Loss Of Heterozygosity Loci Research Articles

Functional loss of tumor suppressor genes detected by loss of heterozygosity, but not driver mutations, predicts aggressive lymph node status in papillary thyroid carcinoma

BackgroundRecognizing aggressive tumor biology is essential to optimizing patient management for papillary thyroid carcinomas (PTC). Aggressive lymph node (ALN) status is one feature that influences decision-making. We evaluated genomic deletions in regions of tumor suppressor genes, detected by loss of heterozygosity (LOH) analysis, to understand causal alterations linked to thyroid cancer aggressiveness and to serve as a molecular diagnostic biomarker for ALN status. MethodsWe analyzed 105 primary PTC enriched for patients with ALN (64% with, 36% without). We also analyzed 39 positive lymph nodes (79% with, 21% without ALN). LOH was determined using a panel of 25 polymorphic microsatellite alleles targeting 10 genomic loci harboring common tumor suppressor genes. Additionally, ThyGeNEXT® and ThyraMIR® assays were performed. ResultsLOH was detected in 43/67 primary PTC from patients with ALN status, compared with only 5/38 primary PTC without ALN (minimal metastatic burden) (P=0.0000003). This is further supported by post hoc analyses of paired primary and metastatic samples. Paired samples from patients with ALN are more likely to harbor LOH, compared to the ALN negative group (P=0.0125). Additionally, 12/31 paired samples from patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. No association was seen between ALN and mutational, translocation, or microRNA data. ConclusionsLOH detected in primary PTC significantly predicts ALN status. Analysis of paired primary and metastatic samples from patients with / without ALN status further supports this relationship. The acquisition of LOH at additional loci is common in lymph nodes from patients with ALN status. Simple summaryA subset of patients with papillary thyroid carcinoma (PTC) will develop recurrent disease. One known predictor of recurrence is the American Thyroid Association category “Aggressive Lymph Node” (ALN) disease, considering metastatic burden. Loss of heterozygosity (LOH) — chromosomal loss in regions of tumor suppressor genes — has yet to be investigated as a possible mechanism driving ALN status in PTC. The ability to predict ALN status prior to surgery can guide the extent of surgery and postoperative treatment options. We found that paired samples from patients with ALN are more likely to harbor LOH, compared to patients without ALN disease. 38% of patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. LOH complements current molecular analysis of thyroid cancer when searching for evidence of aggressive biology.

Abstract 82: Molecular characterization of gallbladder cancer

Abstract Introduction: Gallbladder carcinomas (GBC) are rare and aggressive neoplasms. Previous studies in small GBC cohorts have suggested potential molecular alterations associated with poor prognosis, however, a comprehensive understanding of the recurrent genetic events in this cancer type is very limited. In this study, we aim to characterize in detail the recurrent molecular alterations in GBC and their association with pathologic and clinical characteristics. Material and Methods: We studied the prevalence of somatic mutations and copy number alterations (CNA) in n=244 GBC samples (54% primary, 56% metastatic), collected from 2014 to 2021, and sequenced with the targeted NGS panel MSK-IMPACT. We assessed the correlation of the recurrent genomic variants with several pathologic (e.g., T stage, N stage, grade, histologic subtypes) and clinical characteristics (e.g., clinical stage) using Fisher's exact test. We also used Cox Proportional-Hazards modeling to assess the correlation between genetic variants and patient overall survival (OS). Results: The most common histologic subtypes in this GCB cohort included adenocarcinomas NOS (83%), carcinomas with squamous differentiation (9%), high-grade carcinomas (4%), and carcinomas with neuroendocrine differentiation (3%). Most patients were diagnosed with stage IV (65%) and stage III (11%) disease at the time of biopsy/resection, and the mean OS survival was 29.4 months. The most commonly mutated genes were TP53 (59%), SMAD4 (21%), ARID1A (19%), PIK3CA (10%), KRAS (7%), and ERBB2 (7%). Potentially recurrent oncogenic CNAs included deep deletions in CDKN2A (14%) and CDKN2B (14%), and amplifications in MDM2 (12%), ERBB2 (10%), CCNE1 (9%), MYC (7%), and KRAS (7%). RB1, PBRM1, and CTNNB1 variants were more common in cases with neuroendocrine differentiation, whereas alterations in IKZF1 and AGO2 were enriched in cases with squamous differentiation. The most significant event associated with shorted OS was chromosome 12q13-15 amplification i.e., CDK4 p=0.03 HR=2 [95% CI 1-3.6] and MDM2 p=0.05 HR=1.6 [1-2.5], which associated with a median OS of 20 months (vs. 34 months in the wild-type). Genomic variants associated with longer OS included ERBB2 (p=0.006 HR=0.2 [0.06-0.6]), KMT2C (p=0.03 HR=0.2 [0.1-0.9]) and KMT2D (p=0.01 HR=0.2 [0.04-0.7]) mutations, and CDK12 amplification (p=0.04 HR=0.4 [0.2-0.96]). Although ERBB2 amplification was not associated with prognosis, co-amplification of CDK12 and ERBB2 (chromosome 17q12) was frequently observed (Pearson correlation r=0.8). Conclusions: This large-scale genomic analysis reveals recurrent genomic events potentially associated with prognosis in GBC, including single nucleotide variants in ERBB2, KMT2C, and KMT2D, in addition to CNAs in chromosome 12q13-15 and 17q12 regions. This effort will continue to include a detailed analysis of recurrent structural variants, loss of heterozygosity loci, and analysis of the microbiota in this GBC cohort. Citation Format: Nicolas A. Giraldo, Abhinita Mohanty, Chad Vanderbilt, Rose Brannon, Ryma Benayed, Efsevia Vakiani, Ghassan Abou-Alfa, James Harding, Imane El Dika, Nikolaus Schultz, Bob Li, Michael F. Berger, Marc Ladanyi, Eileen O'Reilly, William Jarnagin, Olca Basturk, Maria Arcila. Molecular characterization of gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 82.

