Publications | DiaCarta, Inc.

Evaluation of a Novel Liquid Biopsy-Based ColoScape Assay for Mutational Analysis of Colorectal Neoplasia and Triage of FIT+ Patients: a Pilot Study

Oct 12, 2018 | Publications

Circulating cell-free tumour derived nucleic acids are becoming recognised as clinically significant and extremely useful biomarkers for the detection of cancer and for monitoring the progression of targeted drug therapy and immunotherapy. Screening programmes for colorectal cancer in Europe use the Fetal Immunochemical Test (FIT) test as a primary screener. FIT+ patients are referred to immediate colonoscopy and the positive predictive value (PPV) is usually 25%. In this article, we report a study employing the ColoScape assay panel to detect mutations in the APC, KRAS, BRAF and CTNNB1 genes, in order to collect preliminary performance indicators and plan a future, larger population study. The assay was evaluated on 52 prospectively collected whole-blood samples obtained from FIT+ patients enrolled in the CRC screening programme of ASL NAPOLI 3 SUD, using colonoscopy as confirmation. The assay’s sensitivity for advanced adenomas was 53.8% and the specificity was 92.3%. The PPV was 70.0% and negative predictive value (NPV) was 85.7%. Workflow optimisation is essential to maximise sensitivity. Of note, four of the six positive cases missed by ColoScape had a less than suboptimal DNA input (data not shown). Had they been ruled out as inadequate, sensitivity would have increased from 53.8% to 69%. However, as stated previously, this is not a clinical trial, but rather an initial, preliminary technical evaluation. In conclusion, this study shows that ColoScape is a promising tool and further studies are warranted in order to validate its use for the triage of FIT+ patients.

Authors: Mauro Scimia, Jinwei Du, Francesco Pepe, Maria Antonia Bianco, Silvana Russo Spena, Farah Patell-Socha, Qing Sun, Michael J Powell, Umberto Malapelle, Giancarlo Troncone

Source: Clin Pathol 2018;0:1–4. doi:10.1136/jclinpath-2018-205412

Reducing Artifactual EGFR T790M Mutations in DNA from Formalin-Fixed Paraffin-Embedded Tissue by Use of Thymine-DNA Glycosylase

Jul 18, 2017 | Publications

Both U:G and T:G lesions in formalinfixed tissue are sources of false-positive EGFR T790M mutations. This is the first report of the use of TDG to reduce sequence artifacts in formalin-fixed DNA and is applicable to the accurate detection of mutations arising at methylated cytosines.

Authors: Hongdo Do, Ramyar Molania, Paul L. Mitchell, Rita Vaiskunaite, John D. Murdoch, and Alexander Dobrovic

Source: Clinical Chemistry 63:9 000–000 (2017) Molecular Diagnostics and Genetics 000–000 (2017), doi:10.1373/clinchem.2017.271932

Human Papillomavirus (HPV) E6/E7 mRNA Detection in Cervical Exfoliated Cells: a Potential Triage for HPV-Positive Women

Feb 8, 2017 | Publications

Cytology triage has been generally recommended for human papillomavirus (HPV)-positive women, but is highly dependent on well-trained cytologists. The present study was designed to explore whether HPV E6/E7 mRNA detection in cervical exfoliated cells can be a potential triage for HPV-positive women from a clinic-based population. Both the primary HPV testing and Papanicolaou (Pap) test were performed on all eligible HPV-positive women. HPV E6/E7 mRNA was detected by QuantiVirus® HPV E6/E7 mRNA assay in cervical exfoliated cells. All HPV-positive women underwent colposcopy and further biopsy if indicated. The data were assessed by Pearson’s Chi-squared test and the receiver operating characteristic curve. A total of 404 eligible HPV-positive women were enrolled. Positive rate of E6/E7 mRNA in high-grade squamous intraepithelial lesion (HSIL) cases was higher than that in low-grade squamous intraepithelial lesion (LSIL) or normal cases. There was no statistical difference found between mRNA and cytological testing with sensitivity (89.52% vs. 86.67%, P=0.671), specificity (48.96% vs. 48.96%, P=1.000), positive predictive value (39.00% vs. 38.24%, P=1.000), and negative predictive value (92.76% vs. 90.97%, P=0.678) for detecting ≥HSIL. HPV E6/E7 mRNA detection in cervical exfoliated cells shows the same performance as Pap triage for HSIL identification for HPV-positive women. Detection of HPV E6/E7 mRNA may be used as a new triage option for HPV-positive women.

