Evidence Of Loss Of Heterozygosity Research Articles

Large cell calcifying Sertoli cell tumour: molecular and immunohistochemical assessment of a series comprising non-metastasising and metastasising neoplasms.

Large cell calcifying Sertoli cell tumour (LCCSCT) is a type of testicular sex cord-stromal tumour that may occur sporadically or in the context of Carney complex and other genetic syndromes. A subset is clinically malignant, and the molecular mechanisms that drive such aggressive behaviour remain unknown. METHODS AND RESULTS: We analysed 21 samples from 20 patients with LCCSCT (12 non-metastasising and eight metastasising) using PRKAR1A immunohistochemistry (IHC) and next-generation sequencing. Alltumours except two (cases 17 and 20, both metastasising) demonstrated loss of PRKAR1A expression. Among 11 cases with interpretable sequencing results, all harboured pathogenic single nucleotide variants of PRKAR1A. Evidence of loss of heterozygosity (LOH) of PRKAR1A was present in all tumours with interpretable zygosity data, but the mechanisms of LOH were different for non-metastasising and metastasising tumours. Non-metastasising tumours demonstrated only copy-neutral LOH, while metastasising tumours demonstrated a spectrum of mechanisms of LOH, including copy-loss LOH, two concurrent mutations or copy-neutral LOH. Relevant molecular findings in non-metastasising LCCSCT were limited to PRKAR1A variants. In contrast, all metastasising LCCSCTs with interpretable data harboured additional pathogenic variants, including (but not restricted to) BRCA2 mutations with evidence of LOH and bi-allelic CDKN2A/B deletions. Three patients harboured PRKAR1A variants of inferred germline origin, including one with Carney complex and two without known syndromic features. CONCLUSIONS: This study further confirms that PRKAR1A IHC is a useful diagnostic tool for both non-metastasising and metastasising tumours and suggests that molecular analyses can be helpful to identify non-metastasising tumours with malignant potential in selected patients. Importantly, these results highlight that germline assessment could be beneficial for all patients presenting with LCCSCT.

Genomic diversity, chromosomal rearrangements, and interspecies hybridization in the Ogataea polymorpha species complex.

The methylotrophic yeast Ogataea polymorpha has long been a useful system for recombinant protein production, as well as a model system for methanol metabolism, peroxisome biogenesis, thermotolerance, and nitrate assimilation. It has more recently become an important model for the evolution of mating-type switching. Here, we present a population genomics analysis of 47 isolates within the O. polymorpha species complex, including representatives of the species O. polymorpha, Ogataea parapolymorpha, Ogataea haglerorum, and Ogataea angusta. We found low levels of nucleotide sequence diversity within the O. polymorpha species complex and identified chromosomal rearrangements both within and between species. In addition, we found that one isolate is an interspecies hybrid between O. polymorpha and O. parapolymorpha and present evidence for loss of heterozygosity following hybridization.

Abstract LB-245: Validating models of imputing germline versus somatic status for variants detected by tumor-only genomic profiling using germline clinical testing data

