Single-strand Conformational Analysis Research Articles

Crude annual incidence rate of medullary thyroid cancer and RET mutation frequency

To determine the frequency and type of RET mutation in Slovenian medullary thyroid cancer (MTC) patients and estimate the crude annual incidence of MTC in Slovenia. This referral-center retrospective analysis involved 186 MTC patients diagnosed between 1995 and 2015 and their relatives who underwent genetic counseling and testing. The crude incidence rate of MTC was estimated with the joinpoint regression analysis. Genomic DNA was isolated, and exons 10, 11, 13, 14, 15, and 16 of the RET proto-oncogene were amplified with polymerase chain reaction. Point mutations of the RET gene were detected by single-strand conformation analysis and DNA sequencing. Detected mutations were confirmed by restriction enzymes. The average crude annual incidence rate of MTC in Slovenia was 0.34/100,000. A germline mutation in the RET proto-oncogene was identified in 25.9% of MTC patients. The most frequently altered codons were codons 634 and 618, followed by codon 790, codon 804, and codon 918. Annual incidence increase and nation-specific frequency of RET mutations justify the future use of genetic counseling and testing of MTC patients in Slovenia.

Comprehensive study for BRCA1 and BRCA2 entire coding regions in breast cancer.

About 5-10% of incidences of breast cancers have been reported as a result of germline mutations of BRCA genes. However, the mutational spectrum of BRCA1 and BRCA2 genes among breast cancer Saudi women patients is inadequate at present. Therefore, the present study aimed to report the specific germinal mutation of BRCA1 and BRCA2 in the entire coding regions, to investigate the prevalence rate of BRCA1 & BRCA2 mutations among Saudi women and the effect of these mutations, both benign and malignant tumors. A total of 270 tissue samples of benign and malignant breast tumors were collected from Saudi women patients, Riyadh, Saudi Arabia. Examination of BRCA1 and BRCA2 germline mutations was performed using heteroduplex DNA analysis (HDA) or single-stranded conformation analysis (SSCA). 177 breast cancer women with malignant tumors and 93 with benign tumors were enrolled in the study. A total of 62 out of 177 breast cancer patients carried a BRCA1 or BRCA2 mutation (54 BRCA1 and 8 BRCA2). The analysis was done using the Sanger sequence assay. Point and frameshift mutations through the entire coding area of the two genes indicated that all the mutations were germline alterations and of early-onset breast cancers. The mean ages of diagnosed breast cancer women for BRCA1 and BRCA2 mutation carriers were 36.3 (± 3.5) and 37.9 (± 3.7)years, whereas that of benign control was 35(± 2.5)years. Point and frameshift mutations across the entire coding region of BRCA1 and BRCA2 are responsible for many breast cancers cases.

Analysis of KRAS, NRAS, BRAF, PIK3CA and TP53 mutations in a large prospective series of locally advanced rectal cancer patients

Little information is available on the clinical significance of cancer-related genes such as KRAS, NRAS, BRAF, PIK3CA and TP53 in nonmetastatic rectal cancer. We investigated mutations of these genes in a large prospective series of locally advanced rectal cancer (LARC) patients who were recruited into two phase II trials. Mutational analyses were performed with diagnostically validated methods including polymerase chain reaction, capillary electrophoresis single-strand conformational analysis, Sanger sequencing and next-generation sequencing. Associations between single or multiple gene mutations and clinicopathological characteristics and treatment outcomes were explored. Of these 269, 210 (78%) patients were assessable. Mutations of KRAS, NRAS, BRAF, PIK3CA and TP53 occurred in 43, 9, 4, 9 and 60% of patients, respectively. Concordance between paired biopsy and resection specimens was 82% for KRAS, 95% for NRAS, 99% for BRAF, 96% for PIK3CA and 63% for TP53. TP53 mutations were associated with extramural venous invasion on baseline MRI (78% vs. 65%, p = 0.04), poor pathological tumour regression (23% vs. 36%, p = 0.05) and a trend toward a worse 5-year progression-free survival (PFS; 60% vs. 74%, HR 1.59, p = 0.06). Patients with tumours harbouring mutation of TP53 and either KRAS or NRAS (32%) had a worse 5-year PFS than those with TP53/KRAS/NRAS wild-type tumours (54% vs. 72%, HR 1.75, p = 0.02). In univariate analysis, BRAF mutation predicted poor 5-year overall survival only among patients treated without cetuximab (20% vs. 73%, HR 3.29, p = 0.03). This is one of the largest biomarker studies in a prospective, largely homogeneous, LARC population. Our findings are hypothesis generating and require validation in independent series.

