High Frequency Of Loss Of Heterozygosity Research Articles

O-095 Assessment of genome editing outcomes in human preimplantation embryos subjected to CRISPR-Cas9 – most loss of heterozygosity (LOH) events are caused by DNA repair deficiency

Abstract Study question What is the cause of loss of heterozygosity, frequently observed in embryos subjected to CRISPR-Cas9, an interhomolog-homologous recombination repair (IH-HR) or deficiency in DNA repair? Summary answer Vast majority of LOH observed in human zygotes subjected to CRISPR-based editing is explained by deficiency in DNA repair prior to genome activation, not IH-HR. What is known already Genome editing (GE) at the preimplantation stage, intended to correct disease-causing mutations, is controversial due to safety and ethical concerns. It has been suggested that a high frequency of LOH seen in zygotes subjected to GE could be explained by resolution of the double-stranded DNA breaks (DSBs) created using CRISPR-Cas9 by interhomolog-homologous recombination repair (IH-HR), a process with potential to correct deleterious heterozygous mutations. However, such hypotheses are controversial and it is unclear whether embryos at this stage development can utilise IH-HR. CRISPR-Cas9 targeted sites are difficult to interrogate with traditional methods, risking a misinterpretation of editing outcomes. Study design, size, duration We hypothesised that the frequently observed LOH affecting the CRISPR-targeted sites in human zygotes is caused by the deficiency in DNA repair at this developmental stage, resulting in complex genotypes which traditional molecular methods have failed to detect, and not IH-HR which uses the second un-cleaved parental allele as a template for repair. DSBs were induced at three genomic sites in embryos microinjected at the time of fertilisation (n = 27) in this IRB-approved study. Participants/materials, setting, methods Research embryos were cultured for 60 hours, then disaggregated. Individual cells underwent an array of genetic tests, including NGS, allowing determination of the repair pathway utilised and revealing any cytogenetic abnormalities. A novel state-of-the-art analytical workflow was developed to interrogate 25kb around CRISPR cleavage sites. This approach provided a means of detecting complex genomic rearrangements as well as unresolved DNA damage, which would appear as LOH when employing traditional PCR-based approaches for assessment. Main results and the role of chance Induction of DSBs using CRISPR-Cas9 was 100% efficient when applied at the time of fertilisation. LOH was confirmed to be common at targeted sites, affecting 69/216 (32%) loci examined in 108 blastomeres. Remarkably, the great majority of LOH events (>90%) were a direct consequence of inappropriate/failed DNA repair (unresolved DNA breaks or complex genomic rearrangements). Strikingly, not a single instance of IH-HR was observed when CRISPR reagents were introduced at fertilisation. This may be explained by the physical separation of the two parental genomes in distinct pronuclei at that time. Allele-drop out (ADO), a technical problem, accounted for <10% of all LOH events. In total, 46% of DSBs failed to undergo appropriate repair, resulting in segmental aneuploidy (21%), large-scale deletions (10%), inversions/translocations (10%), or loss of the targeted chromosome (5%). 45% of DSBs were resolved using error-prone non-homologous end joining (NHEJ), while only 9% utilised the homology directed repair (HDR) pathway required for correction of mutations. Application of CRISPR-Cas9 to embryos on day-3 of development (after embryonic genome activation - EGA) was less efficient, only producing a DSB in 6/37 embryos (16%). However, the mode of repair differed markedly, 100% employing HDR (p = 0.0001). In 4 cases, this involved IH-HR. Limitations, reasons for caution This data provides strong evidence that LOH recorded in earlier studies was incorrectly attributed to IH-HR and was actually due to abnormal/failed repair. CRISPR appears more effective after EGA, with efficient DNA repair dominated by desirable HDR. However, a larger number of embryos is required to corroborate this finding. Wider implications of the findings The use of CRISPR-Cas9 at the time of fertilisation is frequently associated with problematic, unintended outcomes, rendering the technique clinically inapplicable in the current format. Careful study design which allows determination of all possible editing outcomes is essential and advances our understanding of the underlying mechanisms of DNA repair. Trial registration number not applicable

Alterations of chromosome 3p in 24 cases of gastrinomas and their correlations with clinicopathological and prognostic features