Abstract The goal of tumor genomic profiling is to identify somatic mutations. However, most implementations lack patient-matched control DNA sequence to enable determination of variant source (somatic or germline) and resolution of allelic status, including possible loss of heterozygosity (LOH), without additional analyses based on accurate estimates of specimens' tumor purity. Here, we present the validation of an information-theoretic algorithm for identifying germline variants detected in tumor-only sequencing using cancer patient clinical germline test data. 1,631 adult patients consented for the PROFILE study at DFCI between 2014 and 2018, and also underwent clinical germline testing. DNA was isolated from tissue containing >20% tumor nuclei and analyzed using Agilent SureSelect hybrid capture kit. All exons and 191 introns in 447 genes were interrogated for single nucleotide variants, small indels, copy number alterations, and structural variants using standard pipelines. Germline testing was performed at CLIA-certified laboratories. Specimen tumor purity was estimated using computational approaches developed at DFCI and Rutgers with further manual curation. LOHGIC (LOH-Germline Inference Calculator), developed on a model-selection scheme using Akaike Information Criterion weighting, was applied to individual variants to infer the most consistent germline-v-somatic model and LOH status, incorporating biases in clinical sequencing and purity estimation. 427 patients had 676 variants detected in high-penetrance cancer predisposition genes: BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2. Comparison of LOHGIC's results with germline test data showed 91.4% accuracy, 67.6% precision, and 67.0% recall, with other performance measures reported in Table 1. LOHGIC showed evidence of LOH for 35% of variants with concordant inference and germline testing results. Special challenges with TP53 variants will also be discussed. Our results indicate that tumor-only sequencing can provide excellent power for statistical approaches to identify likely germline mutations and infer LOH. Our analyses may be confounded by the presence of reversion mutations, poor specimen quality, inaccurate purity estimates, low sequencing depths, and/or germline call variability. This work further demonstrates the need for a systematic effort to interpret clinical-grade sequencing results. Table 1.Summary of resultsGeneBRCA1BRCA2MLH1MSH2MSH6PMS2OverallGermline Test: Positive/Negative28/10445/1753/6010/6514/12112/39112/564Germline Inference: Positive/Negative/Ambiguous24/94/1448/138/347/46/1013/51/1115/105/154/37/10111/471/94Positive Predictive Value (PPV)79.270.842.969.246.775.067.6Negative Predictive Value (NPV)95.798.6100.0100.096.286.596.9True Positive Rate (TPR)67.975.6100.090.050.025.067.0False Omission Rate (FOR)4.31.40.00.03.813.53.1Sensitivity82.694.4100.0100.063.637.583.3Specificity94.790.792.092.792.797.092.8Accuracy92.491.492.593.890.085.491.4 Citation Format: Israel Gomy, Nahed Jalloul, Samantha Stokes, Alexander Gusev, Shridar Ganesan, Judy Garber, Hossein Khiabanian. Validating models of imputing germline versus somatic status for variants detected by tumor-only genomic profiling using germline clinical testing data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-245.

Genetic and demographic vulnerability of adder populations: Results of a genetic study in mainland Britain.

Genetic factors are often overlooked in conservation planning, despite their importance in small isolated populations. We used mitochondrial and microsatellite markers to investigate population genetics of the adder (Vipera berus) in southern Britain, where numbers are declining. We found no evidence for loss of heterozygosity in any of the populations studied. Genetic diversity was comparable across sites, in line with published levels for mainland Europe. However, further analysis revealed a striking level of relatedness. Genetic networks constructed from inferred first degree relationships suggested a high proportion of individuals to be related at a level equivalent to that of half-siblings, with rare inferred full-sib dyads. These patterns of relatedness can be attributed to the high philopatry and low vagility of adders, which creates high local relatedness, in combination with the polyandrous breeding system in the adder, which may offset the risk of inbreeding in closed populations. We suggest that reliance on standard genetic indicators of inbreeding and diversity may underestimate demographic and genetic factors that make adder populations vulnerable to extirpation. We stress the importance of an integrated genetic and demographic approach in the conservation of adders, and other taxa of similar ecology.

Mosaic-variegated aneuploidy syndrome mutation or haploinsufficiency in Cep57 impairs tumor suppression.

A homozygous truncating frameshift mutation in CEP57 (CEP57T/T) has been identified in a subset of mosaic-variegated aneuploidy (MVA) patients; however, the physiological roles of the centrosome-associated protein CEP57 that contribute to disease are unknown. To investigate these, we have generated a mouse model mimicking this disease mutation. Cep57T/T mice died within 24 hours after birth with short, curly tails and severely impaired vertebral ossification. Osteoblasts in lumbosacral vertebrae of Cep57T/T mice were deficient for Fgf2, a Cep57 binding partner implicated in diverse biological processes, including bone formation. Furthermore, a broad spectrum of tissues of Cep57T/T mice had severe aneuploidy at birth, consistent with the MVA patient phenotype. Cep57T/T mouse embryonic fibroblasts and patient-derived skin fibroblasts failed to undergo centrosome maturation in G2 phase, causing premature centriole disjunction, centrosome amplification, aberrant spindle formation, and high rates of chromosome missegregation. Mice heterozygous for the truncating frameshift mutation or a Cep57-null allele were overtly indistinguishable from WT mice despite reduced Cep57 protein levels, yet prone to aneuploidization and cancer, with tumors lacking evidence for loss of heterozygosity. This study identifies Cep57 as a haploinsufficient tumor suppressor with biologically diverse roles in centrosome maturation and Fgf2-mediated bone formation.