Evaluation of Dietary Supplement Intake (Yeast Tablets) on Pregnant Albino Rats and Their Fetuses (Implications of Yeast Tablets on Rats & Fetuses)

ABSTRACT Histopathological studies in placenta, liver, kidney, apoptotic DNA damage and p53 mutation estimation of pregnant rats and their offspring exposed to dietary supplement yeast tablets, were carried out. Pregnant female albino rats were orally administrated yeast tablets at concentration 41.1 mg/kg during gestation period. Hematoxylin and eosin stained sections were used for examination. Neutral comet assay was performed to assess the apoptotic DNA damage and single strand conformational analysis was performed to screen the mutations inductions in p53 exons 7 and 8. Statistical analysis is applied to found the relation of apoptotic fraction in liver, kidney and placenta of pregnant rats and their offspring. Oral administration of yeast tablets to pregnant rats induced histopathological alterations in the hepatic, kidney tissues of both mothers and fetuses and placenta tissues. In addition, it induced apoptotic DNA damage as revealed by the significant elevations in apoptotic fractions (p<0.001) in liver and kidney tissues of both treated rats and their fetuses and even in the placenta (p<0.001). On contrary, no any mutation was induced in p53 exons 7 and 8 by yeast administration either in pregnant rats or their fetuses examined organs. The histopathological changes and apoptotic DNA damage recorded in female rats and fetus’s tissues which can result in definite hormonal and atrophic effects on adult rats and the fetuses, the possibility of early or late physiological effects in the mature and progeny under the administration of yeast tablets must be taken into consideration.

VITAMIN D RECEPTOR GENE MUTATIONS AND VITAMIN D SERUM LEVELS IN ASTHMATIC CHILDREN.

ABSTRACTObjective: To verify the relationship between polymorphisms of the vitamin D receptor gene (VDR), clinical findings, and serum vitamin D (VD) levels in asthmatics.Methods: A cross sectional study of 77 children aged 7 to 14 years old, who were attended at a specialized clinic. The children were divided into 3 groups: asthmatics who had been using inhaled corticosteroids (ICS) for more than one year; asthmatics who had not been using ICS; non-asthmatics, and children without allergies (according to the International Study of Asthma and Allergies in Childhood ­- ISAAC). Spirometry, skin prick tests, the presence of a VDR promoter CDX2 polymorphism from an allele-specific polimerase chain reaction (PCR), exons 2 and 3 polymorphisms genotyping by PCR-SSCA (single-strand conformational analysis), total immunoglobulin E (IgE) and specific IgE to mites and grass were evaluated in these three groups. Levels of 25-hydroxyvitamin D were determined in asthmatics only.Results: The mean age of the children was 10.8±2.0 years old, 57% were male, 38 were asthmatic and using ICS, 22 were asthmatic and not using ICS, and 17 were non-asthmatic. Allergic rhinitis was present in 90% of asthmatics. Homozygous CDX2 was detected in 23% of the patients and absent in the control group (p=0.03). Lower forced expiratory volume in 1 second (FEV1%) values were observed in CDX2 homozygous asthmatics (p=0.001). Variations in the exon 2 and 3 sequences were not related to asthma or the other tests. VD deficiency or insufficiency was detected in 98% of asthmatics. There was no association between VD levels and genetic polymorphisms from exons 2 and 3.Conclusions: There was a positive association between homozygous CDX2 polymorphism, asthma and lower FEV1% values. CDX2 is capable of modifying cell interaction between VDR and VD, and it could be associated with the prevalence of asthma, and the difficulty in controlling the disease.

Abstract 270: Synergistic effect with APR-246 and standard chemotherapy in small cell lung cancer cells carrying smoking-associated TP53 mutations