Abstract Purpose: The pathogenesis of gastrinomas is largely unknown, and there is a lack of reliable genetic determinants that are useful to distinguish malignant and benign forms of this tumor or predict the prognosis of patients with this disease. Loss of heterozygosity (LOH) on chromosome 3p is reported to occur in pancreatic neuroendocrine tumors (PNETs) as well as in non-PNETs and its presence is reported to correlate with tumor prognosis in non-endocrine tumors. However, little data are available from prospective studies on gastrinomas. Experimental design: We assessed occurrence of 3p LOH in 24 gastrinomas and correlated its presence with tumor biological behavior and other clinicopathological features of gastrinomas. Results: Either 3p LOH or microsatellite instability involving 3p occurred in 11 of 24 tumors (46%). Seven (29%) gastrinomas had 3p LOH. Of the 7 gastrinomas with 3p LOH, 5 (71%) had 3p12 LOH with the marker D3S2406, which was the shortest region of highest overlap (SRO). Chromosome 3p LOH was not associated with aggressive biological behavior of gastrinomas or with poor prognosis of patients with gastrinoma. Similarly, 3p12 LOH (SRO) was not correlated with aggressive growth of tumors and/or liver metastases. Conclusion: Gastrinomas have a relative high frequency of 3p12 LOH suggesting this area may harbor putative tumor suppressor gene(s), which may play a role in the tumorigenesis, but not aggressiveness, of a subset of these tumors.

Early genetic aberrations in patients with sporadic colorectal cancer.

Chromosome instability (CIN) is widely observed in both sporadic and hereditary colorectal cancer (CRC). Defects in APC and WNT signaling are primarily associated with CIN in hereditary CRC, but the genetic causes for CIN in sporadic CRC remain elusive. Using high-density SNP array and exome data from The Cancer Genome Atlas (TCGA), we characterized loss of heterozygosity (LOH) and copy number variation (CNV) in the peripheral blood, normal colon, and corresponding tumor tissue in 15 CRC patients with proficient mismatch repair (MMR) and 24 CRC patients with deficient MMR. We found a high frequency of 18q LOH in tumors and arm-specific enrichment of genetic aberrations on 18q in the normal colon (primarily copy neutral LOH) and blood (primarily copy gain). These aberrations were specific to the sporadic, pMMR CRC. Though in tumor samples genetic aberrations were observed for genes commonly mutated in hereditary CRC (eg, APC, CTNNB1, SMAD4, BRAF), none of them showed LOH or CNV in the normal colon or blood. DCC located on 18q21.1 topped the list of genes with genetic aberrations in the tumor. In an independent cohort of 13 patients subjected to Whole Genome Sequencing (WGS), we found LOH and CNV on 18q in adenomatous polyp and tumor tissues. Our data suggests that patients with sporadic CRC may have genetic aberrations preferentially enriched on 18q in their blood, normal colon epithelium, and non-malignant polyp lesions that may prove useful as a clinical marker for sporadic CRC detection and risk assessment.

Use of laser capture microdissection allows detection of loss of heterozygosity in chromosome 9p in breast cancer.

The present study was designed to determine whether loss of heterozygosity (LOH) in the p arm of chromosome 9 in invasive ductal carcinoma of the breast is detected during the neoplastic progression of the disease. Using laser capture microdissection (LCM) epithelial cells were isolated from 14 invasive ductal carcinoma cases (IDC), ductal carcinomas in situ (DCIS), normal mammary lobules, skin and/or lymph nodes of paraffin embedded tissue sections. LOH analysis of chromosome 9p was performed utilizing the microsatellite markers D9S199, D9S157, D9S171, D9S265 and D9S270. The highest frequency of LOH was observed in invasive ductal carcinomas, which reached a maximum at the 9p22-23 chromosomal location (D9S157). In addition, DCIS lesions presented a high frequency of LOH in 9p22-23 (D9S157), followed by 9p21 (D9S171), D9S199 and D9S265, which were similar in frequency to those observed in IDC. A novel finding was the intralesional heterogeneity in LOH within the same DCIS or IDC case. This is an indication that clones of cells that differ in genetic composition coexist in the same lesion. Notably, phenotypically normal breast tissues adjacent to IDC or DCIS exhibited LOH at D9S157 and/or D9S171. Together, these data indicate that LOH of chromosome arm 9p occurs very early in the progression of cancer and that different clones of cells co-exist within a single tumor.

