scholarly journals Cell-Free DNA Analysis Demonstrates Comprehensive Genomic Profiling and a Facile Method to Sensitively in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5603-5603
Author(s):  
Juan Du ◽  
Jing Lu ◽  
Wanting Qiang ◽  
Lu Li ◽  
Jin Liu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Copy Number ◽  
Genomic Profiling ◽  
Cell Free Dna ◽  
Bone Marrow Biopsies ◽  
Molecular Abnormalities ◽  
Free Dna ◽  
Comprehensive Genomic Profiling

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by complex cytogenetic and molecular abnormalities including translocations involving the immunoglobin heavy chain locus and mutations involving numerous oncogenic signaling pathways. Fluorescence in situ hybridization (FISH) has emerged as the most useful current cytogenetic assessment and provide a new level of insight into the correlation of myeloma prognosis risk model. However, the identification or sorting of malignant cells is required before FISH probes and only involved expansion of the types of probe and number of detectable targets is reached. Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with cancer precision medicine and patient prognosis. Methods: In this retrospective cohort study, we identified 37 patients from 9 relapsed/refractory (RR) and 33 newly diagnosed (ND) patients were analyzed for chromosomal copy number imbalance using the ultrasensitive chromosomal aneuploidy detector (UCAD) platform. Results: Chromosome copy number aberration (CNA) were frequently (82.6%, N=46) detected in MM plasma cell free DNA. Applying UCAD to cfDNA, FISH in CD138 purified bone marrow aspirates, and some matched bone marrow biopsies, we find concordance in copy number alterations (~81%) between liquid and tumor biopsies. Significant copy number changes, including 1q gains, 13q deletion and 17p deletion could be found in 57.89%, 54.05%, and 16.67% in plasma of MM, which is higher percentage than FISH assay (46.81%, 28.26%, and 8.89%), respectively. Besides, chromosome 6p and 6q were determined the higher frequency aberration from UCAD. Moreover, a higher frequency of copy number aberrations and variations was detected in RR patients than ND (100% vs 78.4%, respectively), obviously CNAs heterogeneity displayed in advanced disease. In the inconsistent some samples, UCAD from the plasma and bone marrow showed the similar results, which indicated the FISH is underdetermined and insensitivity in some patients' routine inspection. Conclusion: We conclude that cfDNA analysis as an adjunct to BM biopsy represents a noninvasive and broaden the applicability strategy for comprehensive genomic profiling and therapeutic monitoring of MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals RAS/RAF mutations in tumor samples and cell-free DNA from plasma and bone marrow aspirates in multiple myeloma patients

2020 ◽  
Vol 11 (12) ◽  
pp. 3543-3550
Author(s):  
Qian Li ◽  
Helen J Huang ◽  
Jing Ma ◽  
Yafei Wang ◽  
Zeng Cao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Free Dna ◽  
Free Dna

Observations On Eosinophil Ifiltrates in The Bone Marrow Of Patients With Multiple Myeloma. Clinicopathological Correlates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5338-5338
Author(s):  
Finella MC Brito-Babapulle ◽  
Tanya Cranfield ◽  
Robert B Corser ◽  
Helen Dignum ◽  
Christopher James ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Osteolytic Lesion ◽  
Inverse Correlation ◽  
Eosinophil Infiltration ◽  
Bone Marrow Biopsies ◽  
Lytic Lesions

Abstract Mouse eosinophils have been shown in 2011 to be required for the maintenance of long lasting plasma cells in the bone marrow and in maintaining the bone marrow plasma cell microenvironment. Human eosinophils have been shown by Wong et al to support multiple myeloma cell proliferation via a mechanism independent of IL6. We looked at bone marrow biopsies taken from patients who had a paraprotein and in whom a diagnosis of multiple myeloma was suspected. These samples were taken solely for the purposes of diagnosisng multiple myeloma and were retrospectively reviewed from the point of view of degree of eosinophil infiltration and its correlation with tumour load, bone lytic lesions, plasma cell morphology, whether blastic, crystalline inclusions, Mott cells, flame cells and or lymphoplasmacytoid. There were no cases of IGD or E myeloma or osteosclerotic myeloma.Nonsecretory myeloma and cases of light chain myeloma with or without amyloid were included in the series. Biopsies were not performed from osteolytic lesion unless biopsy was necessary to make a diagnosis of myeloma. Myeloma was diagnosed when plasma cell infiltrate was greater than 10% on bone marrow aspirate with a paraprotein and or lytic lesions. Eosinophil infiltration did not correlate with any of the tumour clinicopathological markers but showed an inverse correlation with degree of plasmacytosis. Eosinophils were hardly ever found in marrow aspirates that had over 70% plasma cells. They were usually found in trephine sections of bone marrow in areas where there was Grade I/II fibrosis and were often found in close proximity to focal areas of plasma cell infiltration. Whether eosinophils play a role in preventing or maintaining malignant plasma cell recurrence is currently being studied. Disclosures: No relevant conflicts of interest to declare.

