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The MCPH1 gene, also known as BRCT-repeat inhibitor of hTERT expression (BRIT1), has three BRCA1 carboxyl-terminal domains which is an important regulator of DNA repair, cell cycle checkpoints and chromosome condensation. MCPH1/BRIT1 is also known as a tumour suppressor in different types of human cancer. The expression level of the MCPH1/BRIT1 gene is decreased at the DNA, RNA or protein level in a number of types of cancers including breast cancer, lung cancer, cervical cancer, prostate cancer and ovarian cancer compared to normal tissue. This review also showed that deregulation of MCPH1/BRIT1 is significantly associated with reduced overall survival in 57% (12/21) and relapsed free survival in 33% (7/21) of cancer types especially in oesophageal squamous cell carcinoma and renal clear cell carcinoma. A common finding of this study is that the loss of MCPH1/BRIT1 gene expression plays a key role in promoting genome instability and mutations supporting its function as a tumour suppressor gene.

Poor-prognosis breast cancers are treated with cytotoxic chemotherapy, but often without any guidance from therapy predictive markers because universally accepted markers are not currently available. Treatment failure, in the form of recurrences, is relatively common. We aimed to identify chemotherapy predictive markers and resistance pathways in breast cancer. Our hypothesis was that tumor cells remaining after neoadjuvant chemotherapy (NAC) contain somatic variants causing therapy resistance, while variants present pre-NAC but lost post-NAC cause sensitivity. Whole-exome sequencing was performed on matched pre- and post-NAC cancer cells, which were isolated by laser microdissection, from 6 cancer cases, and somatic variants selected for or against by NAC were identified. Somatic variant diversity was significantly reduced after therapy (P < 0.05). MUC17 variants were identified in 3 tumors and were selected against by NAC in each case, while PCNX1 variants were identified in 2 tumors and were selected for in both cases, implicating the function of these genes in defining chemoresponse. In vitro knockdown of MUC17 or PCNX1 was associated with significantly increased or decreased chemotherapy sensitivity, respectively (P < 0.05), further supporting their roles in chemotherapy response. Expression was tested for predictive value in two independent cohorts of chemotherapy-treated breast cancers (n = 53, n = 303). Kaplan–Meier analyses revealed that low MUC17 expression was significantly associated with longer survival after chemotherapy, whereas low PCNX1 was significantly associated with reduced survival. We concluded that therapy-driven selection of somatic variants allows identification of chemotherapy response genes. With respect to MUC17 and PCNX1, therapy-driven selection acting on somatic variants, in vitro knockdown data concerning drug sensitivity, and survival analysis of expression levels in patient cohorts all define the genes as mediators of and predictive markers for chemotherapy response in breast cancer.

Aims The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. Background Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. Methods qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. Results The wild type α+/β+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/β− hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/β+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α−/β+ hTERT and α−/β− hTERT (both p = 0.02). A positive association was observed between BRCA1 and α−/β+ hTERT and α−/β− hTERT expression (p = 0.003 and p = 0.04, respectively). Conclusions These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/β+).

The UK led PersonaLising Anal cancer radioTherapy dOse(PLATO) is a complex integrated protocol, consisting of 3 distinct anal cancer trials. Anal cancer IMRT contouring is challenging as there are several nodal areas that are electively irradiated and the primary tumour is not well visualised on CT. We identified FALCON, an ESTRO supported multifunctional online contouring platform (EduCase), aimed at reducing contouring variability, as a method to streamline pre-trial radiotherapy (RT) quality assurance (QA). Investigators contoured a benchmark case using FALCON prior to attending workshops aimed at evaluating contouring variation and refining the trial RT protocol. We present qualitative feedback received from workshop participants. We also present conformity analysis of contours compared with a reference contour.

