Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA.
ABSTRACT: Recent studies suggest that circulating tumor cells and cell-free DNA may represent powerful non-invasive tools for monitoring disease in patients with solid and hematologic malignancies. Here, we conducted a pilot study in 27 myeloma patients to explore the clonotypic V(D)J rearrangement for monitoring circulating myeloma cells and cell-free myeloma DNA. Next-generation sequencing was used to define the myeloma V(D)J rearrangement and for subsequent peripheral blood tracking after treatment initiation. Positivity for circulating myeloma cells/cell-free myeloma was associated with conventional remission status (P<0.001) and 91% of non-responders/progressors versus 41% of responders had evidence of persistent circulating myeloma cells/cell-free myeloma DNA (P<0.001). About half of the partial responders showed complete clearance of circulating myeloma cells/cell-free myeloma DNA despite persistent M-protein, suggesting that these markers are less inert than the M-protein, rely more on cell turnover and, therefore, decline more rapidly after initiation of effective treatment. Positivity for circulating myeloma cells and for cell-free myeloma DNA were associated with each other (P=0.042), but discordant in 30% of cases. This indicates that cell-free myeloma DNA may not be generated entirely by circulating myeloma cells and may reflect overall tumor burden. Prospective studies need to define the predictive potential of high-sensitivity determination of circulating myeloma cells and DNA in the monitoring of multiple myeloma.
Project description:The requirement for bone-marrow aspirates for genomic profiling of multiple myeloma poses an obstacle to enrolment and retention of patients in clinical trials. We evaluated whether circulating cell-free DNA (cfDNA) analysis is comparable to molecular profiling of myeloma using bone-marrow tumour cells. We report here a hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq) method used to sequence all protein-coding exons of KRAS, NRAS, BRAF, EGFR and PIK3CA in 64 cfDNA specimens from 53 myeloma patients to >20,000 × median coverage. This method includes a variant filtering algorithm that enables detection of tumour-derived fragments present in cfDNA at allele frequencies as low as 0.25% (median 3.2%, range 0.25-46%). Using LB-Seq analysis of 48 cfDNA specimens with matched bone-marrow data, we detect 49/51 likely somatic mutations, with subclonal hierarchies reflecting tumour profiling (96% concordance), and four additional mutations likely missed by bone-marrow testing (>98% specificity). Overall, LB-Seq is a high fidelity adjunct to genetic profiling of bone-marrow in multiple myeloma.
Project description:BACKGROUND: Melphalan is one of the most active chemotherapeutic agents in the treatment of multiple myeloma (MM). However, the mechanism underlying differential patient responses to melphalan therapy is unknown. METHODS: Chromatin structure, transcriptional activity and DNA damage response signals were examined following ex vivo treatment with melphalan of both malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of MM patients, responders (n=57) or non-responders (n=28) to melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. RESULTS: In both BMPCs and PBMCs, the local chromatin looseness, transcriptional activity and repair efficiency of the transcribed strand (TS) were significantly higher in non-responders than in responders and lowest in healthy controls (all P<0.05). Moreover, we found that melphalan-induced apoptosis inversely correlated with the repair efficiency of the TS, with the duration of the inhibition of mRNA synthesis, phosphorylation of p53 at serine 15 and apoptosis rates being higher in responders than in non-responders (all P<0.001). CONCLUSIONS: Our findings provide a mechanistic basis for the link between DNA repair efficiency and response to melphalan therapy. Interestingly, the observation of these phenomena in PBMCs provides a novel approach for the prediction of response to anti-myeloma therapy.
