Abstract Introduction Over the past two decades, transcatheter aortic valve replacement (TAVR) has transformed the management of aortic stenosis, initially for high-risk surgical candidates and subsequently for low-risk patients. However, with the expanding indications for TAVR to younger, lower-risk patients, it is crucial to assess not only the short-term clinical outcomes but also the long-term considerations for future interventions. The prevalence of coronary artery disease (CAD) in TAVR patients is relevant, and the optimal timing of percutaneous coronary intervention (PCI) remains a question, given the technical challenges associated with coronary access in TAVR patients. Methods We conducted a systematic literature review and meta-analysis according to Cochrane Collaborations and PRISMA guidelines. We included 19 eligible trials involving 1533 patients who underwent coronary angiography after TAVR. The primary endpoint was the incidence of successful selective coronary re-access. Secondary endpoints included semi-selective and non-selective access rates. The analysis was stratified by balloon-expandable (BEVs) and self-expandable valve (SEVs) types. Results Successful coronary access after TAVR was feasible in the majority of patients, with a higher success rate observed for the left main coronary artery (LM) compared to the right coronary artery (RCA). Balloon-expandable valves demonstrated the highest success rates in coronary ostia cannulation, achieving nearly 100% success for both LM and RCA. Among self-expandable valves, the Evolut R/PRO showed superior success rates in selective coronary access (76,85% (95%CI 53,34-100%) for LM and 57,27% (95%CI 32,8-81,74%) for RCA) compared to the CoreValve (45,62% (95%CI 0-92,78%) for LM and 49,19% (95%CI 0-100%) for RCA). Notably, the majority of coronary angiograms were performed due to acute coronary syndrome, primarily non-ST-segment elevation myocardial infarction (NSTEMI) and unstable angina (UA), 26,5% for NSTEMI and 23,4% for UA, respectively. Conclusions Selective coronary engagement after TAVR is generally achievable, with BEVs demonstrating superior success rates compared to SEVs. Among self-expandable valves, the Evolut R/PRO showed better outcomes than the CoreValve. These findings provide valuable insights into post-TAVR coronary access feasibility, reducing concerns about valve selection based on this criterion

PDF file - 114K, Boxplots of differentially expressed genes between pPCL and MM patients as determined by Q-RT-PCR.

PDF file - 59K, Forty-seven genes differentially expressed in t(11;14), t(4;14) and MAF-associated translocations group.

PDF file - 862K, Boxplot distributions of the expression levels of the 26 progression-associated genes.

PDF file - 114K, Table of the most significant gene sets in GSEA analysis of pPCL versus MM, ordered according to adjusted false discovery rate.

PDF file - 122K, Boxplot distribution of NF-kB and proliferation indexes in the 21 PPCL and the 55 MM patients.

PDF file - 22K, Results of correlation analysis between miRNA expression and copy number.

PDF file - 749K, Histogram of the expression values of MAFA gene in the 21 PPCL and the 55 MM patients.

PDF file - 156K, 503-gene list differentially expressed between pPCL and MM.

We analyzed variations in terms of chromosomal abnormalities (CA) by fluorescence in situ hybridization (FISH) analysis on purified bone marrow plasma cells throughout the progression from monoclonal gammopathy of undetermined significance/smoldering multiple myeloma (MGUS/SMM) to newly diagnosed MM/plasma cell leukemia (NDMM/PCL) at diagnosis and from diagnostic samples to progressive disease. High risk was defined by the presence of at least del(17p), t(4;14), and/or t(14;16). 1p/1q detection (in the standard FISH panel from 2012 onward) was not available for all patients. We analyzed 139 MM/PCL diagnostic samples from 144 patients, with a median follow-up of 71 months: high-risk CA at diagnosis (MGUS/SMM or NDMM) was present in 28% of samples, whereas 37–39% showed high-risk CA at relapse. In 115 patients with NDMM who evolved to relapsed/refractory MM, we identified 3 different populations: (1) 31/115 patients (27%) with gain of new CA (del13, del17p, t(4;14), t(14;16) or 1q CA when available); (2) 10/115 (9%) patients with loss of a previously identified CA; and (3) 74 patients with no changes. The CA gain group showed a median overall survival of 66 months vs. 84 months in the third group (HR 0.56, 95% CI 0.34–0.92, p = 0.023). Clonal evolution occurs as disease progresses after different chemotherapy lines. Patients who acquired high-risk CA had the poorest prognosis. Our findings highlight the importance of performing FISH analysis both at diagnosis and at relapse.