The CUB and sushi multiple domains 1 (CSMD1) gene maps to chromosome 8p23, a region deleted in many cancers. Loss of CSMD1 expression is associated with poor prognosis in breast cancer suggesting that it acts as a tumour suppressor in this cancer. However, the function of CSMD1 is largely unknown. Herein, we investigated CSMD1 functions in cell line models. CSMD1 expression was suppressed in MCF10A and LNCaP cells using short hairpin RNA. Functional assays were performed focusing on the 'normal' MCF10A cell line. Suppression of CSMD1 significantly increased the proliferation, cell migration and invasiveness of MCF10A cells compared to shcontrols. shCSMD1 cells also showed significantly reduced adhesion to Matrigel and fibronectin. In a three-dimensional Matrigel model of MCF10A cells, reduced CSMD1 expression resulted in the development of larger and more poorly differentiated breast acini-like structures that displayed impaired lumen formation. Loss of CSMD1 expression disrupts a model of mammary duct formation while enhancing proliferation, migration and invasion. Our data suggest that CSMD1 is involved in the suppression of a transformed phenotype.

Purpose/Objective(s) Randomized controlled trials for patients with squamous cell carcinoma of the anus have been successfully performed, despite the rarity of the disease. The RTOG 9811 and ACT2 trials demonstrated no improvement in cancer outcomes when neoadjuvant or adjuvant cisplatin 5FU chemotherapy was used. To capture as many anal cancer patients as possible in future clinical trials, we developed an umbrella of trials testing efficacy and acceptable morbidity across different loco-regional risk strata. Materials/Methods We formed a network of UK and international multi-disciplinary trialists and identified the following priorities:- i] the need for national consensus guidance for the introduction of intensity modulated radiation therapy (IMRT) based on the successful principles and fractionation used in the ACT2 protocol prior to future trials; ii] to evaluate a strategy using selective chemoradiation therapy (CRT) after local excision of anal margin tumors; iii] to evaluate reduced dose CRT to the primary tumor in order to limit late effects whilst maintaining elective nodal irradiation in early stage disease; iv] to evaluate dose escalated CRT in locally advanced T3/4 and N+ stage disease. Results The PLATO (personalizing radiotherapy dose in anal cancer) umbrella trial comprising of the ACT3, 4 and 5 trials is funded by Cancer Research UK and is due to commence recruitment in Q3 2016.The ACT 3 trial (n = 90) is a non-randomized phase II study that will evaluate a strategy of local excision for T1N0 anal margin tumors with selective postoperative involved field CRT using 41.4 Gy in 23 fractions (F) and concurrent capecitabine reserved for patients with margins

Objective Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. Design Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. Results 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets ( FGFR2 , ERBB2 ) and also novel genes in gastric cancer ( KLF5 , GATA6 ). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2 -amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2 -amplified gastric cancers. Conclusion The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.

The authors have investigated the expression of the microcephalin (MCPH1) protein to evaluate its prognostic importance in breast cancer. Microcephalin is a damage response protein involved in the regulation of BRCA1 and BRCA2. BRCA1 mutations are often associated with basal-like breast cancer, which are also often negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2. MCPH1 immunohistochemistry was performed on 319 breast cancers prepared as tissue microarray and correlated with pathology, survival, ER, PR, HER2, EGFR, CK5/6, CK14 and BRCA1 expression. After performing continuous data analysis, mean microcephalin expression decreased with increasing grade (P < 0.006). Mean microcephalin expression was lower in ER/PR negative (P < 0.001) and triple negative cancers (P < 0.004). Conversely, an association with HER2-positive cancers was also identified (P < 0.034). Reduced microcephalin also correlated with reduced nuclear BRCA1 staining (P < 0.001). No association was identified with basal markers. After dichotomising the data into low and high microcephalin expression, reduced expression was identified in 29% (93/319) of breast cancers. An association with low expression was identified in invasive ductal carcinomas with breast cancer-specific survival (BCSS) (P = 0.052). Multivariate analysis of ductal carcinomas showed that microcephalin, together with lymph node involvement and tumour size were independent predictors of BCSS (P = 0.037). Microcephalin expression is reduced in 29% of breast cancers, particularly in higher grade tumours and BRCA1-negative cases. Microcephalin is an independent predictor of BCSS in invasive ductal breast cancer patients and may prove to be a useful biomarker for the identification of aggressive breast cancers.

