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Sarcomas are a key feature of Li-Fraumeni and related syndromes (LFS/LFL), associated with germline TP53 mutations. Current penetrance estimates for TP53 mutations are subject to significant ascertainment bias. The International Sarcoma Kindred Study is a clinic-based, prospective cohort of adult-onset sarcoma cases, without regard to family history. The entire cohort was screened for mutations in TP53 using high-resolution melting analysis and Sanger sequencing, and multiplex-ligation-dependent probe amplification and targeted massively parallel sequencing for copy number changes. Pathogenic TP53 mutations were detected in blood DNA of 20/559 sarcoma probands (3.6%); 17 were germline and 3 appeared to be somatically acquired. Of the germline carriers, one appeared to be mosaic, detectable in the tumor and blood, but not epithelial tissues. Germline mutation carriers were more likely to have multiple cancers (47% vs 15% for non-carriers, P = 3.0×10(-3)), and earlier cancer onset (33 vs 48 years, P = 1.19×10(-3)). The median survival of mutation carriers following first cancer diagnosis was not significantly different from non-carriers. Only 10/17 (59%) pedigrees met classical or Chompret criteria for LFS. In summary, germline TP53 mutations are not rare in adult patients with sarcoma, with implications for screening, surveillance, treatment and genetic counselling of carriers and family members.

PCR permits direct genotyping of individuals at the ABO locus. Several methods have been reported for genotyping ABO that rely on differentiating the A, B, and O alleles at specific base substitutions. However, the O allele as defined by serology comprises at least two alleles (O1 and O2) at the molecular level, and most current ABO genotyping methods only take into account the O1 allele. Determining the presence of the O2 allele is critical, as this not-infrequent allele would be mistyped as an A or a B allele by standard PCR typing methods. Furthermore, none of the methods to date distinguish between the A1 and A2 alleles, even though 10% of all white persons are blood group A2. We have developed a method for genotyping the ABO locus that takes the O2 and A2 alleles into account. Typing for A2 and O2 by diagnostic restriction enzyme digestion is a sensitive, nonradioactive assay that provides a convenient method useful for forensic and paternity testing and for clarifying anomalous serological results.

Recurrent and metastatic carcinoma of the colorectum remains a major problem, with survival at 5 years post curative resection still only about 50%. Moreover, up to 30% of patients who present with early stage disease also relapse and die within 5 years, suggesting the presence of micrometastatic disease at diagnosis. One route of metastatic spread is via the blood stream, hence the detection of tumor cells in blood is likely to provide an important predictive tool with respect to relapse of disease. We have developed a sensitive molecular technique to identify tumor cells in blood using mutations in codon 12 of the K-ras gene as a marker. Twenty-seven patients whose tumor carried a mutation in codon 12 of K-ras were studied for the presence of tumor cells in perioperative peripheral blood samples. Immunomagnetic beads, labeled with an epithelial-specific antibody, were used to harvest epithelial cells from blood. K-ras mutations were identified in this selected population using a polymerase chain reaction (PCR)-based analysis (immunobead-PCR). Circulating K-ras mutant cells were detected in 9 of 27 patients; seven of these nine patients have since died due to recurrent or metastatic disease. Mutant cells were not detected in 18 patients, and 16 of 18 have remained disease free (median follow-up: 16 months; range: 7–42 months). Kaplan-Meier analysis showed that detection of K-ras mutant cells in blood was associated with significantly reduced disease-free survival (p = 0.0001). This study indicates that detection of circulating tumor cells perioperatively by immunobead-PCR provides a sensitive prognostic marker for recurrent and metastatic colorectal cancer.

Metastasis is the major cause of cancer mortality. A strong link between metastatic behaviour and epithelial–mesenchymal plasticity (EMP) has been demonstrated in human breast cancer (BC). EMP can provide carcinoma cells with invasive ability to leave the primary tumour, enter into the circulation as circulating tumour cells (CTCs), arrive at a distant organ and ultimately form a metastasis. This project aims to investigate EMP in BC CTCs.