P53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo

Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/− tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data – while cautioning that not all genetic mouse models faithfully represent the human situation – demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.

Analysis of gene expression changes associated with human carcinoma-associated fibroblasts in non-small cell lung carcinoma

BackgroundThis study aimed to investigate the gene expression changes associated with carcinoma-associated fibroblasts (CAFs) involving in non-small cell lung carcinoma (NSCLC).MethodsWe downloaded the GEO series GSE22862, which contained matched gene expression values for 15 CAF and normal fibroblasts samples, and series GSE27289 containing SNP genotyping for four matched NSCLC samples. The differentially expressed genes in CAF samples were identified using the limma package in R. Then we performed gene ontology (GO) and pathway enrichment analysis and protein–protein interaction (PPI) network construction using the identified DEGs. Moreover, aberrant cell fraction, ploidy, allele-specific copy number, and loss of heterozygosity (LOH) within CAF cells were analyzed using the allele-specific copy number analysis.ResultsWe obtained 545 differentially expressed genes between CAF and normal fibroblasts samples. The up-regulated genes are mainly involved in GO terms such as positive regulation of cell migration and extracellular region, while the down-regulated genes participate in the lung development and extracellular region. Multiple genes including bone morphogenetic protein 4 (BMP4) and transforming growth factor, beta 3 (TGFB3) are involved in the TGF-β signaling pathway. Genes including BMP4, TGFBI and matrix Gla protein (MGP) were hub genes. Moreover, no LOH event for BMP4 and MGP was found, that for sphingosine kinase 1 (SPHK1) was 70%, and for TGFBI was 40%.ConclusionOur data suggested that BMP4, MGP, TGFBI, and SPHK1 may be important in CAFs-associated NSCLC, and the abnormal expression and high LOH frequency of them may be used as the diagnosis targets of CAFs in NSCLC.

Genetic features of metachronous esophageal cancer developed in Hodgkin's lymphoma or breast cancer long-term survivors: an exploratory study.

BackgroundDevelopment of novel therapeutic drugs and regimens for cancer treatment has led to improvements in patient long-term survival. This success has, however, been accompanied by the increased occurrence of second primary cancers. Indeed, patients who received regional radiotherapy for Hodgkin’s Lymphoma (HL) or breast cancer may develop, many years later, a solid metachronous tumor in the irradiated field. Despite extensive epidemiological studies, little information is available on the genetic changes involved in the pathogenesis of these solid therapy-related neoplasms.MethodsUsing microsatellite markers located in 7 chromosomal regions frequently deleted in sporadic esophageal cancer, we investigated loss of heterozygosity (LOH) and microsatellite instability (MSI) in 46 paired (normal and tumor) samples. Twenty samples were of esophageal carcinoma developed in HL or breast cancer long-term survivors: 14 squamous cell carcinomas (ESCC) and 6 adenocarcinomas (EADC), while 26 samples, used as control, were of sporadic esophageal cancer (15 ESCC and 11 EADC).ResultsWe found that, though the overall LOH frequency at the studied chromosomal regions was similar among metachronous and sporadic tumors, the latter exhibited a statistically different higher LOH frequency at 17q21.31 (p = 0.018). By stratifying for tumor histotype we observed that LOH at 3p24.1, 5q11.2 and 9p21.3 were more frequent in ESCC than in EADC suggesting a different role of the genetic determinants located nearby these regions in the development of the two esophageal cancer histotypes.ConclusionsAltogether, our results strengthen the genetic diversity among ESCC and EADC whether they occurred spontaneously or after therapeutic treatments. The presence of histotype-specific alterations in esophageal carcinoma arisen in HL or breast cancer long-term survivors suggests that their transformation process, though the putative different etiological origin, may retrace sporadic ESCC and EADC carcinogenesis.

Alternating expression levels of WWOX tumor suppressor and cancer-related genes in patients with bladder cancer.

