PCR Fragment Analysis Research Articles

One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

Frameshift Mutations in Leukemia Associated Genes Correlate with Superior Outcomes in Patients Undergoing Allogeneic Stem Cell Transplant for De Novo Acute Myeloid Leukemia Compared to Other Types of Mutations

Background Allogeneic stem cell transplant (allo-SCT) is a mainstay of treatment for acute myeloid leukemia (AML) and offers the only chance of cure. The success of allo-SCT hinges greatly on the immune response of donor T lymphocytes against leukemia cells, known as graft versus leukemia effect (GvL). One of the key potential drivers of GvL is immune response to mutation derived neoantigens. Previous studies in solid tumors have demonstrated enhanced immunogenicity of frameshift derived peptides vs those from other mutation types (e.g. substitutions) due to the propensity of frameshifts to precipitate long novel amino acid strings distal to the site of mutation. We therefore hypothesized that AML cases bearing somatic frameshift mutations (FS) in leukemia associated genes would be more immunogenic than those with only other types of mutations (non-FS), and thus benefit more from allo-SCT via more robust GvL. Methods We retrospectively searched our institutional electronic medical records for de Novo AML patients who had undergone allo-SCT between 2009 and 2022 and had next generation sequencing data available on diagnostic specimens using a 42 gene hot spot panel. Via the Kaplan-meier method, we analyzed and compared the impact of tumor mutations present at diagnosis on overall survival and relapse free survival based on frameshift versus no frameshift status. Frameshift mutations only in DNMT3A, TET2 or ASXL1 (DTA) were not included in the frameshift group as previous studies have demonstrated DTA mutations to reflect mere clonal hematopoiesis and not to be strongly blast associated. Results 89 de Novo AML patients were identified who had at least one mutation detected by NGS or PCR fragment analysis. Frameshifted genes identified included RUNX1, WT1, TP53, BCOR, CEBPA, STAG2, PHF6, ZRSR2, TET2, ASXL1 and NPM1. Our data shows superior overall survival (p=0.038; HR 2.1) post-transplant in patients who have at least one gene with frameshift mutations (n=22) other than DTA or NPM1versus those with only other types of mutations [FIGURE 1]. Relapse free survival was also significantly better (HR 3.8) in the frameshift group by Gehan-Breslow-Wilcox analysis (p=0.038) but not by the log rank test (p=0.058). Patients with non DTA frameshifts other than NPM1 further showed superior overall survival to patients whose only frameshift was NPM1 (HR 2.3, p=0.045). Three patients showed both non-frameshift and frameshift mutations in the same gene and showed significantly poorer overall survival than those with at least one exclusively frameshifted gene (HR=4.3, p=0.013). Additionally, among patients with RUNX1 mutations (n=15) specifically, those with exclusively frameshifted RUNX1 (n=9) demonstrated superior overall survival (HR 4.8, p=0.042) to those who harbored non-frameshifted RUNX1 (n=6) [Figure 2]. Other mutations occurred with insufficient frequency to individually compare FS vs non-FS survival effects at the level of single genes. Conclusion Though limited by small sample size, our data suggest that frameshift mutated AML cases may benefit more from allo-SCT than those with only non-FS mutations, possibly due to increased generation of immunogenic neoepitopes. If validated in larger studies, incorporation of somatic frameshift mutation status in AML risk stratification models could improve existing patient selection algorithms for bone marrow transplant and thereby lead to superior outcomes.

Streamlining the Detection of FLT3 Internal Tandem Duplication (FLT3-ITD) Using the Cepheid Genexpert® Automated System