Loss of Heterozygosity and Possible Loss of Allele Amplification in Homozygous Short Tandem Repeats Loci in Two ALL Patients with Central Nervous System and Bone Marrow Relapses

Introduction: Loss of heterozygosity (LOH) was described for many forms of cancer including leukemia. LOH may contribute to oncogenesis through the deletion of tumor suppressor genes due to appropriate allelic loss. LOH in short tandem repeats loci (STR) has been reported for solid tumors and is widely used as malignant cells population marker. It should be noted that little is known yet about possible loss of one of certain homozygous alleles in tumour cells and STR analysis might be a promising approach to investigate this phenomenon. STR analysis is used as a routine test for chimerism monitoring after bone marrow transplantation. It could not also be excluded that recipient's chimerism estimation may be distorted in some cases due to LOH issues.The aim: To investigate possible allele loss in homozygous loci in tumour cells and to assess the contribution of LOH to the error in mixed chimerism estimation.Patients and Methods: This study includes analysis of STR DNA-profiles of two ALL patients after allogeneic stem cells transplantation. Patient 1 was 54 years old female with B-ALL, she underwent allogenic BM transplantation but after it she had four central nervous system (CNS) relapses and three bone marrow relapses. Patient 2 was 23 years old male with preT-ALL, two CNS relapses: first before transplantation and second after it. The DNAs were isolated from bone marrow samples of patients in remission before transplantation, from donors blood samples, from cerebrospinal fluid during neuro relapse and from bone marrow samples. The presence of blast cells in bone marrow samples and cerebrospinal fluid was confirmed morphologically and cytogenetically. STR-profiles were assessed by polymerase chain reaction with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software.Results: The STR profile analysis for Patient 1 showed complete recipient chimerism in cerebrospinal fluid with LOH in 10 from 15 heterozygous loci (Fig.1a,b) comparative to control sample of DNA (bone marrow before transplantation). The same LOH pattern was detected in archival DNA sample from first spinal relapse. Mixed chimerism in bone marrow samples estimated by STR analysis was equal to blast cell percentage assessed by cytogenetics. Loss of non-informative allele in D16S539 (Fig.1b) didn't distort chimerism estimation. However decreased levels of mixed chimerism were calculated in two homozygous loci (Fig.1a,c). We suspected that this decrease could be caused by the loss of one allele amplification in homozygous locus. Therefore, we proposed the mathematical model f(x), where x - is donor percentage in chimera, f(x) - difference between real recipient percentage (100-x) and calculated for locus with loss of allele amplification ((100-x)/2:((100-x)/2+x))*100% (Fig.1c) to test this hypothesis. The highest difference in chimerism calculation (about 17%) is expected in about 40% of donor and 60% recipient percentage. For Patient 2 the STR analysis detected only one LOH locus in DNA samples from cerebrospinal fluid during CNS relapse before and after BMT. However this patient has eight homozygous STR loci and complete donor chimerism in bone marrow. To find possible “loss of allele amplification” we used the artificial chimera, the mix of Patient 2 and unrelated random healthy donor DNA (Fig.1d). We used control DNA of Patient 2 (bone marrow before BMT) to check chimerism level deviations in loci of interest. The decreased level of mixed chimerism was detected in two homozygous loci from cerebrospinal fluid DNA-sample. The decreased estimate for chimerism correlated with proposed math model. For the same STR loci in control DNA no chimerism deviations were detected in mixed artificial chimera (Fig.1e).Conclusions: The LOH loci pattern might be preserved during long period of disease for certain malignant clone. Mixed chimerism or artificial mixed chimera can be useful in finding of homozygous loci with loss of one allele amplification. We suppose homozygous loci are the preferable subject for investigating LOH mechanisms. Loci with “loss of allele amplification” might be the used as malignant cells population marker.This study was supported by the Russian Foundation for Basic Research (RFBR) grant 18-015-00399a. DisclosuresRisinskaya:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Chabaeva:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Kulikov:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding.

Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci.

Enhancing Diagnosis, Prognosis, and Therapeutic Outcome Prediction of Gliomas Using Genomics

Malignant gliomas are the most frequent type of primary brain tumors. Patients' outcome has not improved despite new therapeutics, thus underscoring the need for a better understanding of their genetics and a fresh approach to treatment. The lack of reproducibility in the classification of many gliomas presents an opportunity where genomics may be paramount for accurate diagnosis and therefore best for therapeutic decisions. The aim of this work is to identify large and focal copy number abnormalities (CNA) and loss of heterozygosity (LOH) events in a malignant glioma population. We hypothesized that these explorations will allow discovery of genetic markers that may improve diagnosis and predict outcome. DNA from glioma specimens were subjected to CNA and LOH analyses. Our studies revealed more than 4000 CNA and several LOH loci. Losses of chromosomes 1p and/or 19q, 10, 13, 14, and 22 and gains of 7, 19, and 20 were found. Several of these alterations correlated significantly with histology and grade. Further, LOH was detected at numerous chromosomes. Interestingly, several of these loci harbor genes with potential or reported tumor suppressor properties. These novel genetic signatures may lead to critical insights into diagnosis, classification, prognosis, and design of individualized therapies.

Loss of Heterozygosity at BRCA1 Locus Is Significantly Associated with Aggressiveness and Poor Prognosis in Breast Cancer

BRCA1 and BRCA2 are two major tumor suppressor genes for hereditary breast and ovarian cancer. In sporadic breast cancer, although somatic mutations of these genes are rare, loss of heterozygosity (LOH) at BRCA1 and BRCA2 loci is common. LOH at BRCA1 and BRCA2 loci were investigated in 202 Japanese invasive breast cancer patients. The relationships between LOH at these gene loci and clinicopathologic characteristics were analyzed. Among 166 informative cases for both BRCA1 and BRCA2 loci, 69 (41.6%) and 52 (31.3%) tumors revealed LOH at BRCA1 and BRCA2 loci, respectively. LOH at BRCA1 LOH or BRCA2 locus was associated with higher nuclear grade (P < 0.0001, P = 0.0187). LOH at BRCA1 locus was associated with estrogen receptor and progesterone receptor negativity (P = 0.001 and P = 0.015) and significantly shorter disease-free survival (P < 0.0001), distant metastasis-free survival (P < 0.0001), and overall survival (P < 0.0001). In contrast, LOH at BRCA2 locus had no associations with estrogen receptor or progesterone receptor status and prognosis. LOH at BRCA1 locus was independently associated with poor prognosis in terms of disease-free survival (hazard ratio 3.08, 95% confidence interval [CI] 1.58-6.18, P = 0.0009), distant metastasis-free survival (hazard ratio 5.18, 95% CI 2.35-12.19, P < 0.0001), and overall survival (hazard ratio 4.97, 95% CI 1.84-15.1, P = 0.0013). LOH at BRCA1 locus could be an independent prognostic biomarker useful in identifying a subgroup of patients with poor prognosis.

Investigation of tumor suppressor genes apart from VHL on 3p by deletion mapping in sporadic clear cell renal cell carcinoma (cRCC)

