S. E. Hilary Russell

Determination of the replication error phenotype in human tumors without the requirement for matching normal DNA by analysis of mononucleotide repeat microsatellites

Microsatellite instability (MI) characterizing tumors with replication errors (RER+ tumors) was first described in colorectal tumors from hereditary non‐polyposis colorectal cancer (HNPCC) patients as well as in sporadic cases. It has also been observed in subgroups of extracolonic sporadic tumors, but there is no consensus as to the number of microsatellite loci to examine, and the threshold percentage of unstable loci required to classify a tumor as RER+. We have recently shown that BAT‐26, a mononucleotide repeat microsatellite, was quasi‐monomorphic in DNA from normal individuals and from colorectal RER− samples, and showed important size variations in RER+ samples. In the present work, we analyzed BAT‐26 allelic profiles in tumors of the breast (n = 107), brain (n = 78), stomach (n = 59), prostate (n = 49), esophagus (n = 36), thyroid (n = 31), endometrium (n = 12), and cervix (n = 10) whose RER status was already known, thus extending BAT‐26 analysis to a total of 542 human solid tumors. BAT‐26 alleles were quasi‐monomorphic in RER‐ samples (475/481) and shortened in RER+ tumors (57/61), including four tumors shown to have been misclassified on the basis of dinucleotide repeat microsatellite analysis. In 3/481 RER− and 4/61 RER+ cases, BAT‐26 size variation was important enough to attract attention, but not sufficient to establish the RER status of the corresponding tumors. In these cases, the analysis of BAT‐25 and BAT‐34C4, two other mononucleotide repeat microsatellites, was necessary to resolve the ambiguity. There were only 3 false positive cases. In conclusion, BAT‐26 was able to identify the RER status of 539 out of 542 tumors from various origins (99.5% efficiency) in a single‐step experiment without the requirement for matching normal DNA. Genes Chromosomes Cancer 21:101–107, 1998.

The pathobiology of the septin gene family.

Septins are an evolutionarily conserved group of GTP‐binding and filament‐forming proteins that belong to the large superclass of P‐loop GTPases. While originally discovered in yeast as cell division cycle mutants with cytokinesis defects, they are now known to have diverse cellular roles which include polarity determination, cytoskeletal reorganization, membrane dynamics, vesicle trafficking, and exocytosis. Septin proteins form homo‐ and hetero‐oligomeric polymers which can assemble into higher‐order filaments. They are also known to interact with components of the cytoskeleton, ie actin and tubulin. The precise role of GTP binding is not clear but a current model suggests that it is associated with conformational changes which alter binding to other proteins. There are at least 12 human septin genes, and although information on expression patterns is limited, most undergo complex alternative splicing with some degree of tissue specificity. Nevertheless, an increasing body of data implicates the septin family in the pathogenesis of diverse disease states including neoplasia, neurodegenerative conditions, and infections. Here the known biochemical properties of mammalian septins are reviewed in the light of the data from yeast and other model organisms. The data implicating septins in human disease are considered and a model linking these data is proposed. It is posited that septins can act as regulatable scaffolds where the stoichiometry of septin associations, modifications, GTP status, and the interactions with other proteins allow the regulation of key cellular processes including polarity determination. Derangements of such septin scaffolds thus explain the role of septins in disease states. Copyright

Expression profiling the human septin gene family.

The septins are an evolutionarily conserved family of GTP‐binding proteins involved in diverse processes including vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration, and neoplasia. The present paper reports a comprehensive study of septin gene expression by DNA microarray methods in 10 360 samples of normal, diseased, and tumour tissues. A novel septin, SEPT13, has been identified and is shown to be related to SEPT7. It is shown that SEPT13 and the other known human septins are expressed in all tissue types but some show high expression in lymphoid (SEPT1, 6, 9, and 12) or brain tissues (SEPT2, 3, 4, 5, 7, 8, and 11). For a given septin, some isoforms are highly expressed in the brain and others are not. For example, SEPT8_v2 and v1, 1* and 3 are highly expressed in the brain and cluster with SEPT2, 3, 4, 5, 7, and 11. However, a probe set specific for SEPT8_v1 with low brain expression clusters away from this set. Similarly, SEPT4 has lymphoid and non‐lymphoid forms; SEPT2 has lymphoid and central nervous system (CNS) forms; and SEPT6 and SEPT9 are elevated in lymphoid tissues but both have forms that cluster away from the lymphoid forms. Perturbation of septin expression was widespread in disease and tumours of the various tissues examined, particularly for conditions of the CNS, where alterations in all 13 septin genes were identified. This analysis provides a comprehensive catalogue of the septin family in health and disease. It is a key step in understanding the role of septins in physiological and pathological states and provides insight into the complexity of septin biology. Copyright

Altered expression of the septin gene, SEPT9, in ovarian neoplasia.