Project description:Circulating plasma cells at diagnosis, prior to auto-transplant and at relapse have a negative impact on survival in multiple myeloma. However, the impact of kinetics of circulating plasma cells along the course of illness has not been defined. We have analyzed 247 newly diagnosed multiple myeloma patients undergoing early auto-transplant who had paired evaluation of circulating plasma cells at diagnosis and pre-transplant by 6-color flow cytometry. A total of 117 patients had no detectable circulating plasma cells at both time points (CPC-/-), 82 had circulating plasma cells at diagnosis followed by complete eradication after induction (CPC+/-) and 48 had circulating plasma cells at transplant, including persistence of cells (CPC+/+; n=45) or emergence of new cells (CPC-/+; n=3) after induction. The rate of post-transplant stringent complete response was 32% in the CPC-/-, 30% in CPC+/- and 12% in CPC+/+ or -/+ groups (P=0.018). At a median follow up of 58 months from transplantation, the median progression-free survival in the 3 respective groups were 30, 24 and 14 months, and the 5-year overall survival rates were 83%, 70% and 43% (P<0.001 for both comparisons). On a multivariate analysis for overall survival, the risk of mortality was higher in CPC +/- (hazard ratio 2.7, 95%CI: 1.3-5.8; P=0.009) and CPC+/+ or -/+ (hazard ratio 5.7, 95%CI: 2.5-13.1; P<0.001) groups compared to the CPC-/- group. Monitoring for circulating plasma cells before induction therapy and before transplant by 6-color flow cytometry is predictive of survival in newly diagnosed myeloma and should be incorporated into clinical trials.
Project description:miRNA profiling in multiple myeloma - microRNAs represent a class of noncoding regulators of gene expression implicated in several biological and pathophysiological processes, including cancer. We investigate here their role in multiple myeloma using miChip-arrays interrogating 559 miRNAs in 92 purified myeloma-, MGUS-, normal plasma cell- and myeloma cell line samples. Impact on gene expression is assessed by Affymetrix U133 2.0 DNA-microarrays in 741 samples including two cohorts of 332 and 345 myeloma patients; chromosomal aberrations are assessed by iFISH, survival for 247 and 345 patients undergoing up-front high-dose therapy and autologous stem cell transplantation. Compared to normal plasma cells, 67/559 (12%) miRNAs are differentially expressed with fold changes of 4.6 to -3.1 in myeloma-, 20 (3.6%) in MGUS-samples, and three (0.5%) between MGUS- and myeloma-samples. Expression of miRNAs is associated with biological and pathophysiological parameters, i.e. proliferation, chromosomal aberrations, e.g. t(4;14), tumor mass, and gene expression-based high-risk scores. This holds true for target-gene signatures of regulated mRNAs. miRNA-expression confers prognostic significance for event-free (72/559) and overall survival (69/559), as do respective target-gene signatures. In conclusion, the miRNome of myeloma confers a pattern of small changes of individual miRNAs compared to normal plasma cells impacting on gene expression, biological functions, and survival.
Project description:OBJECTIVES:Progress in multiple myeloma treatment allows patients to achieve deeper responses, for which the assessment of minimal residual disease (MRD) is critical. Typically, bone marrow samples are used for this purpose; however, this approach is site-limited. Liquid biopsy represents a minimally invasive and more comprehensive technique that is not site-limited, but equally challenging. METHODS:While majority of current data comes from short-term studies, we present a long-term study on blood-based MRD monitoring using tumor-specific cell-free DNA detection by ASO-qPCR. One hundred and twelve patients were enrolled into the study, but long-term sampling and analysis were feasible only in 45 patients. RESULTS:We found a significant correlation of quantity of tumor-specific cell-free DNA levels with clinically meaningful events [induction therapy (P = .004); ASCT (P = .012)]. Moreover, length of cfDNA fragments is associated with better treatment response of patients. CONCLUSIONS:These results support the concept of tumor-specific cell-free DNA as a prognostic marker.