Despite remarkable advances in the treatment of multiple myeloma in the last decades, the prognosis of patients harboring high-risk cytogenetic abnormalities remains dismal as compared to that of standard-risk patients. Proteasome inhibitors demonstrated to partially ameliorate the prognosis of high-risk patients. We pooled together data from two phase I/II trials on transplant-ineligible patients with multiple myeloma receiving upfront carfilzomib cyclophosphamide and dexamethasone followed by carfilzomib maintenance. The aim of this analysis was to compare treatment outcomes in patients with standard- versus high-risk cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH) analysis. High risk was defined by the presence of at least one chromosomal abnormality, including t(4;14), del17p and t(14;16). Overall, 94 patients were included in the analysis: 57 (61%) in the standard-risk and 37 (39%) in the high-risk group. Median follow-up was 38 months. In standard- vs. high-risk patients, we observed similar progression-free survival (3-year PFS: 52% vs. 43%, respectively; p=0.50), overall survival (3-year OS: 78% vs. 73%; p=0.38), and overall response rate (88% vs 95%; p=0.47), with no statistical differences between the two groups. No difference in terms of progression-free survival was observed between patients with or without del17p. Carfilzomib, used both as induction and maintenance agent for transplant-ineligible newly diagnosed multiple myeloma patients, mitigated the poor prognosis carried by high-risk cytogenetics and resulted into similar progression-free survival and overall survival, as compared to standard-risk patients. ClinicalTrials.gov IDs: NCT01857115 (IST-CAR-561) and NCT01346787 (IST-CAR-506).

Minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is the most effective tool to define a deep response in multiple myeloma (MM). We conducted an MRD correlative study of the EMN02/HO95 MM phase III trial in newly diagnosed MM patients achieving a suspected complete response before maintenance and every 6 months during maintenance. Patients received high-dose melphalan (HDM) versus bortezomib-melphalan-prednisone (VMP) intensification, followed by bortezomib-lenalidomide-dexamethasone (VRd) versus no consolidation, and lenalidomide maintenance. Bone marrow (BM) samples were processed in three European laboratories, applying EuroFlow-based MFC protocols (eight colors, two tubes) with 10 −4 −10 −5 sensitivity. At enrollment in the MRD correlative study, 76% (244/321) of patients were MRD-negative. In the intention-to-treat analysis, after a median follow-up of 75 months, 5-year progression-free survival was 66% in MRD-negative versus 31% in MRD-positive patients (HR 0.39; p < 0.001), 5-year overall survival was 86% versus 69%, respectively (HR 0.41; p < 0.001). MRD negativity was associated with reduced risk of progression or death in all subgroups, including ISS-III (HR 0.37) and high-risk fluorescence in situ hybridization (FISH) patients (HR 0.38;). In the 1-year maintenance MRD population, 42% of MRD-positive patients at pre-maintenance became MRD-negative after lenalidomide exposure. In conclusion, MRD by MFC is a strong prognostic factor. Lenalidomide maintenance further improved MRD-negativity rate.