Objectives: The development of targeted therapies for Acute Myeloid leukemia (AML) demands fast and reliable diagnostic tests. Specifically, FMS-like tyrosine kinase 3 (FLT3-ITD), comprising nearly 25% of all AML cases, is the most common targetable mutation in AML. Current diagnosis of FLT3-ITD by PCR involves numerous laborious steps, with bench-top steps involving time-consuming whole blood processing, DNA isolation, plate-based PCR and subsequent fragment analysis by capillary electrophoresis. Consequently this adds significant overhead, time-consuming personnel training, and numerous steps which could lead to errors, all of which increase the resource burden and time to result. The objective of this study was to create an automated FLT3-ITD diagnostic prototype that is faster, requires less hands-on time that can detect all clinically relevant ITD lengths. Methods: We have developed a simplified FLT3-ITD prototype assay using the Cepheid GeneXpert® automated platform for FLT3-ITD detection. The prototype substantially streamlines the process, using a less than 30 minutes off-board time to prepare a universal blood lysate that is compatible with all Cepheid Hematology Oncology products. Once the sample is loaded into the cartridge and inserted into the instrument, the cartridge performs automated DNA isolation, purification, and qPCR. A Taqman-based probe for ABL is utilized in the qPCR step as a PCR control to ensure that the blood processing and qPCR was successful and avoid wasted downstream steps. Quantitative fragment analysis can easily be performed on the PCR product using capillary electrophoresis (CE) for precise allelic ratio (AR) callout. Additionally, the PCR product is compatible be run on a bioanalyzer or standard Agarose Gel-Electrophoresis if available. Results: The developed automated FLT3-ITD diagnostic prototype demonstrated significant improvements over the existing methodologies with substantially reduced time to result and simplicity in use. To assess the sensitivity of this prototype, we used MV4-11 cancer cells harboring a 30 bp ITD and CRISPR-CAS engineered human cell lines with 3 bp, 21 bp, and 75 bp FLT3 ITD insertions, comprising the most commonly detected ITD lengths spiked into EDTA blood as a test material. For these ITD's, we observed sensitivity and linearity down to 0.01-0.02 by CE for the ITDs tested. Importantly, the prototype detected all lengths of ITD mutations with inserts from 3bp to 300 bp using plasmid test materials. Conclusion: We have successfully developed a novel FLT3-ITD diagnostic prototype that has several major advantages over existing methodologies. The prototype significantly reduces the time and hands-on effort required for analysis, streamlining the diagnostic process for same day results. The larger sample volume used allows improved sensitivity to ensure accurate detection, even at low allelic frequencies. Additionally, the prototype requires fewer reagents in that sample prep and qPCR are performed in a single cartridge and run hands-free. Downstream analysis of amplified products can be easily performed and validated using capillary electrophoresis, bioanalyzer, or gel electrophoresis. Finally, because this prototype uses the same universal blood lysate as other AML tests, it has the potential to improve future diagnostic therapeutic interventions in the clinic.

Deep sequencing reveals recurrent somatic mutations and distinct molecular subgroups in gastric cancer in Mizo population, North East India

In India, Mizoram has the highest incidence of gastric cancer (GC) which might be associated with environmental factors such as diet, Helicobacter pylori (H.pylori) and Epstein-Barr virus (EBV) infections, and somatic genomic alterations. We performed PCR cum sequencing and fragment analysis for detection of H. pylori/EBV infection and microsatellite Instability (MSI) in GC patients (N = 68). Somatic mutations were identified by targeted and exome sequencing. We found 87% of GC patients infected with H. pylori and or EBV. Pathogenic infections were mostly mutually exclusive with only 16% of coinfection. TP53, MUC6, and ARID1A were significantly mutated. Two molecular subgroups with distinctive mutational profiles were identified: (1) patients harboring mutations in TP53 and (2) patients harboring mutations in RTK/RAS/PI3-K signaling pathway and chromatin-remodeling genes. Therefore, EBV and H. pylori infections and somatic mutations in the genes involved in RTK/RAS/PI3K signaling pathway, chromatin-remodeling, and TP53 might drive GC development and progression in Mizo patients.

Determination of polar bear (Ursus maritimus) individual genotype and sex based on DNA extracted from paw-prints in snow