The chromosomal band 1p36 exhibits frequent loss of heterozygosity in a variety of human malignancies, suggesting the presence of an as yet unidentified tumor suppressor gene. The faint terminal subbands often make cytogenetic analysis of 1p36 particularly difficult. Small deletions at this locus may therefore escape detection on analysis by conventional cytogenetics, a hypothesis that we have explored using fluorescence in situ hybridization (FISH) in malignant lymphoma. The study cohort consisted of 20 cases of lymphoma of various subtypes without any 1p abnormality on G-banded karyotyping. FISH was performed using a human chromosome 1 paint and a bacterial artificial chromosome probe RP4-755G5 localizing to 1p36.33, the most telomeric subband of 1p36. Tumors demonstrating 1p36.33 deletions were additionally analyzed by FISH using a second probe from the proximal 1p36.1 subband, to further define the breakpoint. Eight cases of follicular lymphoma (FL), 5 diffuse large B-cell lymphomas (DLBCL), 2 Hodgkin disease, 2 B-cell small lymphocytic lymphomas, 2 T-cell lymphomas, and 1 marginal zone lymphoma were analyzed. FISH identified deletions at 1p36.33 in 5 of the 20 cases: 3 DLBCL and 2 FL. FISH is considerably more sensitive for identifying lymphoma genetic alterations than conventional cytogenetics. Deletion of the distal part of the 1p36 may be a much more common aberration than previously recognized in lymphoma.

Statistics rate colorectal adenocarcinoma as the most common cause of cancer death on exclusion of smoking-related neoplasia. However, the reported accumulation of genetic lesions over the adenoma to adenocarcinoma sequence cannot wholly account for the neoplastic phenotype. Recently, heritable, epigenetic changes in DNA methylation, in association with a repressive chromatin structure, have been identified as critical determinants of tumour progression. Indeed, the transcriptional silencing of both established and novel tumour suppressor genes has been attributed to the aberrant cytosine methylation of promoter-region CpG islands. This review aims to set these epigenetic changes within the context of the colorectal adenoma to adenocarcinoma sequence. The role of cytosine methylation in physiological and pathological gene silencing is discussed and the events behind aberrant cytosine methylation in ageing and cancer are appraised. Emphasis is placed on the interrelationships between epigenetic and genetic lesions and the manner in which they cooperate to define a CpG island methylator phenotype at an early stage in tumourigenesis. Finally, the applications of epigenetics to molecular pathology and patient diagnosis and treatment are reviewed.

ERβ1 is often down-regulated in breast cancer compared to normal breast but mechanisms surrounding this are unclear. We examined whether loss of heterozygosity (LOH) or methylation at ERβ promoters (0N, 0K) and/or untranslated exon 0N were involved in ERβ down-regulation in breast cancer tissues and cell lines and if treatment with the de-methylating agent 5-aza-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A could influence expression in vitro. We found no evidence of correlation between LOH at 14q22-24 (genomic locus containing ERβ/ESR2), and ERβ1 expression in primary breast cancers. A negative correlation between ERβ1 mRNA expression and methylation status was observed for promoter 0N in BT-20, MDA-MB-453 and T47D cells. Promoter 0K was consistently unmethylated. In primary breast tumours, methylation of the untranslated exon 0N, downstream of promoter 0N, but not of promoter 0N itself, correlated with down-regulation of ERβ. In MDA-MB-453 cells, treatment with 5-aza-deoxycytidine was sufficient to induce ERβ1 expression from the 0N promoter while in BT-20 both agents were required. Examination of various sites on ESR2 highlighted epigenetic but not genetic regulation of ERβ1. In particular methylation adjacent to promoter 0N was a key regulatory event for ERβ1 silencing. A combination of de-methylating agents and histone deacetylase inhibitors fully restored ERβ1 expression which may offer a novel therapeutic angle for breast cancer management.

