancer is a heterogeneous disease and divided into five distinct groups (Luminal A, Luminal B, HER2-enriched, Basal-like, and Claudin-low subtypes). DNA copy number studies have suggested that chromosomal aberrations are different among subtypes. Basal-like, and Claudin-low subtypes are mainly ER-, PR- and HER2- (triple negative: TN). Recent study reported that the basal-like subtype was the most distinct with common losses of the regions containing tumor suppressor genes such as RB1, BRCA1, INPP4B, and the greatest overall genomic instability. On the other hand, Claudin-low tumors showed few copy number changes. Luminal A tumors show fewer chromosomal copy number changes than Luminal B and HER2-enriched subtypes. Aims: To investigate the extent of the chromosomal aberrations and to evaluate the relationships between chromosomal aberrations and breast cancer subtypes and other clinicopathological characteristics and prognosis. Methods: Specimens were obtained from 363 invasive breast cancer patients who underwent surgery in our department between 1994 and 2011. Four breast cancer subtypes were determined by the immunohistochemical analysis of ER, PR and HER2; hormone receptor (HR; ER and/or PR)+/HER2-, HR+/HER2+, HR-/HER2+(HER2) and TN. Loss of heterozygosity (LOH) at the tumor suppressor gene (TSGs), BRCA1, BRCA2, TP53, RB1, PTEN and INPP4B, was investigated with microsatellite markers using our microsatellite analysis system. Copy number aberrations (CNAs) were analyzed by SNP-CGH array (Illumina, HumanOmni2.5-8).% Defects, which represents the percent of genome region containing chromosomal aberration, was determined by Karyostudio Software v1.4 (Illumina). Results: The incidence of LOH at BRCA, BRCA2, TP53, RB1, PTEN and INPP4B locus was 37.4, 34.0, 57.1, 41.9, 25.8 and 18.0%, respectively. The incidence of LOH was lowest in hormone receptor (HR)+/HER2- subtype, and higher in HER2 and triple negative (TN) subtypes. LOH at INPP4B locus was significantly associated with TN subtype. The LOH at BRCA1 and TP53 loci was highest in HER2 subtype. The LOH at BRCA1, TP53 and INPP4B loci was significantly associated with poor prognosis in all cases; however, the impact of LOH at these TSGs on the prognosis was different among subtypes. Coexistence of LOH at both BRCA1 and TP53 loci was significantly associated with aggressive phenotype and chromosomal aberrations.%Defect was highest in the tumors with LOH at both BRCA1 and TP53 loci. Coexistence of LOH at both BRCA1 and TP53 loci was significantly associated with poor prognosis, especially in HR+/HER2- subtype. Many cases with TN subtype reveal gross chromosomal aberrations, and these tumors were considered to be basal-like. However, there are some cases with few chromosomal alterations in TN, whose prognosis was very poor. These tumors were considered to be Claudin-low subtype. Conclusions: The incidence and biological significance of LOH at TSGs are different among breast cancer subtypes. Coexistence of LOH at both BRCA1 and TP53 loci was associated with gross chromosomal aberrations and poor prognosis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-08-04.

Pretransplant HLA mistyping in diagnostic samples of acute myeloid leukemia patients due to acquired uniparental disomy

Although acquired uniparental disomy (aUPD) has been reported in relapse acute myeloid leukemia (AML), pretransplant aUPD involving chromosome 6 is poorly documented. Such events could be of interest because loss of heterozygosity (LOH) resulting from aUPD in leukemic cells may lead to erroneous results if HLA typing for hematopoietic stem cell donor searches is performed on blood samples drawn during blastic crisis. We report here six AML patients whose HLA typing was performed on DNA extracted from peripheral blood obtained at diagnosis. We observed LOH involving the entire HLA region (three patients), HLA-A, B, C (two patients) and HLA-A only (one patient). An array-comparative genomic hybridization showed that copy number was neutral for all loci, thus revealing partial aUPD of chromosome 6p21. When HLA typing was performed on remission blood samples both haplotypes were detected. A 3-4% LOH incidence was estimated in AML patients with high blast counts. Based on DNA mixing experiments, we determined by PCR sequence-specific oligonucleotide hybridization on microbeads arrays a detection threshold for HLA-A, B, DRB1 heterozygosity in blood samples with <80% blasts. Because aUPD may be partial, any homozygous HLA result should be confirmed by a second typing performed on buccal swabs or on blood samples from the patient in remission.