Polar bears rely upon sea ice to hunt, travel, and reproduce. Declining sea ice extent and duration has led polar bears to be designated as “threatened” (ESA). Population monitoring is vital to polar bear conservation; but recently, poor sea ice has made traditional aircraft-based methods less viable. These methods largely rely upon the capture and handling of polar bears, and have been criticized over animal welfare concerns. Monitoring polar bears via DNA sampling is a promising option. One common method utilizes biopsy darts delivered from a helicopter to collect DNA, a method that faces similar ice associated challenges to those described above. However, epidermal cells shed from the foot pads of a polar bear into its paw-prints in snow are a source of “environmental DNA” (e-DNA) that can be collected non-invasively on the sea ice or on land for potential use in population monitoring. Mitochondrial DNA (mt-DNA) is used to assess whether polar bear DNA is present within a snow sample, and nuclear DNA (n-DNA) can identify individuals and their sex. The goal of this investigation was to assess the viability of using e-DNA collected from paw-prints in the snow to identify individual polar bears and their sex. Snow was sampled from 13 polar bear trails (10 paw-prints per trail) on the sea ice in the Chukchi and Beaufort seas along the North Slope of Alaska. Species verification was based on a mt-DNA PCR fragment analysis test. Identification of individuals was accomplished by amplifying a multiplex of seven n-DNA microsatellite loci, and sex was determined by the amelogenin gene sex ID marker. Six of the 13 bear trails sampled (46%) yielded consensus genotypes for five unique males and one female. To our knowledge, this is the first time that polar bears have been individually identified by genotype and sex using e-DNA collected from snow. This method is non-invasive, could be integrated into genetic mark-recapture sampling designs, and addresses some of the current challenges arising from poor sea ice conditions. It also can involve, engage, and empower Indigenous communities in the Arctic, which are greatly affected by polar bear management decisions.

Microsatellite Instability in Russian Patients with Colorectal Cancer.

The aim of this study was to determine the characteristics of Russian patients with microsatellite instability (MSI) tumors. MSI in the tumor was determined in 514 patients with colon cancer using PCR and subsequent fragment analysis for five markers (NR21, NR24, BAT25, BAT26, and NR27). In the presence of microsatellite instability, the mismatch repair (MMR) system genes were examined using the NGS and MLPA methods to establish the diagnosis of Lynch syndrome. The overall frequency of MSI tumors was 15%: at stage I—19% (9/48), at stage II—21% (44/213), at stage III—16% (26/160), and at stage IV—2% (2/93). Patients with MSI tumors differed in the age of diagnosis, tumor localization, time of cancer recurrence, and stage of the disease. The overall and disease-free survival of patients whose tumors had MSI status was higher than that of patients with microsatellite-stable status, p = 0.04 and p = 0.02, respectively. Analysis of overall and disease-free survival of patients with Lynch syndrome and patients with sporadic colon cancer, but with MSI status, did not reveal significant differences, p = 0.52 and p = 0.24, respectively. The age of patients with Lynch syndrome was significantly younger than that of patients with sporadic colon cancer whose tumors had MSI status (p < 0.001).

Homozygosity of short VNTR lengths in the CEL gene may confer susceptibility to idiopathic chronic pancreatitis

ObjectiveThe carboxyl-ester lipase (CEL) gene contains a variable number of tandem repeats (VNTR) region. It remains unclear whether the number of repeats in the CEL VNTR is related to the risk of pancreatic diseases. The aim of this study was to investigate whether CEL VNTR length is associated with idiopathic chronic pancreatitis (ICP), alcoholic chronic pancreatitis (ACP), or pancreatic cancer in a cohort of Chinese patients. MethodsCEL VNTRs were genotyped in patients diagnosed with ICP (n = 771), ACP (n = 222), or pancreatic cancer (n = 263), and in healthy controls (n = 927). CEL VNTR lengths were determined using a screening method combining PCR and DNA fragment analysis. ResultsOverall, the CEL VNTR lengths ranged from 5 to 22 repeats, with the 16-repeat allele (‘normal’ size, N) accounting for 73.82% of all observed alleles. The VNTR allele frequencies and genotype distributions were not significantly different between healthy controls and patients with ACP or pancreatic cancer. For the ICP group, allele frequencies did not differ significantly from the controls, while the frequency of the SS genotype (homozygosity for 5–15 repeats) was significantly higher in the patients (4.67%) than in the controls (1.94%) (p = 0.0014; OR = 2.47; 95% CI = 1.39–4.39). ConclusionsThere were no associations between the CEL VNTR length and ACP or pancreatic cancer. However, homozygosity for short VNTR lengths may confer susceptibility to ICP.