Abstract 2990: Evidence for diverse mechanisms of tumorigenesis in breast and ovarian tumors of BRCA1/2 carriers

Abstract Germline mutations in the tumor suppressors BRCA1 and BRCA2 lead to increased risks of breast and ovarian cancers at least in part due to their roles in homologous recombination based double stranded DNA repair. Genetic alterations including loss of the wild-type BRCA1/2 allele, PTEN loss and TP53 mutations are thought to contribute to genomic instability in susceptible tissues and therefore tumor formation. In order to further investigate the mechanisms of tumorigenesis in BRCA1/2 carriers, we performed whole exome sequencing of 39 breast and ovarian tumors and matched blood germline DNA. Sequencing data were analyzed using GATK and MuTECT for variant calls and ngCGH and Sequenza for copy number analysis. Using cellularity corrected tumor versus germline allele frequency calculations, we found no evidence of loss of heterozygosity (LOH) in 17% of BRCA1 tumors (n = 4 of 24) and 40% of BRCA2 tumors (n = 6 of 15). Genomic instability as measured by percentage of genome in the diploid state showed that BRCA1/2 tumors with no evidence of LOH had decreased genomic instability (average% genome diploid 76+27% vs 57+16%, p = 0.01) with no difference in the overall somatic mutation rate (average 50+71 vs 83+156 single nucleotide variants per megabase, p = NS). While there were also no significant differences in the average number of predicted deleterious somatic mutations in BRCA1/2 tumors with no LOH versus tumors with LOH (n = 13+15 vs 13+6, p = NS), tumors without LOH were significantly less likely to have mutations or loss in TP53 or loss of PTEN (50% versus 93%, p = 0.007). Of the ten tumors with no LOH, five were from patients who had received prior cytotoxic chemotherapy. Other tumors with no LOH were from patients with atypical presentations including a mixed histology epithelial ovarian tumor in a BRCA1 carrier, and a triple negative breast cancer in a 70 year old BRCA2 carrier. Of the 29 tumors with evidence of LOH, no patient had received cytotoxic chemotherapy for a prior malignancy or in the neoadjuvant setting. No recurrent driver mutations other than TP53 mutations were identified in the BRCA1/2 tumors with or without LOH. Twenty-six pathogenic likely driver mutations were identified in 25 cancer genes outside of TP53, including NRAS, PIK3CA, and ABL2. Our results indicate that approximately 75% of tumors in BRCA1/2 germline mutation carriers arise via a classic pathway involving LOH of the wildtype allele, loss of function of TP53 and/or PTEN and significant genomic instability. However, approximately 25% of tumors may arise via an alternative pathway, possibly related to prior chemotherapy in some cases. Further studies are needed to determine the molecular and clinical factors associated with this proposed classic versus atypical pathway of tumorigenesis in BRCA1/2 carriers and whether these characteristics are associated with outcomes such as survival and platinum and/or PARP inhibitor sensitivity. Citation Format: Kara N. Maxwell, Daniel De Sloover, Bradley Wubbenhorst, Brandon Wenz, Nicole Lunceford, Lyndsey Emery, Kurt D'Andrea, Robert D. Daber, Michael D. Feldman, Susan M. Domchek, Katherine L. Nathanson. Evidence for diverse mechanisms of tumorigenesis in breast and ovarian tumors of BRCA1/2 carriers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2990. doi:10.1158/1538-7445.AM2015-2990

Evolution of an Experimental Population of Phytophthora capsici in the Field

Populations of the vegetable pathogen Phytophthora capsici are often highly diverse, with limited gene flow between fields. To investigate the structure of a newly established, experimental population, an uninfested research field was inoculated with two single-zoospore isolates of P. capsici in September 2008. From 2009 through 2012, ≈50 isolates of P. capsici were collected from the field each year and genotyped using five microsatellite loci. The same two isolates were also crossed in the lab. High levels of diversity were detected in the research field, with 26 to 37 unique multilocus genotypes detected each year. Through 2012, genotypic diversity did not decline and no evidence of genetic drift was observed. However, during the 2011 and 2012 growing seasons, four new alleles not present in either parental isolate were observed in the field. Selfing (but not apomixis) was observed at low frequency among in vitro progeny. In addition, evidence for loss of heterozygosity was observed in half of the in vitro progeny. These results suggest that recombination, mutation, and loss of heterozygosity can affect the genetic structure observed in P. capsici populations.

