Allele-specific Real-time Polymerase Chain Reaction Research Articles

Accurate quantitation of JAK2 V617F allele burden by array-based digital PCR.

The JAK2 V617F mutation is highly prevalent in patients with myeloproliferative neoplasms (MPN). Several studies have shown that allele burden correlates with hematologic characteristics and clinical end-points in patients with MPN. Allele-specific real-time quantitative polymerase chain reaction (RQ-PCR) is probably the most commonly used technique for detection and quantitation of the JAK2 V617F mutation. Alternatively, digital PCR is an emerging technology for absolute DNA quantitation, which is based on dilution and high-grade partitioning of a sample. We compared array-based digital PCR using the QuantStudio(™) 3D Digital PCR System platform with a RQ-PCR assay, CE-registered for in vitro diagnostic use, regarding JAK2 V617F allele quantitation. This study included 30 samples positive for the JAK2 V617F mutation and additionally 13 samples without the mutation. JAK2 V617F allele burden of samples ranged between well below 1% and more than 90%. Linear regression analysis showed a high correlation between the results obtained from the two techniques (r(2) = 0.9983). Thirteen samples tested negatively for the JAK2 V617F mutation with RQ-PCR were also found negative with digital PCR. We conclude that array-based digital PCR is an appropriate method for the quantitation of JAK2 V617F allele burden demonstrating highest correlation with allele-specific RQ-PCR.

Detection Of MYD88 L265P In Peripheral Blood Of Patients With Waldenström's Macroglobulinemia and IgM Monoclonal Gammopathy Of Undetermined Significance

Waldenström's macroglobulinemia (WM) is characterized by bone marrow (BM) infiltration with lymphoplasmacytic cells and production of an IgM paraprotein. Lymphocytosis is uncommon in WM, and many patients exhibit peripheral B-cell lymphopenia. Recently, by whole genome sequencing, we identified a highly recurrent somatic mutation (L265P) in MYD88. Using real-time allele-specific polymerase chain reaction (AS-PCR), we demonstrated the presence of MYD88 L265P in 93% and 54% of patients with WM and IgM monoclonal gammopathy of undetermined significance (MGUS), respectively (Xu et al, Blood 2013).To clarify the feasibility of using real-time AS-PCR assay to detect MYD88 L265P in peripheral blood, we first examined unselected mononuclear cells from peripheral blood (PB) samples of 88 patients with untreated WM. 81/88 (92%) of these patients were MYD88 L265P positive by bone marrow (BM) examination of CD19 selected B-cells; Of the 81 BM-MYD88 L265P positive patients, 32 (40%) demonstrated MYD88 L265P by AS-PCR in unselected PB mononuclear cells (PBMC) obtained at the time of BM examination. To enhance the ability to detect MYD88 L265P in PB samples, we next used AS-PCR in CD19 selected PBMC obtained from 198 WM patients which included untreated and previously treated patients. Among untreated patients, 106/118 (89.8%) were positive for MYD88 L265P in CD19 selected PB, whereas among previously treated patients, 55/80 (68.8%) were positive for MYD88 L265P (p=0.0002 vs. untreated). Simultaneously analyzed PB and BM samples from 65 untreated WM patients demonstrated positivity for MYD88 L265P in 58/65 (89.2%) and 55/65 (84.6%) of CD19 selected BM and PB samples, respectively. These findings yielded a sensitivity of 94.8%, specificity of 100%, positive and negative predictive values of 100% and 70%, respectively for AS-PCR testing of PB CD19+ cells for MYD88 L265P in untreated WM patients. In addition, we analyzed 12 untreated IgM MGUS patients with paired sampling of CD19 selected PB and BM cells. 6/12 (50%) IgM MGUS patients were positive for MYD88 L265P in BM CD19+ cells, with 5/6 (83%) of these positive patients demonstrating MYD88 L265P by AS-PCR in CD19 selected PB samples.The overall results demonstrate a high concordance of MYD88 L265P status between PB and BM CD19 selected samples, particularly in untreated WM patients. The detection of MYD88 L265P by CD19 selected PB AS-PCR examination provides a convenient and less invasive method to support the diagnosis of WM and IgM MGUS. Disclosures:No relevant conflicts of interest to declare.