Abstract 2217: Primary tumor loss of heterozygosity proximal to M6P/IGF2R locus is predictive for the presence and persistence of disseminated tumor cells in the bone marrow of ovarian cancer patients

Abstract Introduction: Disseminated tumor cells (DTC) in the bone marrow (BM) are present in about 35% of all ovarian cancer patients before surgery and after platinum-based chemotherapy which has been associated with an adverse prognosis. Since invasive BM aspirations are less accepted and often not feasible, it would be highly desirable to unmask a molecular biomarker in the primary tumor predicting tumor cell spread and persistence. It was the purpose of the study to 1) investigate loss of heterozygosity (LOH) incidence at four ovarian cancer relevant chromosomal loci involved in inter alia apoptosis, platinum sensitivity or DNA repair in primary ovarian tumor tissue 2) assess the prognostic value of LOH after a median follow-up time of three years and 3) correlate LOH incidence with the occurrence of DTC before surgery and after platinum-based chemotherapy. Methods: In total, 88 ovarian cancer patients were recruited for this study. Primary tumor DNA was extracted from paraffin embedded tissue and analyzed for LOH status at four polymorphic microsatellite markers including D10S1765 (PTEN locus), D13S218 (BRCA1 locus), D17S855 (BRCA2 locus) and D6S1581 (proximal to M6P/IGF2R locus) using PCR-based fluorescence microsatellite analysis. BM aspirates were feasible in 53 patients before surgery and in 40 patients after chemotherapy. BM cells were isolated from heparinized BM by Ficoll-Hypaque density gradient centrifugation and analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Results: In our cohort of 88 patients, we recorded at least one LOH in 56/88 patients (63%), two LOH in 30/88 patients (34%), three LOH in 8/88 patients (9%) and LOH at all four markers in 3/88 patients (3%), respectively. The occurrence of LOH at the entire marker set significantly correlated with tumor grading (p=0.0001) and histology (p=0.004). The frequency of LOH at the marker D10S1765 correlated with FIGO stage (p=0.046) and grading (p=0.05) whereas LOH at D17S855 was associated with grading (p=0.023) and histology (p=0.012). The occurrence did not correlate with disease free as well as overall survival. Prior to surgery, DTC were detected in 26/53 patients (49%) with a median number of 6 cells (range 1-38 cells). After chemotherapy, 20/40 patients (50 %) revealed DTC in their BM with a median number of 10 cells, ranging from 1 to 35 cells. Interestingly, the LOH incidence proximal to D6S1581 significantly correlated with DTC presence before surgery (p=0.05) as well as DTC persistence after chemotherapy (p=0.022). Conclusion: In primary ovarian tumors, allelic loss proximal to D6S1581 might serve as a potential molecular biomarker for the presence and persistence of DTC in the BM. Further investigations have to prove whether such a molecular marker could replace invasive BM aspirations in the future. 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 2217. doi:10.1158/1538-7445.AM2011-2217

High incidence and frequency of LOH are associated with aggressive features of high-grade HER2 and triple-negative breast cancers

Basal-like and HER2-overexpressing breast carcinomas are histologically undifferentiated, high-grade tumors with a high proliferation rate and associated with a poor outcome. Most basal-like tumors lack the expression of ER, PR, and HER2 (triple-negative; TN). Loss of heterozygosity (LOH) is thought to reflect random chromosomal instability, and recent studies have shown that DNA-copy number alterations or LOH occur with a high frequency in basal-like and HER2-amplified tumors. The levels and patterns of LOH were analyzed by the microsatellite alteration analysis using fluorescence-labeled primers and an automated DNA sequencer at 5 randomly selected loci in 246 Japanese primary breast cancers. Associations between the level of LOH and breast cancer subtypes and tumor aggressiveness were investigated. The incidence and frequency of LOH was significantly higher in HER2 (56.3, 26.7%) and TN groups (44.4, 27.1%) than in luminal A (ER-positive and/or PR-positive and HER2-negative) groups (32.0, 12.2%). The incidence and frequency of LOH increased as nuclear grade was elevated. There were significantly more grade 3 tumors in the HER2 (80.0%) and TN (68.2%) subgroups (p < 0.0001). Even in HER2 and TN cases, the incidence and frequency of LOH was significantly higher in nuclear grade 3 cases than in grade 1 or 2 cases. Relapse-free survival of patients with LOH was significantly shorter than for those without LOH. In addition, the survival time was shorter as the frequency of LOH elevated. The incidence of LOH was an independent prognostic factor for relapse-free survival by multivariate analysis. High incidence and frequency of LOH, which indicate increased genetic instability, were found to be associated with the aggressive features of high-grade HER2 and TN breast cancers.