Waldenström Macroglobulinemia (WM) is defined by the presence of an indolent lymphoplasmacytic lymphoma (LPL) (monoclonal lymphocytes, lymphoplasmacytes and plasma cells (PC) in the bone marrow (BM)) and monoclonal IgM protein secretion.1,2 Therefore, a BM biopsy showing LPL infiltration is currently essential to define WM; nevertheless, sometimes the pathological diagnosis can be troublesome: actually, the prevalence of monoclonal PC might suggest a diagnosis of multiple myeloma (MM), while in other cases, when small lymphocyte infiltration is predominant, the differentiation with other lymphomas can represent a challenge, both at morphologic and at immunophenotypic exam. In addition, the BM biopsy is a rather invasive surgical procedure and might represent a diagnostic limitation, in particular when dealing with elderly or unfit patients. Moreover, both the risk of relapse and the sensitivity to novel drugs are still difficult to predict in WM. Finally, some particular IgM-associated conditions (eg, demyelinating polyneuropathy) are still not well characterized. Recently, a novel method to detect in different tissues the MYD88L265P mutation,3,4 a hallmark of WM, has been described: the droplet digital PCR (ddPCR) assay.5 In this paper clinical reports of useful applications of this sensitive and reliable tool in daily practice are described, in a question & answer form. Might MYD88 be useful for non-invasive differential diagnosis of WM vs IgM-MM? MB, a 62 years-old male, presented with fatigue, dyspnea, headache and tinnitus. Blood exams revealed mild anemia (Hb 11.5 g/dl), an IgM value of 6334 mg/dl and an IgMk M-component (MC) of 3410 mg/dl, so a BM biopsy was performed (Fig. 1A–C). An excess of clonal PCs (nearly 60%) was found, at first suggesting the diagnosis of IgM-MM, while immunophenotype reported indolent B lymphoma infiltration. Interestingly, the clonal PCs did not present the chromosomal translocations typical of MM by fluorescent in situ hybridization (FISH); finally, ddPCR on BM, PB and plasma were positive for the MYD88L265P mutation and a diagnosis of WM was established. The patient then started dexamethasone-rituximab-cyclophosphamide (DRC) treatment preceded by plasmapheresis, achieving partial remission (PR).Figure 1: BM biopsy showing nuomerous lymphoplasmacytic cells (A) that are CD20+ (B) and CD138- (C). BM biopsy showing a diffuse small cell population (D) positive for CD20 (E) and k light chain (F), negative for λ light chain (G).Comment Most of PC dyscrasias are attributable to MM, however, there are some exceptions: an IgG MC can be also attributable to rare cases of IgG-LPL or other indolent lymphomas, and similarly, some cases of IgM MC are due to aggressive IgM-MM and not to WM. In particular, because of its LPL-like pathological and phenotypical features, IgM-MM can be often mistaken for WM; however, as reported also by Treon et al,6 these cases are MYD88 wild-type. So, in this setting, when laboratory findings seem discordant, or the BM biopsy is uncertain or not available, the non-invasive MYD88 evaluation can support the differential diagnosis. Might MYD88 be useful to refine the diagnosis of B-cell lymphoma? AL, a 68-years-old male, presented with multiple lymphadenopathies and an IgMk MC at 680 mg/dl. The cervical lymph node biopsy was inconclusive, showing diffused small B-cell lymphoma CD20+, CD10+, BCL2+, IgD+, CD23+/−, with uncertain differential diagnosis between diffuse follicle center and marginal zone lymphoma, MZL. The BM biopsy revealed a small B-cell population with secretory differentiation and clonal IgMk lymphoplasmacytic population (20%) (Fig. 1D–G). The MYD88L265P detection by ddPCR in BM and plasma finally supported the diagnosis of WM, so the patient underwent a DRC therapy, achieving complete remission. Comment The differential diagnosis of small cell lymphomas with a diffuse pattern can be troublesome. Both the lymph node and the BM biopsy can be inconclusive, even after extensive flow cytometric and immunohistochemical characterization. Therefore, the availability of a sensitive and non-invasive tool, as the ddPCR MYD88L265P assay, might help in choosing the most appropriate therapy. Might MYD88 be used as a non-invasive marker to early identify WM relapse? GT, a 76-years-old female patient, was diagnosed with symptomatic WM and underwent a cyclophosphamide-vincristine-prednisone (CVP) therapy, obtaining PR. Four years later, a mild but progressive increase of IgM value was observed (2489 mg/dl), concurrently with anemia and worsening of general conditions. The patient received R-Bendamustine therapy, again achieving PR. Two years later, the patient presented with fatigue and weight loss, without worsening of blood exams (Hb 12.6 g/dl, IgM 1069 mg/dl); nevertheless, after 6 months, the appearance of pancytopenia and splenomegaly (in absence of IgM increase) suggested to repeat the BM biopsy, with a final diagnosis of progressive WM. A retrospective study of the MYD88L265P mutation on PB by ddPCR showed rising values several months before the insurgence of symptoms (Fig. 2A).Figure 2: Comparison between MYD88 L265P , hemoglobin, and IgM values during the follow-up (A). MYD88L265P levels in BM, PB and cell-free DNA (cfDNA) from diagnosis to the present (B).Comment In pre-treated WM patients, the appearance of cytopenia may be due to different causes (eg, chronic blood loss anemia, myelodisplastic syndrome, acute leukemia), rather than directly related to relapsing disease.7 Moreover, a depletion of the secretory fraction often occurs in heavily treated patients, as well as an isolated IgM suppression may occur independently of cytotoreduction when using mTOR and BTK inhibitors,8 leading to discordant serological/histological results. Therefore, the mere IgM levels are not sufficiently reliable for relapse prediction. Actually, BM biopsy is essential to differentiate among these conditions, but non-invasive evaluation of the MYD88 mutation might act as a diagnostic support. Might MYD88 monitoring be used as an early response predictor to