Evaluation of 2 Real-Time PCR Assays for In Vitro Diagnostic Use in the Rapid and Multiplex Detection of EGFR Gene Mutations in NSCLC

Activating mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer predict for a favorable clinical response to tyrosine kinase inhibitor therapy. Although Sanger sequencing is a conventional method to detect EGFR gene mutations, multiplex real-time allele-specific polymerase chain reaction (PCR) systems are increasingly used in the routine molecular diagnostic setting. We aim to evaluate 2 proprietary real-time PCR assays (cobas and therascreen) against Sanger sequencing in the detection of EGFR gene mutations. The overall concordance rate between cobas and therascreen assays with Sanger sequencing was 89% and 88%, respectively, and increased to 96% and 98%, respectively, if the mutations not covered were excluded. The cobas assay showed a superior coverage of exon 20 mutations, but L861Q was not targeted. The nature of specimen, DNA integrity, and tumor cell content are factors that affect the assay performance. DNA extracted from cell block and clot of pleural fluid gave rise to 1 invalid call and 1 false-negative result by the cobas assay and 1 missed T790M mutation and 1 false-negative result by the therascreen assay. Both assays are around 5 times more expensive compared with Sanger sequencing in terms of reagent cost. We conclude that both assays prove to be a rapid, simple, and validated method in detecting the most common and clinically significant EGFR gene mutations in non-small cell lung cancer. Although less convenient compared with real-time PCR assays, Sanger sequencing is cheaper in terms of reagent cost and allows the detection of rare or novel EGFR gene mutations.

DNMT1 genetic polymorphisms affect breast cancer risk in the central European Caucasian population

IntroductionDNA methylation of CpG islands within the promoter region of genes is an epigenetic modification with an important role in the development of cancer and it is typically mediated by DNA methyltransferases (DNMTs). In cancer cells, global hypomethylation of the genome as a whole and regional hypermethylation of CpG islands have been reported. Four groups of DNMTs have been identified: DNMT1, DNMT2 (TRDMT1), DNMT3A and DNMT3B. DNMT2 uses the catalytic mechanism of DNMTs, but does in fact methylate RNA. Little is known about the significance of these genes in human breast cancer. In the study presented herein, we analyzed five distinct DNMT single SNPs with regard to potential associations with breast cancer risk.Case descriptionIn this study, we genotyped 221 female Caucasian breast cancer patients and 221 female Caucasian healthy controls, and we used five allele-specific real-time polymerase chain reaction (qPCR) assays. We selected one locus within the DNMT1 gene and two loci within the DNMT3A and DNMT3B genes, respectively. Statistics were calculated using the chi-squared and Fisher’s exact tests, and correlated with clinical parameters such as age, diagnosis, histology, TNM stage, hormonal receptor status, human epidermal growth factor receptor 2 (HER2) status, response to treatment and survival. Statistically significant results were obtained for correlations with the DNMT1 gene.Discussion and EvaluationFive genomic loci within the DNMT1, DNMT3A and DNMT3B genes were assessed. Statistical significance (P = 0.030) was identified for DNMT1 SNP (A201G, rs2228612): six women within the control group were GG homozygous (variant), while this mutation was absent in the breast cancer group.ConclusionsWe conclude that women with the DNMT1 SNP (A201G, rs2228612) GG homozygous genotype (variant) have a lower risk of developing breast cancer compared to heterozygous or wildtype genotypes. To date, alterations within the DNMT1 gene have not been reported to be associated with cancer in the Caucasian population.