Keywords: Human papillomavirus (HPV); HPV E6/E7 mRNA; High-grade squamous intraepithelial lesion (HSIL http://dx.doi.org/10.1631/jzus.B1600288

Authors: Ye-li Yao, Qi-fang Tian, Bei Cheng, Yi-fan Cheng, Jing Ye, Wei-guo Lu

Source: J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2017 18(3):256-262

Genetic Alteration and Mutation Profiling of Circulating Cell-free Tumor DNA (cfDNA) for Diagnosis and Targeted Therapy of Gastrointestinal Stromal Tumors

Jul 21, 2016 | Publications

Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wildtype alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived “driver” and “drug-resistant” alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called “liquid biopsy” allows for the dynamic monitoring of the patients’ tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

Keywords: Gastrointestinal stromal tumors, Liquid biopsy, Mutations, Targeted therapy

Authors: Weixin Yan, Aiguo Zhang and Michael J. Powell

Source: Chinese Journal of Cancer, Chin J Cancer (2016) 35:68 DOI 10.1186/s40880-016-0131-1

The Application of Molecular Diagnostics to Stained Cytology Smears

May 1, 2016 | Publications

Detection of mutational alterations is important for guiding treatment decisions of lung non–small-cell carcinomas and thyroid nodules with atypical cytologic findings. Inoperable lung tumors requiring further testing for staging and thyroid lesions often are diagnosed using only cytology material. Molecular diagnostic tests of these samples typically are performed on cell blocks; however, insufficient cellularity of cell blocks is a limitation for test performance. In addition, some of the fixatives used while preparing cell blocks often introduces artifacts for mutation detection. Here, we applied QClamp xenonucleic technology and quantitative RT-PCR to cells microdissected directly from stained cytology smears to detect common alterations including mutations and translocations in non–small-cell carcinomas and thyroid lesions. By using this approach, we achieved a 1% molecular alteration detection rate from as few as 50 cells. Ultrasensitive methods of molecular alteration detection similar to the one described here will be increasingly important for the evaluation of molecular alterations in clinical scenarios when only tissue samples that are small are available.

Authors: Maja H. Oktay, Esther Adler, Laleh Hakima, Eli Grunblatt, Evan Pieri, Andrew Seymour, Samer Khader, Antonio Cajigas, Mark Suhrland, Sumanta Goswami

Source: The Journal of Molecular Diagnostics, May 2016Volume 18, Issue 3, Pages 407–415, DOI: https://doi.org/10.1016/j.jmoldx.2016.01.007

Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas

April 27, 2016 | Publications

Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measurable levels of at least three of the five mutations. HRAS G12D was significantly increased inDCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10−3 to 4.6 × 10−2) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047Rmutant cells have a selective advantage in breast, contribute to breast cancer susceptibility, and drive tumor progression during breast carcinogenesis, even when present as only a subpopulation of tumor cells.

Authors: Meagan B. Myers, Malathi Banda, Karen L. McKim, Yiying Wang, Michael J. Powell and Barbara L. Parsons

Source: Neoplasia (2016) 18, 253–263

An Evaluation of the Cobas4800 HPV Test on Cervico-Vaginal Specimens in Liquid versus Solid Transport Media

