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Abstract Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action of therapeutic monoclonal antibodies (mAbs) such as cetuximab, rituximab and trastuzumab. Fc gamma receptors (FcgR) on human white blood cells are an integral part of the ADCC pathway. Differential response to therapeutic mAbs has been reported to correlate with specific polymorphisms in two of these genes: FCGR2A (H131R) and FCGR3A (V158F). These polymorphisms are associated with differential affinity of the receptors for mAbs. This review critically examines the current evidence for genotyping the corresponding single nucleotide polymorphisms (SNPs) to predict response to mAbs in patients with cancer.

Abstract Background Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. Methods Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput. Results 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays. Conclusions This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.

Abstract Background The aim of this study was to identify critical genes involved in non-small cell lung cancer (NSCLC) pathogenesis that may lead to a more complete understanding of this disease and identify novel molecular targets for use in the development of more effective therapies. Methods Both transcriptional and genomic profiling were performed on 69 resected NSCLC specimens and results correlated with mutational analyses and clinical data to identify genetic alterations associated with groups of interest. Results Combined analyses identified specific patterns of genetic alteration associated with adenocarcinoma vs. squamous differentiation; KRAS mutation; TP53 mutation, metastatic potential and disease recurrence and survival. Amplification of 3q was associated with mutations in TP53 in adenocarcinoma. A prognostic signature for disease recurrence, reflecting KRAS pathway activation, was validated in an independent test set. Conclusions These results may provide the first steps in identifying new predictive biomarkers and targets for novel therapies, thus improving outcomes for patients with this deadly disease.

ARIEL2 (NCT01891344) is a single-arm, open-label phase 2 study of the PARP inhibitor (PARPi) rucaparib in relapsed high-grade ovarian carcinoma. In this post hoc exploratory biomarker analysis of pre- and post-platinum ARIEL2 samples, RAD51C and RAD51D mutations and high-level BRCA1 promoter methylation predict response to rucaparib, similar to BRCA1/BRCA2 mutations. BRCA1 methylation loss may be a major cross-resistance mechanism to platinum and PARPi. Genomic scars associated with homologous recombination deficiency are irreversible, persisting even as platinum resistance develops, and therefore are predictive of rucaparib response only in platinum-sensitive disease. The RAS, AKT, and cell cycle pathways may be additional modulators of PARPi sensitivity.

Abstract Background RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. Results We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR) assays. This single-tube quality control (QC) assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. Conclusions The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

Abstract Background Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations. We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Results Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions. LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. Conclusion LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

Abstract Background Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes that are also known to act as tumour suppressor genes. Increased succinate or fumarate levels as a consequence of SDH and FH deficiency inhibit hypoxia inducible factor-1α (HIF-1α) prolyl hydroxylases leading to sustained HIF-1α expression in tumours. Since HIF-1α is frequently expressed in breast carcinomas, DNA methylation at the promoter regions of the SDHA, SDHB, SDHC and SDHD and FH genes was evaluated as a possible mechanism in silencing of SDH and FH expression in breast carcinomas. Findings No DNA methylation was identified in the promoter regions of the SDHA, SDHB, SDHC, SDHD and FH genes in 72 breast carcinomas and 10 breast cancer cell lines using methylation-sensitive high resolution melting which detects both homogeneous and heterogeneous methylation. Conclusion These results show that inactivation via DNA methylation of the promoter CpG islands of SDH and FH is unlikely to play a major role in sporadic breast carcinomas.

Abstract Background The development of reverse transcription – quantitative real-time PCR (RT-qPCR) platforms that can simultaneously measure the expression of multiple genes is dependent on robust assays that function under identical thermal cycling conditions. The use of a primer optimisation matrix to improve the performance of RT-qPCR assays is often recommended in technical bulletins and manuals. Despite this recommendation, a comprehensive introduction to and evaluation of this approach has been absent from the literature. Therefore, we investigated the impact of varying the primer concentration, leaving all the other reaction conditions unchanged, on a large number of RT-qPCR assays which in this case were designed to be monitored using hydrolysis probes from the Universal Probe Library (UPL) library. Findings Optimal RT-qPCR conditions were determined for 60 newly designed assays. The calculated Cq (Quantification Cycle) difference, non-specific amplification, and primer dimer formation for a given assay was often dependent on primer concentration. The chosen conditions were further optimised by testing two different probe concentrations. Varying the primer concentrations had a greater effect on the performance of a RT-qPCR assay than varying the probe concentrations. Conclusion Primer optimisation is important for improving the performance of RT-qPCR assays monitored by UPL probes. This approach would also be beneficial to the performance of other RT-qPCR assays such as those using other types of probes or fluorescent intercalating dyes.

Abstract Introduction The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells. Results Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specific markers (ELF3, EPHB4, EGFR, MGB1 and TACSTD1) was used to screen for circulating tumour cells in the peripheral blood of 56 breast cancer patients. Twenty patients were positive for two or more RT-PCR markers, including seven patients who were node negative by conventional techniques. Significant increases in the frequency of marker positivity was seen in lymph node positive patients, in patients with high grade tumours and in patients with lymphovascular invasion. A strong trend towards improved disease free survival was seen for marker negative patients although it did not reach significance (p = 0.08). Conclusion Multi-marker immunobead RT-PCR analysis of peripheral blood is a robust assay that is capable of detecting circulating tumour cells in early stage breast cancer patients.