CML-152: Investigation of Short Tandem Repeat Instability of Mismatch Repair Genes in Hematologic Malignancies

Context: In the present study, we sought answers to the following questions: 1. Do mutations in mismatch repair genes contribute substantially toward progression of hematologic malignancies? 2. Are blood cancers solely driven by chromosomal instabilities, or does microsatellite instability play a role in their causation? Objective: Recently, the concept of dark DNA is gaining momentum in cancer research. Short tandem repeats (STRs) are more prevalent in intronic regions, and due to their evolutionary relevance, tandem repeat hypervariability in both protein coding and non-coding regions is being investigated in various cancer subtypes. However, their occurrence in hematologic malignancies is highly debatable. Here, we evaluate the instability of certain STRs in 10 selective repair genes in various hematologic cell lines. Design: We selected MMR proficient/deficient, underemphasized hematologic cell lines (K562, Molt-4, Daudi, KG-1), along with extensively studied microsatellite instable colorectal (HCT116) and breast (MCF7) cancer cells to decipher the hypervariability of TRs. The primers were selected for gene sequence-specific intronic/exonic TRs. The splice sites were analyzed for individual markers. The TR instability was investigated via PCR and capillary electrophoresis-based fragment analysis. The two sample F-test was performed for statistical analysis. Results: The overall results suggest that TR hypermutability alongside chromosomal aberrations could be the underlying cause of myeloid and lymphoid malignancies. As per the ASSP online tool, the PCR products had cryptic splice sites, which could alter with expansion and loss of repeat sequences. A statistically significant TR variation was observed for MSH2, MSH4, and MSH6 (P Conclusion: Taken together, the mutable TRs in intronic and non-intronic regions of DNA repair genes in blood cancer cells might have a tumorigenic role. Although the repeat instabilities are causally reflected in treated patients, primary patient sample studied for such repeat variability can have huge diagnostic relevance.

Epidemiological surveillance of common respiratory viruses in patients with suspected COVID-19 in Southwest China

BackgroundThe outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the peak season of common respiratory viral infections. However, the clinical symptoms of most SARS-CoV-2 infected patients are not significantly different from those of common respiratory viral infections. Therefore, knowing the epidemiological patterns of common respiratory viruses may be valuable to improve the diagnostic and therapeutic efficacy of patients with suspected COVID-19, especially in Southwest China (a mild epidemic area).MethodsA total of 2188 patients with clinically suspected of COVID-19 in Southwest China were recruited from January 21 to February 29, 2020. Nasopharyngeal swabs, throat swabs and sputum specimens were collected to detect SARS-CoV-2 by using real-time reverse transcription-polymerase chain reaction (RT-PCR) and other 12 viruses via PCR fragment analysis combined with capillary electrophoresis. Clinical characteristics and laboratory test findings were acquired from electronic medical records. All data were analyzed to unravel the epidemiological patterns.ResultsOnly 1.1% (24/2188) patients with suspected COVID-19 were eventually confirmed to have SARS-CoV-2 infection, and the most frequently observed symptoms were fever (75.0%, 18/24) and cough (20.8%, 5/24). The overall detection rate of other respiratory pathogens was 10.3% (226/2188). Among them, human rhinovirus (3.2%, 71/2188), human parainfluenza viruses (1.6%, 35/2188), influenza B virus (1.2%, 26/2188) and mycoplasma pneumonia (1.2%, 26/2188) were the predominantly detected pathogens in this study. Moreover, the co-infection was observed in 22 specimens. Notably, one COVID-19 case had a coexisting infection with human parainfluenza virus (4.2%, 1/24) and bocavirus was the most common virus tending to occur in co-infection with other respiratory pathogens.ConclusionsThis study reveals the epidemiological features of common respiratory viruses and their clinical impact during the ongoing outbreak of COVID-19 in a mild epidemic area. The findings highlight the importance of understanding the transmission patterns of the common respiratory virus in COVID-19 regions, which can provide information support for the development of appropriate treatment plans and health policies, while eliminating unnecessary fear and tension.