ObjectivesTo investigate the most recurrent deletion loci on 3p12-p26 by deletion mapping studies by PCR-LOH and BAC array-FISH in sporadic conventional renal cell carcinoma (cRCC) and further, to evaluate the their clinicopathologic significance in cRCC. Comparative allelotyping studies in cRCC and major epithelial carcinomas (MEC) such as lung, breast, and bladder tumors were also carried out to investigate the specificity of the targeted loci in cRCC. Subjects and methodsA total of 40 c-RCC patients were enrolled in this study, categorized in to 2 groups: group I comprises of patients of stages I and II and group II includes patients at stages III and IV. Loss of heterozygosity (LOH) studies were performed by PCR using 15 microsatellite markers of region 3p12-p26 on paired normal-tumor tissues. The recurrent LOH loci found in 27 cRCC tumors were further validated by BAC array-FISH using 23 serially mapped BAC clones. Simultaneously, the allelic deletion status of fragile histidine triad (FHIT) gene was studied by FISH in cRCC and major epithelial carcinoma (MEC) tumors. The numerical aberrations of chromosome 3 were also studied using the centromere enumeration probe (CEP) probe for chromosome 3 to validate the observed allelic losses by BAC array-FISH in cRCC as well as MECs. ResultsOur study revealed 3 affected regions of LOH on 3p in cRCC: 3p12.2-p14.1, 3p14.2-p21.1, and 3p24.2-p26.1 in both group I (stages I and II) and group II (stage III and IV). Comparative allelotyping studies revealed that except for LOH loci D3S2406 (20%), D3S1766 (14%), and D3S1560 (20%), remaining affected loci revealed retention of heterozygosity (ROH) in breast carcinomas. Lung and bladder tumors revealed ROH at all affected LOH loci. FISH with FHIT gene probe revealed deletions in cRCC (88%), breast (30%), and lung tumors (10%). FHIT gene deletions frequency was almost equal in both groups I and II (>70%), whereas a locus 3p13 (D3S2454) revealed the highest LOH in group II (83%) patients in comparison to group I (16%). BAC array-FISH studies in cRCC identified 15 recurrent deletion loci at crucial regions, 3p12.2, 3p14.2, 3p21.3, and 3p24.2-p26 with long continuous deletion of 3p14.1-p26.1 exclusively in patients of stages III and IV. Validation of LOH loci in breast carcinomas by BAC array-FISH with BAC clones mapped at these loci revealed comparatively lower deletion frequency for RP11-59E22 (3p12.2) (30%), RP11-759B7(3p21.1) (12%), and RP11-57D6 (3p25.2, proximal to VHL) (15%) than cRCC. ConclusionMolecular cytogenetic studies by BAC array-FISH was found to be more sensitive over LOH. Deletion patterns on 3p explored that deletion of FHIT and flanking loci may occur as an initiating event followed by deletions at 3p12.2, 3p21.31–3p21.32, and 3p24.2–3p26.1 in the initial stage of development of disease, while continuous large deletions of 3p21.3–3p26.1 and 3p14.1–3p26.1 occur as progressive deletion due to genetic instability. Lack of VHL along with flanking loci in 50% cRCC patients that included both groups I and II supported the hypothesis of both VHL dependent and VHL independent pathways in cRCC tumorigenesis. Comparative allelotyping studies in cRCC and MECs indicated association of specific targeted loci including VHL in cRCC. Further expansion of these studies with characterization of the genes at targeted loci and correlation with clinical outcome will explore the prognostic significance and also provide an insight into the mechanisms of tumor suppressive pathways in genitourinary cancers such as CRCC.

Extensive analysis of D7S486 in primary gastric cancer supports TESTIN as a candidate tumor suppressor gene

BackgroundHigh frequency of loss of heterozygosity (LOH) was found at D7S486 in primary gastric cancer (GC). And we found a high frequency of LOH region on 7q31 in primary GC from China, and identified D7S486 to be the most frequent LOH locus. This study was aimed to determine what genes were affected by the LOH and served as tumor suppressor genes (TSGs) in this region. Here, a high-throughput single nucleotide polymorphisms (SNPs) microarray fabricated in-house was used to analyze the LOH status around D7S486 on 7q31 in 75 patients with primary GC. Western blot, immunohistochemistry, and RT-PCR were used to assess the protein and mRNA expression of TESTIN (TES) in 50 and 140 primary GC samples, respectively. MTS assay was used to investigate the effect of TES overexpression on the proliferation of GC cell lines. Mutation and methylation analysis were performed to explore possible mechanisms of TES inactivation in GC.ResultsLOH analysis discovered five candidate genes (ST7, FOXP2, MDFIC, TES and CAV1) whose frequencies of LOH were higher than 30%. However, only TES showed the potential to be a TSG associated with GC. Among 140 pairs of GC samples, decreased TES mRNA level was found in 96 (68.6%) tumor tissues when compared with matched non-tumor tissues (p < 0.001). Also, reduced TES protein level was detected in 36 (72.0%) of all 50 tumor tissues by Western blot (p = 0.001). In addition, immunohistochemical staining result was in agreement with that of RT-PCR and Western blot. Down regulation of TES was shown to be correlated with tumor differentiation (p = 0.035) and prognosis (p = 0.035, log-rank test). Its overexpression inhibited the growth of three GC cell lines. Hypermethylation of TES promoter was a frequent event in primary GC and GC cell lines. However, no specific gene mutation was observed in the coding region of the TES gene.ConclusionsCollectively, all results support the role of TES as a TSG in gastric carcinogenesis and that TES is inactivated primarily by LOH and CpG island methylation.