Abstract Background: Although smoking increases risk for many tumor types, no malignancy is more closely linked to tobacco exposure than small cell lung cancer (SCLC); 90% of SCLC patients are smokers. Carcinogenic compounds in cigarettes increase cancer susceptibility due to formation of DNA adducts, leading to oncogenic mutations. Etoposide in combination with cisplatin or carboplatin is the standard chemotherapy for SCLC. However, most SCLC patients eventually die of chemotherapy-refractory disease. Mutation in the tumor suppressor gene TP53 is one of the main causes for resistance, and occurs in more than 70% of SCLC patients (http://p53.free.fr). APR-246 (PRIMA-1MET) is the first clinical-stage small molecule that reactivates mutant p53 by inducing its wild-type conformation triggering apoptosis. It has been tested in a First in Human clinical trial in hematological malignancies with encouraging results (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study in combination with platinum-based therapy in recurrent ovarian cancer is ongoing (AACR abstract #CT204, 2015). We have previously observed strong synergy with APR-246 and platinum compounds in TP53-mutant drug-resistant ovarian cancer cells (Mohell et al. Cell Death and Disease 6, 2015). The aim of the present study was to investigate whether APR-246 synergizes with standard chemotherapy in SCLC cells carrying smoking-associated and/or lung cancer-specific TP53 mutations. Methods: Cell viability was tested using CellTiter-Glo assay, TP53 status with Sanger sequencing and single strand conformation analysis, and p53 protein level with Western blotting. Combination Index (CI) was calculated according to Additive model. Results: We observed synergistic (CI&amp;lt;0.8) or strong synergist effect (CI&amp;lt;0.5) with APR-246 and cisplatin in SCLC cell lines carrying smoking-associated (often G&amp;gt;T transversion) and/or lung cancer specific homozygous TP53 mutations, including NCI-H2195 (V157F, 4.3% of all SCLC tumors), NCI-H1048 (R273C, 1.78%) and NCI-H889 (C242S, 1.07%). Synergy was also observed in H196 cells with hotspot R175H mutation (2.14%), while additive effect (CI = 0.8-1.2) was found in NCI-H1882 (R273L, 1.78%) and NCI-H187 (S241C, 0.36%) cells. Synergy was also observed with etoposide in NCI-H2195 cells. Moreover, APR-246 sensitized the NCI-H2195 cells to cisplatin; the IC50 value decreased about 2-fold at clinically relevant concentration of APR-246. All tested SCLC cell lines had medium or high level of p53. Further studies with other conventional drugs are ongoing. Conclusions: Treatment with APR-246 in combination with cisplatin or etoposide resulted in synergy or strong synergy in SCLC cells carrying lung cancer-specific and/or smoking-related TP53 mutations. Our results suggest that combination treatment with APR-246 and standard chemotherapy can provide significantly improved treatment of TP53-mutant SCLC. Citation Format: Nina Mohell, Åsa Fransson, Jessica Alfredsson, Mikael von Euler, Ulf Björklund, Lars Abrahmsen. Synergistic effect with APR-246 and standard chemotherapy in small cell lung cancer cells carrying smoking-associated TP53 mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 270.

Strong synergy with APR-246 and DNA-damaging drugs in primary cancer cells from patients with TP53 mutant High-Grade Serous ovarian cancer

BackgroundMutation in the tumor suppressor gene TP53 is an early event in the development of high-grade serous (HGS) ovarian cancer and is identified in more than 96 % of HGS cancer patients. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 protein by refolding it to wild type conformation, thus inducing apoptosis. APR-246 has been tested as monotherapy in a Phase I/IIa clinical study in hematological malignancies and prostate cancer with promising results, and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. In the present study, we investigated the anticancer effects of APR-246 in combination with conventional chemotherapy in primary cancer cells isolated from ascitic fluid from 10 ovarian, fallopian tube, or peritoneal cancer patients, 8 of which had HGS cancer.MethodsCell viability was assessed with fluorometric microculture cytotoxicity assay (FMCA) and Combination Index was calculated using the Additive model. p53 status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by western blotting.ResultsWe observed strong synergy with APR-246 and cisplatin in all tumor samples carrying a TP53 missense mutation, while synergistic or additive effects were found in cells with wild type or TP53 nonsense mutations. Strong synergy was also observed with carboplatin or doxorubicin. Moreover, APR-246 sensitized TP53 mutant primary ovarian cancer cells, isolated from a clinically platinum-resistant patient, to cisplatin; the IC50 value of cisplatin decreased 3.6 fold from 6.5 to 1.8 μM in the presence of clinically relevant concentration of APR-246.ConclusionThese results suggest that combination treatment with APR-246 and DNA-damaging drugs could significantly improve the treatment of patients with TP53 mutant HGS cancer, and thus provide strong support for the ongoing clinical study with APR-246 in combination with carboplatin and pegylated liposomal doxorubicin in patients with recurrent HGS cancer.

Abstract 2523: Strong synergistic effects with APR-246 and cisplatin in p53-mutant lung cancer cells

