Determining Tumor Load and Biallelic Mutation in Patients with CALR Mutation Using Peripheral Blood Plasma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1818-1818
Author(s):  
Kevin Diep ◽  
Wanlong Ma ◽  
Ferras Albitar ◽  
Ivan De Dios ◽  
Sally Agersborg ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Plasma Dna ◽  
Biallelic Mutation ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tumor Load ◽  
Peripheral Blood Plasma ◽  
Cellular Dna ◽  
Free Plasma ◽  
Calr Mutation

Abstract Background: CALR gene is frequently mutated in patients with myeloproliferative neoplasmas (MPN). Almost all mutations are indel - some with large (>50 bp) deletion. Detecting this type of mutations with acceptable sensitivity is difficult by sequencing. Fragment length analysis (FLA) is a reliable technique in detecting this type of mutations. Furthermore, FLA allows quantifying the mutant DNA and better evaluation of tumor load. Determining tumor load can be confusing and difficult when the mutation is biallelic. Distinguishing between patients with single allele mutation from those with biallelic mutations might add another dimension in predicting clinical behavior and determining the tumor load. We explored using cell free DNA in peripheral blood plasma to test for CALR mutations and determining tumor load. Methods: Using Direct bidirectional sequencing and FLA, we detected CALR indel mutations in 71 of 522 (14%) patients suspected of having MPN and referred for testing for CALR mutation. No sample showed point mutation. DNA from cells and cell free DNA in plasma was available from 31 of the 71 cases. The mutant DNA peak was quantified and the relative percentage of mutant DNA was calculated in both cellular DNA and cell free plasma DNA. FLA and Sanger sequencing data were compared between cellular DNA and cell free plasma DNA. Any ratio >55% was considered as evidence of biallelic mutation. Results: As expected all positive samples by cellular DNA testing also showed the indel mutation in cell free plasma by FLA. However, four of the 31 (13%) positive samples by FLA failed to show the mutation on Sanger sequencing. The most likely cause for failing to detect the mutation by Sanger is low level mutation load and the lower sensitivity of Sanger sequencing. When we compared ratios of mutant peak (tumor load) between cellular DNA and cell free DNA in plasma, mutant CALR DNA was significantly higher (P=0.0002, Wilcoxon matched pairs test) in cell free DNA in plasma than in cellular DNA. More importantly, we were able to determine the presence of homozygous mutation (>55% mutant DNA) in 5 of 31 (16%) patients when cell free DNA in plasma is used. In contrast, only 1 of 31 (3%) patients showed evidence of biallelic mutation when cellular DNA is used. Most of the mutations (25 of 31, 81%) were deletions. As deletions results in smaller size amplicon on the FLA and better amplification efficiency, we set 55% as a cut-off for biallelic mutation to account for the more efficient amplification of the deleted peak. Conclusion: Cell free DNA in plasma is more reliable than cellular DNA for the detection of CALR mutations and for determining tumor load. Testing for CALR must include fragment length analysis. More importantly, biallelic CALR mutation is frequent and the clinical relevance of biallelic CALR mutation needs to be investigated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Deep Sequencing of Peripheral Blood Plasma DNA As a Reliable Test for Confirming the Diagnosis of Myelodysplastic Syndrome

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1909-1909
Author(s):  
Ferras Albitar ◽  
Wanlong Ma ◽  
Kevin Diep ◽  
Ivan De Dios ◽  
Sally Agersborg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Deep Sequencing ◽  
Whole Blood ◽  
Sanger Sequencing ◽  
Cell Free Dna ◽  
Molecular Abnormalities ◽  
Free Dna ◽  
Tumor Load ◽  
Peripheral Blood Plasma