PURPOSE To evaluate the significance of molecular marker-positive cells in a cohort of non-Hodgkin's lymphoma (NHL) patients undergoing high-dose chemotherapy and autologous peripheral-blood stem-cell transplantation (PBSCT). PATIENTS AND METHODS Twenty-eight PBSC transplants have been performed in 24 patients with poor-prognosis NHL. Molecular analysis of the t(14;18) (q32;q21) translocation (bcl-2/immunoglobulin [Ig] heavy-chain joining locus [JH] fusion) or antigen receptor gene rearrangements was performed to determine the presence of lymphoma cells at presentation, in PBSC harvests, and before and after autologous PBSCT. Kaplan-Meier estimates of survival and Cox regression analyses were used to test the effect of bone marrow involvement, tumor-cell contamination of PBSCs, disease stage, and chemotherapy sensitivity at transplantation, and presence of marker-positive cells post-PBSCT on disease-free and overall survival. RESULTS Thirteen of 24 patients (54%) are alive following PBSCT at a median follow-up time of 654 days (range, 193 to 1,908). Nine patients are in complete remission (CR) at day 216 to 1,799 (median, 805) and four are alive following relapse (day 440, 573, 1,188, and 1,908). Eleven patients (46%) have died: three of transplant-related complications at day 0, 1, and 13, and eight of recurrent disease (day 132 to 1,330; median, 451). Longitudinal marker studies post-PBSCT showed that of 16 relapse events, 13 (81%) were positive for the lymphoma marker at or before clinically documented relapse. Marker studies became negative post-PBSCT in nine of nine patients who entered and remained in CR. Disease-free survival (DFS) was significantly shortened in patients in whom marker-positive cells were detected in serial samples posttransplantation (P = .006). Cox regression analysis showed that patients in this group had a 24 times higher risk of relapse (P = .03). CONCLUSION The results show that the reappearance or persistence of marker-positive cells after autologous PBSCT is strongly associated with relapse.

Low-grade serous ovarian cancer (LGSC) is a challenging disease to treat effectively. It often occurs in young women and it is well-recognized to be resistant to standard chemotherapy. The underlying molecular driver mutations are now beginning to be understood and they are distinctly different from the more common counterpart, high-grade serous ovarian cancer. LGSC are characterized by somatic mutations in RAS/RAF genes and a number of new agents have been developed that target these mutations, and related activated pathways. However, it is not yet known which pathway-targeted drugs, or combination of drugs, will be effective in the treatment of LGSC. BRAFV600E mutations have been reported in LGSC and BRAF inhibitors have demonstrated significant improvement in progression-free survival in patients with BRAF-mutant melanoma. However, limited response is seen in other cancer types, such as colorectal cancers harboring the same mutation, suggesting that clinical benefit is tumor-type specific. In this study we aimed to characterize BRAF mutations in LGSC and to determine whether BRAF inhibitors could demonstrate a clinical benefit in ovarian cancer patients. Patients with LGSC were identified through the Australian Ovarian Cancer Study and the Westmead GynBiobank, Sydney, Australia. Tumor mutations were assessed using targeted and exome sequencing, and gene copy number was measured by whole genome SNP arrays. Tumor expression of BRAFV600E protein was also assessed by immunohistochemistry. Dabrafenib monotherapy was trialed in a patient with a somatic BRAFV600E mutation and progress was monitored clinically, biochemically using CA125 tumor marker levels and radiologically with PET imaging. Amongst Grade 1 serous ovarian carcinoma cases, 5/40 (12.5%) were shown to have a BRAFV600E mutation. Tumors with a BRAFV600E mutation had a relatively low degree of gene copy number change and were TP53 wild-type. The BRAF-inhibitor, dabrafenib was trialed in a heavily pre-treated BRAFV600E mutation-positive LGSC patient with progressive chemotherapy-resistant disease (n=1). Whole exome sequencing confirmed the BRAFV600E mutation was the highest frequency variant allele present and also identified deleterious mutations in other cancer-associated genes including CSMD1, BMP1 and DNM1 at lower frequencies, suggestive of sub-clonal events. The patient received dabrafenib monotherapy for 11 months, and demonstrated a substantial clinical, radiological and biochemical response, with complete normalization of her CA125 levels for the first time in six years. These results demonstrate that molecular analysis of low-grade serous ovarian carcinoma can identify targetable mutations and provide effective treatment options. The substantial response to dabrafenib suggests that BRAF inhibition represents a potential therapeutic option for ovarian cancer patients with somatic BRAFV600E mutations and should be tested in future trials. However, the results also highlight the need for novel clinical trial design, as traditional clinical trials are unlikely to be effective in such rare ovarian cancer sub-groups. Citation Format: Anna DeFazio, Tania Moujaber, Dariush Etemadmoghadam, Catherine Kennedy, Yoke-Eng Chiew, Rosemary L. Balleine, Catherine Saunders, Gerard V. Wain, Alexander Dobrovic, Australian Ovarian Cancer Study Group (AOCS), David DL Bowtell, Paul R. Harnett. BRAFV600E mutations in serous ovarian cancer and response to the BRAF inhibitor, dabrafenib. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A25.