Characterization of the Mutational Landscape of Multiple Myeloma Using Comprehensive Genomic Profiling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3418-3418 ◽  
Author(s):  
Christoph Heuck ◽  
Donald Johann ◽  
Brian A Walker ◽  
Caleb K Stein ◽  
Yogesh Jethava ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Mutant Allele ◽  
Genomic Profiling ◽  
Employment Equity ◽  
Advisory Committees ◽  
Comprehensive Genomic Profiling ◽  
Equity Ownership

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disease of the bone marrow characterized by a malignant transformation of plasma cells. Many patients relapse after initial treatment and require additional therapies. Impaired cell cycle regulation and DNA repair mechanisms as well as exposure to genotoxic drugs leads to accumulation of genomic alterations with progressive disease. Pressure from antineoplastic agents, including novel agents, eventually leads to the selection of resistant clones. Assessing acquired somatic mutations in MM patients can identify key genomic drivers and guide the development of a rational, individualized therapy plan for each patient with advanced disease. Here we report on the mutational landscape of cancer-associated genes in 214 patients who underwent comprehensive genomic profiling. Methods: Review of this data was approved by the UAMS institutional review board. DNA and RNA were extracted from CD138+ selected cells from bone marrow aspirates. Adaptor ligated sequencing libraries from extracted nucleic acids were captured by solution hybridization using bait sets targeting 405 cancer-related and 265 frequently rearranged genes (FoundationOne Heme®; Foundation Medicine ). For samples with low cell yield only the DNA portion was performed. All samples were sequenced in a CLIA-certified, CAP-accredited laboratory to an average depth >500x. Results We identified 147 clinically relevant alterations with an average of 3 alterations per patient ranging from 1 to 8. The most frequently altered genes were KRAS (29% of cases), NRAS (23%), TP53 (19%), RB1 (10%), BRAF (8%), TRAF3(8%), CDKN2C (7%), DNMT3A (5%), NF1, FAF1 and TET2 (4% each). While RAS, RAF, RB1 and TP53 mutations are also found in previously untreated patients, albeit in lower frequencies, mutations of DNTM3A and TET2 are rarely reported in the early phase of the disease, arguing for the accumulation of genomic alterations over time. We found concomitant alterations in KRAS and BRAF in 5, KRAS and NRAS in 3, and NRAS and BRAF in 2 patients. The vast majority of RAS alterations occurred at hotspots resulting in activating alterations at codons 12, 13 or 61 with mutant allele frequencies ranging from 0.01 to 0.92 with an average of 0.30. In the 17 patients with BRAF alterations the hotspot mutation V600E was found in 7 with mutant allele frequencies ranging from 0.01 to 0.48 with an average of 0.32. Overall the MAPK pathway was affected in 128 of 214 patients. 61 patients had alterations of genes associated with DNA damage repair. Among the 10 patients with DNMT3A alterations 2 also had alterations of TET2 suggesting significant epigenetic deregulation in a subset of patients. Data on subclonal structure and correlation of mutation status with paired gene expression profiles will be presented as well, as will be selected responses of patients treated on the basis of these results. Conclusion Subjecting CD138 selected bone marrow cells to comprehensive genomic profiling allows for the identification of clinically relevant alterations, which deregulate critical pathways in multiple myeloma. Small molecule inhibitors that target key genes in these affected pathways (MEK, BRAF) have recently been approved for therapy in other cancers or are being actively developed (PI3K, AKT, PARP). This comprehensive genomic characterization allows rational development of individualized clinical strategies using molecular targets for MM patients who are refractory to standard of care therapies. Disclosures Walker: Onyx Pharmaceuticals: Consultancy, Honoraria. van Rhee:Senesco: PI Other. Zangari:Norvartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Morgan:Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees. Barlogie:Celgene: Consultancy, Patents & Royalties, Research Funding; Millenium: Consultancy, Patents & Royalties, Research Funding.

scholarly journals A Pilot Prospective Study of Plasma Cell free DNA Whole Genome Sequencing Identified Chromosome 7p Copy Number Gains is a Specific Biomarker for Early Lung Cancer detection

2020 ◽  
Author(s):  
Hongjie Yu ◽  
Xiaojun Yu ◽  
Jia Tang ◽  
Xun Lu ◽  
Haitao Ma
Keyword(s):  
Lung Cancer ◽  
Plasma Cell ◽  
Cancer Detection ◽  
Copy Number ◽  
Chromosome 7 ◽  
P Value ◽  
Tumor Biomarker ◽  
Cell Free Dna ◽  
Free Dna ◽  
Lung Cancer Detection

Abstract purpose: Chromosome 7 is playing an important role in lung tumorigenesis. Here we investigate whether chromosome 7p copy number gain as a detectable genetic events with plasma cell free DNA for early lung cancer detection biomarker. Methods: Eighteen surgical eligible lung cancer patients and eighteen non-cancer controls were recruited. Peripheral blood was collected before surgery. Cell free DNA was profiled with low coverage whole genome sequencing. Chromosome 7 copy number gains were defined as chr7 normalized coverage≥1.0005 and P value<0.05. Plasma cell-free DNA chr7 copy gains were then compared to pathological examinations on surgical tissues.Results: 83.3% patients were confirmed as malignancy post-operation, with 12 adenocarcinoma and 3 squamous-carcinoma. The other 16.7% were benign lesions. Cell free DNA was successfully extracted from pre-surgical plasma samples, with concentration range from 0.18 to 0.49 ng/ul. Chromosome 7 short arm copy gains were found in 66.7% (10/15) patients, including 66.7% (4/6) T1aN0M0 and 50.0% (1/2) Tis patients, otherwise, chr7p gain was found in 0% (0/3) benign lesions. The specificity was further examined in 18 volunteers who undergoing routine body examinations. Meanwhile, positive CEA and CYFRA21-1 was only found in 1/18 (5.7%) and 4/18 (22.2%). Taking together, UCAD chr7p or UCAD chr7p and tumor biomarker positivity can predict 12/15(80%, 95% CI:49.0-94.3%) early lung cancers. Further analyses showed that chr7p copy number gains tends to be enriched in EGFR/KRAS silent patients (fisher. test, P value=0.077). Conclusions: Chromosome 7p copy gains might be a useful peripheral blood tumor biomarker from lung cancer detection.

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.

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