Integrated analysis of germline and somatic variants in ovarian cancer

We report the first large-scale exome-wide analysis of the combined germline-somatic landscape in ovarian cancer. Here we analyze germline and somatic alterations in 429 ovarian carcinoma cases and 557 controls. We identify 3,635 high confidence, rare truncation and 22,953 missense variants with predicted functional impact. We find germline truncation variants and large deletions across Fanconi pathway genes in 20% of cases. Enrichment of rare truncations is shown in BRCA1, BRCA2, and PALB2. Additionally, we observe germline truncation variants in genes not previously associated with ovarian cancer susceptibility (NF1, MAP3K4, CDKN2B, and MLL3). Evidence for loss of heterozygosity was found in 100% and 76% of cases with germline BRCA1 and BRCA2 truncations respectively. Germline-somatic interaction analysis combined with extensive bioinformatics annotation identifies 237 candidate functional germline truncation and missense variants, including 2 pathogenic BRCA1 and 1 TP53 deleterious variants. Finally, integrated analyses of germline and somatic variants identify significantly altered pathways, including the Fanconi, MAPK, and MLL pathways.

Evidence for loss of heterozygosity (LOH) at chromosomes 9p and 17p in oral granular cell tumors: a pilot study

The objective of this study was to assess loss of heterozygosity (LOH) in tumor suppressor gene loci in oral granular cell tumors (GCTs). We assessed LOH in 8 samples of oral GCT using polymorphic microsatellite markers at chromosome regions 3p, 9p, 11q, and 17p, flanking areas close to tumor suppressor genes. We further performed immunohistochemistry to detect the p53 and Ki-67 proteins and associated these expressions with the molecular results. Five samples showed LOH in 3 markers at chromosomes 9p and 17p (markers P53, AFM238WF2 and D9S162) with fraction of allelic loss of 42.8% for each of these markers. No LOH was identified in any other chromosome. LOH was not associated with the immunohistochemical expression of p53 and Ki-67. The present study shows LOH at chromosomes 9p and 17p in oral GCTs.

Genomic Changes in Gliomas Detected Using Single Nucleotide Polymorphism Array in Formalin-Fixed, Paraffin-Embedded Tissue: Superior Results Compared with Microsatellite Analysis

Deletion or loss of heterozygosity (LOH) in chromosomes 1p and 19q in oligodendrogliomas (ODGs) have diagnostic, prognostic, and therapeutic implications. Current clinical assays are limited because the probes or primers interrogate only limited genomic segments. We investigated the use of single nucleotide polymorphism (SNP) arrays for identifying genomic changes in gliomas from FFPE tissues. DNA was extracted from FFPE tissues of 30 brain tumor cases (15 ODGs and 15 non-ODGs) and assayed on the Illumina array with 300,000 markers. SNP results were compared with standard short tandem repeat (STR) assays of chromosomes 1p and 19q. Fifteen ODGs had LOH by STR and deletion by array on both 1p and 19q. Ten non-ODGs had no evidence of LOH on 1p and 19q by STR, seven of which had no abnormalities for these chromosomes; three had partial deletions by SNP array. Five non-ODG cases had partial LOH or deletion by both assays. No major discordance was found between SNP array and STR results. Advantages of SNP arrays include no need for an accompanying normal sample, the ability to find small segmental deletions, the potential to distinguish between deletions and copy neutral LOH, and whole-genome screening to allow discovery of new, significant loci. Assessment of genomic changes in routine glioma specimens using SNP arrays is feasible and has great potential as an accurate clinical diagnostic test.