Purpose The frequency of BRCA1 and BRCA2 germ-line mutations in women with ovarian cancer is unclear; reports vary from 3% to 27%. The impact of germ-line mutation on response requires further investigation to understand its impact on treatment planning and clinical trial design. Patients and Methods Women with nonmucinous ovarian carcinoma (n = 1,001) enrolled onto a population-based, case-control study were screened for point mutations and large deletions in both genes. Survival outcomes and responses to multiple lines of chemotherapy were assessed. Results Germ-line mutations were found in 14.1% of patients overall, including 16.6% of serous cancer patients (high-grade serous, 22.6%); 44% had no reported family history of breast or ovarian cancer. Patients carrying germ-line mutations had improved rates of progression-free and overall survival. In the relapse setting, patients carrying mutations more frequently responded to both platin- and nonplatin-based regimens than mutation-negative patients, even in patients with early relapse after primary treatment. Mutation-negative patients who responded to multiple cycles of platin-based treatment were more likely to carry somatic BRCA1/2 mutations. Conclusion BRCA mutation status has a major influence on survival in ovarian cancer patients and should be an additional stratification factor in clinical trials. Treatment outcomes in BRCA1/2 carriers challenge conventional definitions of platin resistance, and mutation status may be able to contribute to decision making and systemic therapy selection in the relapse setting. Our data, together with the advent of poly(ADP-ribose) polymerase inhibitor trials, supports the recommendation that germ-line BRCA1/2 testing should be offered to all women diagnosed with nonmucinous, ovarian carcinoma, regardless of family history.

Somatic cell hybrids have been obtained between HPRT Chinese hamster cells and cells from several dasyurid marsupial species. These hybrids show the extensive loss of marsupial chromosomes characteristic of the majority of marsupial-eutherian somatic cell hybrids. Although all of the hybrids expressed the selected marsupial marker, HPRT, the only other markers observed were PGK, GLA, and G6PD, consistent with the conservation of X-linked genes extending to this major group of marsupials. Counterselection confirmed the synteny of PGK and GLA with HPRT, whereas G6PD showed decreased concordance.

Bisulphite pyrosequencing is a quantitative methodology for the investigation of DNA methylation of sequences up to 100-bp in length. Biotin-labelled, single-stranded PCR products generated from bisulphite-treated DNA are used as a template with an internal primer to perform the pyrosequencing reaction. Nucleotides are added in a predetermined order in each pyrosequencing cycle and the amount of incorporated nucleotide results in a proportional emission of light. DNA methylation ratios are calculated from the levels of light emitted from each nucleotide incorporated at individual CpG positions in a strand-dependent manner. The methylation detection limit at individual CpG sites is approximately 5% and the results are displayed as an average methylation level for each CpG position assayed across all amplification products generated during a PCR reaction. As a consequence, bisulphite pyrosequencing allows the identification of heterogeneous DNA methylation patterns but does not provide information at a single allele resolution. This methodology is suited to analyse short DNA sequences such as those typically extracted from formalin-fixed paraffin-embedded specimens. Nevertheless, longer PCR products can be sequenced by serial bisulphite pyrosequencing, which utilises tandem assays along the amplicon. The general information provided is applicable for all formats of current pyrosequencing instruments, however, a specific protocol for the PyroMark Q24 instrument is provided.

Closed-tube PCR methods (sometimes referred to as in-tube PCR methods) for locus-specific DNA -methylation analysis are methodologies in which the amplification and analysis of bisulphite-modified DNA take place in one tube without the need to remove the PCR products for further analysis. Closed-tube methodologies lend themselves to high-throughput applications and molecular diagnostics but are also applicable as a research tool. We review three closed-tube methodologies, methylation-sensitive high-resolution melting (MS-HRM), MethyLight, and sensitive melting after real-time analysis - methylation-specific PCR (SMART-MSP). Closed-tube detection can be performed by simultaneously amplifying both methylated and unmethylated templates and subsequent melting curve analysis (MS-HRM). Alternatively, methylation-specific primers are used in real-time quantitative PCR and monitored either by a fluorescent hydrolysis probe (MethyLight) or using a double-stranded DNA binding fluorescent dye with a subsequent quality control step by melting curve analysis (SMART-MSP).

CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain 4 (CITED4) inhibits HIF-1α transactivation by binding to CBP/p300. We hypothesised that either somatic mutation or hypermethylation of the CITED4 gene underlies CITED4 down-regulation and thus enhanced HIF-1α expression in some breast tumours. DNA sequencing was used to screen for somatic mutations. Methylation-sensitive high resolution melting was performed to identify CITED4 methylation. RT-qPCR was carried out to measure the expression of CITED4 and selected HIF downstream targets. HIF-1α and downstream gene expression was assessed with immunohistochemistry. No somatic mutations of CITED4 were identified in 10 tumour cell lines and 100 breast carcinomas. However, CITED4 promoter methylation was identified in 5/168 breast carcinomas (four infiltrating ductal carcinomas and one infiltrating lobular carcinoma) and in 3/10 breast cancer cell lines (MDA-MB-453, MDA-MB-231 and Hs578T). CITED4 mRNA expression in cell lines was inversely correlated with DNA methylation. CITED4 mRNA expression was significantly increased in all three cell lines after 5-aza-2-deoxycytidine (DAC) treatment. Treatment of the MDA-MB-231 cell line with DAC followed by hypoxia (0.1% O²) resulted in down-regulation of expression of the HIF-1α downstream genes VEGFA and SLC2A1 (P = 0.0029). HIF-1α downstream SLC2A1 was decreased (P = 0.021) after CITED4 was re-expressed under hypoxia. Loss of expression of CITED4 in breast cancer may be due to DNA methylation but is unlikely to be due to mutation. Demethylation and histone modification can potentially reactivate CITED4 gene expression in some breast cancers and lead to changes in tumour behaviour. Strategies such as HDAC inhibitors may overcome this effect.