Abstract Background: In patients presenting with cytopenia, myelodysplastic syndrome (MDS) should be considered, but confirmation of diagnosis requires bone marrow biopsy and morphologic and cytogenetic evaluation. It is extremely difficult to rely on subjective morphologic features to confirm the diagnosis of MDS, when the karyotype is normal and blasts are not increased. Objective criteria for the diagnosis of MDS are needed in these cases. With recent advances in the characterization of molecular abnormalities in MDS, diagnosis of early MDS is becoming more objective by documenting the presence of MDS-specific molecular abnormalities in cases with appropriate clinical presentation. Since MDS is a disease of excessive apoptosis in bone marrow, DNA resulting from the apoptosis is abundant in circulation. We explored the potential of using cell free DNA in peripheral blood plasma using next generation sequencing (NGS) to confirm the diagnosis of early MDS without the need for marrow biopsy. Methods: Total nucleic acid was extracted from the plasma of 16 patients presenting with cytopenia and confirmed diagnosis of early MDS (blasts <5%) by the presence of mutations in one or more MDS-specific genes in DNA from cells in bone marrow. Plasma samples from 4 age-matched normals were used as negative controls. We performed targeted sequencing of 14 genes (581 amplicons) using Illumina MiSeq platform. This panel included the following genes: ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. NGS and Sanger sequencing was used for testing. Results of cell free DNA in plasma were compared to that from cells or whole blood. Results: Deep sequencing of cell free DNA in plasma from the 16 patients with early MDS showed at least one or more mutated gene confirming the diagnosis of MDS. Three patients (19%) showed mutation in one gene and the remaining 13 patients (81%) showed mutations in two or more genes. Cell free DNA in plasma from normal controls showed no evidence of mutations. When NGS data of cell free DNA from plasma was compared with Sanger sequencing data of DNA from cells in bone marrow, 10 of the 16 patients (63%) showed mutations in cell free DNA in plasma not detected by Sanger sequencing in DNA from cells in bone marrow. All mutations detected by NGS in cell free DNA in plasma were below the detection level of the Sanger technique and most likely represent subclones. NGS allowed the measurement of relative tumor load in plasma. Tumor load in plasma as detected by NGS was significantly (P=0.008) higher than that detected in cellular DNA, suggesting higher sensitivity of the former in detecting minimal residual disease and a better tool for monitoring therapy. Without exception, all detected mutations showed higher tumor load in plasma as compared with DNA from cells or whole blood, supporting the concept that plasma is enriched in tumor-specific DNA. Conclusion: NGS of cell free DNA in plasma using limited number of MDS-specific genes, when used in patients with cytopenia, presents an objective test for the confirmation of the diagnosis of MDS. Plasma is enriched in tumor-specific DNA in patients with MDS. Furthermore, mutation analysis of cell free DNA in plasma can detect subclones with mutations and can predict the emergence of new clones. Analysis of cell free DNA in plasma using NGS provides important data on tumor load, which can be used to monitor therapy, and predict progression, and also reduces the need for performing bone marrow biopsies. Disclosures No relevant conflicts of interest to declare.

Impact Of Cell-Free Plasma DNA In Metastatic And Non-Metastatic Prostate Cancer

2021 ◽  
Vol 21 ◽  
Author(s):  
Abdelraouf A. Abonar ◽  
Shymaa E. Ayoub ◽  
Ibrahim A. Tagreda ◽  
Marwa N. Abdelhafez ◽  
Mohammed M Khamiss ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
Metastatic Prostate Cancer ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Group Iii ◽  
Free Dna ◽  
Group I ◽  
Total Psa ◽  
Free Plasma

: Increased cell-free DNA (cfDNA) is observed in many diseases such as cancer, myocardial infarction, and autoimmune diseases. It has the ability to alter the receptor cell phenotype, triggering events related to malignant transformation. Our study aims at assessing the use of Cell-free plasma DNA in the diagnosis of metastatic and non-metastatic prostate cancer. The study included 180 subjects who were classified into four groups: Group I (GI) included 50 in perfect health subjects as the control group, Group II (GII) included 40 patients with prostatitis, group III (GIII) included 40 patients with benign prostatic hyperplasia (BPH) and Group IV (GIV) included 50 patients with pre-operative prostate cancer (PC). Evaluation of the plasma level of circulating cell-free DNA by real-time PCR and measurement of total PSA (tPSA) and free to total PSA percent (f/tPSA%) were done for all groups. Our study revealed that the level of tPSA was significantly higher in prostate cancer patients while levels of f/t PSA were found to be significantly lower. The level of cfDNA was significantly higher in prostate cancer patients (399.9±88.6ng/ul) when compared to that of the group I (12.1±1.5ng/ul) (p<0.01), group II (14.7±2.4 ng/ul) (p<0.01), and group III (26.6±45.6 ng/ul) (p<0.01) respectively. There was a statistically significant difference in yields of cfDNA between metastatic and non- metastatic groups (P=0.03) with a higher level in the metastatic group.