Comparison of Delta- PCR and Conventional Fragment Analysis for the Detection of FLT3-ITD Mutations in Paired Diagnosis-Relapse Samples of Patients with Acute Myeloid Leukemia

Comparison of Delta- PCR and Conventional Fragment Analysis for the Detection of FLT3-ITD Mutations in Paired Diagnosis-Relapse Samples of Patients with Acute Myeloid Leukemia

Диференціація генотипів сосни звичайної за поліморфізмом довжини інтронів генів дефензинів

Наведено результати дослідження генотипів сосни звичайної (Pinus sylvestris L.) із використанням молекулярних маркерів на підстаі генетичного поліморфізму довжини інтронів генів дефензинів PsDef1-4. Виявлено високу інформативність маркерів на підстаі інтрону гена PsDef2 (IPL-PsDef2) для дослідження внутрішньовидового поліморфізму в сосни звичайної. Встановлено межі осередка інфекції кореневої губки (Heterobasidion annosum (Fr.) Bref.) в сосновому насадженні за допомогою полімеразно-ланцюгової реакції (ПЛР) із використанням видоспецифічних праймерів. Проведено ДНК-профілювання модельних дерев способом електрофоретичного розділення ампліконів IPL-PsDef2 маркерів. За результатами поліморфізму довжини інтронів генів дефензинів здійснено кластеризацію генотипів сосни звичайної із використанням алгоритму UPGMA. на підстаі дендрограми виділено два кластери генотипів зі 100 % бутстреп підтримкою, які відрізняються за стійкістю до кореневої губки. Методом К-середніх визначено значущість кожного з ампліконів для диференціації генотипів сосни на групи. Виявлено амплікони, які можуть бути генетичними маркерами для оцінки потенційної стійкості генотипів сосни звичайної до кореневої губки. Отримані дані щодо поліморфізму локусів генів дефензинів, а також результати кластерного аналізу вказують на перспективність використання IPL-PsDef2 маркерів для генотипування сосни звичайної.

A Broad Test Based on Fluorescent-Multiplex PCR for Noninvasive Prenatal Diagnosis of Cystic Fibrosis

Background: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF). Methods: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele. Results: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis. Conclusions: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests.

Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients.

Microsatellite instability (MSI) testing identifies patients who may benefit from immune checkpoint inhibitors. We developed an MSI assay that uses data from a commercially available next‐generation sequencing (NGS) panel to determine MSI status. The assay is applicable across cancer types and does not require matched samples from normal tissue. Here, we describe the MSI‐NGS method and explore the relationship of MSI with tumor mutational burden (TMB) and PD‐L1. MSI examined by PCR fragment analysis and NGS was compared for 2189 matched cases. Mismatch repair status by immunohistochemistry was compared to MSI‐NGS for 1986 matched cases. TMB was examined by NGS, and PD‐L1 was determined by immunohistochemistry. Among 2189 matched cases that spanned 26 cancer types, MSI‐NGS, as compared to MSI by PCR fragment analysis, had sensitivity of 95.8% (95% confidence interval [CI] 92.24, 98.08), specificity of 99.4% (95% CI 98.94, 99.69), positive predictive value of 94.5% (95% CI 90.62, 97.14), and negative predictive value of 99.2% (95% CI, 98.75, 99.57). High MSI (MSI‐H) status was identified in 23 of 26 cancer types. Among 11,348 cases examined (including the 2189 matched cases), the overall rates of MSI‐H, TMB‐high, and PD‐L1 positivity were 3.0%, 7.7%, and 25.4%, respectively. Thirty percent of MSI‐H cases were TMB‐low, and only 26% of MSI‐H cases were PD‐L1 positive. The overlap between TMB, MSI, and PD‐L1 differed among cancer types. Only 0.6% of the cases were positive for all three markers. MSI‐H status can be determined by NGS across cancer types. MSI‐H offers distinct data for treatment decisions regarding immune checkpoint inhibitors, in addition to the data available from TMB and PD‐L1.

Standardization and validation of PCR-fragment analysis based assay for detection of mixed donor chimerism following bone marrow transplant

Standardization and validation of PCR-fragment analysis based assay for detection of mixed donor chimerism following bone marrow transplant

Detection of Immunoglobulin Heavy Chain Gene Clonality by Next-Generation Sequencing for Minimal Residual Disease Monitoring in B-Lymphoblastic Leukemia.

