Droplet digital polymerase chain reaction for the assessment of disease burden in hairy cell leukemia.

BRAFV600E mutation is the pathogenic driver of hairy cell leukemia (HCL) found in the vast majority of cases both at onset and during recurrences. The identification of the mutated allele in blood and marrow correlates with the presence of neoplastic cells and can be considered a marker of active disease. Likewise, the absence of the mutation after treatment may indicate a state of deep response. The BRAFV600E burden was measured by droplet digital polymerase chain reaction (ddPCR) and expressed as fractional abundance in 35 HCL patients at different stages of disease (onset, relapse, complete response [CR] after treatment, long-term remission) in peripheral blood and/or bone marrow (when available). Mean values of fractional abundance for patients at diagnosis, relapse and response, respectively, were 12.26%, 16.52% and 0.02% in peripheral blood and 23.51%, 13.96% and 0.26% in bone marrow. Four patients out of 6 evaluated at response were molecularly negative for BRAFV600E in peripheral blood. Mean fractional abundance in peripheral blood tested in 14 patients with long lasting CR was 0.05%, and 10 patients were BRAFV600E negative. These preliminary results suggest that ddPCR permits to assess the active tumor burden in HCL at different disease phases and support the hypothesis that some patients in CR qualify for a molecular CR.

Identification of a Maturation Plasma Cell Index through a Highly Sensitive Droplet Digital PCR Assay Gene Expression Signature Validation in Newly Diagnosed Multiple Myeloma Patients.

DNA microarrays and RNA-based sequencing approaches are considered important discovery tools in clinical medicine. However, cross-platform reproducibility studies undertaken so far have highlighted that microarrays are not able to accurately measure gene expression, particularly when they are expressed at low levels. Here, we consider the employment of a digital PCR assay (ddPCR) to validate a gene signature previously identified by gene expression profile. This signature included ten Hedgehog (HH) pathways' genes able to stratify multiple myeloma (MM) patients according to their self-renewal status. Results show that the designed assay is able to validate gene expression data, both in a retrospective as well as in a prospective cohort. In addition, the plasma cells' differentiation status determined by ddPCR was further confirmed by other techniques, such as flow cytometry, allowing the identification of patients with immature plasma cells' phenotype (i.e., expressing CD19+/CD81+ markers) upregulating HH genes, as compared to others, whose plasma cells lose the expression of these markers and were more differentiated. To our knowledge, this is the first technical report of gene expression data validation by ddPCR instead of classical qPCR. This approach permitted the identification of a Maturation Index through the integration of molecular and phenotypic data, able to possibly define upfront the differentiation status of MM patients that would be clinically relevant in the future.

A real-world efficacy and safety analysis of combined carfilzomib, lenalidomide, and dexamethasone (KRd) in relapsed/refractory multiple myeloma.

Carfilzomib-lenalidomide-dexamethasone (KRd) has been approved for the treatment of relapsed/refractory multiple myeloma (RRMM). We conducted a retrospective analysis of 197 RRMM patients (pts) between January 2016 and March 2018 in six Italian hematologic centers, with the aim to evaluate efficacy and safety of KRd in real-life. At KRd initiation 27% carried high risk cytogenetic abnormalities (HRCA) [del17p and/or t(4;14) and/or t(14;16)], median number of prior lines of therapy was 2 (1-8), nearly all pts (96%) received prior bortezomib (18% refractory) while 45% were exposed to lenalidomide (R; 22% refractory). At the median of 12.5 months, 52% of the pts had discontinued treatment, mainly (66%) for progression. Main grade 3-4 adverse events were neutropenia (21%), infections (11%), and hypertension (6%). Overall, the response rate was 88%. The median progression-free survival (PFS) was 19.8 months and 1-year overall survival (OS) rate was 80.6%. By subgroup analysis, extended PFS and OS were observed for pts who received ≤2 prior lines of therapy (HR = 0.42, p < 0.001 and HR = 0.35, p = 0.001, respectively), not refractory to prior R (HR = 0.37, p < 0.001, and HR = 0.47, p = 0.024), without HRCA (HR = 0.33, p = 0.005 and HR = 0.26, p = 0.016) and achieving ≥ very good partial response (VGPR; HR = 0.17, p < 0.001 and HR = 0.18, p < 0.001). In conclusion, KRd demonstrated to be effective in RRMM pts treated in real-world setting, without new safety concerns. Better survival outcomes emerged for pts with ≤2 prior lines of therapy, achieving at least a VGPR, and without HRCA.