describe the activity of new treatments? DM, a 43-years-old male, in 1997 was diagnosed with symptomatic WM and underwent high dose sequential (HDS) therapy followed by autologous stem cell transplantation. After 5 years, a slow but progressive increase of the MC was observed, leading to large lymphadenopathies and massive BM infiltration 9 years later. Therefore, a rituximab-citarabine-bortezomib therapy was started, resulting in PR. Again, after 3 years, an increase of the abdominal lymphadenopathies was observed, with anemia and rising MC: the patient started ibrutinib therapy, with complete resolution of anemia, MC reduction >50%, lymphadenopathies stability. The patient is now in good health and has been on ibrutinib treatment for 30 months. A retrospective analysis of the MYD88L265P levels in BM by ddPCR showed persistent positivity during the follow-up, with a transient, deep reduction after the bortezomib-containing therapy and a slighter but constant decrease during ibrutinib (detectable in PB and plasma, too) (Fig. 2B). Comment Although WM is traditionally managed as an indolent and constantly relapsing disease, modern chemo-immunotherapies containing rituximab, bendamustine, bortezomib, as well as the new drugs carfilzomib and venetoclax9,10,11 resulted in major cytoreduction. Therefore, MRD analysis might provide a more accurate evaluation of the efficacy of novel treatments, rather than the simple clinical response. Actually, MYD88L265P ddPCR assay can overcome the limited feasibility of the IGH-based approach,5,12 providing a stable molecular marker virtually to all WM patients. Moreover, the data on cell-free DNA (cfDNA) seem to nicely reflect the BM status, thus representing a non-invasive alternative for MRD detection. Nevertheless, the role of MRD in WM is not as well characterized as it is in other indolent lymphomas, so far, and its clinical impact is still under evaluation.13 Might ddPCR be useful to identify MYD88 mutation in pre-treated patients? LF, a 62-years-old male, presented with diffused lymphadenopathy and anemia (Hb 10.9 g/dl). The serum protein electrophoresis showed a MC of 1585 mg/dl (IgM value 2730 mg/dl), so lymph node and BM biopsy were performed and a diagnosis of WM was made. The MYD88L265P screening by ddPCR was positive on BM. The patient underwent a DRC therapy, but at the end of treatment the CT scan revealed a SD, along with no serological response (IgM 2438 mg/dl); actually, MYD88L265P resulted negative on PB, but still positive on plasma. Finally, the patient was referred to bendamustine-rituximab-bortezomib (BRB)12 experimental therapy. Comment In patients pre-treated with rituximab, non-invasive MYD88L265P evaluation in PB is not reliable because of the high rate of false negative results,5 likely due to the high clearance of circulating lymphoma cells. Therefore, BM or plasma analysis is advisable in pre-treated cases to identify the mutation. Actually, in paired analysis the median mutational load in PB samples is 1 log lower, compared to BM; conversely, between BM and plasma-cfDNA no statistically significant differences were reported.5 Moreover, a similar underestimation of MYD88L265P was described in PB samples of pre-treated vs rituximab-naïve patients, both in terms of mutational detection rates (about 40% of false negatives) and of median quantitative burden (about 1 log lower).5 This clue is particularly relevant when MYD88 mutational status is investigated as response predictor to targeted therapies, such as to prescribe BTK-inhibitors vs a different relapse treatment.14 Might MYD88 be useful to supplement the diagnosis of anti-mag polyneuropathy? DF, a 54-years-old male, presented with lower limbs paresthesia. An electroneurography (ENG) showed the presence of a demyelinating polyneuropathy. The evaluation for anti-myelin-associated glycoprotein antibodies (MAG) resulted positive and the IgM value was 307 mg/dl (no MC at protein electrophoresis nor at immunofixation). A BM biopsy revealed the presence of a LPL, and the MYD88L265P screening by ddPCR was positive on BM, PB and plasma. Based on the presence of LPL and a progressive anti-MAG polyneuropathy, the patient underwent 4 rituximab infusions. At the end of therapy, the IgM value was 175 mg/dl, the MYD88 screening was negative on PB and plasma (BM biopsy was not repeated) and the ENG revealed a clear reduction of the demyelinization signs. Comment Anti-MAG demyelinating polyneuropathy is a rare, disabling and still under-characterized disease that can be associated either to WM/LPL or to IgM-MGUS. The prevalence of MYD88L265P mutations in anti-MAG neuropathy patients has been recently shown to be comparable to those observed in WM and MGUS control groups. Since there is actually no consensus on the optimal treatment strategy for anti-MAG neuropathies, detecting MYD88L265P mutation even in non-invasive tissues might help to reveal a smoldering WM, identifying patients for which rituximab treatment may be of benefit.15,16,17 In conclusion, the ddPCR MYD88L265P assay might have several clinical applications (Fig. 3): 1) driving the differential diagnosis with IgM-MM and small lymphocytes, Ig-secreting disorders; 2) easy-to use molecular marker for MRD, particularly to measure the efficacy of new drugs; 3) predictive biomarker of response to ibrutinib treatment; 4) supporting the diagnosis of WM as underlying disease for rare IgM-related disorders (eg, anti-MAG polyneuropathy). Figure 3: Clinical applications of the ddPCR MYD88 L265P assay in WM.The ddPCR MYD88L265P assay also presents important advantages compared to other available techniques (as qPCR or NGS): actually, it is non-invasive, cheap, fast, easily applicable to clinical routine and clinical trials, standardizable and promptly scalable to other mutations of interest (eg, CXCR4). Therefore, this assay is rapidly finding its role both for mutational screening and for MRD monitoring in ongoing (BRB, EudraCT Number: 2013-005129-22 and BIO_WM, ID: NCT03521516) and future (ECWM-2, EudraCT Number: 2017-004362-95) clinical trials.