Abstract 4647: Clinical Implications of Rabphillin-3A-Like (RPH3AL/NOC2) Gene Alterations in Breast Cancer

Abstract Background: The Rabphillin-3A-like (RPH3AL) gene, located at the 17p13.3 locus, has been reported to be involved in progression and metastasis of colorectal cancer. Since there have been no studies of this gene in breast cancer (BC), mutations and loss of heterozygosity (LOH) of RPH3AL in BCs were evaluated. Methods: BCs (n=104) and matching benign tissues were evaluated for LOH by use of two locus-specific polymorphic markers (D17S1866 and D17S643) and ABI-3100 sequencing technology. Of 104 cases, 16 had corresponding snap-frozen BCs and benign epithelial tissues. The cDNA samples of these frozen cases were analyzed for RPH3AL mutations by RT-PCR and direct sequencing. Since most of the missense mutations were located in the exon-6 region, the genomic DNA samples of 68 (of 104) BCs and their corresponding benign tissues were assessed only for exon 6 by the use of exon-6 specific primers. Additionally, 96 (of 104) BCs and the corresponding control tissues were analyzed for germ line mutations at 43 base pairs downstream from exon-6. Mutations and LOH status were correlated with clinicopathologic features of BCs and patient overall survival by chi-square and Kaplan-Meier analyses, respectively. Results: LOH analysis demonstrated that 69 of 99 (70%) informative cases exhibited LOH in at least one marker. In total, 19% (13 of 68) of BCs exhibited missense point mutations in the coding region (exon-6) of RPH3AL. Six BCs exhibited mutations at codon 175, two BCs at codon 200, one BC had double mutations at codon 178 and 189, and four BCs had single mutations at codons 165, 174, 181 and 196. There was a germ-line mutation (G&amp;gt;T) at 43 base pairs downstream from exon-6. Genotyping of this alteration showed that 31% (30 of 96) of BCs were homozygous for the G/G allele, 56% (54 of 96) were heterozygous for G/T, and 13% (12 of 96) were homozygous for T/T. In our correlative analysis, most BCs with mutations were associated with lymph-node metastases (9 of 13, 69%) and with an allelic loss (8 of 13, 62%). The G/G alleles were more commonly found in African-Americans than Caucasians (χ2, p=0.003), in ER/PR positive BCs than in ER/PR negative BCs (χ2, p=0.03), and in BCs with advanced stage (χ2, p=0.02). The incidence of LOH increased with nodal metastasis (χ2, p=0.04) and tumor size (χ2, p=0.05) of the BCs. Furthermore, LOH was significantly associated with poor patient survival (log rank, p=0.002). Conclusions: The results suggest that genetic alterations in the RPH3AL gene are associated with aggressive behavior of breast cancer. (Grant Support: UAB-Breast SPORE-Pilot and Susan G. Komen Breast Cancer Foundation, POP138306). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4647.