Abstract Background Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of CDKN2B can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable. Results MS-HRM was used to assess CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the CDKN2B promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM. Conclusion dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.

Abstract Primer design for PCR-based methylation analysis following bisulfite conversion of DNA is considerably more complex than primer design for regular PCR. The choice of the optimal primer set is critical to the performance and correct interpretation of the results. Most methodologies in methylation analysis utilize primers that theoretically amplify methylated and unmethylated templates at the same time. The proportional amplification of all templates is critical but difficult to achieve due to PCR bias favouring the amplification of the unmethylated template. The focus of this brief communication is to point out the important criteria needed for the successful choice of primers that will enable the control of PCR bias in bisulfite based methylation-screening protocols.

Abstract Background Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45–60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples. Results We developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29) were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results. Conclusion HRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases.

Abstract Background Epithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case. Methods We developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues. Results All 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive. Conclusion This is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings.

Abstract Background A recurrent somatic mutation, E17K, in the pleckstrin homology domain of the AKT1 gene, has been recently described in breast, colorectal, and ovarian cancers. AKT1 is a pivotal mediator of signalling pathways involved in cell survival, proliferation and growth. The E17K mutation stimulates downstream signalling and exhibits transforming activity in vitro and in vivo. Findings We developed a sensitive high resolution melting (HRM) assay to detect the E17K mutation from formalin-fixed paraffin-embedded tumours. We screened 219 non-small cell lung cancer biopsies for the mutation using HRM analysis. Four samples were identified as HRM positive. Subsequent sequencing of those samples confirmed the E17K mutation in one of the cases. A rare single nucleotide polymorphism was detected in each of the remaining three samples. The E17K was found in one of the 14 squamous cell carcinomas. No mutations were found in 141 adenocarcinomas and 39 large cell carcinomas. Conclusion The AKT1 E17K mutation is very rare in lung cancer and might be associated with tumorigenesis in squamous cell carcinoma. HRM represents a rapid cost-effective and robust screening of low frequency mutations such as AKT1 mutations in clinical samples.

Abstract Background Germline inactivating mutations in BRCA1 and BRCA2 underlie a major proportion of the inherited predisposition to breast and ovarian cancer. These mutations are usually detected by DNA sequencing. Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population. High resolution melting (HRM) analysis is a rapid screening methodology with very low false negative rates. We therefore evaluated the use of HRM as a mutation scanning tool using, as a proof of principle, the three recurrent BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population in addition to other mutations that occur in the same regions. Methods We designed PCR amplicons for HRM scanning of BRCA1 exons 2 and 20 (carrying the founder mutations185delAG and 5382insC respectively) and the part of the BRCA2 exon 11 carrying the 6174delT founder mutation. The analysis was performed on an HRM-enabled real time PCR machine. Results We tested DNA from the peripheral blood of 29 individuals heterozygous for known mutations. All the Ashkenazi founder mutations were readily identified. Other mutations in each region that were also readily detected included the recently identified Greek founder mutation 5331G>A in exon 20 of BRCA1. Each mutation had a reproducible melting profile. Conclusion HRM is a simple and rapid scanning method for known and unknown BRCA1 and BRCA2 germline mutations that can dramatically reduce the amount of sequencing required and reduce the turnaround time for mutation screening and testing. In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results. This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).

Abstract Background High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL). Results We adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood (PB). PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation (DGC), while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC (RS+DGC). The purity of CLL cells after DGC averaged 74.1% (range: 15.9 – 97.4%). Using RS+DGC, the purity averaged 93.8% (range: 80.4 – 99.4%) with 23 of 29 (79%) samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis. Conclusion This study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.

Abstract Background p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. Methods We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. Results One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. Conclusion HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.

Abstract Background The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis (HRM) to screen for KRAS mutations in clinical cancer samples. In non-small cell lung cancer, KRAS mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important. Methods We developed a high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for KRAS mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in EGFR exons 18–21. Results Known KRAS mutations in cell lines (A549, HCT116 and RPMI8226) were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5–6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had KRAS mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in KRAS and EGFR were mutually exclusive. Conclusion HRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.

Abstract Background Assessment of array quality is an essential step in the analysis of data from microarray experiments. Once detected, less reliable arrays are typically excluded or "filtered" from further analysis to avoid misleading results. Results In this article, a graduated approach to array quality is considered based on empirical reproducibility of the gene expression measures from replicate arrays. Weights are assigned to each microarray by fitting a heteroscedastic linear model with shared array variance terms. A novel gene-by-gene update algorithm is used to efficiently estimate the array variances. The inverse variances are used as weights in the linear model analysis to identify differentially expressed genes. The method successfully assigns lower weights to less reproducible arrays from different experiments. Down-weighting the observations from suspect arrays increases the power to detect differential expression. In smaller experiments, this approach outperforms the usual method of filtering the data. The method is available in the limma software package which is implemented in the R software environment. Conclusion This method complements existing normalisation and spot quality procedures, and allows poorer quality arrays, which would otherwise be discarded, to be included in an analysis. It is applicable to microarray data from experiments with some level of replication.