Evaluation of the fidelity of multiple displacement amplification from small number of cells

To evaluate the fidelity of multiple displacement amplification (MDA) from small number of cells (1-10 cells) by 10K 2.0 SNP mapping array. A fibroblast cell line (Tri-18; GM02732, 47, XY, +18) was used as the template, and 6 groups were set up in the study. Groups A and B were positive and negative control, respectively; groups C-F were experimental groups involving the MDA products from 1, 2, 5 and 10 cells respectively. In combination of single nucleotide polymorphism (SNP) array, the product of each group was assessed based on the genome coverage, loss of heterozygosity (LOH) rate and allele dropout (ADO) rate. The nonspecific product of negative control presented an average call rate of 3.2%. The genome coverage of the MDA product increased from 86.4% to 96.4% with the increasing number of template from 1 to 10 cells, while the LOH rate and ADO rate decreased significantly (P<0.05). MDA is a highly efficient and reliable method for whole genome amplification. The fidelity of MDA will be improved significantly with the increasing number of template cells. 10K 2.0 SNP mapping array is a quick, accurate and comprehensive method to evaluate the fidelity of amplified DNA products, but the ADO SNPs should be distinguished from those of preferential amplification from the LOH loci to avoid errors.

Prevalence of copy-number neutral LOH in glioblastomas revealed by genomewide analysis of laser-microdissected tissues

We have employed a laser-capture microdissection technique and single-nucleotide polymorphism arrays to characterize genomic alterations associated with the development of glioblastoma multiforme (GBM). Combined analysis of loss of heterozygosity (LOH) and copy number revealed that more than half (56.3%) of the 254 identified LOH loci showed no copy-number alteration, indicating the presence of copy-number neutral LOH (cnLOH). Furthermore, we found a GBM case that showed cnLOH in 18 of the 22 autosomes. These results were confirmed by quantitative real-time PCR, microsatellite analysis, and fluorescence in situ hybridization. The high rate of cnLOH suggests that epigenetic abnormalities of many genes are involved in the development and progression of GBMs.

Fingerprinting genomic instability in oral submucous fibrosis

Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7-13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation. In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray technique to 'fingerprint' global genomic instability in the form of LOH in 15 patient-matched OSF-blood genomic DNA samples. This rapid high-resolution mapping technique has revealed for the first time that a small number of discrete hot-spot LOH loci appeared in 47-53% of the OSF tissues studied. Many of these LOH loci were previously identified regions of genomic instability associated with carcinogenesis of the HNSCC. To our knowledge, this is the first evidence that genomic instability in the form of LOH is present in OSF. We hypothesize that the genomic instability detected in OSF may play an important role in malignant transformation. Further functional association studies on these putative genes may reveal potential predictive oral cancer markers for OSF patients.

Genetic changes in Slovenian patients with gastric adenocarcinoma evaluated in terms of microsatellite DNA

Adenocarcinoma of the stomach is a relatively frequent malignant disease in Slovenia. We investigated the frequency of microsatellite instability (MSI) and loss of heterozygosity (LOH) in gastric carcinomas from the Slovenian population to determine their prognostic significance. We evaluated MSI of mismatch repair associated loci and LOH on loci associated with following tumour suppressors: APC, nm23, Rb and p53. Results of the multiplex-PCR amplifications were correlated with clinicopathological factors for 73 patients. LOH was found in 52% of informative samples (20.5% LOH-H; 31.5% LOH-L). We found correlation of MSI with low-frequency LOH (LOH-L) in 11% of cases and with high-frequency LOH (LOH-H) tumours in 4% of cases. LOH-H and high-frequency MSI (MSI-H) were not associated. LOH was found in APC 36%, p53 33%, Rb 24% and nm23 33% of informative samples, whereas MSI was found in 30% of samples (12% MSI-H; 18% MSI-L). LOH-H status was associated with ulceration (P=0.029). LOH-N status was associated with diagnosis at higher TNM status (0.074) and infiltrative growth (P=0.006). Interestingly, in 6% of samples we found MSI on LOH loci as well. MSI-H was associated with higher age at diagnosis (r=0.24; P=0.04), antral location (r=0.252; P=0.04), intestinal type (P=0.044), expansive growth (P=0.001), tubular type (0.014), better differentiation (P=0.01), less nodal involvement (0.006) and better survival (P=0.022). The poorest prognosis was found in patients with both low-frequency MSI (MSI-L) and low-frequency LOH (LOH-L) tumours. The experimental design presented in the study may be of potential value for clinicians: at least five relevant markers for both MSI and LOH analysis may be needed to evaluate a gastric cancer (GC) patient's clinical status.

Acquisition of New In Vivo Genomic Change over Time in Multiple Myeloma.