Identification of Loss of Heterozygosity on Circulating Free DNA in Peripheral Blood of Prostate Cancer Patients: Potential and Technical Improvements

Accurate identification of loss of heterozygosity (LOH) on circulating free DNA is often restricted by technical limitations such as poor quality and quantity of tumor-specific DNA and contamination by normal DNA. However, plasma DNA may harbor tumor-specific genetic alterations and could therefore be an interesting target for noninvasive examinations of tumor DNA. By PCR-based fluorescence microsatellite analysis using 12 polymorphic markers, we investigated LOH on cell-free DNA in blood plasma from 59 patients with localized prostate cancer (PCa) and 12 with metastatic disease (MPCa). In addition, plasma DNA from 21 PCa patients was fractionated into high- and low-molecular-weight DNA by 2 different column systems. To avoid appearance of artificial allelic loss and stabilize the amplification, TMAC (tetramethylammonium chloride) was added to each PCR. Overall incidences of LOH at all markers analyzed were 10% in PCa and 12% in MPCa samples. Highest frequencies were found at markers D11S898 (28%) in PCa and D6S1631 (27%) in MPCa. Statistical evaluation showed significant associations between LOH and increasing Gleason scores for the marker combinations D6S1631*D8S286*D9S171 (P = 0.03) and D8S286*D9S171 (P = 0.05). Fractionation of plasma DNA resulted in a higher overall LOH frequency in the low-molecular-weight DNA fraction (23%) compared with the high-molecular-weight DNA (7%). LOH analysis of circulating DNA can provide tumor-specific genetic information on PCa patients. Fractionation of plasma DNA and addition of TMAC improved LOH detection and general assay performance.

Investigation of allelic imbalances on chromosome 3p in nasopharyngeal carcinoma in Tunisia: High frequency of microsatellite instability in patients with early-onset of the disease

Tunisia is one of the world's intermediate risk areas for nasopharyngeal carcinoma (NPC). Loss of heterozygosity (LOH) on the short arm of chromosome 3 (3p) is the most frequent genetic change reported in NPC from endemic areas. In the present study, we investigate the incidence of LOH and microsatellite instability (MSI) on chromosome 3p in 49 microdissected primary NPC specimens and corresponding non-cancerous tissues from Tunisian patients using six microsatellite polymorphic markers. LOH at one or more markers was observed in 40 out of 48 informative cases (83.3%). The markers D3S1038 at 3p25.2-26.1 and D3S1076 at 3p21.1-21.2 have showed the highest frequency of LOH (51.3%), followed by D3S1067 at 3p14.3-21.1 (48.7%), D3S1568 at 3p21.3 (47.4%), D3S659 at 3p13 (15.3%), and D3S1228 at 3p14.1-14.2 (11%). Interestingly, MSI at one or more microsatellite markers was observed in 15 cases (31.2%). The highest frequency of MSI was presented by D3S1568 (18.4%), D3S1067 (17.9%), and D3S1038 (12.8%). With regard to clinicopathological features, LOH was found to be less common in young patients (under 25 years) than in adults (p=0.04), whereas MSI was found to be more frequent in patients under 45 years than in older patients (p=0.006). No significant correlation was found between LOH or MSI and the other clinicopathological features investigated including, gender, tumor size, lymph node metastasis, UICC clinical stage, and histological subtype. This study revealed different patterns of allelic imbalance on chromosome 3P in NPC between age groups in Tunisia, and suggests an alteration in the DNA mismatch repair machinery that may be, in part, responsible of the early age onset form of this disease in North African populations. More attention should be given to the mismatch repair system in the juvenile form of this disease in future studies.

A critical evaluation of loss of heterozygosity detected in tumor tissues, blood serum and bone marrow plasma from patients with breast cancer

IntroductionThe aim of the study was to perform a comparative analysis of LOH (loss of heterozygosity) in primary tumors as well as peripheral blood and bone marrow (BM) of patients with breast cancer (BCa).MethodsPerforming PCR-based fluorescence microsatellite analysis and using a panel of seven polymorphic microsatellite markers, we compared the profiles of LOH in primary tumors, peripheral blood and BM plasma from patients with primary BCa (n = 40, stage M0) as well as tumor tissues and blood serum from metastatic BCa patients (n = 48, stage M1). During the course of systemic treatment blood samplings from 12 M0 and 16 M1 patients were at least once repeated.ResultsThe overall incidences of LOH in tumor tissues, blood and BM samples were 27.5%, 9.0% and 5.0%, respectively. The marker D3S1255 was the only one in the panel that showed similar frequencies of LOH ranging from 19.0 to 24.5% in tumor, blood and BM samples. Both M0 blood serum and BM plasma samples displayed the same rate of 19.0%, whereas tumor and M1 serum showed a rate of 24.5% and 24.0%, respectively, at this locus. This marker also showed the highest frequency of LOH in serum and BM samples, whereas in tumor samples LOHs at the markers D13S218 (38%) and D17S855 (36%) were more frequent. Statistical analysis of the tumor samples showed that occurrence of LOH at the markers D3S1255 (P < 0.04), D9S171 (P < 0.05) and D17S855 (P < 0.03) correlated with undifferentiated nuclear grade. Additionally, significant associations of the number of LOH recorded at D17S250 with estrogen receptor (P < 0.02), progesterone receptor (P < 0.03) expression and high proliferation score (Ki-67 expression, P = 0.009) were observed. In blood serum samples a relationship between positive lymph node status and LOH at the marker D3S1255 was revealed (M0 stage, P = 0.05; M0+M1 stage, P = 0.004).ConclusionOur study demonstrates heterogeneous profiles and low rates of LOH, particularly on free DNA in BM and blood samples. However, the significant associations of LOH with some risk factors and the demonstrated possibility of monitoring free DNA in patients undergoing systemic therapy suggest that LOH analysis may be developed into a useful diagnostic tool.