Abstract The FANCA gene is one of the genes in which mutations lead to Fanconi anaemia, a rare autosomal recessive disorder characterised by congenital abnormalities, bone marrow failure, and predisposition to malignancy. FANCA is also a potential breast and ovarian cancer susceptibility gene. A novel allele was identified which has a tandem duplication of a 13 base pair sequence in the promoter region. Methods We screened germline DNA from 352 breast cancer patients, 390 ovarian cancer patients and 256 normal controls to determine if the presence of either of these two alleles was associated with an increased risk of breast or ovarian cancer. Results The duplication allele had a frequency of 0.34 in the normal controls. There was a non-significant decrease in the frequency of the duplication allele in breast cancer patients. The frequency of the duplication allele was significantly decreased in ovarian cancer patients. However, when malignant and benign tumours were considered separately, the decrease was only significant in benign tumours. Conclusion The allele with the tandem duplication does not appear to modify breast cancer risk but may act as a low penetrance protective allele for ovarian cancer.

Abstract Background Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells. Methods In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested. Results MUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR. Conclusions ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment.

Abstract Background The NUP98 gene is involved in multiple rearrangements in haematological malignancy. The leukemic cells in an acute myeloid leukemia (AML) patient with a t(9;11)(p22;p15) were recently shown to have a fusion between the NUP98 gene and the LEDGF gene but it was not demonstrated that this fusion was recurrent in other leukaemia patients with the same translocation. Results We used RT-PCR to analyse the leukemic cells from an AML patient who presented with a cytogenetically identical translocation as the sole chromosomal abnormality. A NUP98-LEDGF fusion transcript was observed and confirmed by sequencing. The reciprocal transcript was also observed. The fusion transcript was not detectable during remission and recurred at relapse. The breakpoints in the NUP98 and LEDGF genes were different to those previously reported. The NUP98 breakpoint occurs in the intron between exons 8 and 9. It is the most 5' breakpoint reported in a translocation involving the NUP98 gene. All of the LEDGF gene is included in the fusion except for exon 1 which codes for the first 24 amino terminal amino acids. Conclusions Our results show that fusion of the NUP98 and LEDGF genes is a new recurrent translocation in AML.

Background: Despite initial response to platinum-based chemotherapy and PARP inhibitor therapy (PARPi), nearly all recurrent high-grade serous ovarian cancer (HGSC) will acquire lethal drug resistance; indeed, ~15% of individuals have de novo platinum-refractory disease. Objectives: To determine the potential of anti-microtubule agent (AMA) therapy (paclitaxel, vinorelbine and eribulin) in platinum-resistant or refractory (PRR) HGSC by assessing response in patient-derived xenograft (PDX) models of HGSC. Design and methods: Of 13 PRR HGSC PDX, six were primary PRR, derived from chemotherapy-naïve samples (one was BRCA2 mutant) and seven were from samples obtained following chemotherapy treatment in the clinic (five were mutant for either BRCA1 or BRCA2 (BRCA1/2), four with prior PARPi exposure), recapitulating the population of individuals with aggressive treatment-resistant HGSC in the clinic. Molecular analyses and in vivo treatment studies were undertaken. Results: Seven out of thirteen PRR PDX (54%) were sensitive to treatment with the AMA, eribulin (time to progressive disease (PD) ⩾100 days from the start of treatment) and 11 out of 13 PDX (85%) derived significant benefit from eribulin [time to harvest (TTH) for each PDX with p < 0.002]. In 5 out of 10 platinum-refractory HGSC PDX (50%) and one out of three platinum-resistant PDX (33%), eribulin was more efficacious than was cisplatin, with longer time to PD and significantly extended TTH (each PDX p < 0.02). Furthermore, four of these models were extremely sensitive to all three AMA tested, maintaining response until the end of the experiment (120d post-treatment start). Despite harbouring secondary BRCA2 mutations, two BRCA2 -mutant PDX models derived from heavily pre-treated individuals were sensitive to AMA. PRR HGSC PDX models showing greater sensitivity to AMA had high proliferative indices and oncogene expression. Two PDX models, both with prior chemotherapy and/or PARPi exposure, were refractory to all AMA, one of which harboured the SLC25A40-ABCB1 fusion, known to upregulate drug efflux via MDR1. Conclusion: The efficacy observed for eribulin in PRR HGSC PDX was similar to that observed for paclitaxel, which transformed ovarian cancer clinical practice. Eribulin is therefore worthy of further consideration in clinical trials, particularly in ovarian carcinoma with early failure of carboplatin/paclitaxel chemotherapy. [ABSTRACT FROM AUTHOR]