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.

Closed-tube PCR methods (sometimes referred to as in-tube PCR methods) for locus-specific DNA -methylation analysis are methodologies in which the amplification and analysis of bisulphite-modified DNA take place in one tube without the need to remove the PCR products for further analysis. Closed-tube methodologies lend themselves to high-throughput applications and molecular diagnostics but are also applicable as a research tool. We review three closed-tube methodologies, methylation-sensitive high-resolution melting (MS-HRM), MethyLight, and sensitive melting after real-time analysis - methylation-specific PCR (SMART-MSP). Closed-tube detection can be performed by simultaneously amplifying both methylated and unmethylated templates and subsequent melting curve analysis (MS-HRM). Alternatively, methylation-specific primers are used in real-time quantitative PCR and monitored either by a fluorescent hydrolysis probe (MethyLight) or using a double-stranded DNA binding fluorescent dye with a subsequent quality control step by melting curve analysis (SMART-MSP).

Bisulphite pyrosequencing is a quantitative methodology for the investigation of DNA methylation of sequences up to 100-bp in length. Biotin-labelled, single-stranded PCR products generated from bisulphite-treated DNA are used as a template with an internal primer to perform the pyrosequencing reaction. Nucleotides are added in a predetermined order in each pyrosequencing cycle and the amount of incorporated nucleotide results in a proportional emission of light. DNA methylation ratios are calculated from the levels of light emitted from each nucleotide incorporated at individual CpG positions in a strand-dependent manner. The methylation detection limit at individual CpG sites is approximately 5% and the results are displayed as an average methylation level for each CpG position assayed across all amplification products generated during a PCR reaction. As a consequence, bisulphite pyrosequencing allows the identification of heterogeneous DNA methylation patterns but does not provide information at a single allele resolution. This methodology is suited to analyse short DNA sequences such as those typically extracted from formalin-fixed paraffin-embedded specimens. Nevertheless, longer PCR products can be sequenced by serial bisulphite pyrosequencing, which utilises tandem assays along the amplicon. The general information provided is applicable for all formats of current pyrosequencing instruments, however, a specific protocol for the PyroMark Q24 instrument is provided.

We describe the localization of the chromosome 11 breakpoint of a T-ALL translocation, t(4;11)(q21;p14–15), to sub-band 11p15.5. The breakpoint is located between the genes for insulin-like growth factor II (IGFII) and the M1 subunit of ribonucleotide reductase (RRM1). This region does not include any previously cloned genes involved in cancer.

The ABO blood group has been used extensively as a marker in population studies, epidemiology, and forensic work. However, until the cloning of the gene, it was not possible to determine the genotype of group A and B individuals without recourse to family studies. We have developed a method to determine the ABO genotype directly from human DNA using multiplex PCR and restriction enzyme analysis. Two PCR fragments spanning positions 258 and 700 of the cDNA sequence are amplified. The site at position 258 allows us to differentiate the O allele from the A and B alleles. The site at position 700 allows us to distinguish the B allele from the A and O alleles. Analysis at the two sites thus allows us to distinguish the three alleles. The multiplex PCR product is digested separately with four enzymes, two for each of the sites. The pair of enzymes for each site cut in a reciprocal fashion. Whereas one enzyme for each site is theoretically sufficient for genotyping, the use of complementary pairs of enzymes prevents the assignment of a false genotype as a result of false negative or partial digestion. This method is fast and reliable, does not rely on probing of blots, and should be widely applicable.