Feb 1, 2016 | Publications

Objectives: Determine the ability of the Cobas 4800 assay to detect high-risk human papillomavirus (HrHPV) and high-grade cervical lesions when using cervico-vaginal samples applied to liquid medium and solid media cards compared to a direct cervical sample. Methods: Two cervico-vaginal specimens (pseudo self-collected) were obtained from 319 women. One was applied to an iFTA Card (FTA) then the brush placed in liquid-based medium (LSELF); the other was applied to a new solid media: POI card (POI). The clinical performance of Cobas4800 assay using the three aforementioned specimens was compared to direct collected endocervical specimens in liquid media (LDOC). Results: The overall agreements of HrHPV detection were 84.2% (LSELF vs. LDOC), 81.0%(FTA vs. LDOC), and 82.3% (POI vs. LDOC). LSELF, FTA and POI identified 98.0%, 79.6%, and 97.5% positive cases of LDOC. Sensitivity to identify CIN2+ were 98.4% (LSELF), 73.8% (FTA), 95.1%(POI), and 93.4%(LDOC) respectively. FTA had 78.1% and 90.4% agreement with the LSELF samples for all HrHPV and HPV16/18 detection respectively, while POI had 91.6%for both. Conclusions: Cobas4800 HPV test combined with cervico-vaginal specimens applied to both liquid media and POI solid card are accurate to detect HrHPV infection and high-grade cervical lesions as compared with direct endocervical samples in liquid media.

Authors: Hongxue Luo, Hui Du, Kathryn Maurer, Jerome L. Belinson, Guixiang Wang, Zhihong Liu, Lijie Zhang, Yanqiu Zhou, Chun Wang, Jinlong Tang, Xinfeng Qu, Ruifang Wu

Source: PLOSONE|DOI:10.1371/journal.pone.0148168

Evaluation of a New Solid Media Specimen Transport Card for High Risk HPV Detection and Cervical Cancer Prevention

Dec 29, 2015 | Publications

Background: Solid media transport can be used to design adaptable cervical cancer screening programs but currently is limited by one card with published data.Objective: To develop and evaluate a solid media transport card for use in high-risk human papilloma virus detection (HR–HPV).Study design: The Preventative Oncology International (POI) card was constructed using PK 226 paper®treated with cell-lysing solution and indicating dye. Vaginal samples were applied to the POI card and the indicating FTA (iFTA) elute card. A cervical sample was placed in liquid media. All specimens were tested for HR–HPV. Color change was assessed at sample application and at card processing. Stability of the POI card and iFTA elute card was tested at humidity. Results: 319 women were enrolled. Twelve women had at least one insufficient sample with no difference between media (p = 0.36). Compared to liquid samples, there was good agreement for HR-HPV detection with kappa of 0.81 (95% CI 0.74–0.88) and 0.71 (95% CI 0.62–0.79) for the POI and iFTA elute card respectively. Sensitivity for ≥CIN2 was 100% (CI 100–100%), 95.1% (CI 92.7–97.6%), and 93.5% (CI 90.7–96.3%)for the HR–HPV test from the liquid media, POI card, and iFTA elute card respectively. There was no color change of the POI card noted in humidity but the iFTA elute card changed color at 90% humidity. Conclusions: The POI card is suitable for DNA transport and HR–HPV testing. This card has the potential to make cervical cancer screening programs more affordable worldwide.

Keywords: Cervical cancer screening, Solid media transport, High-risk HPV testing

Authors: Kathryn Maurer, Hongxue Luo, Zhiyong Shen, Guixiang Wang, Hui Du,Chun Wang, Xiaobo Liu, Xiamen Wang, Xinfeng Qu,Ruifang Wu,Jerome Belinson

Source: Journal of Clinical Virology 76 (2016) 14–19

High Sensitivity Detection of Tumor Gene Mutations

Feb 16, 2015 | Publications

To reduce the wild-type background and improve sensitivity, a molecular clamp has been designed to hybridize selectively to wild-type template DNA and block its amplification. This molecular clamp consists of a synthetic, sequence-specific Xeno-nucleic acid (XNA) probe. It is called QClamp™. In the presence of a mutation such as a single nucleotide polymorphism (SNP) gene deletion, insertion, or rearrangement in the region of the XNA probe sequence, the XNA probe molecule melts off the mutant template DNA during the PCR cycling process, and only mutant templates are amplified efficiently. QClamp has been shown to be a sensitive and precise quantitative PCR (qPCR) technology. It is able to block the amplification of wild-type DNA from samples. In addition, it can detect low-frequency genetic mutations (<0.05%) in DNA samples obtained from patient tumor biopsy or whole blood samples. This level of sensitivity enables detection of gene mutations in the oncology therapeutic clinical setting utilizing patient biopsy, surgical tissue, or formalin fixed, paraffin-embedded (FFPE) tissue.