Minimal residual disease (MRD) following B-lymphoblastic leukemia (B-ALL) treatment has gained prognostic importance. Clonal immunoglobulin heavy chain (IGH) gene rearrangement is a useful follow-up marker in B-ALL owing to its high positivity rate. We evaluated the performance and clinical applicability of a next-generation sequencing (NGS) assay for IGH rearrangement in B-ALL MRD monitoring. IGH rearrangement was tested by using fluorescence PCR-fragment analysis and the NGS assay in eight B-ALL patients. The NGS assay was run on two platforms: the Ion Torrent PGM (Thermo Fisher Scientific, USA) (18 samples from 1st to 7th patients) and the MiSeq system (Illumina, USA) (four samples from 8th patient). All initial diagnostic samples and four follow-up samples were positive for clonal IGH rearrangement with fluorescence PCR-fragment analysis and the NGS assay, and six follow-up samples were positive only with NGS. In one case with BCR-ABL1 translocation, BCR-ABL1 quantitative PCR was negative but the NGS IGH assay was positive just prior to full-blown relapse, suggesting the high sensitivity and clinical utility of the NGS assay. The NGS assay is proposed for MRD monitoring in B-ALL Additional studies are needed to confirm the clinical implications of cases showing positive results only in NGS.

Clinical and hematological relevance of JAK2 V617F and CALR mutations in BCR-ABL-negative ET patients

ABSTRACTBackground: Classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis frequently harbor JAK2, MPL, and CALR somatic mutations.Methods: AS-PCR for JAK2 V617F, pyrosequencing for MPL W515L/K, and PCR-fragment analysis for CALR exon 9 mutations were established to analyze genomic DNA isolated from peripheral blood samples of 58 newly diagnosed ET patients in Thailand.Results: JAK2 V617F was detected in 41 patients (71%) and CALR exon 9 mutation was positive in eight patients (14%), whereas no mutation of MPL W515L/K was observed in this study. Patients with CALR mutation were older (p = 0.023) and exhibited lower number of platelet count (p = 0.041) than patients without CALR mutation. Two previously known CALR mutation types were identified in this study (six patients with CALR-type 1 and two patients with CALR-type 2). Additionally, no co-existence of JAK2 V617F and CALR mutations was identified in this work.Conclusion: We reported the frequency of JAK2 V617F, MPL W515L/K, and CALR mutations in Thai patients with ET. Clinical and hematological phenotypes of patients were associated with JAK2 and CALR mutation statuses. The combination of laboratory testing for the detection of JAK2, CALR, and MPL mutations is necessary to improve the diagnosis and classification of BCR-ABL1-negative MPN.

MP91-13 THE ASSOCIATION BETWEEN TESTOSTERONE AND VITALITY: THE ROLE OF GENETIC VARIATION IN THE ANDROGEN RECEPTOR

You have accessJournal of UrologySexual Function/Dysfunction: Medical, Hormonal & Non-surgical Therapy I1 Apr 2017MP91-13 THE ASSOCIATION BETWEEN TESTOSTERONE AND VITALITY: THE ROLE OF GENETIC VARIATION IN THE ANDROGEN RECEPTOR Matthew Panizzon, Tung-Chin Hsieh, Franz Carol, Richard Hauger, and William Kremen Matthew PanizzonMatthew Panizzon More articles by this author , Tung-Chin HsiehTung-Chin Hsieh More articles by this author , Franz CarolFranz Carol More articles by this author , Richard HaugerRichard Hauger More articles by this author , and William KremenWilliam Kremen More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2853AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Controversy still exists regarding the efficacy of testosterone replacement therapy (TRT) for symptomatic hypogonadism. A recent multi-site randomized controlled study of men undergoing TRT failed to show an improvement in several symptom domains, among them is vitality. We hypothesized that differences in androgen sensitivity would obscure symptom improvement for a subset of individuals. Androgen sensitivity is related to the number of trinucleotide (CAG) repeats on exon 1 of the androgen receptor (AR) gene. We sought to examine the regulatory role of this genetic polymorphism on the relationship between testosterone and vitality in middle-aged men METHODS Participants were men from the Vietnam Era Twin Study of Aging (N=696). Average waking, salivary free-testosterone level was acquired on 3 non-consecutive days. The AR gene CAG repeat length was derived using a combination of PCR fragment analysis and Sanger sequencing. Vitality was evaluated with the SF-36Vitality scale. We tested the interaction between low testosterone (low-T) and CAG repeat length while controlling for age, ethnicity, smoking status, BMI, heart disease, hypertension, diabetes, and the correlated nature of the twin data. Low-T was defined as being = 1 SD below the mean for the 3-day average value RESULTS Mean age of the participants was 56.4 years (SD = 2.6) with average CAG repeat length of 22 (range: 8-37). We observed a significant interaction effect between low-T and AR CAG repeat length. In men with a short variant of the AR gene (< 21 repeats), representing greater androgen sensitivity, there was a significant effect of low-T on vitality (p < 0.01). Men with low-T and the short AR gene were on average 10 points lower on the Vitality scale relative to men with normal T and the same genotype. This effect was not observed in men with either medium (21-23 repeats) or long (= 24 repeats) variants of the AR gene. Without the interaction effect in the model, neither low-T nor the AR gene had a significant effect on vitality CONCLUSIONS These results demonstrate that the relationship between low-T and vitality is regulated, in part, by variation in the AR gene. The effect of low-T on vitality was observed only when the genetically-determined sensitivity of the androgen receptor is at its greatest. Variations in the AR gene, bestowing differential androgen sensitivity, are potential targets for personalized therapy of hypogonadism. Future studies are needed to determine how genetic information can be used to improve the efficacy of TRT © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1223 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Matthew Panizzon More articles by this author Tung-Chin Hsieh More articles by this author Franz Carol More articles by this author Richard Hauger More articles by this author William Kremen More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The recipient's heme oxygenase-1 promoter region polymorphism is associated with cardiac allograft vasculopathy.