In chronic lymphocytic leukemia (CLL), the hypoxia-inducible factor 1 (HIF-1) regulates the response of tumor cells to hypoxia and their protective interactions with the leukemic microenvironment. In this study, we demonstrate that CLL cells from TP53-disrupted (TP53dis) patients have constitutively higher expression levels of the α-subunit of HIF-1 (HIF-1α) and increased HIF-1 transcriptional activity compared to the wild-type counterpart. In the TP53dis subset, HIF-1α upregulation is due to reduced expression of the HIF-1α ubiquitin ligase von Hippel-Lindau protein (pVHL). Hypoxia and stromal cells further enhance HIF-1α accumulation, independently of TP53 status. Hypoxia acts through the downmodulation of pVHL and the activation of the PI3K/AKT and RAS/ERK1-2 pathways, whereas stromal cells induce an increased activity of the RAS/ERK1-2, RHOA/RHOA kinase and PI3K/AKT pathways, without affecting pVHL expression. Interestingly, we observed that higher levels of HIF-1A mRNA correlate with a lower susceptibility of leukemic cells to spontaneous apoptosis, and associate with the fludarabine resistance that mainly characterizes TP53dis tumor cells. The HIF-1α inhibitor BAY87-2243 exerts cytotoxic effects toward leukemic cells, regardless of the TP53 status, and has anti-tumor activity in Em-TCL1 mice. BAY87-2243 also overcomes the constitutive fludarabine resistance of TP53dis leukemic cells and elicits a strongly synergistic cytotoxic effect in combination with ibrutinib, thus providing preclinical evidence to stimulate further investigation into use as a potential new drug in CLL.