Multiple myeloma (MM) cells present with significant genetic abnormality. This genetic instability is considered responsible for not only development of malignant phenotype but also progression of myeloma as well as development of drug resistance. We have previously demonstrated that MM cell lines have elevated homologous recombination activity that leads to acquisition of new genomic changes over time and is associated with development of drug resistance (Blood 2004; 104: 3409). However, such genomic evolution in patient samples has not been documented. Here, we have performed a genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci, and identify areas of amplification and losses to determine ongoing development of new changes that may reflect instability. We have evaluated 17 MM patients with purified myeloma cells obtained at two time points, at least six months apart. CD138–expressing myeloma cells from these patients were purified and their peripheral blood mononuclear cells were also obtained. Genomic DNA from these cells was digested with StyI, PCR amplified and hybridized to 250K SNP array. Results from CD138+ myeloma cells from two time points or more were compared with each other using the dChip software for LOH and copy number analysis. During the LOH analysis, we have adapted a tumor vs. tumor comparison to track all the new SNPs affected. We have observed that myeloma cells acquired new LOH loci in the subsequent samples. These new areas of genetic changes were recurrent; for example on chromosome 13, an average area of recurrent LOH of 87 Mb was found to be present among three patients. We were able to observe recurrence of significant copy changes on several chromosomes with/without LOH. The reproducible area of new acquisition of LOH and/or significant copy changes interestingly involved areas on chromosomes 1p, 1q, 8p, 9q, 13q, 15, 16q, 20p, 21q, and X that may have significant role in the pathogenesis and progression of the disease. We are currently analyzing the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary we demonstrate continued acquisition of new genomic changes in vivo in MM over time and provide a molecular basis for evolution of more aggressive disease able to escape therapeutic interventions. The mechanism governing such evolution is an important target to not only inhibit progression of the disease but also development of drug resistance.

Loss of Heterozygosity Analysis Identifies Genetic Abnormalities in Mycosis Fungoides and Specific Loci Associated With Disease Progression

Mycosis fungoides (MF) exhibits a variety of underlying molecular defects. Loss of heterozygosity (LOH) is a technique used to detect chromosomal imbalances in neoplastic disorders using archival tissue. We analyzed skin biopsies of MF in different stages for the presence of LOH at specific loci to evaluate underlying genetic aberrations involved in MF and its progression. Twenty-five skin biopsies (15 plaque stage and 10 tumor stage) from 19 patients were evaluated. LOH was examined at 1p22 (D1S2766), 9p21 [IFNA, p15 (D9S1748), p16 (D9S171)], 10q23 [PTEN (D10S185, D10S541, D10S2491)], and 17p13 [p53 (TP53)]. Abnormal lymphocytes were microdissected from formalin-fixed, paraffin-embedded tissue sections. Sixteen of the 25 (64%) specimens evaluated had at least one abnormal LOH locus and LOH was identified in 7 of 15 (47%) plaque and in 9 of 10 (90%) tumor stage lesions, respectively. All 3 patients with sequential biopsies (plaque followed by tumor lesions) had additional LOH abnormalities in tumor specimens compared with plaque stage lesions. LOH most frequently involved chromosome 10, including 7 of 10 (70%) tumor stage lesions. Loss of multiple alleles was only identified in tumor stage cases, with 3 tumors undergoing allelic losses at 3 separate loci. Our results suggest that LOH studies are a robust method for evaluating genetic abnormalities in MF. Tumor stage lesions manifest increasing allelic losses compared with plaque stage. Further, in this series, several loci associated with the tumor suppressor gene PTEN on chromosome 10 appear to be associated with progression from plaque to tumor stage.

Genomic Evolution of Multiple Myeloma In Vivo over Time.

Genetic instability is an important feature of malignant cells, specifically in multiple myeloma (MM). This instability, by activation of oncogene or deletion of tumor suppressor genes, plays an important role in oncogenesis. The ongoing genetic instability is responsible for tumor progression, acquisition of invasiveness, and development of drug resistance, as well as eventual mortality. We have previously demonstrated that MM cells have elevated homologous recombination activity that leads to acquisition of new genomic changes over time and is associated with development of drug resistance (Blood 2004; 104: 3409). However, such genomic evolution in patient samples has not been documented. Here we have performed genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 250K single nucleotide polymorphisms (SNP) loci and able to analyze small areas of gains or losses as an indicator of genomic instability to determine ongoing development of new changes that may reflect instability. We have evaluated nine MM patients with purified myeloma cells obtained at two time points, atleast six months apart. CD138-expressing myeloma cells from these patients were purified and their peripheral blood mononuclear cells were also obtained. Genomic DNA from these cells was digested with StyI, PCR amplified and hybridized to 250K SNP array. Results from CD138+ myeloma cells from two time points were compared with each other as well as with sample from normal peripheral blood mononuclear cells using the dChip software for LOH and copy number analysis and areas of deletions and amplifications. The changes at the first time point were considered as base line, and the subsequent sample was considered as test sample where acquisition of new changes was evaluated compared to base line. In two patients we had three samples available on a serial basis. We have observed that myeloma cells from the second time point demonstrated significant new areas of genomic change with acquisition of mean 4467 (range 79 to 18998) new LOH loci from base line sample. Of theses new areas of change, 41 SNP loci were found to be present in more than three patient samples indicating recurrent loci of interest in regards to progression and/or development of drug resistance. In 2 patients with 3 serial samples, we observed serial increase in acquisition of new SNPs, 4947 and 18998 at first follow up and 531 and 5275 at second follow up suggesting cumulative accumulation of new genetic change. The reproducible area of new acquisition interestingly involves the area with chromosome 1 and 8 that may have significant role in the pathogenesis and progression of the disease. We are currently obtaining the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary our results confirm genomic evolution of myeloma in vivo over time and provide means to identify molecular targets associated with progression of MM that can be utilized to inhibit progression of the disease and possibly development of drug resistance.