Heme oxygenase-1 (HO-1) catalyses the degradation of heme to biliverdin, free iron, and carbon monoxide. The promoter region contains a highly polymorphic (GT)n repeat, where shorter (GT)n repeat sequences are linked to higher transcriptional activity, which was shown to correlate with a cytoprotective effect. Higher HO-1 levels may protect from cardiac allograft vasculopathy. Cardiac allograft recipients transplanted between 1988 and 2012 were analyzed for the HO-1 (GT)n repeat polymorphism using PCR and DNA fragment analysis with capillary electrophoresis. A relation to cardiac allograft vasculopathy (CAV) was analyzed using Cox regression including common risk factors for CAV and the occurrence of rejection episodes as explanatory variables. A total of 344 patients were analyzed, of which 127 patients were positive for CAV (36.9%). In our multivariable Cox regression analysis, the short homozygous HO-1 (GT)n genotype with <27 repeats (S/S) revealed a higher risk for CAV (P = 0.032). Donor age (P = 0.001) and donor weight (P = 0.005) were significant predictors for CAV. A potential risk for CAV was associated with rejection episodes (P = 0.058) and history of smoking (P = 0.06). The recipient HO-1 (GT)n genotype may contribute to CAV development. This finding has to be evaluated in larger series including studies targeting the underlying disease mechanism.

Abstract B09: Molecular phenotyping of colorectal tumors in clinical practice: Assignment of extended prognostic subtypes by direct testing of endoscopic specimens