The aim of the present study was to determine the roles of the WWOX tumor suppressor and cancer-related genes in bladder tumor carcinogenesis. Reverse transcription-quantitative polymerase chain reaction was used to analyze the status of WWOX promoter methylation (using MethylScreen™ technology) and loss of heterozygosity (LOH) in papillary urothelial cancer tissues. The associations between the expression levels of the following tumorigenesis-related genes were also assessed: The WWOX tumor suppressor gene, the MKI67 proliferation gene, the BAX, BCL2 and BIRC5 apoptotic genes, the EGFR signal transduction gene, the VEGF vascular endothelial growth factor gene, and the CCND1 and CCNE1 cell cycle genes. The results reveal a high frequency of LOH in intron 1 in the WWOX gene, as well as an association between reduced WWOX expression levels and increased promoter methylation. In addition, the present study demonstrates that in bladder tumors, apoptosis is inhibited by increased expression levels of the BCL2 gene. A correlation between the proliferation indices of the MKI67 and the BIRC5 genes was also revealed. Furthermore, the expression levels of VEGF were identified to be positively associated with those of the EGFR gene.

Androgen receptor CAG repeats, non-random X chromosome inactivation, and loss of heterozygosity at Xq25 in relation to breast cancer risk

BackgroundThe aim of this study was to examine the association of non-random X chromosome inactivation (XCI) and loss of heterozygosity (LOH) at Xq25 with breast cancer development.MethodsSeventy-nine breast cancer patients, 39 female lung cancer patients, 30 other cancer patients and 77 healthy females were analysed for LOH using a panel of 11 microsatellite markers spanning Xq25. The androgen receptor (AR) gene was chosen as an XCI marker.ResultsLOH of at least one microsatellite locus at Xq25 was identified in 46/65 breast cancers examined, while only 10/25 cancers of other origins demonstrated LOH in this region (p = 0.014). The critical deletion region in breast cancer was around marker DXS1047 (47.23%). Moreover, we found that tissues from eight breast cancers showed LOH at all of the informative loci tested at Xq25, while the other 38 showed partial (interstitial or telomeric) alterations at Xq25. Interestingly, the pattern of XCI of these eight breast cancers tended to be non-random. We estimated the frequencies of AR alleles and found that women with two long AR alleles (≥21 CAG repeats) had an increased risk of developing breast cancer, while those with two short AR alleles (<21 CAG repeats) were likely to be normal (p = 0.00069).ConclusionsThe extraordinary high frequencies of LOH at Xq25 found in this study strongly imply that there might be one or more tumour suppressor genes (TSGs) related to the development of breast cancer at Xq25 in the Taiwanese female population.

Single cell analysis exposes intratumor heterogeneity and suggests that FLT3-ITD is a late event in leukemogenesis

FMS-like tyrosine kinase 3 receptor-internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia and is considered rare in acute lymphocytic leukemia. Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic polymerase chain reaction and have recently incorporated GeneScan (Applied Biosystems, Foster City, CA) to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed acute myeloid leukemia (n = 5) and acute lymphocytic leukemia (n = 3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. Seven patients, considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis. Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested provides evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity of the normal allele, affecting 25%-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.

KCTD11 tumor suppressor gene expression is reduced in prostate adenocarcinoma.

Prostate cancer is the most common noncutaneous cancer among men in the United States. A genetic contribution to prostate cancer risk has been documented, but knowledge of the molecular mechanisms involved in prostate cancer initiation is still not well understood. Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In human prostate cancer, several chromosomal regions demonstrating a high frequency of LOH have been previously identified. KCTD11 (REN) is a tumor suppressor gene mapping on human chromosome 17p13.2, whose expression is frequently lost in human medulloblastoma and in several other cancer types. KCTD11 acts as a negative regulator of the Hedgehog (Hh) signaling. Here, we demonstrated that KCTD11 LOH is a common genetic lesion in human prostate adenocarcinoma. Indeed, nuclear KCTD11 protein expression is strongly reduced in primary prostate cancer, and this event correlated with overexpression of proteins acting into the Hedgehog pathway. Low levels of KCTD11 mRNA have been also observed in prostatic cancer cells, and ectopic overexpression of KCTD11 led to growth arrest. Our study demonstrates and supports that KCTD11, as well as negatively regulated downstream effectors belonging to Hh signaling, plays a role in prostate cancer pathogenesis. This could be suitable to characterize new diagnostic and therapeutic markers.