The septin family of genes has been implicated in a variety of cellular processes including cytokinesis, membrane transport and fusion, exocytosis, and apoptosis. One member of the septin family maps to chromosome 17q25.3, a region commonly deleted in sporadic ovarian and breast tumours, and has also been identified as a fusion partner of MLL in acute myeloid leukaemias. The present study demonstrates that the pattern of expression of multiple splice variants of this septin gene is altered in ovarian tumours and cell lines. In particular, expression of the zeta transcript is detectable in the majority of tumours and cell lines, but not in a range of non‐malignant adult and fetal tissues. Zeta expression is accompanied by loss of the ubiquitous beta transcript. Somatic mutations of the gene were not detected in ovarian tumours, but it was demonstrated that beta expression in tumour cell lines can be reactivated by 5‐azacytidine treatment, suggesting a role for methylation in the control of expression of this gene. Copyright

Multimodality expression profiling shows SEPT9 to be overexpressed in a wide range of human tumours

Septins are an evolutionarily conserved family of GTPases with diverse functions including roles in cytokinesis that have been implicated in neoplasia. To address the potential role of SEPT9 in tumorigenesis, we assessed the expression of SEPT9 in 7287 fresh frozen human tissue samples and 292 human cell lines by microarray analysis. In addition, we used a sensitive RT–PCR strategy to define the expression of SEPT9 isoforms in archival formalin-fixed and paraffin-embedded normal human tissues. The mRNA data were further confirmed by immunohistological analyses of SEPT9 protein expression in normal human tissues using antisera that detect SEPT9 isoforms. Using these complementary approaches, we demonstrate that SEPT9 mRNA and protein are expressed ubiquitously, with the isoforms showing tissue-specific expression. The microarray analysis indicates that there is consistent overexpression of SEPT9 in diverse human tumours including breast, CNS, endometrium, kidney, liver, lung, lymphoid, oesophagus, ovary, pancreas, skin, soft tissue and thyroid. Since tumours are commonly associated with enhanced cell proliferation, we examined the possible correlation of Ki67 and SEPT9 expression in normal tissues and tumours. Our data indicate that the overexpression of SEPT9 in neoplasia is not simply a proliferation-associated phenomenon, despite its role in cytokinesis.

A multistep model for ovarian tumorigenesis: the value of mutation analysis in the KRAS and BRAF genes.

Epithelial ovarian tumours represent a complex group of histological subtypes and there has long been controversy over the question of a precursor lesion for these neoplasms. The application of mutation analysis of the KRAS and BRAF genes (members of the RAS‐RAF‐MEK‐ERK‐MAP kinase pathway) is consistent with the model for progression of mucinous carcinomas and a subset of serous carcinomas (the so‐called low‐grade serous carcinomas) through benign and borderline lesions. The relatively high incidence of BRAF and KRAS mutations in serous borderline tumours and low‐grade serous carcinomas, and their extremely low incidence/absence in high‐grade serous carcinomas, provide strong evidence that high‐grade carcinomas do not arise through this intermediate step. Copyright

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5′ splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3′ terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3′ UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3′ UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT–PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.

Altered patterns of transcription of the septin gene, SEPT9, in ovarian tumorigenesis