Abstract Introduction: Since the pioneering work of Fearon and Vogelstein in 1990, detailed mechanisms of colorectal neoplasia have intensively been studied (1). Only recently, however, importance of molecular subtypes for prognosis has been confirmed on large cohorts (2,3). The so-called Jass classification is set to become an part of diagnostics complementary to the companion predictive genotyping (4). The diagnosis is mostly based on examination of tumor tissue from endoscopy. It was an intention of this work to evaluate feasibility of molecular phenotyping in addition to the standard testing performed from the same source material. Patients and Methods: A total of 145 carcinomas (105 fresh biopsies or 40 FFPE sections, all stages) have been acquired over a 3-year period. The molecular subtypes were assigned based upon results of CIMP/MSI/BRAF/RAS (5). To partition the most common type arising from traditional adenoma-carcinoma pathway, we have additionally examined MLH1 methylation and APC and TP53 mutations (6). The methodology was based on previously validated protocols including MS-MLPA for the evaluation of CIMP, PCR fragment analysis MSI and high-sensitive denaturing CE assay (DCE) for somatic mutations in RAS, BRAF, TP53 and APC genes. By extending Jass classification we recognize 6 molecular subtypes with distinct features: CIMP status &amp;gt; negative &amp;gt; TP53 &amp;gt; positive &amp;gt; Traditional CIMP (median prognosis) &amp;gt; positive &amp;gt; MLH1 ------------&amp;gt; negative &amp;gt; KRAS -----------------------&amp;gt; positive &amp;gt; Traditional CIN antiEGFR resistant (median prognosis) -----------------------&amp;gt; negative &amp;gt; APC&amp;gt; positive &amp;gt; Traditional CIN antiEGFR sensitive (median prognosis) ------------&amp;gt; positive &amp;gt; BRAF --------------------&amp;gt; negative&amp;gt; MSI &amp;gt; positive &amp;gt; Familial MSI (good prognosis) --------------------&amp;gt; positive &amp;gt; MSI --------------------------------&amp;gt; negative &amp;gt; Serrated CIMP (poor prognosis) --------------------------------&amp;gt; positive&amp;gt; Serrated CIMP+MSI (best prognosis) Results: A total of 105/105 (100%) of fresh tissue samples and 33/45 (73.3%) of FFPE samples provided high-quality DNA for subsequent completion of the complete molecular testing panel. The distribution of the six types was (i) 3x Serrated CIMP (ii) 9x Serrated CIMP+MSI; (iii) 2x Familial MSI, (iv) 26x Traditional CIN antiEGFR resistant (v) 12x Traditional CIN antiEGFR sensitive and (vi) 18x Traditional CIMP. The remaining 68 carcinomas were resulting from other combinations. Conclusions: Methodology as well as logistics of sample processing has been optimized for use at endoscopy unit. The distribution of Jass molecular subtypes corresponded to the larger foreign cohorts with the dominating contribution from the Traditional CIN/CIMP subtypes. Until CIMP/CIN/MSI panels covering all markers have been developed a multi-tier testing according to the above algorithm is the most cost efficient. Assignment of molecular subtypes by an optimized protocol is useable in clinical management of patients and feasible in routine practice. Supported by IGA Ministry of Health project No. NT 14383 Literature 1. Fearon ER, Vogelstein B. Cell. 1990 Jun 1;61(5):759-67. 2. Phipps AI et al. Gastroenterology 2015; 148: 77-87.e2 3. Sinicrope FA et al. Gastroenterology 2015; 148: 88-99. 4. Modest DP et al. Ann Oncol. 2016 Jun 29. pii: mdw261. [Epub ahead of print] 5. Jass JR. Int J Colorectal Dis 1999; 14: 194-200 6. Kim JH, Bae JM, Cho NY, Kang GH. Oncotarget 2016; Epub ahead of print Citation Format: Marek Minarik, Tereza Halkova, Petra Minarikova, Barbora Belsanova, Stepan Suchanek, Jiri Cyrany, Jan Bures, Miroslav Zavoral, Lucie Benesova. Molecular phenotyping of colorectal tumors in clinical practice: Assignment of extended prognostic subtypes by direct testing of endoscopic specimens. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B09.

Association of FOXE1 polyalanine repeat region with thyroid cancer is dependent on tumour size.

Polymorphisms in the thyroid transcription factor forkhead factor E1 (FOXE1) gene have been implicated in the genetic susceptibility to differentiated thyroid cancer, but little is known about their effect on tumour characteristics. The objective of this study was to determine the contribution of the FOXE1 polyalanine repeat region to the susceptibility to thyroid cancer and to its clinical characteristics. A total of 500 patients with sporadic thyroid cancer (440 papillary and 60 follicular thyroid carcinoma) and 502 healthy controls were included in this case-control association study. The number of FOXE1 alanine repeats in each subject was determined by PCR and multiplex fragment analysis by capillary electrophoresis. FOXE1 genotype and allele frequencies among groups were compared by logistic regression and adjusted for sex and age at diagnosis. Data were analysed according to cancer subtype, tumour size and the presence of lymph node or distant metastasis. FOXE1 alleles with 16 or more alanine repeats were more frequent in patients with tumour size > 1 cm compared to tumour size ≤ 1 cm (adjusted OR 1·44; 95% CI 1·05-1·88; P = 0·019). Genotypes containing at least one allele with 16 or more alanine repeats were associated with larger tumour size (adjusted OR 1·71; 95% CI 1·15-2·57; P = 0·009). No significant differences were observed between cancer subtypes or the presence/absence of metastasis. FOXE1 polyalanine repeat polymorphisms are associated with thyroid cancer, but only for tumours larger than 1 cm, suggesting a role in disease progression.