New Quantitative Method to Identify NPM1 Mutations in Acute Myeloid Leukaemia

Somatic mutations in the NPM1 gene, which encodes for nucleophosmin, have been reported to be the most frequent genetic abnormalities found in acute myeloid leukaemia (AML). Their identification and quantification remain crucial for the patients' residual disease monitoring. We investigated a new method that could represent a novel reliable alternative to sequencing for its identification. This method was based on high-resolution melting analysis in order to detect mutated patients and on an allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) for the identification and quantification of the transcripts carrying NPM1 mutations (NPM1m). Few patients carrying known NPM1m enabled us to set up a table with the different primers' ΔCT values, identifying a profile for each mutation type. We then analysed a series of 337 AML patients' samples for NPM1 mutational status characterization and confirmed the ASO-RQ-PCR results by direct sequencing. We identified some mutations in 86 samples, and the results were fully correlated in 100% of the 36 sequenced samples. We also detected other rare NPM1m in two samples, that we confirmed by direct sequencing. This highly specific method provides a novel quick, useful, and costless tool, easy to use in routine practice.

MYD88 L265P in Waldenström macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction

AbstractBy whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia (WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from splenic marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors (0/40; P < 1.5 × 10−5 for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis.

Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population

Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population. Materials and Methods. Three major TPMT single-nucleotide polymorphisms (TPMT∗2, ∗3B, and ∗3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing. Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency of TPMT∗3C was 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. No TPMT∗2, ∗3A, or ∗3B alleles were observed in this Korean population. Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.

Reductions in JAK2 V617F Allele Burden with Ruxolitinib Treatment in Comfort-II, a Phase 3 Study Comparing the Safety and Efficacy of Ruxolitinib with Best Available Therapy (BAT)