Ovarian carcinoma represents the most lethal gynaecological malignancy. A variety of morphological subtypes are recognised (e.g. serous, mucinous, endometrioid), which may be benign, borderline or malignant. While their relationship is controversial, knowledge of the molecular mechanisms of ovarian tumorigenesis may help resolve this issue and perhaps identify early markers of disease. Perturbed patterns of expression of the SEPT9 gene on chromosome 17q25.3 have been implicated in a variety of tumour types including both breast and ovarian neoplasia. In preliminary studies, we showed that SEPT9 mRNA was upregulated in a bank of ovarian tumours, which included benign, borderline and malignant tumours, and reported increased levels of one splice variant, SEPT9_v4*. We now describe a comprehensive analysis of SEPT9 expression specifically in serous and mucinous ovarian tumours (benign, borderline and malignant), using cDNA microarray, semi‐ and quantitative RTPCR of microdissected archival tumour material. Our data show consistent and specific overexpression of both SEPT9_v1 and SEPT9_v4* transcripts in the epithelial component of ovarian tumours. These transcripts show highest levels of expression in serous and mucinous borderline tumours. SEPT9_v1 is also upregulated in both serous and mucinous carcinomas. Interestingly, highest levels of expression are observed in serous borderline and low‐grade tumours rather than high‐grade in keeping with a model of progression of benign, borderline and low‐grade serous tumours.

Barrett's oesophagus: microsatellite analysis provides evidence to support the proposed metaplasia-dysplasia-carcinoma sequence.

The development of adenocarcinoma in Barretts oesophagus is proposed to occur via a stepwise progression recognised histologically as a metaplasia‐dysplasia‐carcinoma sequence. In order to identify chromosomal loci involved in the malignant transformation of Barretts epithelium and the development of oesophageal adenocarcinoma, microsatellite analysis was carried out on 17 cases of Barretts‐associated oesophageal adenocarcinoma. Samples of premalignant Barretts epithelium adjacent to adenocarcinoma were obtained from seven of these cases. Allelic imbalance was detected in >45% of informative cases of oesophageal adenocarcinoma on chromosome arms 3q (65%), 4q (71%), 5q (59%), 6q (59%), 9p (50%), 9q (47%), 12p (47%), 12q (65%), 17p (76%), and 18q (75%). Allelic imbalance at 4q, 17p, and 18q was significantly higher than the upper 95% confidence interval for background allelic imbalance. Allelic imbalance was detected at several loci in the premalignant epithelium from five of the seven cases studied. These loci included several chromosomal arms that had demonstrated high levels of allelic imbalance in oesophageal adenocarcinoma, namely, 4q (one case), 5q (two cases), 9 (three cases), 12q (five cases), 17p (four cases), and 18q (two cases). Novel microsatellite alleles were detected in both premalignant and malignant Barretts epithelium. In three cases, dysplastic Barretts epithelium and adjacent adenocarcinoma demonstrated the same pattern of novel microsatellite alleles at a number of loci. In conclusion, these data indicate chromosomal loci which may be specifically involved in the histological progression of Barretts epithelium. The detection of shared novel microsatellite alleles in premalignant and malignant Barretts epithelium is consistent with a process of clonal expansion underlying this progression. Genes Chromosomes Cancer 21:49–60, 1998.

The Septin-Binding Protein Anillin Is Overexpressed in Diverse Human Tumors

Anillin is an actin-binding protein that can bind septins and is a component of the cytokinetic ring. We assessed the anillin expression in 7,579 human tissue samples and cell lines by DNA microarray analysis. Anillin is expressed ubiquitously but with variable levels of expression, being highest in the central nervous system. The median level of anillin mRNA expression was higher in tumors than normal tissues (median fold increase 2.58; 95% confidence intervals, 2.19-5.68, P < 0.0001) except in the central nervous system where anillin mRNA levels were lower in tumors. We developed a sensitive reverse transcription-PCR strategy to show that anillin mRNA is expressed in cell lines and in cDNA panels derived from fetal and adult tissues, thus validating the microarray data. We compared anillin with Ki67 mRNA expression and found a significant linear relationship between anillin and Ki67 mRNA expression (Spearmann r ∼ 0.6, P < 0.0001). Anillin mRNA expression was analyzed during tumor progression in breast, ovarian, kidney, colorectal, hepatic, lung, endometrial, and pancreatic tumors and in all tissues there was progressive increase in anillin mRNA expression from normal to benign to malignant to metastatic disease. Finally, we used anti-anillin sera and found nuclear anillin immunoreactivity to be widespread in normal tissues, often not correlating with proliferative compartments. These data provide insight into the existence of nonproliferation-associated activities of anillin and roles in interphase nuclei. Thus, anillin is overexpressed in diverse common human tumors, but not simply as a consequence of being a proliferation marker. Anillin may have potential as a novel biomarker.