Genetic pathways and mutation profiles of human cancers: site- and exposure-specific patterns.

Cancer is a complex disease that involves the accumulation of both genetic and epigenetic alterations of numerous genes. Data in the Genetic Alterations in Cancer database for gene mutations and allelic loss [loss of heterozygosity (LOH)] in human tumors (e.g. lung, oral, esophagus, stomach and colon/rectum) were reviewed. Results for the genes and pathways implicated in tumor development at these sites are presented. Mutation incidence, spectra and codon specificity are described for lung, larynx and oral tumors. LOH occurred more frequently than gene mutations in tumors from all sites examined. The cell cycle gene, TP53 (all sites), and cell signaling gene, APC (colorectal and gastric cancers), were the only genes with similar incidences of LOH and mutation. Alterations of one or more cell cycle and cell signaling genes were reported for tumors from each site. Site-specific activation was apparent in the cell signaling mitogen-activated protein kinase oncogenes (KRAS in lung, HRAS in oral cancers and BRAF in esophageal and colorectal cancers). Analysis of genetic changes in lung tumors showed that the incidence of mutations in the TP53 and KRAS genes and the incidence of LOH in the FHIT gene were significantly greater in smokers versus non-smokers (P < 0.01). In lung and oral cancers, the TP53 GC --> TA transversion frequency increased with tobacco smoke exposure (P < 0.05). Furthermore, the TP53 mutational hot spots for lung and laryngeal cancers from smokers included codons 157, 245 and 273, whereas for oral tumors included codons 280 and 281.

Comparison of Genetic Alterations Detected in Circulating Microsatellite DNA in Blood Plasma Samples of Patients with Prostate Cancer and Benign Prostatic Hyperplasia

Prostate cancer is the most frequent malignant disease and the second most frequent cause of death due to cancer in men in the Western world. Since serum prostate-specific antigen (PSA) and its subforms show poor specificity in clinical practice, a molecular marker for the detection and discrimination of prostate cancer (PCa) could be of great interest. To investigate the potential significance of genetic aberrations, such as loss of heterozygosity (LOH), in PCa we identified and characterized allelic losses in circulating tumor-associated DNA in blood from patients with localized PCa. Genomic DNA extracted from cell-free plasma of blood samples drawn from 65 PCa patients was analyzed using a panel of 15 polymorphic microsatellite markers mapping to known tumor-suppressor genes. Comparative analyses were performed with a control group of 36 patients with benign prostatic hyperplasia (BPH). In the current study, we demonstrate that PCa patients had higher DNA concentrations in their blood circulation than BPH patients. In the marker panel studied, LOH was more frequently detected in PCa patients (34%) than in BPH patients (22%). The incidence of LOH in the plasma DNA of PCa patients was highest at chromosomal regions 3p24 (THRB, 22%) and 8p21 (D8S360, 22%) in comparison to the BPH control cohort, which frequently showed LOH at loci 8q21, 8p21, 9p21, and 11q22 (D8S286, D8S360, D9S1748, and D11S898, each 6%). These results indicate that microsatellite analysis using plasma DNA may be an interesting tool for molecular screening of PCa patients.