PB Samples Research Articles

Comparison of three digestion methods for determination of lead and cadmium in milk and dairy products

Background: Toxic metals enter the human food chain through water, soil, and plants. High consumption of dairy products makes it necessary to measure their concentrations in milk and its products. Methods: In this study, four samples of dairy products, including milk, dough, yogurt, and cream were selected. They were spiked with concentrations of 0, 20, 40, and 60 μg/kg of lead (Pb) and cadmium (Cd) separately. In all samples, the concentration of these metals was determined using graphite furnace atomic absorption spectroscopy (GFAAS) after microwave, wet, and dry ashing digestion methods. To select the best digestion method, recovery percentage, linearity of increasing concentrations, relative standard deviation (RSD), and the limit of detection were used. Results: According to the results, the RSD of all measurements was less than 5%. The instrument detection limit for Pb and Cd were 0.188 and 0.157 μg/L, respectively. The recovery efficiency of all digested samples by three methods was between 75.7% and 120%. According to the linearity index and R2 , the microwave digestion method with 90 to 110% efficiency was the best for Pb-spiked samples, and the dry digestion method was the best for Cd-spiked samples. Conclusion: Considering all indexes, microwave digestion was the best method for Pb and Cd samples.

Effect of fecal preservation method on captive southern white rhinoceros gut microbiome

AbstractThe southern white rhinoceros (Ceratotherium simum simum) faces an uncertain future in the wild due to increased poaching pressure and habitat fragmentation, thus the management of reproductively successful populations is of critical importance. Successful reproductive outcomes in rhinoceros may be mediated by diet and gut microbial diversity; therefore, understanding gut microbial dynamics within and between captive and wild populations may help improve conservation efforts. Accordingly, gut microbiome preservation methods are needed that are practical for in situ field sampling of wild populations. We evaluated the efficacy of 3 different preservation methods over 2 timepoints for stabilizing microbial communities in feces from southern white rhinoceros (n = 10) at the North Carolina Zoo in Asheboro, North Carolina, USA, during July–September 2020 and January–March 2021. Samples were immediately frozen at −80°C, stored in PERFORMAbiome™·GUT (PB) tubes or stored in 95% ethanol at ambient temperatures (to simulate field conditions), and processed after 14 or 230 days post‐collection. We quantitatively compared alpha and beta diversity across microbial communities and identified taxa that were enriched in each treatment group. Samples preserved in 95% ethanol consistently harbored lower Shannon diversity index (SHDI) and Simpson's diversity (SDI) values compared to Frozen and PB samples. This trend was apparent in both Ethanol day‐14 samples (SHDI 4.94; SDI 0.98) versus Frozen day‐14 (SHDI 5.19; W = 518, P < 0.001; SDI 0.99; W = 476, P < 0.001) and PB day‐14 (SHDI 5.15; W = 430, P < 0.01; SDI 0.99; W = 1075, P = 1) samples, and in Ethanol day‐230 samples (SHDI 4.48; SDI 0.97) versus Frozen day‐230 (SHDI 5.18; W = 0, P < 0.05; SDI 0.99; W = 0, P = 0.032) and PB day‐230 (SHDI 5.23; W = 0, P < 0.05; SDI 0.99; W = 0, P = 0.032) samples. Ethanol day 230 samples differed (P < 0.05) from all other treatments in both alpha and beta diversity indices. Notably, frozen and PB preservation methods maintained compositionally similar microbial communities across both time points. Our results indicate that PB tubes stored at ambient temperatures perform similarly to freezing at −80°C, highlighting their utility for microbiome fieldwork applications. Identifying optimal and versatile microbiome preservation techniques will enable future studies of the gut microbiome in reproductively‐successful wild populations, an effort central to conservation efforts in the southern white rhinoceros and other threatened species.

Stratigraphy, provenance, and timing of Neogene sedimentation in the western Valdés Basin, Patagonia. Accurate paleogeographic reconstructions as a key piece for andean-passive margin integration

With the aim to better understand the timing and geological evolution of the late Cenozoic stratigraphic record of the onshore, western Valdés Basin in northeast Chubut province (Patagonia, Argentina), we perform sedimentological, stratigraphic, and zircon U–Pb geochronologic analyses. The study includes the terrestrial Early Miocene Trelew Member of the Sarmiento Formation, the shallow marine Early Miocene Gaiman Formation, the shallow marine to estuarine Late Miocene Puerto Madryn Formation, and the Plesitocene fluvial conglomerates of the Rodados Patagónicos. Based on our data set, together with the recognition of relevant stratigraphic surfaces, we divide the succession into stratigraphically bounded units and define three cycles of sedimentation. The Lower Cycle rests on top of Surface 1, which comprise a long hiatus including the entire Oligocene Epoch. This cycle is in turn divided into two subunits separated by the transgressive, ravinement Surface 2. The Lower Cycle is separated from the overlying Middle Cycle (=Puerto Madryn Formation) by the flat and regionally extensive Surface 3. Finally, the latter is separated from the Upper Cycle (=Rodados Patagónicos) by Surface 4, a complex surface at the base of gravel deposits forming several terrace levels. Maximum depositional ages calculated from 10 zircon U–Pb samples indicate the succession between surface 1 and 4 extends from 21.5 to 2.7 Ma (Aquitanian-Piacenzian). The Lower Cycle is restricted to the 21.5–16 Ma interval (Aquitanian-Burdigalian); the Middle Cycle between 12 and 2.7 Ma (latest Serravalian-Piacenzian), with most of the new and previous ages clustering in the Tortonian; whereas the Upper Cycle is restricted to the Pleistocene. Although most of the analyzed sediments show fresh volcanic glass particles, older zircon ages are dominant, representing detrital components with a dominant peak of Permian-aged grains, with subordinated Devonian, Jurassic, Cretaceous, and Paleogene peaks. We interpret the analyzed succession as a condensed section compared to the offshore Valdés Basin depocenter area. Sedimentation in the study area was controlled by periods of slow subsidence, probably related to thermal subsidence and sediment compaction in the depocenter. Two main sources of sediments for the analyzed succession are envisaged: one represented by explosive volcanism located either in the Andes or in the North Patagonian Massif, the other through erosion of older rocks surrounding the North Patagonian Massif where late Paleozoic plutonic rocks are abundant. Particularly for the Middle and Upper cycles, an increase in Mesozoic contribution suggests erosion from sediments of the Cañadón Asfalto Basin. The new ages and stratigraphic analyses here reported are of major relevance for the integration of the onshore and offshore stratigraphies of the Valdés Basin which is key to connect the geologic evolution of the Andean and passive margin areas of southern South America. They are also of major relevance for the calibration of fossil communities, particularly for the abundant and diverse marine vertebrates preserved in the Burdigalian Gaiman Formation.