Project description:The presence of circulating plasma cells in patients with multiple myeloma is considered a marker for highly proliferative disease. In the study herein, the impact of circulating plasma cells assessed by cytology on survival of patients with multiple myeloma was analyzed. Wright-Giemsa stained peripheral blood smears of 482 patients with newly diagnosed myeloma or plasma cell leukemia were reviewed and patients were classified into 4 categories according to the percentage of circulating plasma cells: 0%, 1-4%, 5-20%, and plasma cell leukemia with the following frequencies: 382 (79.2%), 83 (17.2%), 12 (2.5%) and 5 (1.0%), respectively. Median overall survival according to the circulating plasma cells group was 47, 50, 6 and 14 months, respectively. At multivariate analysis, the presence of 5 to 20% circulating plasma cells was associated with a worse overall survival (relative risk 4.9, 95% CI 2.6-9.3) independently of age, creatinine, the Durie-Salmon system stage and the International Staging System (ISS) stage. Patients with ?5% circulating plasma cells had lower platelet counts (median 86×10<sup>9</sup>/L <i>vs</i> 214×10<sup>9</sup>/L, <i>P</i><0.0001) and higher bone marrow plasma cells (median 53% <i>vs</i> 36%, <i>P</i>=0.004). The presence of ?5% circulating plasma cells in patients with multiple myeloma has a similar adverse prognostic impact as plasma cell leukemia.
Project description:Multiple myeloma is an incurable cancer of bone marrow plasma cells, with a 5-year survival rate of 43%. Its incidence has increased by 126% since 1990. Treatment typically involves high-dose combination chemotherapy, but therapeutic response and patient survival are unpredictable and highly variable-attributed largely to the development of multidrug resistance (MDR). MDR is the simultaneous cross-resistance to a range of unrelated chemotherapeutic agents and is associated with poor prognosis and survival. Currently, no clinical procedures allow for a direct, continuous monitoring of MDR. We identified circulating large extracellular vesicles (specifically microparticles (MPs)) that can be used to monitor disease burden, disease progression and development of MDR in myeloma. These MPs differ phenotypically in the expression of four protein biomarkers: a plasma-cell marker (CD138), the MDR protein, P-glycoprotein (P-gp), the stem-cell marker (CD34); and phosphatidylserine (PS), an MP marker and mediator of cancer spread. Elevated levels of P-gp+ and PS+ MPs correlate with disease progression and treatment unresponsiveness. Furthermore, P-gp, PS and CD34 are predominantly expressed in CD138- MPs in advanced disease. In particular, a dual-positive (CD138-P-gp+CD34+) population is elevated in aggressive/unresponsive disease. Our test provides a personalised liquid biopsy with potential to address the unmet clinical need of monitoring MDR and treatment failure in myeloma.
Project description:Circulating tumor DNA is a promising biomarker to monitor tumor load and genome alterations. We explored the presence of circulating tumor DNA in multiple myeloma patients and its relation to disease activity during long-term follow-up. We used digital droplet polymerase chain reaction analysis to monitor recurrent mutations, mainly in mitogen activated protein kinase pathway genes <i>NRAS</i>, <i>KRAS</i> and <i>BRAF</i> Mutations were identified by next-generation sequencing or polymerase chain reaction analysis of bone marrow plasma cells, and their presence analyzed in 251 archived serum samples obtained from 20 patients during a period of up to 7 years. In 17 of 18 patients, mutations identified in bone marrow during active disease were also found in a time-matched serum sample. The concentration of mutated alleles in serum correlated with the fraction in bone marrow plasma cells (r=0.507, n=34, <i>P</i><0.002). There was a striking covariation between circulating mutation levels and M protein in ten out of 11 patients with sequential samples. When relapse evaluation by circulating tumor DNA and M protein could be directly compared, the circulating tumor DNA showed relapse earlier in two patients (3 and 9 months), later in one patient (4 months) and in three patients there was no difference. In three patients with transformation to aggressive disease, the concentrations of mutations in serum increased up to 400 times, an increase that was not seen for the M protein. In conclusion, circulating tumor DNA in myeloma is a multi-faceted biomarker reflecting mutated cells, total tumor mass and transformation to a more aggressive disease. Its properties are both similar and complementary to M protein.
Project description:Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.