Isolated familial somatotropinoma: 11q13-loh and gene/protein expression analysis suggests a possible involvement of aip also in non-pituitary tumorigenesis

OBJECTIVENon-pituitary tumors have been reported in a subset of patients harboring germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene. However, no detailed investigations of non-pituitary tumors of AIP-mutated patients have been reported so far.PATIENTSWe examined a MEN1- and p53-negative mother-daughter pair with acromegaly due to somatotropinoma. Subsequently, the mother developed a large virilizing adrenocortical carcinoma and a grade II B-cell non-Hodgkin’s lymphoma.DESIGNMutational analysis was performed by automated sequencing. Loss-of-heterozygosity (LOH) analysis was carried out by sequencing and microsatellite analysis. AIP expression was assessed through quantitative PCR (qPCR) and immunohistochemistry.RESULTSThe functional inactivating mutation c.241C>T (R81X), which blocks the AIP protein from interacting with phosphodiesterase 4A (PDE4A), was identified in the heterozygous state in the leukocyte DNA of both patients. Analyzing the tumoral DNA revealed that the AIP wild-type allele was lost in the daughter’s somatotropinoma and the mother’s adrenocortical carcinoma. Both tumors displayed low AIP protein expression levels. Low AIP gene expression was confirmed by qPCR in the adrenocortical carcinoma. No evidence of LOH was observed in the DNA sample from the mother’s B-cell lymphoma, and this tumor displayed normal AIP immunostaining.CONCLUSIONSOur study presents the first molecular analysis of non-pituitary tumors in AIP-mutated patients. The finding of AIP inactivation in the adrenocortical tumor suggests that further investigation of the potential role of this recently identified tumor suppressor gene in non-pituitary tumors, mainly in those tumors in which the cAMP and the 11q13 locus are implicated, is likely to be worthwhile.

High incidence of allelic loss at 16q12.2 region spanning RB2/p130 gene in retinoblastoma

Retinoblastoma (Rb) is the most common intra-ocular tumor that manifests in early childhood. It is initiated by the inactivation of RB1/p105 gene, a prototype tumor suppressor gene. However, observed recurrent chromosomal aberrations accompanying RB1/p105 mutations suggest the involvement of additional mutational events. Chromosome 16q is one of the loci with recurrent losses which are likely to contain tumor suppressor genes. In this study, allelic loss was demonstrated at a second locus for retinoblastoma, RBL2/p130 on 16q12.2. Using intragenic single nucleotide polymorphisms (SNPs) (g.7085556A>C and g.7118919T>C) and flanking extragenic microsatellite markers (D16S411 and D16S408), 40 retinoblastoma tumor samples were analyzed. Loss of heterozygosity (LOH) of these markers was found in 11 (57.9%) out of 19 informative tumors at the RBL2/p130 gene locus and while a total of 15 (78.9%) tumors showed LOH in at least one marker. Deletions extending more than 13 cM across the pericentromeric region of 16q12.1-q13 were inferred from 4 tumors. Microsatellite instability was observed in two other tumors at the flanking markers. No mutations were found in RBL2/p130 exons 19-22 coding for the protein domain critical for biological activity. This is the first evidence of LOH within RBL2/p130 gene in retinoblastoma. The high frequency of allelic loss provides further evidence on the implication of this gene in retinoblastoma development and/or progression.

Somatic alterations of the NF1 gene in an NF1 individual with multiple benign tumours (internal and external) and malignant tumour types