An allelotype analysis of transitional cell carcinoma of the bladder identified loss of heterozygosity (LOH) on chromosome arm 4p in 22% of tumours. In a more detailed LOH study of 178 bladder carcinomas, a 750 kb common region of deletion was identified between the markers D4S43 and D4S127 just telomeric to the Huntington disease locus. To refine this region of deletion at 4p16.3, we have carried out detailed fluorescence in situ hybridisation (FISH) analysis of 12 bladder cancer cell lines by using a chromosome 4 centromeric probe combined with a series of cosmid probes from contigs spanning the 750 kb region of deletion. A common 30 kb region of deletion was identified at 4p16.3 in over one-third of the bladder cancer cell lines analysed. The present study has refined the localisation of the critical region of deletion from 750 kb to approximately 30 kb, providing a precise starting point for positional cloning of the gene(s) involved in bladder cancer from within a very gene-rich region on chromosome band 4p16.3. This study demonstrates that FISH can be used for fine deletion mapping of potential tumour suppressor gene regions. The utilisation of FISH analysis to map chromosomal deletions should facilitate positional cloning of other genes as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) contigs of the human genome are established.

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon. NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis. Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008). NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.

Background: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers. Methods: Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data. Results: A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival. Conclusion: Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

Background: Mutations in Ki- ras codon 12 and the p53 gene are common abnormalities in colorectal cancer. The occurrence of p53 overexpression and/or Ki- ras codon 12 mutations were analyzed in 100 colorectal adenomas to determine if they were related to patient survival. Methods: p53 overexpression was identified by immunohistochemistry, and Ki- ras codon 12 mutations were detected using the polymerase chain reaction and a restriction enzyme digestion method. Results: p53 overexpression was identified in 45% of tumors, with a higher frequency identified in DNA aneuploid and left-sided tumors than in DNA diploid and right-sided tumors. Mutations in Ki- ras codon 12 were identified in 24% of carcinomas. Individually, mutations in Ki- ras codon 12 or p53 overexpression were not prognostic indicators of survival. However, a statistically significant difference in survival was identified when these two oncogenic abnormalities were analyzed together. The median survival of patients whose tumors contained both oncogenic abnormalities was less than half of that of patients with either alteration alone or without either abnormality. Conclusions: Screening for multiple genetic abnormalities in colorectal cancers excised at surgery may prove to be a useful tool in determining prognosis.

CUB and SUSHI multiple domain protein 1 (CSMD1) is a candidate tumour suppressor gene that maps to chromosome 8p23, a region deleted in many tumour types including 50% of breast cancers. CSMD1 has homologies to proteins implicated in carcinogenesis. We aimed to study the expression pattern of the CSMD1 protein and evaluate its prognostic importance in invasive ductal carcinoma (IDC). An anti-CSMD1 antibody was developed and validated. The expression pattern of CSMD1 in normal breast and IDC samples was investigated by immunohistochemistry in 275 patients. Univariate and multivariate Cox regression analyses were performed. In normal breast duct epithelial cells, luminal, membranous and cytoplasmic CSMD1 staining was identified. Reduced expression of CSMD1 was detected in 79/275 (28.7%) of IDC cases. Low CSMD1 expression was significantly associated with high tumour grade (P = 0.003). CSMD1 expression was associated with overall survival (OS; HR = 0.607, 95%CI: 0.4-0.91, P = 0.018) but not with disease-free survival (DFS; HR = 0.81, 95%CI: 0.46-1.43, P = 0.48). Multivariate analysis showed that CSMD1, together with Nottingham Prognostic Index, was considered an independent predictor of OS (HR = 0.607, 95%CI: 0.4-0.91, P = 0.018) but not DFS (HR = 0.84, 95%CI: 0.46-1.5, P = 0.573). Reduction of CSMD1 expression was significantly associated with high tumour grade and decreased OS. Therefore, our results support the idea that CSMD1 is a tumour suppressor gene and suggest its possible use as a new prognostic biomarker. The membrane expression pattern of CSMD1 suggests that it may be a receptor or co-receptor involved in the process of signal transduction. © Springer Science+Business Media, LLC. 2009.