High‐resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to non‐hodgkin's lymphoma

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin's lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

High frequency of allelic loss at the BRCA1 locus in ovarian cancers: clinicopathologic and molecular associations

BRCA1 dysfunction may occur by different mechanisms that are rarely evaluated concomitantly. We aimed to analyze BRCA1 germline mutations, loss of heterozygosity (LOH) and promoter methylation in unselected ovarian carcinomas in the context of their clinicopathologic characteristics and other molecular changes. BRCA1 mutations were analyzed in 257 carcinomas using single-strand conformation polymorphism (SSCP), heteroduplex, and sequencing methods. LOH at the BRCA1 locus was screened for in 180 cancers. Methylation analysis was performed for 241 tumors using quantitative methylation specific PCR(qMSP). BRCA1 alterations, comprising germline mutations, allelic loss, and/or aberrant promoter methylation, were found in 77.6% (125/161) of ovarian carcinomas. Patients with germline mutations were younger than non-carriers (P < 0.0001). Germline mutations and LOH were associated with advanced stages (P=0.009, P < 0.0001), high tumor grade (P=0.005, P<0.0001), and TP53 mutations (P=0.003, P < 0.0001, for mutations and LOH, respectively). LOH was also associated with the serous histological type (P=0.004) and PIK3CA amplification (P=0.003). Aberrant promoter methylation was associated with LOH (P=0.017) and absence of germline mutations (P=0.037). The high frequency of LOH at the BRCA1 locus suggests that LOH may be an important mechanism of BRCA1 deficiency in ovarian carcinomas. Tumors with various BRCA1 alterations have a similar phenotype of high-grade, high-stage carcinomas with frequent TP53 mutations.

Detecting and investigating the significance of high-frequency LOH chromosome regions for endometriosis-related candidate genes

To detect the high-frequency loss of heterozygosity (LOH) chromosome regions for ectopic endometrium of ovarian endometriosis (EMs) and to investigate the significance of high-frequency LOH chromosome regions in EMs, we obtained ectopic endometrium by laser capture microdissection (LCM (22 samples)), manual capture microdissection (MCM (18 samples)), and routine dissection (14 samples), respectively. After restriction and circularization-aided rolling circle amplification (RCA-RCA), LOH was detected at 12 microsatellite (MS) loci. The frequency of LOH was 59.09% (13/22) in LCM group, 61.11% (11/18) in the MCM group and 21.43% (3/14) in the routine dissection group. The latter was significantly lower when compared with the former two (p < 0.05). In the LCM group, candidate chromosome regions 17q21.31 and 9p21.3 had LOH frequencies of 23.8 and 13.6%, respectively. The highest LOH frequency was detected at the locus AAAT2 on chromosome 17q21.31 (40%). The chromosome region with the highest frequency of LOH for ectopic endometrium was 17q21.31, especially at the AAAT2 locus, which prompted that down regulation of the candidate genes nearby the locus might be one of the mechanisms of EMs pathogenesis. LCM combined with RCA-RCA is a reliable technique for analyzing endometrial LOH at multiple MS loci. MCM combined with RCA-RCA, which provided similar results, was more cost-effective.

Chromosome 17q25 genes, RHBDF2 and CYGB, in ovarian cancer

It has been proposed that the frequent loss of heterozygosity (LOH) of an entire chromosome 17 contig in epithelial ovarian cancers (EOC) is the consequence of the inactivation of multiple tumour suppressor genes on this chromosome. We report the characterization of a 453 Kb 17q25 locus shown previously to exhibit a high frequency of LOH in EOC samples. LOH analysis further defined the minimal region of deletion to a 65 Kb interval flanked by D17S2239 and D17S2244, which contains RHBDF2, CYGB and PRCD as tumour suppressor gene candidates. Tissue specific expression excluded PRCD as a candidate. RHBDF2 was expressed at low levels in the majority of benign and low malignant potential (LMP) tumours, and in a subset of malignant ovarian tumour samples, as compared with primary cultures of normal ovarian surface epithelial cell (NOSE) samples. CYGB was expressed at low levels in the majority of LMP and malignant samples compared with benign and NOSE samples. In contrast to CYGB expression, RHBDF2 was expressed at low or undetectable levels in EOC cell lines exhibiting tumourigenic characteristics and up-regulated in a genetically modified EOC cell line rendered non-tumourigenic. DNA sequence analysis identified variants but no apparent deleterious mutations in either gene. Methylation-specific PCR analysis suggested that promoter methylation of CYGB but not RHBDF2 occurred in 6 of 31 malignant samples. The results combined suggest that RHBDF2 and CYGB may play distinctive roles in ovarian cancer and could be added to the growing roster of chromosome 17 genes implicated in this disease.