Abstract 802 Background: Ruxolitinib is a potent oral JAK1 & JAK2 inhibitor that has demonstrated superiority over traditional therapies for the treatment of MF. In the two phase 3 studies, ruxolitinib demonstrated rapid and durable reductions in splenomegaly and improved disease-related symptoms and quality of life compared with placebo (COMFORT-I) and best available therapy (BAT; COMFORT-II) for pts with or without the JAK2 V617F mutation. Change in JAK2 V617F allele burden (%V617F) as a metric of molecular response to treatment in JAK2 V617 F–positive pts was investigated as an exploratory endpoint. Previously, we reported allele burden reductions in pts receiving ruxolitinib in the COMFORT-II study and demonstrated a positive correlation with reduction in spleen volume after 24 and 48 wk of treatment (Vannucchi, et al. Haematologica.2012); here, we evaluate the correlation between changes in %V617F and spleen size reduction after 72 wk of ruxolitinib therapy. Methods: COMFORT-II is a randomized (2:1), open-label, phase 3 study evaluating the safety and efficacy of ruxolitinib (n = 146) compared with BAT (n = 73) in pts with primary MF (PMF), post–polycythemia vera MF (PPV-MF), and post–essential thrombocythemia MF (PET-MF). Allele burden was measured from blood samples using allele-specific quantitative real-time polymerase chain reaction (qPCR) using the method outlined in Levine et al, 2006, using an Applied Biosystems ABI 7900 real-time PCR analyzer. Pts were stratified by absolute reduction in %V617F ( Results: Overall, 110 (76%) pts in the ruxolitinib group and 49 (71%) pts in the BAT group were JAK2V617 F–positive at baseline. More pts in the ruxolitinib arm had ≥ 10% V617F reductions compared with BAT at wk 48 (Table; 41% \(2868\) vs 5% \(122\) ) and at wk 72 (40% \(2153\) vs 0). The majority of reductions ≥ 20% were gradual and progressive over the course of the study (Figure); 2 pts had rapid initial reductions in allele burden that were sustained over 72 wk, from absolute %V617F of 48% and 45% at baseline to 20% reduction at wk 48 but the data were not available at the time of the 48-wk analysis, 2 pts did not have data at wk 48, and 1 pt achieved a 15% reduction at wk 48 that improved to a 21% reduction by wk 72. Among pts who achieved a ≥ 20% reduction in allele burden, 39% had PMF, 39% had PPV-MF, and 22% had PET-MF; this distribution was similar to that of the overall study population. In the ruxolitinib arm, significantly more pts with a ≥ 20% V617F reduction achieved a 9 35% reduction from baseline in spleen volume compared with pts with a Conclusions: Patients who received ruxolitinib had larger reductions in JAK2 V617F allele burden compared with BAT. %V617F reductions were gradual over the course of the study and continued between wk 48 and 72 in some pts. In JAK2 V617 F–positive pts, reductions in %V617F were associated with spleen responses; in pts with a ≥ 20% reduction, spleen volume reductions were sustained out to 72 wk. These results, along with findings from COMFORT-I and the phase 1/2 study, suggest that ruxolitinib has the potential to alter the course of disease through a reduction in the burden of JAK2V671 F–mutated cells. Disclosures: Vannucchi:Novartis: Membership on an entity9s Board of Directors or advisory committees. Passamonti:Sanofi: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Al-Ali:Sanofi Aventi: Consultancy, Honoraria; celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria. Harrison:Shire: Honoraria, Research Funding; Sanofi: Honoraria; YM Bioscience: Consultancy, Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau. Sirulnik:Novartis: Employment. Stalbovskaya:Novartis: Employment, Equity Ownership. Squires:Novartis : Employment. Burn:Incyte: Employment, Equity Ownership. Knoops:Novartis: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Cervantes:Bristol-Myers Squibb: Speakers Bureau; Teva Pharmaceuticals: Advisory Board, Advisory Board Other; Pfizer: Advisory Board, Advisory Board Other; Celgene: Advisory Board, Advisory Board Other; Sanofi-Aventis: Advisory Board, Advisory Board Other; Novartis: AdvisoryBoard Other, Speakers Bureau. Barbui:Novartis: Honoraria. Gisslinger:Celgene: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau; AOP Orphan Pharma AG: Consultancy, Speakers Bureau. Kiladjian:Incyte: Membership on an entity9s Board of Directors or advisory committees; Shire: Membership on an entity9s Board of Directors or advisory committees; Novartis: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

Simple and rapid detection method for the mutations in SLC22A12 that cause hypouricemia by allele-specific real-time polymerase chain reaction

BackgroundHypouricemia is a disorder that serum urate level is less than 2.0mg/dl, and relatively common in the Japanese population, where the main genetic cause of hypouricemia is W258X and R90H mutations in human urate trasnsporter 1(SLC22A12). Small scale screening has relied on time-consuming traditional ways like polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Therefore, it is beneficial that we have an easy and rapid detection method for these mutations. MethodsIn this report, we established a touchdown allele-specific real-time polymerase chain reaction (ASPCR) assay for detecting W258X and R90H mutations in SLC22A12, respectively. ResultsQuantifiable discrimination was successfully achieved by ∆Ct value. Furthermore, we conducted W258X and R90H screening against 120 control genome sets, whereby frequency was 2.92% for W258X, and not detected for R90H, respectively. ConclusionsThe two mutations, W258X and R90H in SLC22A12 were successfully genotyped by an easy and rapid ASPCR assay.

Rapid Quantitative Detection of the T315I Mutation in Patients With Chronic Myelogenous Leukemia

Acquired resistance to tyrosine kinase inhibitors (TKIs) in the treatment of chronic myelogenous leukemia (CML) is frequently caused by point mutations in the ABL kinase domain of the BCR-ABL fusion gene. The T315I mutation is the most common mutation found in the kinase domain and leads to complete resistance to existing TKIs. Sensitive and specific approaches for detecting this mutation in patient specimens can provide valuable information to guide treatment decisions and monitor their effectiveness. Here, we describe an allele-specific real-time polymerase chain reaction method to distinguish and quantify wild type or T315I mutant ABL transcripts. This approach has high specificity in identifying mutant transcripts and shows minimal interference from wild-type transcripts. As few as 5 copies of the T315I mutant transcript or 0.025% (2.5×10(-4)) T315I mutant transcripts could be detected by this method. This approach requires no additional specialized reagents other than those used in standard real-time polymerase chain reaction and therefore may be easily incorporated as an effective strategy for the early detection and monitoring of TKI resistance in patients with CML.