CUB and Sushi multiple domain protein 1 (CSMD1) is a candidate tumour suppressor gene of unknown function. CSMD1 maps to chromosome 8p23, a region deleted in 50% of breast cancers (BC). We have examined the contribution of CSMD1 to the tumorigenic phenotype of mammary acini and evaluated its prognostic value in BC patients.

Interest has focused on a recently identified gene, BCL10, thought to play an important role in the genesis of extranodal, marginal zone (MALT) lymphomas. This gene belongs to a family containing caspase recruitment domains (CARD), that are involved in the apoptotic pathway. Translocations of the BCL10 gene to the immunoglobulin heavy chain locus at 14q32 have been described. We report herein a case of MALT lymphoma showing t(1; 2)(p22; p12). The translocation was shown to involve the BCL10 gene and the immunoglobulin kappa light chain locus by fluorescence in situ hybridization.

One hundred and nine samples comprising carcinomas, adenomas, dysplastic, inflamed and normal mucosa from patients with sporadic colon cancer and ulcerative colitis (UC) were analysed for c-Ki-ras mutations. DNA was extracted from archival paraffin-embedded material, amplified using the polymerase chain reaction (PCR) and the PCR products analysed using restriction enzyme digestion. Forty-two per cent (14/33) of the sporadic carcinoma controls contained Ki-ras codon 12 mutations in contrast to 24% (8/33) of ulcerative colitis carcinomas. A significantly higher c-Ki-ras mutation rate was observed in rectal carcinomas (72%) in comparison to colonic carcinomas (28%) in control patients (P less than 0.04), while the opposite was observed in UC patients. The difference between the incidence of c-Ki-ras mutations in rectal carcinomas in UC (9%) and in sporadic rectal carcinomas (72%) was also significant (P less than 0.01). This lower prevalence rate and different site distribution of c-Ki-ras mutations in UC carcinomas compared to sporadic carcinomas suggests that specific genetic differences may underlie the causation of carcinomas arising in these situations. Images Figure 2 Figure 3 Figure 4 Figure 5

The coumermycin antibiotic novobiocin is currently under investigation as an agent that can modify the toxicity of various anti-cancer drugs, potentially via one of its many pharmacological effects: namely, the interference with type II DNA topoisomerase function. This paper investigates the ability of novobiocin to modify the cellular/nuclear accumulation and toxicity of two types of DNA binding agents (the minor groove ligand Hoechst 33342 and the intercalating anthracycline Adriamycin). We report that novobiocin reduces the cytotoxicity of both agents and that this can be attributed to a reduction in cellular and, consequently, nuclear accumulation of these agents rather than to any effect on cellular export. The antibiotic was also active (at non-toxic concentrations) in delaying the progression of cells into S phase and G2 phase. This potential for novobiocin to effect rescue from toxicity by disturbance of the delivery of a drug to a potentially important intracellular target, together with the provision of an extended period of cellular recovery prior to the commitment of cells to G2 + M phase, should be recognised in the design of combination chemotherapy.