Disease-Specific Derangement of Circulating Endocannabinoids and N-Acylethanolamines in Myeloproliferative Neoplasms.

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family-palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC-MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

Novel agent-based salvage autologous stem cell transplantation for relapsed multiple myeloma.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard frontline therapy for multiple myeloma (MM). Therapeutic options for patients with relapsed MM after ASCT include novel agents in different combos, salvage ASCT (sASCT), and allogeneic transplant, with no unique standard of care. We retrospectively analyzed 66 MM patients who relapsed after up-front single or double ASCT(s) and received novel agent-based sASCT at five Italian centers. Median event-free survival from up-front ASCT(s) to first relapse (EFS1) was 44 months. Seventy-three percent of patients received sASCT at first disease progression. Re-induction regimens were bortezomib based in 87% of patients. Response to re-induction therapy included complete response (CR) 18%, ≥ very good partial response (VGPR) 48%, and overall response rate (ORR) 83%. Response to sASCT included CR 44%, ≥ VGPR 77%, and ORR 94%. With a median follow-up of 24 months after sASCT, 39 patients experienced disease progression. Median EFS from sASCT (EFS2) was 17 months. Median overall survival from ASCT (OS1) and sASCT (OS2) was 166 and 43 months, respectively. EFS2 and OS2 were significantly shorter in patients with EFS1 ≤ 24 months, in patients who did not receive sASCT at first disease progression and in patients with extramedullary disease (EMD). In multivariate analysis, EFS1 ≤ 24 months was associated with shorter EFS2 and OS2, EMD was associated with shorter EFS2, and < CR after sASCT was associated with shorter OS2. Novel agent-based sASCT is a safe and effective procedure for relapsed MM.

HIF-1α inhibition blocks the cross talk between multiple myeloma plasma cells and tumor microenvironment.

Multiple myeloma (MM) is a malignant disorder of post-germinal center B cells, characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). The reciprocal and complex interactions that take place between the different compartments of BM and the MM cells result in tumor growth, angiogenesis, bone disease, and drug resistance. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of Hypoxia-Inducible transcription Factor-1 alpha (HIF-1α) in the PCs-bone marrow stromal cells interplay. To test this hypothesis, we used EZN-2968, a 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. Herein, we provide evidence that the interaction between MM cells and BM stromal cells is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BM stromal cells were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed a down-modulation of cytokine-induced signaling cascades and a reduction of MM cells adhesion capability to the extracellular matrix proteins in EZN-2968-treated samples. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between bone BM cells and PCs in Multiple Myeloma.

Bortezomib- and thalidomide-induced peripheral neuropathy in multiple myeloma: clinical and molecular analyses of a phase 3 study.

A subanalysis of the GIMEMA-MMY-3006 trial was performed to characterize treatment-emergent peripheral neuropathy (PN) in patients randomized to thalidomide-dexamethasone (TD) or bortezomib-TD (VTD) before and after double autologous transplantation (ASCT) for multiple myeloma (MM). A total of 236 patients randomized to VTD and 238 to TD were stratified according to the emergence of grade ≥2 PN. Gene expression profiles (GEP) of CD138+ plasma cells were analyzed in 120 VTD-treated patients. The incidence of grade ≥2 PN was 35% in the VTD arm and 10% in the TD arm (P < 0.001). PN resolved in 88 and 95% of patients in VTD and TD groups, respectively. Rates of complete/near complete response, progression-free and overall survival were not adversely affected by emergence of grade ≥2 PN. Baseline characteristics were not risk factors for PN, while GEP analysis revealed the deregulated expression of genes implicated in cytoskeleton rearrangement, neurogenesis, and axonal guidance. In conclusion, in comparison with TD, incorporation of VTD into ASCT was associated with a higher incidence of PN which, however, was reversible in most of the patients and did not adversely affect their outcomes nor their ability to subsequently receive ASCT. GEP analysis suggests an interaction between myeloma genetic profiles and development of VTD-induced PN.

Multi-dimensional scaling techniques unveiled gain1q&loss13q co-occurrence in Multiple Myeloma patients with specific genomic, transcriptional and adverse clinical features.