Abstract Background: Platinum compounds have been used as first-line treatment for many solid tumors including non small cell (NSCLC) and small cell (SCLC) lung cancer. However, patients with lung cancer often develop resistance to platinum compounds and eventually die of chemotherapy refractory disease. Mutation in the tumor suppressor protein p53 is common in lung cancer, ranging from 33% in adenocarcinomas to 70% in SCLC (The p53 website, http://p53.free.fr), and is one of the main causes for resistance to chemotherapy. APR-246 (PRIMA-1MET) is the first compound in clinical development that reactivates mutant p53 by inducing its wild type conformation thus triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has yielded promising results in a first-in-human clinical trial in patients with hematological malignancies and prostate cancer (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. Previously we have shown strong synergy with APR-246 and platinum compounds in p53-mutant drug-resistant ovarian cancer cells (AACR abstract # 3448, 2013). Moreover, APR-246 completely restored the sensitivity of cisplatin to resistant p53-mutant ovarian cancer cells (AACR abstract #1801, 2014). The aim of the current study was to investigate whether strong synergistic effect can also be observed in p53-mutant lung cancer cells. Methods: Cell viability was determined with FMCA or Cell Titer-Glo assay, p53 gene status by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Combination Index (CI) was calculated according to Additive model. Results: We observed strong synergistic effect (CI&amp;lt;0.5) with APR-246 and cisplatin in lung cancer cell lines carrying homozygous p53 hotspot mutations; NCI-H1770 (R248W), NCI-H1975 (R273H), NCI-H596 (G245C), PC-14 (R248W) and PC-14/CDDP (R248W). All these cell lines expressed a high level of p53. Synergistic (CI&amp;lt;0.8) or strong synergistic effect was found in lung cancer cell lines NCI-H378 (Y163C) and NCI-2087 (V157F) with homozygous mutations that occur frequently but are not hotspot mutations. NCI-2087 cells with the smoking-related V157F mutation express a high level of p53, while NCI-H378 cells express lower level. Mixed antagonist/additive/synergistic effects were observed in the p53 null cell line HOP-62, which does not express p53. Conclusions: Treatment with APR-246 in combination with cisplatin resulted in strong synergistic effect in both NSCL and SCLC cancer cells. Strongest synergies were observed in lung cancer cells with p53 hotspot mutations expressing a high level of p53. Studies to investigate the molecular mechanisms underlying the synergistic effects are ongoing. Our results suggest that combination treatment with APR-246 and platinum drugs may allow an improved therapy of p53-mutant lung cancer. Citation Format: Nina Mohell, Åsa Fransson, Jessica Alfredsson, Mikael von Euler, Ulf Björklund, Lars Abrahmsen. Strong synergistic effects with APR-246 and cisplatin in p53-mutant lung cancer cells. [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 2523. doi:10.1158/1538-7445.AM2015-2523

Abstract 1639: Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells

Abstract Background: Mutations in the TP53 gene occur in at least 60% of ovarian tumors and are associated with chemoresistance and poor prognosis. APR-246 (PRIMA-1MET) is the first mutant p53-reactivating compound in clinical development and has been tested as monotherapy in hematological malignancies and prostate cancer with promising results (Lehmann et al. J Clin Oncol 30, 2012). The aim of this study was to investigate the anticancer effects of APR-246 in combination with conventional chemotherapy in cancer cells isolated from ascites fluid from ovarian cancer patients. Methods: Ascites cells were purified by Ficoll and viably frozen, and the quality and purity were confirmed by May Grünwald/Giemsa staining. For some samples, immunocytochemical stainings with anti-Ber-EP4 and anti-calretinin antibodies were used to distinguish between mesothelial and cancer cells. Cell viability was assessed with FMCA assay and Combination Index (CI) calculated using Additive model. CI &amp;lt; 0.8 indicates synergy and CI &amp;lt; 0.5 strong synergy. TP53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Results: Eight of ten samples tested were from patients with recurrent ovarian cancer previously treated with platinum drugs. Cancer cells from seven patients possessed TP53 core domain missense mutations L111Q, C135Y, P151H, Y163H, C238F, P278R and R280K, respectively; two had nonsense mutations E346* and E204*, and one was wild type. All the missense mutations have been predicted to severely affect p53 tumor suppressor function. Missense mutant p53 proteins were expressed at high levels while no p53 expression was detected in cells with wild type or nonsense mutant p53. Synergistic or strong synergistic effects with APR-246 and cisplatin were observed in all ten samples tested. Synergy was also observed with the platinum analogue carboplatin and the anthracycline doxorubicin. The IC50 values for cisplatin ranged from 3 to 40 μM and for APR-246 from 5 to 37 μM. We also tested the ability of APR-246 to sensitize the primary ovarian cancer cells carrying Y163H mutant p53, to cisplatin; the IC50-value of cisplatin decreased from 10 to 2.6 μM in the presence of 6 μM APR-246. Conclusions: We observed striking synergy with APR-246 and platinum drugs or doxorubicin. These results are consistent with our previous results in ovarian cancer cell lines showing synergy not only in p53 mutant but also in p53 null cancer cell lines. In these cells, the synergy may be related to the fact that APR-246 decreases intracellular glutathione level. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with carboplatin and doxorubicin in patients with recurrent ovarian cancer and suggest that combination treatment with APR-246 and DNA-damaging drugs could allow significantly improved treatment for ovarian cancer carrying mutant p53. Citation Format: Åsa Fransson, Daria Glaessgen, Jessica Alfredsson, Klas G. Wiman, Svetlana Bajalica Lagercrantz, Nina Mohell. Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells. [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 1639. doi:10.1158/1538-7445.AM2015-1639

Expression and mutational analysis of Cip/Kip family in early glottic cancer.