Neurofibromatosis type 1 is a common familial cancer syndrome, affecting about 1 in every 4,000 individuals worldwide. We have carried out NF1 gene mutation analysis on DNA isolated from 25 tumours (dermal and plexiform neurofibromas, malignant peripheral nerve sheath tumour, MPNST), obtained at post-mortem from an NF1 patient. Macro and micro sequence alterations of the NF1 gene were studied by dHPLC, microsatellite, RFLP markers and multiplex ligation probe amplification (MLPA). The underlying germline mutation involves a deletion of exons 2 and 3. Of the 25 tumours studied from this patient, characterised somatic mutations were identified in 9 tumours, these were six small deletions (748del T, 2534-2557 del 24bp, 2843delA, 3047-3048 del GT, 4743del G, 7720-7721 delAA), an insertion 649 ins 73 bp, a non-sense mutation R1513X and a single splice site mutation, IVS4C-1 G>A, eight of these represent novel sequence changes in the gene. Evidence for loss of heterozygosity (LOH) was identified in DNA from 7 of the tumours. Each of the tumours analysed contained a different somatic NF1 mutation, indicating that each tumour is the result of an independent somatic event. The somatic mutation detection rate in this study is 64% (16/25), is one of the highest rates in genomic DNA reported so far in a single NF1 patient. Only 68 characterised NF1 somatic mutations have so far been reported and so our data will contribute to NF1 somatic mutational spectrum of the NF1 gene and will be important for understanding the molecular basis of NF1 tumorigenesis.

Germline and somaticNF1 gene mutation spectrum in NF1-associated malignant peripheral nerve sheath tumors (MPNSTs)

About 10% of neurofibromatosis type 1 (NF1) patients develop malignant peripheral nerve sheath tumors (MPNSTs) and represent considerable patient morbidity and mortality. Elucidation of the genetic mechanisms by which inherited and acquired NF1 disease gene variants lead to MPNST development is important. A study was undertaken to identify the constitutional and somatic NF1 mutations in 34 MPNSTs from 27 NF1 patients. The NF1 germline mutations identified in 22 lymphocytes DNA from these patients included seven novel mutations and a large 1.4-Mb deletion. The NF1 germline mutation spectrum was similar to that previously identified in adult NF1 patients without MPNST. Somatic NF1 mutations were identified in tumor DNA from 31 out of 34 MPNSTs, of which 28 were large genomic deletions. The high prevalence (>90%) of such deletions in MPNST contrast with the =or<20% found in benign neurofibromas and is indicative of the involvement of different mutational mechanisms in these tumors. Coinactivation of the TP53 gene by deletion, or by point mutation along with NF1 gene inactivation, is known to exacerbate disease symptoms in NF1, therefore TP53 gene inactivation was screened. DNA from 20 tumors showed evidence for loss of heterozygosity (LOH) across the TP53 region in 11 samples, with novel TP53 point mutations in four tumors.

Loss of Heterozygosity (LOH) for HumACTBP2 (SE33) Is Detected in Philadelphia (Ph)-Positive and Ph-Negative Hematopoietic Cells of Some Patients with Ph-Positive CML.

Introduction: In the majority of patients with Philadelphia (Ph)-positive chronic myelogenous leukemia (CML), the non-leukemic hematopoiesis is considered polyclonal. Occasionally, clonal cytogenetic abnormalities have been reported in Ph-negative cells from patients in cytogenetic remission after IFN-alpha or imatinib. In the present project we determined clonality of flow-sorted hematopoietic cells from untreated and treated patients with Ph-positive CML in first chronic phase by analyzing the two alleles of short tandem repeats (STR) of given loci. The loss of one of the two alleles of STR identifies loss of heterogzygosity (LOH) and clonal expansion. These results were compared to conventional cytogenetics and correlated with response to therapy.Patients and methods: A total of 25 consecutive patients (13 m/12 f, median age 58 y) treated at the University of Leipzig from April to June 2005 were included. Conventional cytogenetics were analyzed by R-banding. FISH using the LSI BCR/ABL ES probe in unsorted as well as in flow-sorted monocytes (CD14+), granulocytes (CD14-/CD33+), T- lymphocytes (CD3+), B-lymphocytes (CD19+), and CD34+-cells was performed. For LOH analysis, DNA was extracted from flow-sorted cells. 8 autosomal loci were analysed: D3S1358 (3p), vWA (12p12-pter), D8S1179 (8q), D21S11 (21q11.2-q21), D18S51 (18q21.3), TH01 (11p15.5), FGA (4q28), SE33 (6q15). PCR products were detected by capillary electrophoresis. Loss of, or a strong decrease in the signal intensity of one allele was considered as evidence for LOH. An allelic imbalance factor (AIF) was calculated by the quotient of the peak ratios from constitutional and flow-sorted cell DNA (N1:N2/T1:T2). An AIF > 1.8 was considered as LOH. Buccal swabs were used as controls.Results: Median time from diagnosis to analysis was 60 months. Of the 25 patients, twenty (80%) received Imatinib for a median of 26 months, three (12%) were newly diagnosed and two (8%) were treated with IFN-alpha. Both patients with IFN-alpha as well as 8 (40%) patients treated with Imatinib had sustained complete cytogenetic remission (CCR). Furthermore, 8 (40%) responded to Imatinib with hematological remission (HR) only, 3 (15%) patients with major CR (MCR) and one patient (5%) with minor CR. Conventional cytogenetics revealed additional abnormalities [t(7;17), del (20q), trisomy 8, and -Y] in Ph- negative cells in 4 (16%) patients. No LOH was detected in these patients. Ph-positive B-cells were detected in 4 (16%) with HR. Interestingly, the median quotient of Ph-positive CD34+ and Ph-positive unsorted cells was 0.65. LOH for SE33 in CD34+ cells, monocytes and granulocytes was detected in 6 (24%) patients [3 in CCR (2 patients treated with IFN-alpha and 1 with imatinib)], 1 patient in MCR under imatinib, and 2 patients with newly diagnosed CML. LOH was not detected in buccal swabs.Conclusions: We confirm the occurrence of clonal cytogenetic abnormalities in Ph-negative cells. LOH did not parallel abnormal cytogenetics and LOH for SE33 only was detected in patients with Ph-negative and Ph-positive cytogenetics. The detection of LOH which could be a sign of inactivation of tumor suppressor genes in treated and untreated patients with CML argues against a treatment-induced event and raises speculations on the role of this finding in the pathogenesis of at least some cases of CML.