The complexity of Multiple Myeloma (MM) is driven by several genomic aberrations, interacting with disease-related and/or -unrelated factors and conditioning patients' clinical outcome. Patient's prognosis is hardly predictable, as commonly employed MM risk models do not precisely partition high- from low-risk patients, preventing the reliable recognition of early relapsing/refractory patients. By a dimensionality reduction approach, here we dissect the genomic landscape of a large cohort of newly diagnosed MM patients, modelling all the possible interactions between any MM chromosomal alterations. We highlight the presence of a distinguished cluster of patients in the low-dimensionality space, with unfavorable clinical behavior, whose biology was driven by the co-occurrence of chromosomes 1q CN gain and 13 CN loss. Presence or absence of these alterations define MM patients overexpressing either CCND2 or CCND1, fostering the implementation of biology-based patients' classification models to describe the different MM clinical behaviors.

Circulating Multiple Myeloma Cells (CMMCs) as Prognostic and Predictive Markers in Multiple Myeloma and Smouldering MM Patients.

In recent years, liquid biopsy has emerged as a promising alternative to the bone marrow (BM) examination, since it is a minimally invasive technique allowing serial monitoring. Circulating multiple myeloma cells (CMMCs) enumerated using CELLSEARCH® were correlated with patients' prognosis and measured under treatment to assess their role in monitoring disease dynamics. Forty-four MM and seven smouldering MM (SMM) patients were studied. The CMMC medians at diagnosis were 349 (1 to 39,940) and 327 (range 22-2463) for MM and SMM, respectively. In the MM patients, the CMMC count was correlated with serum albumin, calcium, ß2-microglobulin, and monoclonal components (p < 0.04). Under therapy, the CMMCs were consistently detectable in 15/40 patients (coMMstant = 1) and were undetectable or decreasing in 25/40 patients (coMMstant = 0). High-quality response rates were lower in the coMMstant = 1 group (p = 0.04), with a 7.8-fold higher risk of death (p = 0.039), suggesting that continuous CMMC release is correlated with poor responses. In four MM patients, a single-cell DNA sequencing analysis on residual CMMCs confirmed the genomic pattern of the aberrations observed in the BM samples, also highlighting the presence of emerging clones. The CMMC kinetics during treatment were used to separate the patients into two subgroups based on the coMMstant index, with different responses and survival probabilities, providing evidence that CMMC persistence is associated with a poor disease course.

Single-Cell DNA Sequencing Reveals an Evolutionary Pattern of CHIP in Transplant Eligible Multiple Myeloma Patients.

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the phenomenon where a hematopoietic stem cell acquires fitness-increasing mutation(s), resulting in its clonal expansion. CHIP is frequently observed in multiple myeloma (MM) patients, and it is associated with a worse outcome. High-throughput amplicon-based single-cell DNA sequencing was performed on circulating CD34+ cells collected from twelve MM patients before autologous stem cell transplantation (ASCT). Moreover, in four MM patients, longitudinal samples either before or post-ASCT were collected. Single-cell sequencing and data analysis were assessed using the MissionBio Tapestri® platform, with a targeted panel of 20 leukemia-associated genes. We detected CHIP pathogenic mutations in 6/12 patients (50%) at the time of transplant. The most frequently mutated genes were TET2, EZH2, KIT, DNMT3A, and ASXL1. In two patients, we observed co-occurring mutations involving an epigenetic modifier (i.e., DNMT3A) and/or a gene involved in splicing machinery (i.e., SF3B1) and/or a tyrosine kinase receptor (i.e., KIT) in the same clone. Longitudinal analysis of paired samples revealed a positive selection of mutant high-fitness clones over time, regardless of their affinity with a major or minor sub-clone. Copy number analysis of the panel of all genes did not show any numerical alterations present in stem cell compartment. Moreover, we observed a tendency of CHIP-positive patients to achieve a suboptimal response to therapy compared to those without. A sub-clone dynamic of high-fitness mutations over time was confirmed.

Correlation between eight-gene expression profiling and response to therapy of newly diagnosed multiple myeloma patients treated with thalidomide-dexamethasone incorporated into double autologous transplantation.