Authors: Michael Powell J, Madhuri Ganta, Elena Peletskaya, Larry Pastor, Melanie Raymundo

Source: et al. (2015) High Sensitivity Detection of Tumor Gene Mutations. BAOJ Cancer 001.

Diagnostic Validity of Human Papillomavirus E6/E7 mRNA Test in Cervical Cytological Samples

Oct 22, 2013 | Publications

Human papillomavirus (HPV) DNA tests tend to show high sensitivity, but poor specificity in detecting high-grade cervical lesions. This study aimed to explore the clinical performance of QuantiVirus®HPVE6/E7 mRNA in identifying ≥Grade 2 cervical intraepithelial neoplasia. Thin-prep®liquid based cytology test (LBC) samples were collected from October 2009 to October 2011 from women who underwent out-patient hospital-based gynecological screening. LBC samples were processed for E6/E7 mRNA detection and HPV DNA detection. Of 335 patients, 135 (40.3%) were HPV E6/E7 mRNA positive for high-risk HPV subtypes. The positivity rate of HPV E6/E7 mRNA increased with the severity of cytological and histo-logical evaluation. An optimal cut-off value of ≥567 copies/ml was determined using receiver operating characteristic (ROC) curve, and positive predictive value and negative predictive value of cut-off value(≥567 copies/ml) were higher than those of E6/E7 mRNA positivity only, but not significant. QuantiVirus®HPV E6/E7 mRNA testing may be a valuable tool in triage for identifying ≥Grade 2 cervical intraepithe-lial neoplasia. A high specificity and a low positivity rate of E6/E7mRNA testing as a triage test in HPVDNA-positive women can be translated into a low referral for colposcopy. Studies composed of large population-based samples of women and with rigorous disease ascertainment, are needed to establish the optimal cut-off point based on ROC curve analysis.

Keywords: HPV E6/E7 mRNA, Human papillomavirus, Cervical intraepithelial neoplasia

Authors: Tong-Yu Liu, Rong Xie, Li Luo, Kathleen H. Reilly, Cheng He, Yu-Zhen Lin, Gang Chen, Xiong-Wei Zheng, Lu-Lu Zhang, Hai-Bo Wang

Source: J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.10.032

QuantiVirus® HPV E6/E7 RNA 3.0 Assay (bDNA) is as Sensitive, but Less Specific Than Hybrid Capture 2 Test

Nov 11, 2012 | Publications

Human papillomavirus (HPV) infection is the primary cause of cervical cancer. The Quantivirus® HPV E6/E7 RNA 3.0 assay (DiaCarta, CA, USA) detects E6/E7 mRNA of 13 high-risk subtypes and 6 low-risk subtypes. Cervical specimens collected in PreservCyt were processed for HPV detection. Cervical biopsies were taken only from those women with abnormal colposcopy. 200 out of 272 (73.5%) cases were mRNA positive. The percentage of HPV E6/E7 mRNA positive samples increases with the severity of the cytological diagnosis, but not in histological diagnosis. In 146 patients with both tests, the E6/E7 mRNA assay had significant higher positivity rate than the Hybrid Capture 2 assay (75.3% versus 62.3%). The HPV mRNA assay and the HC2 assay had the same sensitivity of high grade cervical intraepithelial neoplasia (CIN 2+), 82.4% (14/17) (95% confidence interval [CI], 64.3, 100). However, the specificity of CIN 2+ for the HPV mRNA assay was significantly lower than HC2 assay. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the E6/E7 mRNA and HC2. E6/E7 mRNA achieved 58.8% sensitivity with 74.1% specificity, HC2, achieved 47.1% sensitivity with 70.7% specificity. The overall performance of HPV E6/E7 mRNA assay for detecting CIN 2+ was lower than HC2. This study does not support the use of this assay in screening for cervical cancer prevention alone.