Elevated Apurinic/Apyrimidinic Endonuclease Activity Significantly Contributes to DNA Instability in Multiple Myeloma.

Apurinic/apyrimidic (AP) sites are the commonest DNA damage lesions and are highly mutagenic and cytotoxic requiring immediate repair. AP endonucleases (APE) 1 and 2 are the key proteins involved in the repair of AP sites and therefore play an important role in maintaining genomic integrity. However, if APE activity is dysregulated, it may lead to increase in DNA breaks leading to genomic rearrangements. In this study we demonstrate that endonuclease activity as measured by a novel plasmid based assay is elevated over 5 fold in myeloma cell lines and primary myeloma cells relative to normal plasma cells. APE1 and 2 were identified as the major endonuclease in MM by gene expression profiling and protein studies as well as using methoxamine (MX), which specifically inhibits APE activity by reacting with the aldehyde group of an abasic site. To further investigate the role of elevated APE activity in MM, we cultured MM cells in the presence of MX and demonstrate a significant inhibition of DNA recombination activity, as well as acquisition of new mutations as assessed by change in genome-wide loss of heterozygosity (LOH) using the 100K single nucleotide polymorphism arrays. Conversely, in cell line with lower APE activity we demonstrate a significant increase in genomic rearrangement with acquisition of significant number of new LOH loci. These data suggest that dysregulated AP endonuclease activity plays an important role in ongoing accrual of mutational changes, which may be associated with progression of myeloma and/or development of drug resistance. Moreover APE1 and APE2 provide a novel target to prevent disease progression and drug resistant phenotype associated with genomic instability.

Loss of heterozygosity on chromosome 3p in breast cancer and precancerous lesion

To study the loss of heterozygosity (LOH) on chromosome 3p in breast cancers and precancerous lesion. LOH at 11 microsatellite loci was detected in 41 cases of breast cancers and 12 cases of precancerous lesion by polymerase chain reaction and silver stain. The expressions of ER, PR, FHIT and hMLH1 were detected in breast cancer by immunohistochemistry. LOH on 3p was detected in 97% of breast cancers. D3S1295, D3S1029 and D3S1038 located at 3p14, 3p21-p22 and 3p25 were identified as the loci with most frequent LOH (53.1%, 43.6% and 52.5%). LOH of D3S1038 and expression of hMLH1 protein correlated with several clinicopathological features. LOH of D3S1295 had significant negative correlation with the expression of FHIT. In breast precancerous lesions, LOH on 3p was detected in 41.7% lesions. D3S1295 and D3S1029 were also identified as the most frequent LOH locus (27.3% and 16.7%). The smallest common LOH region seems likely lie between 3p14 and 3p25. The smallest LOH region indicates the existence of breast tumor related genes and some of them affect gene expression.

Genome-Wide Analysis for Loss of Heterozygosity in Primary and Recurrent Phyllodes Tumor and Fibroadenoma of Breast using Single Nucleotide Polymorphism Arrays

Phyllodes tumors of the breast are biphasic stromal and epithelial tumors histologically similar to benign fibroadenomas, but with a neoplastic stromal component. In contrast to fibroadenoma, phyllodes tumors can recur and be locally aggressive or be malignant. This study uses SNP array analysis to present a genome-wide map of loss of heterozygosity (LOH) in a cohort of phyllodes tumors and fibroadenomas. LOH is frequent and sometimes extensive in phyllodes tumors, but is rarely seen in fibroadenomas. There is heterogeneity between phyllodes tumors of different patients and no one LOH marker identifies a majority of these lesions. However, a subset of LOH loci occur in multiple cases of phyllodes tumors and are not found in fibroadenomas. Primary phyllodes tumors and paired recurrences from the same patient share common regions of LOH. In contrast, metachronous fibroadenomas from the same patient have different LOH patterns with no indication of a shared origin. Specific LOH loci may be associated with pathologic progression in recurrent phyllodes tumors. In a single case of phyllodes tumor containing a malignant epithelial component the malignant epithelium and stroma partially share an LOH genotype, suggesting a common precursor cell for the biphasic malignant components.