Determina KRAS: A consolidated project.

428 Background: The identification of KRAS mutation status as a predictive biomarker for the activity of anti-EGFR in mCRC patients, was the start of the personalize medicine. ASCO recognized it as a major advance in 2008 and it also gave a clinical opinion so every candidate for anti-EGFR therapy should be tested for KRAS mutations. NCCN guidelines recommends to do it at the first diagnose of metastatic disease. KRAS status testing was no widely available in Spain by 2008. The project Determina KRAS was developed in order to provide access to this test throughout the country to any mCRC patient. Methods: Five well known Spanish centres were trained and provided with equipment so they could analyze KRAS status in a five working day period. First centre was initiated in July 2008, being the last one in October 2008. A validated KRAS mutation kit (DxS Ltd, Manchester, UK) which identifies 7 different somatic mutations located in codons 12 and 13 using allele-specific real-time polymerase chain reaction was used. Results: 15.330 samples from mCRC patients have been analyzed since July 2008. 53,7% samples showed wild type KRAS status. G12D mutation was the most frequent mutation observed in general (35,1% of all mutation). G13D mutation was the most frequent of the codon 13 mutation, with a 20,2% of the global mutations. Conclusions: KRAS status testing allows to patients with mCRC receive a personalized medicine. KRAS status testing is essencial for treatment decision-making in mCRC and access to it must be guaranteed for any patient. The determination of biomarkers to choose the most effective treatment for each patient will be the future of oncology. These futures assessments must be available for all patients through national Networks, as the Determina KRAS project has made with KRAS testing.

Association of polymorphism of IL12B gene and predisposition to psoriasis among the populationof the Northwestern region of Russia

The IL12B gene encodes protein р40 being a common subunit of interleukin-12 and interleukin-23 playing an important part in the pathogenesis of psoriasis. The IL12B gene has polymorphism rs12188300, which can be associated with the risk of psoriasis development. The goal of the study was to assess the distribution of alleles of polymorphism rs12188300 in psoriatic patients in the Northwestern region of Russia. Genotyping was carried out using the real-time allele-specific polymerase chain reaction. An increased occurrence of a rare allele T in psoriatic patients as compared to healthy people was observed (OR = 1.96, р = 0.0007). The occurrence of the rare allele T in patients with psoriatic arthritis was higher than in the control group (OR = 3.49, р = 0.005). These results suggest that rs12188300 polymorphism of the IL12B gene is a new genetic marker of psoriasis and psoriatic arthritis.

Development of in vitro atovaquone-resistant Babesia gibsoni with a single-nucleotide polymorphism in cytb

An atovaquone (ATV)-resistant Babesia gibsoni was developed by in vitro exposure of uncloned wild type (WT) B. gibsoni to 800nM ATV for 6 days. Sequence analysis of mitochondrial genes showed a single-nucleotide polymorphism (SNP) at cytb nt363 (G to T) that resulted in the substitution of methionine with isoleucine (M121I), which is one of the SNPs reported in a previous in vivo study. 363T or 363G allele-specific real-time polymerase chain reaction (PCR) revealed that an M121I variant was present in over 99% of the ATV-resistant population. As neither ATV resistance nor gene polymorphisms appeared in the B. gibsoni WT sibling clones, the expression of ATV resistance in this study was suspected to be because of selective multiplication of the B. gibsoni M121I variant. This ATV-resistant B. gibsoni displayed the same sensitivity as the WT B. gibsoni against 5 other drugs, including diminazene aceturate, azithromycin, doxycycline, clindamycin, and proguanil. This is the first report on the in vitro establishment of an ATV-resistant B. gibsoni with gene polymorphisms.