Women carrying germline mutations in BRCA1 are at a substantially elevated risk of breast cancer and their tumors typically have distinctive morphologic features. We hypothesized that constitutional methylation of the BRCA1 promoter region could give rise to such breast cancers in women. We selected 255 women diagnosed with breast cancer before the age of 40 years for whom BRCA1 germline mutations had not been identified. Of them, 52 had five or more of nine BRCA1 mutation-associated morphologic features (group 1), 39 had four (group 2), and 164 had three or less (group 3). The prevalence of detectable BRCA1 promoter methylation in peripheral blood DNA decreased from 31% to 10% to 5% across groups 1–3, respectively (P = 0.000002), and was significantly greater than the 4% frequency in unaffected controls (P = 0.004). Peripheral blood methylation was associated with a 3.5-fold (95% CI, 1.4–10.5) increased risk of having early onset breast cancer. Methylation was consistently mosaic in the peripheral blood where the estimated allelic frequency of BRCA1 promoter methylation ranged from 0.1% to 17%. Group 1 women, but not group 3 women, with detectable methylation of peripheral blood DNA had high levels of BRCA1 promoter methylation of their tumor DNA, indicating that constitutional BRCA1 methylation strongly predisposes toward the development of BRCA1 methylated tumors that then have features resembling BRCA1 mutated tumors. Screening peripheral blood for BRCA1 promoter methylation might thus predict early-onset breast cancers. This raises the possibility of chemoprevention or other intervention to diminish the risk of developing breast cancer in these women. Cancer Prev Res; 4(1); 23–33. ©2010 AACR. Cancer Prev Res; 4(1); 23–33. ©2010 AACR.

As DNA methylation analysis enters the mainstream of biomedical research, there is increasing interest in methodologies that can be used for rapid analysis of a wide variety of biological and clinical samples. The methods that have been commonly used for methylation analysis are usually time-consuming and require multiple steps. The methylation-sensitive high-resolution melting (MS-HRM) assay is an in-tube assay which was developed to assess promoter methylation in sodium bisulphite-modified DNA and is particularly useful for assessing methylation in short fragments of DNA derived from formalin-fixed paraffin-embedded biopsies.

As DNA methylation analysis enters the mainstream of biomedical research, there is increasing interest in methodologies that can be used for rapid analysis of a wide variety of biological and clinical samples. The methods that have been commonly used for methylation analysis are usually time-consuming and require multiple steps. The methylation-sensitive high-resolution melting (MS-HRM) assay is an in-tube assay which was developed to assess promoter methylation in sodium bisulphite-modified DNA and is particularly useful for assessing methylation in short fragments of DNA derived from formalin-fixed paraffin-embedded biopsies.

Hypoxia-inducible factor-1 (HIF-1) activity is regulated by prolyl hydroxylase (PHD1, PHD2, PHD3) and factor inhibiting HIF-1 (FIH) that target the α subunit of HIF-1 (HIF-1α) for proteosomal degradation. We hypothesised that the elevated HIF-1α level is due in some tumours to epigenetic silencing by DNA hypermethylation of the promoter region of one or more of the PHDs and FIH genes. The aims were to define the presence or absence of promoter methylation of PHDs and FIH in cell lines of various sources and breast carcinomas and, if present, determine its effect on mRNA and protein expression.Tumour cell lines (n = 20) and primary invasive breast carcinomas (n = 168) were examined for promoter region DNA methylation using methylation-sensitive high-resolution melting. There was evidence of PHD3 but not of PHD1, PHD2 or FIH DNA methylation in breast cancer (SkBr3) and leukaemic (HL60 and CCRF-CEM) cell lines, but there was no evidence of methylation in any of 168 breast cancers. Only the high-level PHD3 methylation seen in leukaemic cell lines correlated with absent mRNA and protein expression.Methylation-induced epigenetic silencing of PHD1, PHD2, PHD3 and FIH is unlikely to underlie up-regulated HIF-1α expression in human breast cancer but may play a role in other tumour types.

KIT has been identified as a of biological importance in melanoma, and mutations (and/or amplification) appear to be largely confined to acral and mucosal subtypes and those associated with chronic sun damage (Curtin et al, 2006; Beadling et al, 2008; Satzger et al, 2008). Clinical experience in patients with gastrointestinal stromal tumour (GIST) indicates that sensitivity and resistance patterns to the KIT kinase inhibitors can be predicted from the presence and location of specific KIT mutations (Heinrich et al, 2006). Patients with melanomas containing activating mutations in KIT have been reported to respond to imatinib therapy (Lutzky et al, 2008; Quintas-Cardama et al, 2008; Satzger et al, 2010); however, it remains uncertain whether mutations considered to be predictive of imatinib resistance can respond to second-generation KIT kinase inhibitors. This report describes the frequency of KIT mutations in a prospectively selected group of Australian melanoma patients identified as ‘at risk' of harbouring a KIT mutation based on clinical subtype. Four case reports illustrating significant clinical responses to kinase inhibitors highlight the therapeutic potential of KIT mutation screening in advanced melanoma. However, central nervous system (CNS) progression following systemic responses in three patients raises an important issue pertaining to treatment efficacy in the setting of brain metastases.