Analysis of loss of heterozygosity and X chromosome inactivation in spleens with myeloproliferative disorders and acute myeloid leukemia

Neoplastic myeloid proliferations are seen in the spleens of some patients with acute and chronic myeloproliferative disorders. Both acute myeloid leukemia (AML) and chronic myeloproliferative disorders have a variety of underlying cytogenetic defects that can be evaluated by loss of heterozygosity (LOH) studies. LOH studies have advantages over conventional cytogenetics by allowing the use of archival tissues. We evaluated the spleens in AML and chronic myeloproliferative disorders with neoplastic myeloid proliferations for the presence of LOH at several chromosome loci, and X-chromosome inactivation. A total of 17 spleens were evaluated (chronic myelogenous leukemia=6; chronic idiopathic myelofibrosis=6; essential thrombocythemia=1; AML arising from previous chronic myeloproliferative disorders=4). We examined LOH loci 7q (D7S2554), 8q (D8S263), 9p (D9S157, D9S161), 13q (D13S319), common sites of genetic abnormality in chronic myeloproliferative disorders, and TP53. In six cases, spleen LOH findings were compared to those of concurrent or preceding bone marrow biopsies. Five spleens of female patients were evaluated for the presence of clonality using X-chromosome inactivation. Of the 16 cases analyzed, 14 (88%) had at least one abnormal LOH locus, with 6/16 with two abnormal loci. The abnormalities were distributed as follows: D9S161–7/15 (47%), TP53–6/16 (38%), D7S2554–5/16 (31%), D9S157–5/15 (33%), D8S263–3/14 (21%), and D13S319–2/14 (14%). Of the six bone marrows, 4/6 showed concordance in bone marrow and spleen specimens, with additional LOH abnormalities being identified in the spleen specimens of all four cases. X-chromosome inactivation studies were showed nonrandom (clonal) patterns in two cases. Our results show that allelic losses were common in the neoplastic extramedullary hematopoiesis found in spleens of chronic myeloproliferative disorders and AML. Comparison of spleen and bone marrow specimens by LOH demonstrated additional abnormalities in the spleen compared to the marrow.

Molecular Basis of Genomic Instability and Progression in Multiple Myeloma: Potential Role of Apurinic (Apyrimidinic) Endonuclease.

Genetic instability is a prominent feature of most cancers including multiple myeloma (MM) and is responsible for ongoing accrual of mutational changes which may lead to development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in MM is therefore extremely important to understand carcinogenesis and to identify novel targets for treatment. As genomic rearrangements require excision of DNA, we hypothesized that an elevated endonuclease activity may induce recombination and subsequent genomic instability in cancer cells. We developed a plasmid degradation assay that confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease the degradation assay was carried out in the presence of specific endonuclease inhibitors, which identified apurinic/apyrimidinic endonuclease (Ape1 and Ape2) as the predominant endonucleases in mediating increased endonuclease activity in MM. Gene expression analysis confirmed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Both immunocytochemistry and western blot analyses confirmed upregulation of Ape1 protein in all MM cell lines and patient samples. Next, we investigated the role of elevated APE endonuclease activity in DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. To investigate the role of elevated AP endonuclease activity in MM, we cultured myeloma cells in the presence of methoxyamine (MX), which specifically inhibits AP endonuclease activity, and evaluated its effect on HR activity and genome-wide appearance of new mutations. Exposure of intact myeloma cells to MX resulted in > 90% inhibition of HR activity and a significant (71±10.9%; p<0.05) reduction in the appearance of new mutations compared to untreated cells, as assessed by genome-wide loss of heterozygosity (LOH) assay (Affymetrix). We also evaluated the effects of overexpression of Ape1 & 2 in normal fibroblasts which have low endonuclease activity. The transgenic upregulation of AP endonucleases (Ape1 and Ape2) in normal cells led to a significant increase in the lecombination activity, leading to a marked mutational instability as indicated by the appearance of over 20,063 and 20,143 new LOH loci per 100,000 polymorphic regions examined throughout the genome, at population doublings 25 and 50 respectively. Mutational instability was also associated with chromosomal instability confirmed by spectral karyotyping of these cells showing significant numerical and structural chromosomal abnormalities. These changes were associated with indefinite growth of cells and formation of tumors when injected in SCID mice. These data suggest that elevated AP endonuclease may be responsible for mutational and chromosomal instabilities, leading to progression of myeloma.