Genetic alteration of cyclin-dependent kinase inhibitors has been associated with carcinogenesis mechanisms in various organs. This study aimed to evaluate the expression and mutational analysis of Cip/Kip family cyclin-dependent kinase inhibitors (p21CIP1/WAF1, p27KIP1 and p57KIP2) in early glottic cancer. Expressions of Cip/Kip family and p53 were determined by quantitative reverse transcription polymerase chain reaction and densitometry. For the analysis of p21 inactivation, sequence alteration was assessed using single-strand conformational polymorphism polymerase chain reaction. Additionally, the inactivation mechanism of p27 and p57 were investigated using DNA methylation analysis. Reduced expression of p27 and p57 were detected in all samples, whereas the expression of p21 was incompletely down-regulated in 6 of 11 samples. Additionally, single-strand conformational polymorphism polymerase chain reaction analysis showed the p53 mutation at exon 6. Methylation of p27 and p57 was detected by DNA methylation assay. Our results suggest that the Cip/Kip family may have a role as a molecular mechanism of carcinogenesis in early glottic cancer.

Abstract 3518: TP53 mutations and MDM2 single nucleotide polymorphism 309T-G predicts outcome and treatment resistance in acute myeloid leukemia

Abstract The tumor-suppressor gene TP53, encoding the p53 protein, is vital for normal cellular processes including cell cycle regulation, apoptosis and senescence. Mutations in TP53 are frequent in many cancers, and have been associated with treatment resistance and inferior prognosis in acute myeloid leukemia (AML). Altered expression of MDM2 (mouse double minute 2), a negative regulator of p53, may also attenuate normal p53 signaling, thereby enhancing tumor transformation and resistance to apoptosis. The single nucleotide polymorphism (SNP) 309 (T&amp;gt;G, rs2279744) has been reported to increase MDM2 expression and impair normal p53 response. We investigated the frequency and impact of TP53 mutations (TP53mut) and MDM2SNP309 on treatment response and overall survival (OS) in 207 Swedish AML patients. To detect the TP53mut in exons 5-8, the single strand conformation analysis (SSCA) was performed. Samples with mobility shift were sequenced to determine the exact nucleotide sequence and compared to the corresponding TP53 reference sequence (NM_000546.4). MDM2SNP309 was analyzed using Pyrosequencing. We found a high frequency (22%) of TP53mut in patients with cytogenetic aberrations, with strong association to high risk cytogenetics (p&amp;lt;0.001). TP53mut patients had markedly lower response rates compared to TP53 wild-type (wt) patients (21% and 76% CR, respectively, p&amp;lt;0.001) and a significantly reduced OS (5 and 21 months, respectively, p&amp;lt;0.001). In TP53wt patients with abnormal karyotype, the MDM2SNP309 conferred an impaired outcome, with patients carrying the alternative G allele having shorter OS compared to T/T patients (13 and 29 months, p=0.031). This effect was neither observed in OS analysis of the entire group, nor in the normal karyotype patients, suggesting that the SNP G-allele carriers may suppress function of wild-type p53 in cytogenetically altered tumors. Allo-SCT in first complete remission improves the prognosis in AML patients with intermediate or high risk. However, some patients, including those with monosomal karyotype, and, as seen in our study, patients with TP53mut, are unlikely to reach CR with standard treatment. These patients may be considered for clinical trials with emerging novel therapies targeting the p53 signaling pathway. In conclusion, our results show that TP53mut identifies a subgroup of AML patients with dramatically impaired outcome. TP53 analysis, and potentially also MDM2SNP309 genotyping, should be implemented in prognostication, risk stratification and selection of patients most likely to benefit from new drugs such as the nutlins (MDM2-inhibitors) and PRIMA-1 (“p53 Reactivation and Induction of Massive Apoptosis”). Citation Format: Ingrid Jakobsen Falk, Kerstin Willander, Roza Chaireti, Johan Lund, Monica Hermanson, Henrik Greén, Peter Söderkvist, Kourosh Lotfi. TP53 mutations and MDM2 single nucleotide polymorphism 309T-G predicts outcome and treatment resistance in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2014-3518

Abstract 1801: APR-246, a clinical-stage mutant p53-reactivating compound, resensitizes ovarian cancer cells to platinum compounds and doxorubicin