We performed a molecular study aimed at identifying a gene expression profile (GEP) signature predictive of attainment of at least near complete response (CR) to thalidomide-dexamethasone (TD) as induction regimen in preparation for double autologous stem cell transplantation in 112 younger patients with newly diagnosed multiple myeloma. A GEP supervised analysis was performed on a training set of 32 patients, allowing to identify 157 probe sets differentially expressed in patients with CR versus those failing CR to TD. We then generated an eight-gene GEP signature whose performance was subsequently validated in a training set of 80 patients. A correct prediction of response to TD was found in 71 % of the cases analyzed. The eight genes were downregulated in patients who achieved CR to TD. Comparisons between post-autotransplantation outcomes of the 44 non-CR-predicted patients and of the 36 CR-predicted patients showed that this latter subgroup had a statistically significant benefit in terms of higher rate of CR after autotransplant(s) and longer time to progression, event-free survival, and overall survival. These results can be an important first step to identify at diagnosis those patients who will respond more favourably to a particular treatment strategy.

High levels of CRBN isoform lacking IMiDs binding domain predicts for a worse response to IMiDs-based upfront therapy in newly diagnosed myeloma patients.

In recent years, the immunoderivative (IMiD) agents have been extensively used for the treatment of multiple myeloma (MM). IMiDs and their newer derivatives CRBN E3 ligase modulator bind the E3 ligase substrate recognition adapter protein cereblon (CRBN), which has been recognized as one of the IMiDs' direct target proteins, and it is essential for the therapeutic effect of these agents.High expression of CRBN was associated with improved clinical response in patients with MM treated with IMiDs, further confirming that the expression of IMiDs' direct target protein CRBN is required for the anti-MM activity. CRBN's central role as a target of IMiDs suggests potential utility as a predictive biomarker of response or resistance to IMiDs therapy. Additionally, the presence of alternatively spliced variants of CRBN in MM cells, especially those lacking the drug-binding domain for IMiDs, raise questions concerning their potential biological function, making difficult the transcript measurement, which leads to inaccurate overestimation of full-length CRBN transcripts. In sight of this, in the present study, we evaluated the CRBN expression, both full-length and spliced isoforms, by using real-time assay data from 87 patients and RNA sequencing data from 50 patients (n = 137 newly diagnosed MM patients), aiming at defining CRBN's role as a predictive biomarker for response to IMiDs-based induction therapy. We found that the expression level of the spliced isoform tends to be higher in not-responding patients, confirming that the presence of a more CRBN spliced transcript predicts for lack of IMiDs response.

Long term follow-up of humoral and cellular response to mRNA-based vaccines for SARS-CoV-2 in patients with active multiple myeloma.

Long-term kinetics of antibody (Ab) and cell-mediated immune (CMI) response to full anti-SARS-CoV-2 vaccine schedule and booster doses in Multiple Myeloma (MM) patients remain unclear. We prospectively evaluated Ab and CMI response to mRNA vaccines in 103 SARS-CoV-2-naïve MM patients (median age 66, 1 median prior line of therapy) and 63 health-workers. Anti-S-RBD IgG (Elecsys®assay) were measured before vaccination and after 1 (T1), 3 (T3), 6 (T6), 9 (T9) and 12 (T12) months from second dose (D2) and 1 month after the introduction of the booster dose (T1D3). CMI response (IGRA test) was evaluated at T3 and T12. Fully vaccinated MM patients displayed high seropositivity rate (88.2%), but low CMI response (36.2%). At T6 the median serological titer was halved (p=0.0391) in MM patients and 35% reduced (p=0.0026) in controls. D3 (94 patients) increased the seroconversion rate to 99% in MM patients and the median IgG titer in both groups (up to 2500 U/mL), maintained at T12. 47% of MM patients displayed a positive CMI at T12 and double-negativity for humoral and CMI (9.6% at T3) decreased to 1%. Anti-S-RBD IgG level ≥346 U/mL showed 20-times higher probability of positive CMI response (OR 20.6, p<0.0001). Hematological response ≥CR and ongoing lenalidomide maintenance enhanced response to vaccination, hindered by proteasome inhibitors/anti-CD38 monoclonal antibodies. In conclusion, MM elicited excellent humoral, but insufficient cellular responses to anti-SARS-CoV-2 mRNA vaccines. Third dose improved immunogenicity renewal, even when undetectable after D2. Hematological response and ongoing treatment at vaccination were the main predictive factors of vaccine immunogenicity, emphasizing the role of vaccine response assessment to identify patients requiring salvage approaches.

The ALLgorithMM: How to define the hemodilution of bone marrow samples in lymphoproliferative diseases.