Keywords: Human papillomavirus, E6/E7 mRNA, HPV DNA, Cervical lesion, Liquid-based cytology triage

Authors: Yong Shen, Jiaomei Gong, Yanxia He, Guomei Cheng, Paul Okunieff, Xiaofu Li

Source: of Virological Methods 187 (2013) 288– 293

LOH and Copy Neutral LOH (cnLOH) Act as Alternative Mechanism in Sporadic Colorectal Cancers with Chromosomal and Microsatellite Instability

Feb 4, 2011 | Publications

Background and aims: Tumor suppressor genes are often located in frequently deleted chromosomal regions of colorectal cancers (CRCs). In contrast to microsatellite stable (MSS) tumors, only few loss of heterozygosity (LOH) studies were performed in microsatellite instable (MSI) tumors, because MSI carcinomas are generally considered to be chromosomally stable and classical LOH studies are not feasible due to MSI. The single nucleotide polymorphism (SNP) array technique enables LOH studies also in MSI CRC. The aim of our study was to analyse tissue from MSI and MSS CRC for the existence of (frequently) deleted chromosomal regions and tumor suppressor genes located therein. Methods and results. We analyzed tissues from 32 sporadic CRCs and their corresponding normal mucosa(16 MSS and 16 MSI tumors) by means of 50K SNP array analysis. MSS tumors displayed chromosomal instability that resulted in multiple deleted (LOH) and amplified regions and led to the identification of MTUS1 (8p22)as a candidate tumor suppressor gene in this region. Although the MSI tumors were chromosomally stable, we found several copy neutral LOHs (cnLOH) in the MSI tumors; these appear to be instrumental in the inactivation of the tumor suppressor gene hMLH1 and a gene located in chromosomal region 6pter–p22. Discussion. Our results suggest that in addition to classical LOH, cnLOH is an important mutational event in relation to the carcinogenesis of MSS and MSI tumors, causing the inactivation of a tumor suppressor gene without copy number alteration of the respective region; this is crucial for the development of MSI tumors and for some chromosomal regions in MSS tumors.

Authors: Ralph Melcher, Elena Hartmann, Waltraud Zopf, Sabine Herterich, Philipp Wilke, Ludwig Muller, Eduard Rosler, Theodor Kudlich, Oliver Al-Taie, Andreas Rosenwald, Tiemo Katzenberger, Bettina Scholtka, Stefan Seibold, Dorothee Rogoll, Wolfgang Scheppach, Michael Scheurlen and Hardi Luhrs

Source: Carcinogenesis vol.32 no.4 pp.636–642, 2011 doi:10.1093/carcin/bgr011

A Gene Marker Panel Covering the Wnt and the Ras-Raf-MEK-MAPK Signalling Pathways Allows to Detect Gene Mutations in 80% of Early (UICC I) Colon Cancer Stages in Humans

May 30, 2009 | Publications

Background: Very recently a gene marker panel that allows the mutation analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/ or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early-stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.

Keywords: APC B-RAF CTNNB1 Colorectal carcinomas K-RAS Microsatellite instability Oncogenes Tumour suppressor genes

Authors: Bettina Scholtka, Mandy Schneider, Ralph Melcher, Tiemo Katzenberger, Daniela Friedrich, Kornelia Berghof-Jager, Wolfgang Scheppach, Pablo Steinberg

Source: Cancer Epidemiology 33 (2009) 123–129

Detection of the Hereditary Hemochromatosis Gene Mutation by Real-Time Fluorescence Polymerase Chain Reaction and Peptide Nucleic Acid Clamping

Feb 10, 1998 | Publications

Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new doublestranded DNA-selective fluorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that fluorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.

Authors: Erich M. Kyger, Mark D. Krevolin, and Michael J. Powell

Source: Analytical Biochemistry 260, 142–148 (1998) Article No. AB982687

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