Abstract Background: Platinum-based drugs are since decades used as first-line treatment for many solid tumors. Patients with ovarian cancer often respond well to platinum compounds but a majority of patients rapidly develop resistance and die of chemotherapy refractory disease. The mechanisms underlying resistance are multifactorial, but two of the main causes are mutations in the tumor suppressor p53 and elevated intracellular glutathione (GSH) levels. Mutations in p53 occur in about 60% of ovarian tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53. APR-246 is a prodrug that accumulates in cancer cells and is converted to the active form MQ, a Michael acceptor that binds to mutant p53, refolds it to wild type conformation and triggers apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has been tested in a Phase I/IIa clinical trial with promising results (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study with platinum-based combination therapy in recurrent p53 mutant ovarian cancer is underway. Methods: Cell viability was assessed with WST-1, MTT or FMCA assay. p53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Intracellular GSH levels were assessed with GSH kit (Cayman). Results: We have previously shown outstanding synergistic anticancer effects with APR-246 in combination with platinum compounds in p53 mutant solid cancer cell lines, including cisplatin resistant ovarian cancer cells. Synergistic effects were also observed ex vivo as well as in vivo in mice carrying human tumor xenografts. Here we show that APR-246 not only reactivates mutant p53 but also decreases intracellular GSH levels in a dose-dependent manner, presumably via adduct formation between MQ and GSH. APR-246 resensitized cisplatin resistant p53 mutant ovarian A2780-CP20 carcinoma cells to cisplatin, as shown by a decrease in the IC50 value from 52 to 2.9 µM, similar to the IC50 in parental A2780 cells. APR-246 also restored the sensitivity of resistant A2780ADR cells to doxorubicin. A2780-CP20 and A2780ADR were developed from the parental A2780 line with wild type p53 by chronic exposure to the respective drug. Moreover, APR-246 resensitized the p53 mutant OVCAR-3 cell line, established from a drug resistant patient, to cisplatin. Conclusions: Our results show that APR-246 not only reactivates mutant p53 but also decreases intracellular glutathione levels. We propose that this unique dual mechanism of action accounts for the resensitization and strong synergistic effects with APR-246 and platinum drugs. Our results provide strong rationale for the planned clinical study in ovarian cancer and suggest that combination treatment with APR-246 and DNA damaging drugs could have broad applicability in the treatment of drug resistant p53 mutant human tumors. Citation Format: Nina Mohell, Jessica Alfredsson, Åsa Fransson, Vladimir Bykov, Mikael von Euler, Klas Wiman, Ulf Björklund. APR-246, a clinical-stage mutant p53-reactivating compound, resensitizes ovarian cancer cells to platinum compounds and doxorubicin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1801. doi:10.1158/1538-7445.AM2014-1801

A Comparison of Methods for EGFR Mutation Testing in Non–Small Cell Lung Cancer

EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non-small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

NOTCH1 mutations influence survival in chronic lymphocytic leukemia patients

BackgroundNOTCH1 PEST domain mutations in chronic lymphocytic leukemia have recently been shown to be of prognostic relevance. Both NOTCH1 and NOTCH2 are constitutively activated in B-cell CLL but not expressed in normal B cells and may be involved in survival and resistance to apoptosis in CLL. We screened for mutations in different parts of both NOTCH1 and NOTCH2 genes and related the changes to survival and other known risk factors.MethodsIn a cohort of 209 CLL patients, we used single strand conformation analysis to determine which of the samples carrying the NOTCH mutations and direct dideoxy sequencing was used to determine the exact nucleotide changes. Kaplan-Meier curves and log rank test were used to determine overall survival for NOTCH1 mutated cases and Cox regression analysis was used to calculate hazardous ratios.ResultsIn the present study, we found NOTCH1 PEST domain mutations in 6.7% of the cases. A shorter overall survival was found in patients with NOTCH1 mutations compared to wildtype (p = 0.049). Further, we also examined the extracellular and the heterodimerisation domains of the NOTCH1 gene and the PEST domain and heterodimerisation domain of the NOTCH2 gene, but no mutations were found in these regions. NOTCH1 mutations were most commonly observed in patients with unmutated IGHV gene (10/14), and associated with a more aggressive disease course. In addition, NOTCH1 mutations were almost mutually exclusive with TP53 mutations. In the combined group of NOTCH1 (6.7%) or TP53 (6.2%) mutations, a significant difference in overall survival compared to the wildtype NOTCH1 and TP53 was found (p = 0.002).ConclusionsBoth NOTCH1 and TP53 mutations seem to be independent predictive markers for worse outcome in CLL-patients and this study emphasizes the contention that NOTCH1 mutations is a novel risk marker.

Abstract 3448: Strong synergistic effects with cisplatin and APR-246, a novel compound reactivating mutant p53, in ovarian cancer cell lines and primary cells from patients.