Introduction: Minimal residual disease (MRD) is commonly assessed in bone marrow (BM) aspirate. However, sample quality can impair the MRD measurement, leading to underestimated residual cells and to false negative results. To define a reliable and reproducible method for the assessment of BM hemodilution, several flow cytometry (FC) strategies for hemodilution evaluation have been compared. Methods: For each BM sample, cells populations with a well-known distribution in BM and peripheral blood - e.g., mast cells (MC), immature (IG) and mature granulocytes (N) - have been studied by FC and quantified alongside the BM differential count. Results: The frequencies of cells' populations were correlated to the IG/N ratio, highlighting a mild correlation with MCs and erythroblasts (R=0.25 and R=0.38 respectively, with p-value=0.0006 and 0.0000052), whereas no significant correlation was found with B or T-cells. The mild correlation between IG/N, erythroblasts and MCs supported the combined use of these parameters to evaluate BM hemodilution, hence the optimization of the ALLgorithMM. Once validated, the ALLgorithMM was employed to evaluate the dilution status of BM samples in the context of MRD assessment. Overall, we found that 32% of FC and 52% of Next Generation Sequencing (NGS) analyses were MRD negative in samples resulted hemodiluted (HD) or at least mildly hemodiluted (mHD). Conclusions: The high frequency of MRD-negative results in both HD and mHD samples implies the presence of possible false negative MRD measurements, impairing the correct assessment of patients' response to therapy and highlighs the importance to evaluate BM hemodilution.

Next-Generation Sequencing for Clinical Management of Multiple Myeloma: Ready for Prime Time?

Personalized treatment is an attractive strategy that promises increased efficacy with reduced side effects in cancer. The feasibility of such an approach has been greatly boosted by next-generation sequencing (NGS) techniques, which can return detailed information on the genome and on the transcriptome of each patient's tumor, thus highlighting biomarkers of response or druggable targets that may differ from case to case. However, while the number of cancers sequenced is growing exponentially, much fewer cases are amenable to a molecularly-guided treatment outside of clinical trials to date. In multiple myeloma, genomic analysis shows a variety of gene mutations, aneuploidies, segmental copy-number changes, translocations that are extremely heterogeneous, and more numerous than other hematological malignancies. Currently, in routine clinical practice we employ reduced FISH panels that only capture three high-risk features as part of the R-ISS. On the contrary, recent advances have suggested that extending genomic analysis to the full spectrum of recurrent mutations and structural abnormalities in multiple myeloma may have biological and clinical implications. Furthermore, increased efficacy of novel treatments can now produce deeper responses, and standard methods do not have enough sensitivity to stratify patients in complete biochemical remission. Consequently, NGS techniques have been developed to monitor the size of the clone to a sensitivity of up to a cell in a million after treatment. However, even these techniques are not within reach of standard laboratories. In this review we will recapitulate recent advances in multiple myeloma genomics, with special focus on the ones that may have immediate translational impact. We will analyze the benefits and pitfalls of NGS-based diagnostics, highlighting crucial aspects that will need to be taken into account before this can be implemented in most laboratories. We will make the point that a new era in myeloma diagnostics and minimal residual disease monitoring is close and conventional genetic testing will not be able to return the required information. This will mandate that even in routine practice NGS should soon be adopted owing to a higher informative potential with increasing clinical benefits.

Integrative analysis of the genomic and transcriptomic landscape of double-refractory multiple myeloma.

In multiple myeloma, novel treatments with proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) have prolonged survival but the disease remains incurable. At relapse, next-generation sequencing has shown occasional mutations of drug targets but has failed to identify unifying features that underlie chemotherapy resistance. We studied 42 patients refractory to both PIs and IMiDs. Whole-exome sequencing was performed in 40 patients, and RNA sequencing (RNA-seq) was performed in 27. We found more mutations than were reported at diagnosis and more subclonal mutations, which implies ongoing evolution of the genome of myeloma cells during treatment. The mutational landscape was different from that described in published studies on samples taken at diagnosis. The TP53 pathway was the most frequently inactivated (in 45% of patients). Conversely, point mutations of genes associated with resistance to IMiDs were rare and were always subclonal. Refractory patients were uniquely characterized by having a mutational signature linked to exposure to alkylating agents, whose role in chemotherapy resistance and disease progression remains to be elucidated. RNA-seq analysis showed that treatment or mutations had no influence on clustering, which was instead influenced by karyotypic events. We describe a cluster with both amp(1q) and del(13) characterized by CCND2 upregulation and also overexpression of MCL1, which represents a novel target for experimental treatments. Overall, high-risk features were found in 65% of patients. However, only amp(1q) predicted survival. Gene mutations of IMiD and PI targets are not a preferred mode of drug resistance in myeloma. Chemotherapy resistance of the bulk tumor population is likely attained through differential, yet converging evolution of subclones that are overall variable from patient to patient and within the same patient.