A mutant murine cell line has previously been reported to be resistant to the AT-specific DNA minor groove ligand 2',5'-bi-1H-benzimidazole, 2',(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl), trichloride (Ho33342), due to an enhanced capacity to remove ligand molecules from cellular DNA via a pathway which can be blocked by DNA topoisomerase poisons. We have studied the relationship between ligand resistance and DNA topoisomerase II activity. The cross-sensitivity patterns of the mutant were examined for covalently (anthramycin) and non-covalently (distamycin A) binding minor groove ligands, and DNA intercalating [adriamycin, mitoxantrone and 4'-(9-acridinylamino)methanesulphon-m-anisidide (mAMSA)] and non-intercalating (VP16-213) topoisomerase II poisons. The mutant was cross-resistant to distamycin A alone. The mutant showed no abnormality in: (i) the in vitro decatenation activity of topoisomerase II, (ii) VP16-213 or mAMSA induced protein-DNA cross-linking activities in nuclear extracts, (iii) 'cleavable complex' generation (or DNA strand scisson) in intact cells exposed to topoisomerase poisons. Ho33342 and the topoisomerase II inhibitor novobiocin were found to disrupt both the in vitro binding of nuclear extracted proteins, from mutant and parental cells, to plasmid DNA and the formation of drug-induced cleavable complexes in vitro. Unexpectedly, Ho33342 induced significant levels of DNA-protein crosslinking in both parental and mutant cells. We conclude that: (i) resistance of the mutant is limited to non-covalently binding minor groove ligands, (ii) Ho33342 can block the trapping of DNA topoisomerase II by enzyme poisons in vitro, (iii) Ho33342 can induce a novel form of DNA-protein cross-link in intact cells, and (iv) the resistance of the mutant is not dependent upon some abnormality in topoisomerase II function.

Epigenetic mechanisms such as DNA methylation are now recognized to play an important role in neoplasia. The aim of this study is to relate the pattern of expression of multiple cancer genes known to undergo epigenetic inactivation by promoter hypermethylation in breast cancer with histologic and outcome data. We used immunohistochemistry to study expression of the tumor suppressor gene p16, estrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR), and the DNA repair gene MGMT (O6 -methylguanine-DNA methyltransferase) in a panel of 200 breast cancers. Methylation-specific polymerase chain reaction was used to confirm MGMT promoter methylation. Loss of expression of MGMT, ERalpha, ERbeta, PR, and p16 was observed in 19%, 24%, 13%, 40%, and 50% of cases, respectively. A significant correlation was seen between grade III tumor and loss of expression of ERalpha, ERbeta, PR (all P.0001), and MGMT (P = .04), whereas loss of expression of p16 was associated with grades I and II tumors (P.001). Cases that expressed 3 or less of the 5 proteins studied had significantly reduced survival (P = .0016). Methylation-specific polymerase chain reaction in a subset of 20 cancers showed DNA methylation associated with the loss of MGMT expression (P.001). In conclusion, there is silencing of several key genes in breast cancer affecting molecular pathways involved in cell immortalization, DNA repair, and hormonal regulation, and this correlates significantly with risk of cancer-specific death. This expression profile could be linked to epigenetic events, and if so, these pathways have potential as targets for therapeutic strategies based on reversal of epigenetic silencing.

To gain insights into the possible role of oestrogen receptor (ER) beta in breast carcinogenesis, immunohistochemical analysis of ER beta was performed on 512 breast specimens encompassing normal (n = 138), pure ductal carcinoma in situ (n = 16), invasive cancers (n = 319), lymph node metastases (n = 31), and recurrences (n = 8). Real-time polymerase chain reaction (PCR) was used to investigate the methylation status of the ER beta gene in the ER beta negative breast cancer cell lines SkBr3 and MDA-MB-435. A gradual reduction in, but not a complete loss of, ER beta expression was observed during the transition from normal and pre-invasive lesions to invasive cancers, where ER beta was lost in 21% of cases. This was more pronounced in invasive ductal than in lobular carcinomas, a significantly higher proportion of which were ER beta-positive (74% compared with 91%, respectively, p = 0.0004). Examination of paired primary cancers with their axillary lymph node metastases showed that if ER beta was present in the primary tumour, it persisted in the metastasis. Treatment of ER beta-negative cell lines with DNA methyl transferase inhibitors restored ER beta expression, providing experimental evidence that silencing of ER beta in breast carcinomas could be due to promoter hypermethylation. These results suggest that loss of ER beta expression is one of the hallmarks of breast carcinogenesis and that it may be a reversible process involving methylation.