Citation for published version (APA): Op Bruinink, D. H., Oliva, S., Rihova, L., Schmitz, A., Gilestro, M., Te Marvelde, J., Kralova, R., Høholt, H., Broijl, A., Johnsen, H. E., Hajek, R., Boccadoro, M., Sonneveld, P., Omedè, P., Van der Velden, V. H. J., & European Myeloma Network (2021). Standardization of flow cytometric minimal residual disease assessment in international clinical trials a feasibility study from the European Myeloma Network. Haematologica, 106(5), 1496-1499. https://doi.org/10.3324/haematol.2020.267831

Several new drugs are approved for treatment of patients with multiple myeloma (MM), but no validated biomarkers are available for the prediction of a clinical outcome. We aimed to establish whether pretreatment blood and bone marrow plasma concentrations of major cytokines and angiogenic factors (CAFs) of patients from a phase 3 trial of a MM treatment could have a prognostic and predictive value in terms of response to therapy and progression-free and overall survival and whether these patients could be stratified for their prognosis. Blood and bone marrow plasma levels of Ang-2, FGF-2, HGF, VEGF, PDGF-β, IL-8, TNF-α, TIMP-1, and TIMP-2 were determined at diagnosis in MM patients enrolled in the GIMEMA MM0305 randomized controlled trial by an enzyme-linked immunosorbent assay (ELISA). These levels were correlated both reciprocally and with the type of therapy and patients’ characteristics and with a group of non-MM patients as controls. No significant differences were detected between the blood and bone marrow plasma levels of angiogenic cytokines. A cutoff for each CAF was established. The therapeutic response of patients with blood plasma levels of CAFs lower than the cutoff was better than the response of those with higher levels in terms of percentage of responding patients and quality of response. FGF-2, HGF, VEGF, and PDGF-β plasma levels at diagnosis have predictive significance for response to treatment. The stratification of patients based on the levels of CAFs at diagnosis and their variations after therapy is useful to characterize different risk groups concerning outcome and response to therapy. Clinical trial information can be found at the following link: NCT01063179

Background:In Waldenström Macroglobulinemia (WM) whole genome sequencing has revealed recurring somatic mutations, affecting the NFK‐B and Bruton tyrosine kinase pathways, which are responsible for drug response prediction. MYD88L265P is the highly prevalent mutation, detected in up to 95% of patients, and represents a suitable molecular marker. Mutational analysis currently performed by ASqPCR has some technical limitations, in particular: marker identification failure at low tumor level, especially after chemo‐immunotherapy, and requirement of CD19+ cells selection in routine diagnostic laboratories. Furthermore, 1.5–2% of monoclonal gammopathy of undetermined significance (IgM‐MGUS) patients, per year, progress to WM and there are not reliable biological markers able to distinguish between these two conditions. Recently, droplet digital PCR (ddPCR) demonstrated to be highly suitable for WM screening and MRD monitoring.Aims:In this study we aim to compare ASqPCR and ddPCR in CD19+ selected and unselected samples from WM and IgM‐MGUS patients, to assess whether: 1) cell selection could be avoided in diagnostic routine, for MYD88L265P detection; 2) the MYD88L265P quantitative level might discriminate WM from IgM‐MGUS.Methods:We analyzed two retrospective samples series from WM and IgM‐MGUS patients collected at the Hematology Units of Torino and Varese. All patients provided written informed consent in accordance with Helsinki's declaration. Both ddPCR and ASqPCR were performed on CD19+ selected cells (Whole Blood CD19 MicroBeads‐MACS, Miltenyi Biotec) collected in Varese and on unselected mononuclear cells (MNC) collected in Torino. The experiments were performed in parallel and in blind on the same series, by Varese (ASqPCR) and Torino (ddPCR), as previously described (Hunter Z et al, Drandi D et al).Results:Method comparison was performed on 130 samples from 110 patients (66 WM and 34 IgM‐MGUS), 75 CD19+ selected samples (53 BM, 22 PB at baseline) and 55 unselected MNC (30 BM, 25 PB, both at baseline and follow‐up).As reported in literature, ddPCR detected MYD88L265P in 96% (23/24) of baseline CD19+ BM WM samples while ASqPCR in 83% (20/24). If considering the whole series, 31% (41/130) of samples were discordant between the two techniques mostly (39/41) mutated by ddPCR and WT by ASqPCR and regardless of disease status. Focusing on CD19+ selected samples, 36% (27/75) were discordant, mostly (26/27) mutated by ddPCR and WT by ASqPCR, with a median MYD88L265P MUT/WT ratio of 2.36E‐03 (range:3.62E‐04–4.78E‐02). Concordantly, 25% (14/55) of MNC samples were discordant, mostly (13/14) mutated by ddPCR and WT by ASqPCR, with a median MYD88L265P MUT/WT ratio of 5.53E‐04 (range:4.33E‐04–9.00E‐04) (Figure1).Moreover, ddPCR detected MYD88L265P in 86% (25/29) of IgM‐MGUS baseline CD19+ BM, while ASqPCR only in 52% (15/29). Interestingly, compared to WM, IgM‐MGUS showed a lower median mutational level: 1.65E‐03 (range:1.0E‐05–2.6E‐01) vs 1.68E‐01 (range:2.2E‐04–9.0E‐1) (p < 0.0001).Summary/Conclusion:This study highlights ddPCR as a feasible and highly sensitive approach for MYD88L265P mutational detection, particularly in samples harboring low concentrations of tumor cells. These results suggest that the implementation of ddPCR assay in routine diagnostic laboratories might avoid the need of CD19+ selection for MYD88L265P screening. Moreover, significantly different MYD88L265P mutational levels identified between WM and IgM‐MGUS cases highlighted the need for further studies on larger patients’ series to identify correlations between mutational levels and risk of progression to WM.image