Abstract Background: The tumor suppressor protein p53 is frequently mutated in cancer, and cancer cells carrying defects in p53 are generally more resistant to conventional chemotherapy. About 60% of patients with ovarian cancer have p53 mutations. Thus, restoration of wild type function of p53 is a promising strategy for cancer therapy. APR-246 (PRIMA-1MET) belongs to a class of small molecules (quinuclidinones) that reactivate mutated or otherwise non-functional p53 by promoting its correct wt folding thus triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). In various in vitro, ex vivo and in vivo cancer models APR-246 has shown good antitumor activity and a unique pharmacological profile. In a Phase I/II clinical dose-finding study on hematological malignancies and prostate cancer APR-246 had a good safety profile, and both biological and clinical responses were observed (Lehmann et al. J Clin Oncol 30, 2012). A Phase II Proof of Concept study in p53 mutant ovarian cancer patients is currently under way. Here results from combination studies with APR-246 and platinum compounds in p53 mutant ovarian cancer cell lines, and primary cells from patients, are presented. Methods: Cell viability/proliferation was assessed with WST-1, MTT and/or FMCA assay. p53 gene status was determined with Sanger sequencing and single strand conformation analysis. p53 expression was determined with Western immunoblotting. Results: In the ovarian cancer cell line OVCAR-3, with homozygous “hot spot” p53 core domain mutation (R248Q), strong/outstanding synergistic effects with APR-246 and cisplatin were observed (CI &amp;lt; 0.5). This cell line was established from a patient resistant to clinically relevant concentrations of cisplatin, doxorubicin and melphalan. The OVCAR-3 cell line expresses a high level of p53. In the in vitro cisplatin resistant ovarian cancer cell lines (A2780-CP20, IGROV-1/CDDP, IGROV-1/Pt-1) harboring heterozygous frequently occurring p53 core domain mutations, synergistic (CI &amp;lt; 0.8) or strong synergistic effects were observed. Also these cell lines express p53 but at lower level. These resistant cell lines have been developed from parental cisplatin sensitive cell lines derived from untreated patients with wt p53, by chronic in vitro exposure to cisplatin. Carboplatin and doxorubicin showed cross-resistance in cisplatin resistant cell lines, while the sensitivity of APR-246 did not change. Primary ovarian cancer cells were also investigated and strong synergistic effect was found in all samples. In vivo efficacy (xenograft) combination studies using cisplatin resistant ovarian cancer cell lines in nude mice are ongoing. Conclusion: These results further support the Phase II Proof of Concept study of APR-246 in p53 mutated ovarian cancer patients who are candidates for further platinum-based chemotherapy. Citation Format: Nina Mohell, Jessica Alfredsson, Maria Uustalu, Åsa Fransson, Vladimir J.N. Bykov, Klas G. Wiman, Ulf Björklund. Strong synergistic effects with cisplatin and APR-246, a novel compound reactivating mutant p53, in ovarian cancer cell lines and primary cells from patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2013-3448

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.

MDM2 SNP309 promoter polymorphism and p53 mutations in urinary bladder carcinoma stage T1

BackgroundUrinary bladder carcinoma stage T1 is an unpredictable disease that in some cases has a good prognosis with only local or no recurrence, but in others can appear as a more aggressive tumor with progression to more advanced stages. The aim here was to investigate stage T1 tumors regarding MDM2 promoter SNP309 polymorphism, mutations in the p53 gene, and expression of p53 and p16 measured by immunohistochemistry, and subsequently relate these changes to tumor recurrence and progression. We examined a cohort of patients with primary stage T1 urothelial carcinoma of the bladder and their tumors.MethodsAfter re-evaluation of the original slides and exclusions, the study population comprised 141 patients, all with primary stage T1 urothelial carcinoma of the bladder. The hospital records were screened for clinical parameters and information concerning presence of histologically proven recurrence and progression. The paraffin-embedded tumor material was evaluated by immunohistochemistry. Any mutations found in the p53 gene were studied by single-strand conformation analysis and Sanger sequencing. The MDM2 SNP309 polymorphism was investigated by pyrosequencing. Multivariate analyses concerning association with prognosis were performed, and Kaplan-Meier analysis was conducted for a combination of changes and time to progression.ResultsOf the 141 patients, 82 had at least one MDM2 SNP309 G allele, and 53 had a mutation in the p53 gene, but neither of those anomalies was associated with a worse prognosis. A mutation in the p53 gene was associated with immunohistochemically visualized p53 protein expression at a cut-off value of 50%. In the group with p53 mutation Kaplan-Meier analysis showed higher rate of progression and shorter time to progression in patients with immunohistochemically abnormal p16 expression compared to them with normal p16 expression (p = 0.038).ConclusionsMDM2 SNP309 promoter polymorphism and mutations in p53 were not associated with worse prognosis in this cohort of patients with primary stage T1 urinary bladder carcinoma. However, patients with abnormal p16 expression and a mutated p53 gene had a higher rate of and a shorter time to progression, and p53 gene mutation was associated with an abnormal immunohistochemistry for p53 at a cut-off of 50%.