High Sensitivity Testing Shows Multiclonal Mutations in Patients with CLL Treated with BTK Inhibitor and Lack of Mutations in Ibrutinib-Naive Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 716-716 ◽  
Author(s):  
Adam Albitar ◽  
Wanlong Ma ◽  
Ivan De Dios ◽  
Jeffrey Estrella ◽  
Mohammed Farooqui ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Sanger Sequencing ◽  
High Sensitivity ◽  
Resistance Mechanisms ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Sensitivity Testing ◽  
Cell Free Dna ◽  
Free Dna ◽  
Cellular Dna

Abstract Background: Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton's tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). Mutations in BTK at the C481S hotspot alter the active site of the mutant BTK to the effect that Ibrutinib is reversibly bound. PLCγ2 is downstream of BTK in the B-cell signaling pathway; mutations in PLCγ2 at either of the R665W, L845F, or S707Y hotspots result in a constitutively activated PLCγ2. In order to better understand the development of these resistance mechanisms in patients with CLL, we developed a high sensitivity (HS) assay utilizing branched and locked nucleic acids (BNA and LNA, respectively). We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell free DNA (cf-DNA) from patients with CLL. Methods: Using custom BNA or LNA oligos in a wild-type blocking polymerase chain reaction, then sequencing by Sanger and NGS methods, we achieved 100x greater sensitivity than Sanger. Sanger sequencing was capable of detecting <1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. The assay is designed to cover BTK and PLCγ2 hotspots. Using this assay we tested peripheral blood samples from 44 Ibrutinib-naïve patients (Ib-) with CLL and 7 samples from CLL patients being treated with Ibrutinib (Ib+), that showed clinical evidence of disease progression. The same wild-type blocking was also used in NGS approach for confirmation. We performed wild-type blocking in a Nextera Rapid Capture Enrichment workflow for our custom 315 gene panel. Results: No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. In contrast, all (N=7) tested patients with progressive disease on Ibrutinib showed one or more mutation in BTK or PLCγ2 using the HS method. Without the HS testing only 4 patients (57%) showed a mutation in BTK or PLCγ2. Two patients showed multiple mutant clones. One patient with double mutations in PLCγ2 (R665W and L845F) also showed triple independent mutations in BTK at codon C481 with HS testing. These mutations give rise to two distinct mutant proteins C481R (TGC>CGC) and C481S (TGC>AGC and TGC>TCC). NGS analysis confirmed that the the three BTK mutations are in three independent clones A second patient showed initially a mutation in BTK (C481S), but subsequent sample showed a mutation in PLCg2 (R665W), in addition to the BTK mutation. All mutations detected in the peripheral blood cells were also detectable in cell-free DNA (cfDNA) using HS testing. However, without using HS testing, a BTK mutation was detected in cfDNA from a patient and this mutation was not detectable when cellular DNA was used. Conclusions: Our data suggests that ibrutinib-naïve patients with CLL do not have BTK or PLCγ2 mutations even when a highly sensitive assay is used. Emerging BTK or PLCγ2 mutant clones can be seen after therapy with the possibility of multiple clones emerging at the same time and may involve both BTK and PLCγ2 genes in the same patient. Furthermore, testing cfDNA is not only as informative as cellular DNA, but might show mutations earlier than cellular DNA. This may have clinical relevance in patients with lymphoma when only few lymphoma cells are in circulation. Figure 1. Figure 1. Disclosures Wiestner: Pharmacyclics: Research Funding.