Background:Multiple myeloma (MM) is a clinically heterogeneous disease and several classification systems have been developed to stratify patients in low, intermediate or high risk. Tumor Circulating Plasma Cells (TCPC) are considered a marker of high‐risk disease in all plasma cell disorders. To date, different methods are available to detect TCPC in peripheral blood, but they are barely reproducible and time consuming.Aims:For the first time the amount of TCPC was quantified in newly diagnosed MM patients with a single platform flow cytometric method and was evaluated their correlation with patients‘ baseline characteristics.Methods:Whole peripheral blood (PB) samples from 413 newly diagnosed young MM patients enrolled in the UNITO‐MM‐01/FORTE trial were included in the study. For the single platform technique, the antibody combination CD38PC7/CD138PC5.5/CD45KO/ CD56PE/CD19PB was mixed with 100 μL of EDTA PB, dispensed with reverse pipetting, and incubated for 15 min at RT, added with 500 μL of lysing solution and, after 15 min, 100 μL of flow count fluorospheres were dispensed with reverse pipetting and cells acquired with Navios flow cytometer. Intracytoplasmic kappa and lambda were tested to confirm the TCPC clonality.Results:Circulating Plasma Cells (CPC) were found in 390 out of 413 samples (94.4%), with median values of 0.03% (range: 0%>51%) and 2.37/mm3 (range: 0/mm3–6272/mm3). White blood cells were 5710/mm3 (range: 1752/mm3–26102/mm3); median CPC events 58 (range: 0–441.000); cellular events acquired were 190.000 (range: 4.428–1.300.000).TCPC were found in 272 samples (66%) with a median value of 1.24/mm3 (range 0.06/mm3 ‐ 6272/mm3).Patients were stratified according to different baseline characteristics and median values of absolute TCPC were compared. The most significant statistical differences (p < 0.001) were: Hb <10 (12.9/mm3) vs ≥10 (0.81/mm3); ISS I (0.30/mm3) vs ISS II (2.85/mm3) vs ISS III (5.14/mm3); R‐ISS I (0.25/mm3) vs II (2.76/mm3) vs III (5.14/mm3); Albumin <3.5 g/dL (2.76/mm3) vs ≥3.5 g/dL (1.05/mm3); β2‐microglobulin <3.5 mg/dL (0.67/mm3) vs 3.5 mg/dL‐5.5 mg/dL (3.88/mm3) vs >5.5 mg/dL (16.47/mm3); LDH ≤upper limit (1.14/mm3) vs >upper limit (7.36/mm3); PC in biopsy <60% (0.60/mm3) vs ≥60% (2.76/mm3); with 1q gain (3.18/mm3) vs without (1.18/mm3); Morgan risk Standard (1.16/mm3) vs High (3.00/mm3) (presence of del17 and/or t(4;14) and/or t(14;16) and/or 1q gain).Moreover, the TCPC were sorted in quartiles according to the absolute values (0/mm3–0/mm3, 0/mm3–1.24/mm3, 1.24/mm3–8.3/mm3, 8.3/mm3–6272/mm3) and a correlation between the increasing levels of TCPC and the parameters mentioned above was observed.The most significant correlations expressed by Cramer's V were observed between TCPC and: hemoglobin (V = 0.41), ≥60% of plasma cells in biopsy (V = 0.28), ISS (V = 0.26), β2‐microglobulin <3.5 mg/dL vs 3.5 mg/dL‐5.5 mg/dL vs >5.5 mg/dL (V = 0.26), R‐ISS (V = 0.24), Morgan risk (V = 0.24 p < 0.001), 1q gain (V = 0.24) all with a p < 0.001.Summary/Conclusion:The single platform flow cytometric method allowed us to quantify TCPC in a high number of MM patients and the values obtained were strongly related to the worst clinical and cytogenetic features. This method is fast, not expensive, feasible with a small amount of PB and can be easily performed in a high number of patients.