Research on the mechanisms of PTEN gene inactivation in ovarian cancer

To investigate the mechanisms of PTEN gene inactivation starting from DNA, mRNA and protein levels in ovarian cancers. Tumor tissue samples were obtained from 48 patients with epithelial ovarian cancers. Using four polymorphic markers (D10s541, D10s583, D10s1687 and D10s2491) within and flanking the PTEN gene located in chromosome 10q 23.3, polymerase chain reaction (PCR) and loss of heterozygosity (LOH) were introduced to examine LOH of PTEN gene; PCR-single strand conformation polymorphism (PCR-SSCP) was introduced to examine mutations of the fifth, sixth, seventh, and eighth exons of PTEN. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (SP method) were applied to detect PTEN mRNA and PTEN protein expressions, respectively. LOH of PTEN gene was observed in 19 of 48 (39.6%) ovarian cancers. PTEN mutations were found only in 2 (4.2%) of the cases. Absence of PTEN mRNA expression was 18.8% (9 of 48). Immunostaining of 48 cancer samples revealed that 13 (27.1%) were PTEN immunostain negative. Of these 13 samples, only 2 (15.4%) had structural, biallelic inactivation by intragenic PTEN mutations and loss of the remaining wild-type allele; 7 (53.8%) showed evidence of LOH, 5 of these 7 samples showed deletion of PTEN mRNA expression, another 2 samples showed positive expression of PTEN mRNA; 4 (30.8%) tumors had neither PTEN gene mutation nor LOH but exhibited no PTEN protein expression, 2 of these 4 cases showed deletion of PTEN mRNA expression, another 2 showed positive expression of PTEN mRNA. For the cases of PTEN protein absent staining, the rate of LOH was 69.2% (9 of 13), higher than 28.6% (10 of 35) for the positive staining (P < 0.05). PTEN gene inactivation may contribute to epithelial ovarian carcinogenesis. There may be several mechanisms of PTEN gene inactivation in ovarian cancers. Protein expression deletions may be a significant mechanism.

Identification of Krüppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer.