Minimal Residual Disease Assessment Within the Bone Marrow of Multiple Myeloma: A Review of Caveats, Clinical Significance and Future Perspectives.

There is an increasing clinical interest in the measure and achievement of minimal residual disease (MRD) negativity in the bone marrow of Multiple Myeloma (MM) patients, as defined equally either by Multicolor Flow Cytometry (MFC) or by Next Generation Sequencing (NGS) technologies. At present, modern technologies allow to detect up to one on 104 or on 105 or even on 106 cells, depending on their throughput. MFC approaches, which have been progressively improved up to the so-called Next Generation Flow (NGF), and NGS, which proved clear advantages over ASO-PCR, can detect very low levels of residual disease in the BM. These methods are actually almost superimposable, in terms of MRD detection power, supporting the lack of unanimous preference for either technique on basis of local availability. However, some technical issues are still open: the optimal assay to use to detect either phenotype (e.g., next generation multidimensional flow cytometry, imaging) or genotype aberrations (e.g., ASO-RQ PCR, digital droplet PCR, NGS) and their standardization, the sample source (BM or peripheral blood, PB) and its pre-processing (red-cell lysis vs. Ficoll, fresh vs. frozen samples, requirement of CD138+ cells enrichment). Overall, MRD negativity is considered as the most powerful predictor of favorable long-term outcomes in MM and is likely to represent the major driver of treatment strategies in the near future. In this manuscript, we reviewed the main pitfalls and caveats of MRD detection within bone marrow in MM patients after front-line therapy, highlighting the improving of the currently employed technology and describing alternative methods for MRD testing in MM, such as liquid biopsy.

Prognostic impact of serial measurements of serum-free light chain assay throughout the course of newly diagnosed multiple myeloma treated with bortezomib-based regimens.

We retrospectively investigated the role of serial serum-free light chain (sFLC) evaluations in 150 multiple myeloma (MM) patients treated with first-line bortezomib-based regimens. Baseline sFLC ratio (sFLCR) identified three groups of patients - normal, lightly abnormal (<100), and highly abnormal (≥100) - with different progression-free survival (PFS: 3-year estimate 72% versus 61% versus 44%, respectively, p = 0.03). Moreover, the achievement of a normal sFLCR correlated with extended PFS (49 versus 17 months, p < 0.0001) and overall survival (75 versus 43 months, p < 0.0001) as compared with abnormal sFLCR, a gain maintained in a multivariate analysis for PFS. At relapse, a high sFLCR was associated with earlier start of salvage therapy compared with sFLCR <100 (3-month probability: 89% versus 64%, p = 0.0426). In 20% of patients, sFLC escape preceded the conventional relapse by a median of 3.8 months. Our results highlight the role of sFLC assay in the prognosis and follow-up of MM.

The genetic and genomic background of multiple myeloma patients achieving complete response after induction therapy with bortezomib, thalidomide and dexamethasone (VTD).

The prime focus of the current therapeutic strategy for Multiple Myeloma (MM) is to obtain an early and deep tumour burden reduction, up to the level of complete response (CR). To date, no description of the characteristics of the plasma cells (PC) prone to achieve CR has been reported. This study aimed at the molecular characterization of PC obtained at baseline from MM patients in CR after bortezomib-thalidomide-dexamethasone (VTD) first line therapy.One hundred and eighteen MM primary tumours obtained from homogeneously treated patients were profiled both for gene expression and for single nucleotide polymorphism genotype. Genomic results were used to obtain a predictor of sensitivity to VTD induction therapy, as well as to describe both the transcription and the genomic profile of PC derived from MM with subsequent optimal response to primary induction therapy.By analysing the gene profiles of CR patients, we identified a 5-gene signature predicting CR with an overall median accuracy of 75% (range: 72%-85%). In addition, we highlighted the differential expression of a series of genes, whose deregulation might explain patients' sensitivity to VTD therapy. We also showed that a small copy number loss, covering 606Kb on chromosome 1p22.1 was the most significantly associated with CR patients.