We and others previously identified a region of hemizygous or homozygous deletion at chromosome band 8p23 in oral and oropharyngeal squamous cell carcinomas (OSCCs) and many other cancer types, suggesting the presence of a tumor-suppressor gene (TSG) in this region. Recently, based on a single region of homozygous deletion in head and neck squamous cell carcinomas (HNSCC), a putative TSG, CUB and sushi multiple domains-1 (CSMD1), has been identified. In the present study, we mapped three OSCC cell lines with previously described homozygous deletions at a high resolution onto a detailed physical map. Critically, this map covered a wider region than that used in previous studies, and in contrast to these studies, our results revealed multiple regions of homozygous deletion within a small interval on 8p23. To investigate this deletion pattern further, we generated a panel of 34 sequence tagged site (STS) markers spanning the region and tested these three cell lines and an additional 34 OSCC cell lines, identifying homozygous deletions in a further four. Combining the results from all seven deleted cell lines identified three non-overlapping regions of homozygous deletion. This complex pattern could be consistent with the presence of multiple TSGs or one very large TSG in this region, and/or specific chromosomal instability. CSMD1 spans two of the three deleted regions and, therefore, would appear to be an excellent candidate for a TSG. However, deletion mapping with STSs corresponding to the exons of CSMD1 shows that some of the deletions do not interrupt its coding region, and in other cell lines the coding region is interrupted by two discontinuous homozygous deletions, suggesting the presence of redundant deletions. These results call into question whether the CSMD1 gene is the 8p23 TSG or whether this or any other genes at this locus are involved in the development of OSCC.

In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).

The frequency of p53 overexpression and K-ras codon 12 mutation was investigated in a series of colorectal adenomas. p53 was detected by immunohistochemistry in only 5% of tumours, whereas K-ras mutation was found in eight of 30 adenomas examined. In vitro, mutant p53 and ras genes cooperate to transform primary rat cells into a tumourigenic cell line. The presence of both p53 overexpression and K-ras mutation in a benign tubulovillous polyp in the present series suggests that in vivo this combination of events is insufficient to cause malignant transformation of a large bowel adenoma.

CUB and Sushi multiple domains protein 1 (CSMD1) maps to 8p23, a region deleted in many cancers, and thought to be a tumour suppressor gene. Loss of CSMD1 expression is associated with reduced survival in breast cancer patients. CSMD1’s function is unknown; however, CSMD1‘s structure suggests it is involved in signal transduction. Here, we have investigated the function of CSMD1. CSMD1 expression was silenced in MCF10A, MDA-MB435 and LNCaP cell lines by shRNA and functional assays were performed. Loss of CSMD1 expression disrupted cell morphology and caused 30% (p < 0.001), 32% (p = 0.03) and 56% (p < 0.001) increase in cell proliferation of MDA-MB-435, LNCaP and MCF10A, respectively, compared to controls. Also MDA-MB-435 and MCF10A shCSMD1 cells showed reduced adhesion to matrigel (32%, p = 0.0005 and 44%, p = 0.0006, respectively), and to fibronectin (39%, p = 0.004 and 32%, p < 0.001, respectively). Moreover, loss of CSMD1 expression enhanced cell migration of MDA-MB-435 and MCF10A and caused 33% (p < 0.001) increase in cell invasion of MCF10A, compared to control. The MCF10A 3D model revealed that loss of CSMD1 expression resulted in the development of larger poorly differentiated breast acini and impaired lumen formation. Loss of CSMD1 expression induced behaviour consistent with cellular transformation. Our data supports the concept that CSMD1 participates in signaling pathways that regulate a range of key cellular processes involved in the suppression of a transformed phenotype.

Previously we identified MCPH1, a DNA damage response protein involved in the regulation of BRCA1 and BRCA2, as the defective protein in one form of microcephaly. BRCA1 mutations are associated with basal-like breast cancer, which are often also negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2. Our data indicate that MCPH1 plays a role in response to chemotherapeutic agents used in the treatment of breast cancer due to its role in DNA repair and the spindle checkpoint.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com