scholarly journals A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 650
Author(s):  
Selena Y. Lin ◽  
Yue Luo ◽  
Matthew M. Marshall ◽  
Barbara J. Johnson ◽  
Sung R. Park ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Amplification Efficiency ◽  
Normal Donor ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Pcr Product ◽  
Cleanup Procedure ◽  
Dna Profiles ◽  
The Impact

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA than did kits M or Q (p < 0.001) from urine, and similar amounts from plasma (p = 0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p < 0.001) and plasma (p ≤ 0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p = 0.05). We demonstrate that DNApure can provide an efficient means of improving the yield and purity of cfDNA and minimize the effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

scholarly journals Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma

2018 ◽  
Vol 115 (46) ◽  
pp. E10925-E10933 ◽  
Author(s):  
Peiyong Jiang ◽  
Kun Sun ◽  
Yu K. Tong ◽  
Suk Hang Cheng ◽  
Timothy H. T. Cheng ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Circulation ◽  
Sequence Information ◽  
Sequencing Analysis ◽  
Plasma Samples ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tumor Dna

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.

scholarly journals Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients

2015 ◽  
Vol 112 (11) ◽  
pp. E1317-E1325 ◽  
Author(s):  
Peiyong Jiang ◽  
Carol W. M. Chan ◽  
K. C. Allen Chan ◽  
Suk Hang Cheng ◽  
John Wong ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B ◽  
Molecular Diagnostic ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Size Profile ◽  
A Genome ◽  
Circulating Cell Free Dna

The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-levelz-score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.

scholarly journals Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

2021 ◽  
Vol 118 (50) ◽  
pp. e2114937118
Author(s):  
Stephanie C. Y. Yu ◽  
Peiyong Jiang ◽  
Wenlei Peng ◽  
Suk Hang Cheng ◽  
Y. T. Tommy Cheung ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Analysis ◽  
Large Population ◽  
Maternal Plasma ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Dna Molecule ◽  
Free Dna ◽  
Monogenic Diseases

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

scholarly journals Methods for Separate Isolation of Cell-Free DNA and Cellular DNA from Urine-Application of Methylation-Specific PCR on both DNA Fractions

2011 ◽  
Vol 4 (1) ◽  
pp. 15-17 ◽  
Author(s):  
Agnes Beermann ◽  
Foued Ghanjati ◽  
Thomas Hermanns ◽  
Cedric Poyet ◽  
Joana Pereira ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Room Temperature ◽  
Proteinase K ◽  
Kinetics Analysis ◽  
Cell Free Dna ◽  
Methylation Specific Pcr ◽  
Specific Pcr ◽  
Free Dna ◽  
Cellular Dna ◽  
Reliable Methods

The analysis of genomic DNA is widely-used for research, forensic and diagnostic purposes. Here we describe reliable methods for isolation of cell-free DNA and cellular DNA from urine. Both DNA fractions are suitable for PCR and Methylation-Specific PCR (MSP) amplification, leading to consistent and reproducible results. A kinetics analysis describes the decline of efficiency of MSP performed with urinary DNA which had been stored at room temperature with and without proteinase K for various time periods.

scholarly journals A new method for improving extraction efficiency and purity of urine and plasma cell-free DNA

2021 ◽  
Author(s):  
Selena Y. Lin ◽  
Yue Luo ◽  
Matthew M. Marshall ◽  
Barbara J. Johnson ◽  
Sung R. Park ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Amplification Efficiency ◽  
Normal Donor ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Pcr Product ◽  
Cleanup Procedure ◽  
Dna Profiles ◽  
The Impact

AbstractThis study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples, and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA compared with kit M or Q (p<0.001) from urine, and similar amounts from plasma (p=0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p<0.001) and plasma (p≤0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p=0.05). We conclude that DNApure provides an efficient means of improving the yield and purity of cfDNA and minimizing effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

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