Atypical Spitzoid melanocytic tumors: A morphological, mutational, and FISH analysis

Identification of the clinical behavior of atypical Spitzoid tumors with conflicting histopathologic features remains controversial. We sought to assess whether molecular findings may be helpful in the diagnostic and prognostic assessment of atypical Spitzoid tumors. A total of 38 controversial, atypical Spitzoid lesions (≥ 1 mm in thickness) were analyzed for clinicopathological features, chromosomal alterations by fluorescence in situ hybridization (FISH) analysis (RREB1/MYB/CCND1/CEP6), BRAF(V600E) mutation by allele-specific real-time polymerase chain reaction confirmed by sequencing, and H-RAS gene mutation by direct sequencing. Atypical Spitzoid lesions developed in 21 female and 17 male patients (mean age 22 years). Nine patients underwent sentinel lymph node biopsy and a sentinel lymph node micrometastasis was detected in 4 of these 9 cases. Four additional patients, who did not receive a sentinel lymph node biopsy, experienced bulky lymph node metastases and one experienced visceral metastases and death. Lesions from patients with lymph node involvement showed more deep mitoses (P < .01), less inflammation (P = .05), and more plasma cells (P = .04). FISH analysis demonstrated the presence of chromosomal alterations in 6 of 25 cases. Correlation with follow-up data showed that the only case with fatal outcome showed multiple chromosomal alterations by FISH analysis. BRAF(V600E) mutation was detected in 12 of 16 cases (75%) and H-RAS mutation on exon 3 was found in 3 of 11 cases (27%). Our results require validation in a larger series with longer follow-up information. FISH assay may be of help in the prognostic evaluation of atypical Spitzoid tumors. Diagnostic significance of BRAF(V600E) and H-RAS mutations in this setting remains unclear.

The Role of Transplacental Microtransfusions of Maternal Lymphocytes in In Utero HIV Transmission

The mechanisms of HIV transmission from mothers to infants are poorly understood. A possible mechanism of in utero transmission is transplacental transfer of HIV-infected maternal leukocytes into the fetal circulation during pregnancy. To determine if the frequency of in utero HIV infection correlates with presence or levels of maternal cells (MCs) in placenta-derived cord blood. DNA was extracted from dried cord blood spots (DBS) from newborns born to HIV+ mothers and corresponding maternal DBS specimens. Paired mother-infant samples were probed to identify unique maternal sequences targeted by 24 allele-specific real-time polymerase chain reaction assays. Infant DBS-derived DNA was then probed in replicate analyses for noninherited maternal allelic sequences. Rates of detection and levels of MCs in DBS samples of HIV(+) and HIV(-) newborns were compared. Of 114 mother-infant pairs with informative alleles, 38 newborns were HIV(+) and 76 HIV(-), based on detection of HIV DNA/RNA at birth. MC were detected in 23 of 38 HIV(+) newborns (60.5%) and in 47 of 76 HIV(-) newborns (61.8%). The mean and median concentrations of nucleated MCs in DBS for the HIV(+)/MC(+) newborns (n = 23) were 0.33% and 0.27%, respectively, compared with 0.09% and 0.10% for the HIV(-)/MC(+) newborns (n = 47) (2-sample T test for means: P = 0.78). There was no significant difference in rates of detection or concentrations of MC in DBS between HIV(+) and HIV(-) newborns. Therefore, we could not demonstrate a correlation between MC in DBS, assumed to reflect levels of in utero maternal-fetal cell trafficking, and the risk of in utero HIV transmission.