Phyllodes tumours and cellular fibroadenomas are both fibroepithelial tumours of the breast. Phyllodes tumours, unlike fibroadenomas, have the ability to recur and metastasise. Although these lesions can be distinguished by their stromal cellularity, mitotic index, presence or absence of stromal overgrowth and cellular atypia, there is overlap and not infrequently a definitive diagnosis cannot be made, particularly on biopsy. We sought to evaluate whether DNA promoter methylation profiling using selected genes known to be methylated in cancer would allow us to learn more about the biology of these tumours, and whether it could identify methylation markers that could differentiate phyllodes tumours from fibroadenomas and/or distinguish phyllodes tumours of different grades. Methylation-sensitive high resolution melting (MS-HRM) was used to screen promoter DNA methylation changes in 86 phyllodes tumours (15 benign, 28 borderline, 43 malignant) and 26 fibroadenomas. A panel of 11 genes (RASSF1A, TWIST1, APC, WIF1, MGMT, MAL, RARβ, CDKN2A, CDH1,TP73 and MLH1) was tested. Methylation status was correlated with histology and with clinicopathological parameters. Five of the gene promoters showed some methylation in a proportion of phyllodes tumours; RASSF1A, 45.3%; TWIST1, 10.7%; APC, 4.1%; WIF1, 2.9% and MGMT, 1.3%. Only two genes showed any methylation in fibroadenomas usually at background levels; RASSF1A, 53.8% and MGMT, 8.3%. No CDKN2A methylation was observed in either tumour type, contrary to previous reports. Overall, the methylation patterns differed little from that which might be seen in normal cells. However, significant levels of methylation of RASSF1A (24.4%) and TWIST1 (7.1%) was observed in some phyllodes tumours. Elevated RASSF1A and/or TWIST1 methylation was significantly associated with phyllodes tumours compared with fibroadenomas (P = 0.02), TWIST1 methylation correlated with increasing malignancy in phyllodes tumours (P < 0.001). In conclusion, assessment of methylation of RASSF1A and TWIST1 may aid in the diagnosis of phyllodes tumours. The absence of frequent methylation in fibroadenomas supports a non-neoplastic origin.

In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non-acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non-acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.

Ludwig Institute of Cancer Research National Breast Cancer Foundation Introduction: Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.Methods: We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.Results: Most brain metastases were triple negative and basal-like. the brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. in particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.Conclusions: Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. the need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules. Univ Queensland, Clin Res Ctr, Brisbane, Qld 4029, Australia Queensland Inst Med Res, Brisbane, Qld 4006, Australia Universidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, Brazil Griffith Univ, Brisbane, Qld 4011, Australia Univ Queensland, Ctr Magnet Resonance, Brisbane, Qld 4072, Australia Eijkman Inst, Jakarta 10430, Indonesia Inst Nacl Canc, Dept Patol, BR-20230130 Rio de Janeiro, Brazil Lab Salomao & Zoppi, Dept Patol, BR-04104000 São Paulo, Brazil Charles Univ Prague, Fac Med, Dept Pathol, Plzen 30605, Czech Republic Univ Sydney, Inst Clin Pathol & Med Res, Sydney W Area Hlth Serv, Sydney, NSW 2145, Australia Univ Sydney, Westmead Millennium Inst, Sydney W Area Hlth Serv, Sydney, NSW 2145, Australia Peter MacCallum Canc Ctr, Dept Pathol, Melbourne, Vic 3002, Australia Univ Queensland, Queensland Brain Inst, Brisbane, Qld 4072, Australia Royal Brisbane & Womens Hosp, Brisbane, Qld 4029, Australia Universidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, Brazil Web of Science

Evidence that activating mutations of the KRAS oncogene abolish the response to anti-epidermal growth factor receptor therapy has revolutionized the treatment of advanced colorectal cancer. This has resulted in the urgent demand for KRAS mutation testing in the clinical setting to aid choice of therapy. The aim of this study was to evaluate six different KRAS mutation detection methodologies on two series of primary colorectal cancer samples. Two series of 80 frozen and 74 formalin-fixed paraffin-embedded tissue samples were sourced and DNA was extracted at a central site before distribution to seven different testing sites. KRAS mutations in codons 12 and 13 were assessed by using single strand conformation polymorphism analysis, pyrosequencing, high resolution melting analysis, dideoxy sequencing, or the commercially available TIB Molbiol (Berlin, Germany) or DxS Diagnostic Innovations (Manchester, UK) kits. In frozen tissue samples, concordance in KRAS status (defined as consensus in at least five assays) was observed in 66/80 (83%) cases. In paraffin tissue, concordance was 46/74 (63%) if all assays were considered or 71/74 (96%) using the five best performing assays. These results demonstrate that a variety of detection methodologies are suitable and provide comparable results for KRAS mutation analysis of clinical samples.

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.

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.