Krüppel-like factor 4 (KLF4 or GKLF) is an inhibitor of the cell cycle. The gene encoding KLF4 is localized on chromosome 9q, previously shown to exhibit allelic loss in colorectal cancer (CRC). In this study, we show that the mean level of KLF4 mRNA in a panel of 30 CRC was 52% that of paired normal colonic tissues. Similarly, the levels of KLF4 mRNA and protein in a panel of six established CRC cell lines were significantly lower than those of an untransformed colonic epithelial cell line. Using highly polymorphic DNA markers that flank the KLF4 locus, we found evidence for loss of heterozygosity (LOH) in two of eight surgically resected CRC specimens. In addition, LOH was observed in five of six CRC cell lines with one additional cell line exhibiting hemizygous deletion in the KLF4 gene. We also found that the 5'-untranslated region of KLF4 was hypermethylated in a subset of resected CRC specimens and cell lines. Lastly, the open-reading frame of KLF4 in two of three CRC cell lines examined contained several point mutations that resulted in a diminished ability to activate the p21(WAF1/Cip1) promoter. These findings indicate that KLF4 is a potential tumor suppressor gene in CRC.

Multiple roles of the tuberous sclerosis complex genes.

Soon after proposing the "two-hit" hypothesis for tumorigenesis, Knudson pursued further experimental validation of the concept by using a rat model of dominantly inherited renal tumor. Today, the Eker rat is one of the best characterized models of tuberous sclerosis complex (TSC) and has been used extensively for study of the function of the TSC2 tumor suppressor gene. Along with TSC1, these two genes behave as expected for tumor suppressor genes with evidence for loss of heterozygosity in tumors and suppression of growth when expressed in proliferating cells. Despite much experimental work, the mechanisms of these genes have remained elusive until recently. This review summarizes some of the current concepts in our understanding of the biological and biochemical function of the TSC genes.

The parathyroid hormone-responsive B1 gene is interrupted by a t(1;7)(q42;p15) breakpoint associated with Wilms' tumour.

Wilms' tumour (WT) has a diverse and complex molecular aetiology, with several different loci identified by cytogenetic and molecular analyses. One such locus is on chromosome 7p, where cytogenetic abnormalities and loss of heterozygosity (LOH) indicate the presence of a Wilms' tumour suppressor gene. In order to isolate a candidate gene for this locus, we have characterized the breakpoint regions at a novel constitutional chromosome translocation (t(1;7)(q42;p15)), found in a child with WT and skeletal abnormalities. We identified two genes that were interrupted by the translocation: the parathyroid hormone-responsive B1 gene (PTH-B1) at 7p and obscurin at 1q. With no evidence for LOH at 1q42, we focused on the characterization of PTH-B1. We detected novel alternately spliced isoforms of PTH-B1, which were expressed in a wide range of adult and foetal tissues. Importantly, expression of two isoforms were disrupted in the WT of the t(1;7) patient. We also identified an additional splice isoform expressed only in 7p LOH tumours. The disruption of PTH-B1 by the t(1;7), together with aberrant splicing in sporadic WTs, suggests that PTH-B1 is a candidate for the 7p Wilms' tumour suppressor gene.

Mutations and molecular variants of the NBS1 gene in non-Hodgkin lymphoma.

Non-Hodgkin lymphomas (NHLs) are characterized by chromosomal translocations that juxtapose loci encoding lymphoid antigen receptors with cellular proto-oncogenes. These translocations are thought to arise from inaccurate processing of DNA breaks created during physiologic recombination of the antigen receptor genes in lymphocytes. The inherited disorders ataxia-telangiectasia and Nijmegen breakage syndrome are caused by mutations in the ATM and NBS1 genes, respectively, and are characterized by generalized genomic instability and a high incidence of lymphoid cancers. Lymphoid cells from patients with either disorder frequently have chromosomal translocations involving T-cell-receptor or immunoglobulin loci. To investigate the potential role of the NBS1 gene in the pathogenesis of NHL, we screened tumor DNA samples from 91 sporadic cases of NHL and genomic DNA from 154 control individuals for mutations in all 16 exons of the NBS1 gene and in flanking intronic sequences. One NHL case with a truncating mutation in NBS1 and a second NHL case with a putative missense mutation were detected. Neither mutation was observed among controls. Three additional putative missense mutations were observed only in the normal control samples. A panel of six common polymorphisms spanning the NBS1 gene was genotyped and provided no evidence for loss of heterozygosity in the NHL cases with mutations or in the NHL population overall. These results suggest that mutations in NBS1 do not play a major role in the development of NHL in the United States.