High-throughput β-thalassemia carrier screening by allele-specific Q-primer real-time polymerase chain reaction

Based on a novel Q-primer real-time polymerase chain reaction (PCR) system, we designed allele-specific Q-primers for the detection of three β-thalassemia mutations [Cd41/42(-TCTT), IVSI nt5 (G>C), and IVSII nt654 (C>T)] that have a high carrier frequency in Southeast Asia. With clear distinction between heterozygote and wild-type, Δ C t (threshold cycle) values were defined. The results of evaluating 139 blinded samples by our system match perfectly with those obtained by the conventional reverse dot blot (RDB) method. With a 384-well plate that included replicates in the same analysis, our throughput reached 190 reactions per run with a turnaround time as short as 130 min, and the cost of consumables was as low as $1 (US) for each test.

Prognostic Role of KRAS and BRAF in Stage II and III Resected Colon Cancer: Results of the Translational Study on the PETACC-3, EORTC 40993, SAKK 60-00 Trial

Mutations within the KRAS proto-oncogene have predictive value but are of uncertain prognostic value in the treatment of advanced colorectal cancer. We took advantage of PETACC-3, an adjuvant trial with 3,278 patients with stage II to III colon cancer, to evaluate the prognostic value of KRAS and BRAF tumor mutation status in this setting. Formalin-fixed paraffin-embedded tissue blocks (n = 1,564) were prospectively collected and DNA was extracted from tissue sections from 1,404 cases. Planned analysis of KRAS exon 2 and BRAF exon 15 mutations was performed by allele-specific real-time polymerase chain reaction. Survival analyses were based on univariate and multivariate proportional hazard regression models. KRAS and BRAF tumor mutation rates were 37.0% and 7.9%, respectively, and were not significantly different according to tumor stage. In a multivariate analysis containing stage, tumor site, nodal status, sex, age, grade, and microsatellite instability (MSI) status, KRAS mutation was associated with grade (P = .0016), while BRAF mutation was significantly associated with female sex (P = .017), and highly significantly associated with right-sided tumors, older age, high grade, and MSI-high tumors (all P < 10(-4)). In univariate and multivariate analysis, KRAS mutations did not have a major prognostic value regarding relapse-free survival (RFS) or overall survival (OS). BRAF mutation was not prognostic for RFS, but was for OS, particularly in patients with MSI-low (MSI-L) and stable (MSI-S) tumors (hazard ratio, 2.2; 95% CI, 1.4 to 3.4; P = .0003). In stage II-III colon cancer, the KRAS mutation status does not have major prognostic value. BRAF is prognostic for OS in MS-L/S tumors.

Persistent Minority K103N Mutations among Women Exposed to Single‐Dose Nevirapine and Virologic Response to Nonnucleoside Reverse‐Transcriptase Inhibitor–Based Therapy

We investigated whether there are long-lasting effects of exposure to single-dose nevirapine (sdNVP) treatment on virologic response to nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based therapy among human immunodeficiency virus (HIV)-infected women. An observational epidemiologic study was conducted in Johannesburg, South Africa. Initial and sustained virologic response to NNRTI-based therapy was compared between 94 HIV-infected women who had received sdNVP 18-36 months earlier and 60 unexposed, HIV-infected women who had been pregnant 12-36 months earlier. Viral load was measured every 4 weeks up to week 24 and then every 12 weeks up to week 78. Time to viral suppression (viral load, <50 copies/mL) and confirmed rebound in the viral load (viral load, >400 copies/mL) were compared. Drug resistance was assessed using K103N allele-specific real-time polymerase chain reaction assay and population sequencing. Almost all women (97.5% of sdNVP-exposed women and 91.3% of sdNVP-unexposed women; P = .21) achieved viral suppression by week 24, and similar percentages of sdNVP-exposed and -unexposed women (19.4% and 15.1%, respectively) experienced viral rebound within 78 weeks after treatment (P = .57). K103N was detected with the K103N allele-specific real-time polymerase chain reaction assay among sdNVP-exposed and -unexposed women before treatment; detection was strongly predictive of inadequate viral response: 60.9% of women for whom K103N was detected in either viral RNA or DNA did not experience viral suppression or experienced viral rebound, compared with 15.1% of women for whom K103N was not detected (P < .001). After treatment, the M184V mutation occurred less frequently among sdNVP-exposed women than among sdNVP-unexposed women, but the frequency of NNRTI-associated mutations was similar between these groups of women with inadequate virologic response. Exposure to sdNVP in the prior 18-36 months was not associated with a reduced likelihood of achieving and sustaining viral suppression while receiving NNRTI-based therapy. However, women with minority K103N mutations before treatment had a reduced durability of virologic suppression.