The ability of sodium bisulfite to modify cytosines in a methylation-dependent manner allows the conservation of DNA methylation information during PCR amplification. PCR products amplified from bisulfite-modified DNA have significantly different base compositions according to whether they originate from methylated or unmethylated variants of the target template. Different base compositions give rise to different thermal properties of the PCR products. Hence, melting analysis of amplification products in methylation studies allows the determination of whether the PCR products originate from methylated or unmethylated templates. Here, we briefly review recent advances in methodologies based on melting analyses of PCR products derived from bisulfite-modified templates and provide a methodology for methylation-sensitive high-resolution melting.

The ability of sodium bisulfite to modify cytosines in a methylation-dependent manner allows the conservation of DNA methylation information during PCR amplification. PCR products amplified from bisulfite-modified DNA have significantly different base compositions according to whether they originate from methylated or unmethylated variants of the target template. Different base compositions give rise to different thermal properties of the PCR products. Hence, melting analysis of amplification products in methylation studies allows the determination of whether the PCR products originate from methylated or unmethylated templates. Here, we briefly review recent advances in methodologies based on melting analyses of PCR products derived from bisulfite-modified templates and provide a methodology for methylation-sensitive high-resolution melting.

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.

Udgivelsesdato: 2009-Jan Hereditary cancer syndromes caused by germline mutations give rise to distinct spectra of cancers with characteristic clinico-pathological features. Many of these hereditary cancer genes are silenced by methylation in a similar spectrum of sporadic cancers. It is likely that the initiating event in some of those cases of sporadic cancer is the somatic epigenetic inactivation (epimutation) of the same hereditary cancer gene. Recently, it has been shown that epimutations of certain hereditary cancer genes can be constitutional i.e. present throughout the soma. These epimutations may be inherited or arise very early in the germline. The heritability of these epimutations is very low as in most cases they are erased by passage through the germline. In other cases, predisposition to epimutations rather than the epimutations themselves can be inherited. These cases are characterised by Mendelian inheritance and are likely to be associated with sequence variants. Other sequence variants and environmental influences may also affect methylation propensity at a global level.

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.

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.

Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS. Hum Mutat 0,1–6, 2008. © 2008 Wiley-Liss, Inc.

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).

The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown samples with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR amplification of bisulfite-modified DNA with primers designed to proportionally amplify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.

De novo presentation of acute myeloid leukemia (AML) expressing the Philadelphia (Ph) chromosomal abnormality is rare and is associated with a dismal prognosis. To date, reported cases of Ph+ AML have expressed either the e13a2 or e14a2 BCR-ABL fusion transcripts. We report a unique case of de novo AML expressing the e6a2 fusion transcript and describe disease sensitivity to both imatinib before allogeneic stem-cell transplantation and dasatinib for AML relapse after allogeneic stem-cell transplantation. Furthermore, we report that sustained molecular remission has been achieved despite withdrawal of tyrosine kinase inhibitor (TKI) therapy.

We examined the concept that individuals vary in their propensity to methylate promoter CpG islands by measuring such methylation in their peripheral blood. Individuals more prone to methylate CpG islands may be more susceptible to breast or other cancers. Scope: Our objective was to test peripheral blood for promoter methylation of selected genes known to become methylated in breast cancer using sensitive and specific quantitative assays. Major findings/ up-to-date report of the progress in terms of results and significance: We developed several powerful new methodologies based on high resolution melting for sensitive detection of DNA methylation and to genotype DNA for SNPS potentially involved in methylation. Promoter region methylation was observed in all tested blood DNA samples for CDH1 and HIC1, in the majority of DNA samples for TWIST1 and DAPK1 and in a substantial proportion of the DNA samples for MGMT, APC and RARB. MGMT methylation was associated with a single nucleotide polymorphism. No BRCA1, CDKN2A, GSTP1 or RASSF1A promoter methylation was found in this sample set. Several individuals had higher levels of methylation at several loci suggestive of a methylator phenotype. These findings will be implemented to establish case control studies to determine breast cancer risk.

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.

The point mutation 1849 (G→T) V617F in the JAK2 gene occurs at high frequency in several chronic myeloproliferative diseases. Although a number of V617F mutation detection methods have been described, few are readily implemented in a diagnostic setting. We developed a simple and sensitive allele-specific competitive blocker polymerase chain reaction (ACB-PCR) assay to detect the V617F mutation. DNA was extracted from peripheral whole blood samples of 26 patients with chronic myeloproliferative disease. The ACB-PCR limit of detection was 1%. All positive samples detected by sequencing were detected by ACB-PCR. In 3 patients with essential thrombocythemia, the V617F mutation was readily detected by ACB-PCR but was near the detection limit of sequencing, confirming that ACB-PCR is more effective at detecting V617F when the mutant cell population is low. Detection of the monomorphic JAK2 V617F mutation using the ACB-PCR assay is easy to perform, rapid, sensitive, and cost-effective, which are key features of an ideal diagnostic method.