Aberrant protein expression and frequent allelic loss of MSH3 in colorectal cancer with low-level microsatellite instability

High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC. We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3. Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors. Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

Expression and clinical significance of HCCS1 in non-small cell lung cancer

Aim of the studyHepatocellular carcinoma suppressor 1 (HCCS1) has been identified as a tumor suppressor gene in the high-frequency loss of heterozygosity (LOH) region on chromosome 17p13.3 in hepatocellular carcinoma (HCC). There was also a high frequency of LOH on chromosome 17p13.3 in non-small cell lung cancer (NSCLC). Therefore, the aim of this study was to explore the expression of HCCS1 in NSCLC as well as its clinical significance.Material and methodsReal-time PCR and immunohistochemistry were performed to detect the expression level of HCCS1 mRNA and protein in NSCLC and noncancerous tissues, respectively. Further, we explored the relationship between HCCS1 expression and various clinical features in NSCLC.ResultsThe mRNA and protein expression of HCCS1 were both significantly lower in NSCLC samples than those in noncancerous tissues. That is, the mRNA level of HCCS1 was 0.0044 ±0.0036 and 0.0067 ±0.0054 in NSCLC samples and noncancerous tissues, respectively. The protein level of HCCS1 was 4.67 ±1.15 and 6.13 ±1.24 in NSCLC samples and noncancerous tissues, respectively. Importantly, this difference in expression was significantly correlated with tumor lymph node metastasis (TNM) in NSCLC (p < 0.05), but not with gender and age of the patients, pathological types, TNM stages, or grades of cancers (p > 0.05).ConclusionOur results suggest that HCCS1 may be involved in NSCLC carcinogenesis.

Loss of Heterozygosity (LOH) of PTPRJ in Non-Hodgkin`s Lymphoma (NHL)

Abstract 5231The identification of Tumor Suppressor Genes (TSGs) in tumor tissue lacking of its normal germ-line counterpart has been limited due to the absence of proper statistical approaches that allow inferring the specific germ-line heterozygous status of a patient. Nonetheless, the implementation of Hidden Markov model (HMM) or cohort heterozygosity comparisons (CHC) approaches in the analysis of SNP array data from un-paired tumor DNA samples have permitted the detection of LOH regions and possible TSGs that might be implicated in the origin and development of cancer. Non-Hodgkin's Lymphoma (NHL) is the most common type of lymphoma. Nevertheless, its aetiology and pathogenesis remain unclear because of heterogeneity in biology, genetics and chemoresponse.Here, we employed high-density SNP array analysis of tumors from two of the most frequent subtypes of NHL, Follicular Lymphoma (FL) and Diffuse Large B-cell Lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region of 11p11.2, containing the gene encoding the receptor-type protein tyrosine phosphatase, PTPRJ. LOH of this gene has been previously described in colorectal, lung, thyroid and breast cancer and the gene product regulates key survival pathways in B-cells, such as MAPK, PLCγ1, Scr and BCR signalling.We validated LOH of the PTPRJ region by microsatellite analysis of an extended cohort of NHL tumors and found significantly increased rates of homozygosity compared to a patient-matched control population. Furthermore, sequencing of NHL cases revealed a strong association between LOH and the presence of a non-synonymous SNP in the PTPRJ coding region. Together, these results show the utility of LOH screening in identifying novel tumor suppressor genes, and implicate LOH of the PTPRJ gene as a potential lymphomagenic mechanism. Disclosures:Aya-Bonilla:Cancer Council of Queensland: Research Funding.