Minority Quasispecies of Drug‐Resistant HIV‐1 That Lead to Early Therapy Failure in Treatment‐Naive and ‐Adherent Patients

Early virological failure of antiretroviral therapy associated with the selection of drug-resistant human immunodeficiency virus type 1 in treatment-naive patients is very critical, because virological failure significantly increases the risk of subsequent failures. Therefore, we evaluated the possible role of minority quasispecies of drug-resistant human immunodeficiency virus type 1, which are undetectable at baseline by population sequencing, with regard to early virological failure. We studied 4 patients who experienced early virological failure of a first-line regimen of lamivudine, tenofovir, and either efavirenz or nevirapine and 18 control patients undergoing similar treatment without virological failure. The key mutations K65R, K103N, Y181C, M184V, and M184I in the reverse transcriptase were quantified by allele-specific real-time polymerase chain reaction performed on plasma samples before and during early virological treatment failure. Before treatment, none of the viruses showed any evidence of drug resistance in the standard genotype analysis. Minority quasispecies with either the M184V mutation or the M184I mutation were detected in 3 of 18 control patients. In contrast, all 4 patients whose treatment was failing had harbored drug-resistant viruses at low frequencies before treatment, with a frequency range of 0.07%-2.0%. A range of 1-4 mutations was detected in viruses from each patient. Most of the minority quasispecies were rapidly selected and represented the major virus population within weeks after the patients started antiretroviral therapy. All 4 patients showed good adherence to treatment. Nonnucleoside reverse-transcriptase inhibitor plasma concentrations were in normal ranges for all 4 patients at 2 separate assessment times. Minority quasispecies of drug-resistant viruses, detected at baseline, can rapidly outgrow and become the major virus population and subsequently lead to early therapy failure in treatment-naive patients who receive antiretroviral therapy regimens with a low genetic resistance barrier.

Viral Dynamics and In Vivo Fitness of HIV-1 in the Presence and Absence of Enfuvirtide

To estimate the in vivo fitness cost of enfuvirtide (ENF) resistance, we analyzed dynamic shifts in the HIV-1 quasispecies under changing selective pressure in 3 subjects on failing ENF-based regimens who interrupted ENF while maintaining stable background regimens. Subsequently, ENF was readministered for 4 weeks as "pulse intensification." The proportion of plasma virus carrying the V38A mutation in gp41 was quantified by allele-specific real-time polymerase chain reaction in serial samples collected from 3 subjects at 1- to 4-week intervals. Fitness differences were calculated using a method that corrected for time dependence of the viral replication rate. The V38A mutant made up >or=85% of the quasispecies at baseline and decayed to <5% over 12-24 weeks; plasma HIV-1 RNA levels remained stable during this time. Fitness differences for mutant versus wild type ranged from -25% to -65%, providing in vivo evidence for the reduced fitness of ENF-resistant HIV-1. The V38A mutant virus reemerged rapidly during the ENF pulse. These results demonstrate that the HIV-1 quasispecies undergoes dynamic changes in response to withdrawal and reinitiation of fusion inhibitor therapy. The relative stability of plasma HIV-1 titers during decay of V38A suggests that factors other than viral fitness likely define viral load set-point in patients with advanced disease.