Introduction The LRH-1 gene (NR5A2) located on chromosome 1q codes for a transcription factor implicated in promoting cell proliferation and migration in breast cancer. The gene contains five CpG islands and codes for several mRNA isoforms. However, little is known about its expression in breast cancer tissues. In this study, we examine LRH-1 expression patterns in breast cancer tissue by immunohistochemistry, its association with phenotypic features, and the relationship of DNA methylation with LRH-1 expression. Methods LRH-1 immunohistochemical (IHC) staining was performed on tissue microarray sections containing 329 cases of breast cancer (175 ductal carcinoma in situ (DCIS), 154 invasive carcinomas). Methylation was assessed using methylation sensitive high resolution melting assays targeted towards regions in the second and fifth CpG islands of LRH-1. These CpG islands are situated immediately upstream of the transcription start sites of mRNA transcripts 1 and 3, respectively. CpG island 2 (CpG2) methylation was assessed in 96 cases (65 DCIS, 31 invasive carcinoma), methylation of CpG island 5 (CpG5) was assessed in 99 cases (66 DCIS, 33 invasive carcinoma), and methylation data for both CpG islands was obtained in 85 cases (55 DCIS, 30 invasive carcinoma). Results IHC staining revealed four patterns of nuclear staining in breast cancer tissues. Nuclear staining was present as either in a finely granular pattern (pattern 1), a sparse punctate pattern (pattern 2), a dense punctate pattern (pattern 2+), or as a coarse granular pattern (pattern 3). Patterns 1 and 2 were considered LRH-1 low and patterns 2+ and 3 were considered LRH-1 high. LRH-1 high staining was significantly associated with adverse phenotypic features including high grade (p Conclusions This study identifies, for the first time, distinct nuclear IHC staining patterns in breast cancer tissue and the association of LRH-1 IHC-high patterns with aggressive phenotypic features. Differential DNA methylation of two regions is associated with LRH-1 IHC staining pattern, indicating a role for DNA methylation in the control of differential mRNA isoform expression and thus protein expression. Citation Format: Jia-Min Pang, Ashwini Chand, Kevin Knower, Elena Takano, David Byrne, Evelien Sprenkeler, Ramyar Molania, Ewan Millar, Soon Lee, Sandra O'Toole, Colin Clyne, Alexander Dobrovic, Stephen Fox. LRH-1 expression in breast cancer tissue and its association with phenotype and DNA methylation. [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 2962. doi:10.1158/1538-7445.AM2015-2962

Background: The 2447 A>T pathogenic variation at codon 816 of exon 17 (D816V) in the KIT gene, occurring in systemic mastocytosis (SM), leads to constitutive activation of tyrosine kinase activity and confers resistance to the tyrosine kinase inhibitor imatinib mesylate. Thus detection of this variation in SM patients is important for determining treatment strategy, but because the population of malignant cells carrying this variation is often small relative to the normal cell population, standard molecular detection methods can be unsuccessful. Methods: We developed 2 methods for detection of KIT D816V in SM patients. The first uses enriched sequencing of mutant alleles (ESMA) after BsmAI restriction enzyme digestion, and the second uses an allele-specific competitive blocker PCR (ACB-PCR) assay. We used these methods to assess 26 patients undergoing evaluation for SM, 13 of whom had SM meeting WHO classification criteria (before variation testing), and we compared the results with those obtained by direct sequencing. Results: The sensitivities of the ESMA and the ACB-PCR assays were 1% and 0.1%, respectively. According to the ACB-PCR assay results, 65% (17/26) of patients were positive for D816V. Of the 17 positive cases, only 23.5% (4/17) were detected by direct sequencing. ESMA detected 2 additional exon 17 pathogenic variations, D816Y and D816N, but detected only 12 (70.5%) of the 17 D816V-positive cases. Overall, 100% (15/15) of the WHO-classified SM cases were codon 816 pathogenic variation positive. Conclusion: These findings demonstrate that the ACB-PCR assay combined with ESMA is a rapid and highly sensitive approach for detection of KIT D816V in SM patients.

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.

1.1. Introduction Although assessing DNA methy-lation has not yet become commonplace in the diagnostic molecular pathology laboratory, many tests involving methyla-tion analysis will become part of routine practice. In this chapter, a practical approach will be taken toward evaluating DNA methylation. The principal question likely to be asked in the pathology laboratory is whether a specific gene or region is methylated in a specific pathological situation; for example, is the MLH1 gene promoter methylated in a colorectal cancer specimen showing microsatellite instability? This chapter will deal with methods that examine specific sequences for methy-lation rather than those examining total genomic methylation or those screening for new sites of recurrent methylation.

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.

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.