Using loss of heterozygosity of microsatellites to distinguish high-grade dysplastic nodule from early minute hepatocellular carcinoma

Background It is practical significant to seek new applicable adjuvant diagnostic biomarkers to differentiate high-grade dysplastic nodule (HGDN) from well-differentiated minute hepatocellular carcinoma (w-MHCC) due to their closely overlapping morphology. Methods In the present study, by using microdissection-based paraffin-embedded tissues, loss of heterozygosity (LOH) patterns of a panel of 22 microsatellite (MS) markers was examined in 8 HGDN, 14 w-MHCC (≤ 1 cm) and 35 larger HCC (LHCC, > 1 cm). Results The results revealed a stepwise increasing fractional allelic loss from HGDN, w-MHCC and LHCC (0.166 ± 0.141, 0.377 ± 0.198, 0.471 ± 0.264, respectively, P = 0.005). Loci-specific analyses showed that LOH on D4S415 (66.7% vs 0.0%, P = 0.04), D1S507 (50.0% vs 0.0%, P = 0.098), and D9S1752 (50.0% vs 0.0%, P = 0.33) occurred more frequently than 50% in w-MHCC, but not in HGDN. On the other hand, LOH on D17S960, D17S1796 and D9S1749 occurred in HGDN, but not in w-MHCC. When compared with w-MHCC, LHCC had a higher LOH frequency on D17S720 (73.9% vs 36.4%, P = 0.04), D17S960 (68.8% vs 0.0%, P = 0.03) and D17S1796 (81.8% vs 0.0%, P = 0.01). Conclusions The present study suggests MS-LOH is a simple and specific assay for routinely diagnostic pathology. We recommend that D4S415, D1S507, D9S1752, D17S960, D17S1796 and D9S1749 can be used as the first-line markers for differential diagnosis between HGDN and w-MHCC, and D9S1748, D17S921 and D17S520 with a LOH frequency of 40%–50% in w-MHCC, but netative in HGDN, can be regarded as the second-line candidate markers.

Prognostic relevance of genetic aberrations in gastrointestinal stromal tumors.

49 Background: Gastrointestinal stromal tumours (GISTs) contain oncogenic KIT or PDGFRA tyrosine kinase (TK) mutations leading to disturbance of downstream signaling pathways that contribute to GIST pathogenesis. Additional genetic aberrations were found in GISTs, demonstrating the involvement of other genes important in tumor progression. The aim of the study was to evaluate the prognostic relevance of different TK mutations in GISTs and to analyze the additional genetic aberrations in GISTs according to mutational status. Methods: 180 GIST patients were examined for KIT (9, 11, 13, 17 exons) and PDGFRA mutations (12, 14, 18 exons) by direct sequencing of DNA obtained from microdissected tumor sections. Tumor tissue DNA from 44 GISTs patients was screened for loss of heterozygosity (LOH) at 11 microsatellite loci on 1p, 9p, 14q, 15q and 22q arms. Results: KIT and PDGFRA mutations were identified in 76.1% and 10% of GISTs, respectively. 13.9% GISTs had no mutations (wild type). Most of KIT mutations were located in exon 11 (65%) and exon 9 (9.4%). Prognostic significance of TK mutations was evaluated. There was a trend of better survival for patients with PDGFRA mutation then with KIT mutation or wild type GISTs. The higher overall survival prior to target therapy was shown for patients with duplication or point mutation in KIT exon 11 in comparison to exon 11 deletion. Analysis of LOH revealed allelic deletions in 85% of GISTs, more frequently at 14q arm. There was a different LOH pattern in subgroups of GISTs. GISTs with PDGFRA mutation and with KIT exon 11 point mutation had LOH at 14q, while GISTs with KIT exon 11 deletions revealed high LOH frequency also on 22q, 15q and 1p arms. LOH in wild type GISTs occurred on all chromosomes with low frequency. LOH on 9p was found exclusively in metastatic and recurrent GISTs. Specific gene loci of frequent LOH were identified on 9p, 15q and 22q that may be contributed to GIST progression. Conclusions: Specific mutations are associated with GISTs prognosis. Tumors with point mutations and duplications in KIT exon 11 are associated with a better survival then GISTs with other KIT mutations. Specific pattern of allelic deletions was identified in subgroups of GISTs according to type of mutation and tumor progression. No significant financial relationships to disclose.