Incidence of BRAF p.Val600Glu and p.Val600Lys mutations in a consecutive series of 183 metastatic melanoma patients from a high incidence region

Approximately 40-60% of melanomas from Caucasian populations carry activating mutations in the BRAF oncogene, with the most common being the p.Val600Glu (V600E) hotspot mutation in exon 15. The aim of the present study was to investigate the frequency of the less common p.Val600Lys (V600K) mutation in metastatic melanoma from a high incidence region. Dideoxy sequencing and fluorescent single strand conformation analysis were used to screen for mutations in exon 15 of BRAF in 183 cases of metastatic melanoma. The overall incidence of BRAF mutation (89/183, 49%) was very similar to other large studies of Caucasian populations. However, the frequency of the p.Val600Lys mutation was higher than in most other studies and comprised almost one-third of all BRAF mutations in our cohort (27/89, 30%). BRAF p.Val600Lys mutations were present at a relatively high frequency in this cohort of metastatic melanoma patients (27/183, 15%). Assays used to screen for BRAF mutations in the clinic should be robust enough to detect the p.Val600Lys mutation, as this may have therapeutic implications.

BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimen

Introduction Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the V600E BRAF mutation in HCL makes it a valuable molecular diagnostic marker. Objective To compare the ability of Sanger sequencing (SS), fluorescent single-strand conformational analysis (F-SSCA) and high resolution melting (HRM) analysis to detect BRAF mutations in HCL with DNA extracted predominantly from archival material. Methodology 20 cases of HCL consisting of four archival Roma-novsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) sple-nectomy samples and one fresh peripheral blood sample were reviewed. DNA was amplified by PCR and BRAF mutation status determined by the three methods above. Results V600E mutation was identified in 95%, 87% and 76% of HCL cases by F-SSCA, HRM and SS, respectively. In one HCL case, in addition to the V600E mutation, a K601T mutation was identified. One of the Romanowsky stained samples was negative for mutation by all three methods. Three mercury fixed cases were negative for mutation by sequencing but positive for the mutation using F-SSCA and HRM. This discrepancy could be due to mutations below the limit of sensitivity of SS. Conclusion DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity especially using HRM/F-SSCA. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL. The presence of the mutation may provide a target for therapy. Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the V600E BRAF mutation in HCL makes it a valuable molecular diagnostic marker. To compare the ability of Sanger sequencing (SS), fluorescent single-strand conformational analysis (F-SSCA) and high resolution melting (HRM) analysis to detect BRAF mutations in HCL with DNA extracted predominantly from archival material. 20 cases of HCL consisting of four archival Roma-novsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) sple-nectomy samples and one fresh peripheral blood sample were reviewed. DNA was amplified by PCR and BRAF mutation status determined by the three methods above. V600E mutation was identified in 95%, 87% and 76% of HCL cases by F-SSCA, HRM and SS, respectively. In one HCL case, in addition to the V600E mutation, a K601T mutation was identified. One of the Romanowsky stained samples was negative for mutation by all three methods. Three mercury fixed cases were negative for mutation by sequencing but positive for the mutation using F-SSCA and HRM. This discrepancy could be due to mutations below the limit of sensitivity of SS. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity especially using HRM/F-SSCA. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL. The presence of the mutation may provide a target for therapy.

DNA Variation in myoMIRs of the 1, 133, and 208 Families in Hypertrophic Cardiomyopathy

MicroRNAs (miRNAs) are small RNAs that bind to mRNAs and regulate gene expression. MyoMirs are miRNAs implicated in cardiogenesis. Some MyoMirs have been found deregulated in hearts from patients with left ventricular hypertrophy (LVH). DNA variants at these miRNAs could contribute to the risk of developing hypertrophic cardiomyopathy (HCM). To test this hypothesis we used single strand conformation analysis and direct sequencing to search for DNA variants in the mir-208a, miR-208b, miR-133a-1, miR-133a-2, miR-133b, miR-1-1, and miR-1-2 genes in patients with HCM (n=245), LVH secondary to hypertension (n=120), and healthy controls (n=250). We found several nucleotide variants. Genotyping of patients and healthy controls showed significantly associations between a 133a-1 polymorphism and HCM and a 133b polymorphism and hypertensive- LVH. We concluded that rare variants in these mature miRNAs would be rarely found among HCM patients, but miR-133a-1 and 133b polymorphisms could contribute to the risk of developing cardiac hypertrophy.