Low-cost and sensitive method for Pb isotope determination using a novel β-Si3N4 emitter by thermal ionization mass spectrometry

Thermal ionization mass spectrometry (TIMS) is the workhorse for lead isotopic ratio analysis due to its excellent precision. Silica gel as ionization activator on Re filament is proved to the best emitter that can provide excellent sensitivity even small Pb sample size. However, the price of Re filament is three times that of Ta filament that leads to high experimental cost for TIMS laboratory. Here, we first present a novel silicon nitride (β-Si3N4) emitter on the Ta filament with good sensitivity for Pb isotopic ratio measurements. Hence, the cost of filament material is cut down ∼70%. The β-Si3N4 emitter can yield stable and long-life Pb+ signal, about 2–3 V 208Pb and 0.65–0.90 V 208Pb for 20 ng and 5 ng NIST SRM981 sample size that is applicable to the most geological materials for bulk analysis. A suite of silicate reference materials were analyzed to verify the reliability and accuracy of our method. For 206Pb/204Pb, 207Pb/204Pb and 208Pb/204Pb isotope ratios of geological samples, excellent internal precisions (2 SE) of ±0.005%–0.013% are achieved. Replicate digestions and analyses of the basalt standard BCR-2 and coal fly ash standard GBW08401 demonstrate that good external precision is obtainable that is 0.10–0.18% (n = 6, 2 SD) for 206Pb/204Pb, 207Pb/204Pb and 208Pb/204Pb ratios.

Conventional dendritic cells type 1 are strongly enriched, quiescent and relatively tolerogenic in local inflammatory arthritis.

Dendritic cells (DC) are crucial for initiating and shaping immune responses. So far, little is known about the functional specialization of human DC subsets in (local) inflammatory conditions. We profiled conventional (c)DC1, cDC2 and monocytes based on phenotype, transcriptome and function from a local inflammatory site, namely synovial fluid (SF) from patients suffering from a chronic inflammatory condition, Juvenile Idiopathic Arthritis (JIA) as well as patients with rheumatoid arthritis (RA). Paired PB and SF samples from 32 JIA and 4 RA patients were collected for mononuclear cell isolation. Flow cytometry was done for definition of antigen presenting cell (APC) subsets. Cell sorting was done on the FACSAria II or III. RNA sequencing was done on SF APC subsets. Proliferation assays were done on co-cultures after CD3 magnetic activated cell sorting (MACS). APC Toll-like receptor (TLR) stimulation was done using Pam3CSK4, Poly(I:C), LPS, CpG-A and R848. Cytokine production was measured by Luminex. cDC1, a relatively small DC subset in blood, are strongly enriched in SF, and showed a quiescent immune signature without a clear inflammatory profile, low expression of pathogen recognition receptors (PRRs), chemokine and cytokine receptors, and poor induction of T cell proliferation and cytokine production, but selective production of IFNλ upon polyinosinic:polycytidylic acid exposure. In stark contrast, cDC2 and monocytes from the same environment, showed a pro-inflammatory transcriptional profile, high levels of (spontaneous) pro-inflammatory cytokine production, and strong induction of T cell proliferation and cytokine production, including IL-17. Although the cDC2 and monocytes showed an overlapping transcriptional core profile, there were clear differences in the transcriptional landscape and functional features, indicating that these cell types retain their lineage identity in chronic inflammatory conditions. Our findings suggest that at the site of inflammation, there is specific functional programming of human DCs, especially cDC2. In contrast, the enriched cDC1 remain relatively quiescent and seemingly unchanged under inflammatory conditions, pointing to a potentially more regulatory role.

Collaborative estimation of heavy metal stress in wheat seedlings based on LIBS-Raman spectroscopy coupled with machine learning

Systematic research procedures of this research. (a) Step 1 is wheat culture, heavy metal Pb stressed and sample preparation; (b) step 2 is spectral data preprocessing, and discriminant models foundation and analysis.

Significantly improved thermoelectric performance of SnSe originating from collaborative adjustment between valence and conduction bands, mass fluctuations, and local strain.

In this study, environmentally friendly, low cost, and easy to synthesize InSn and VSn co-doped SnSe materials was designed and prepared via vacuum melting and spark plasma sintering technology, which avoids the shortcomings of high-performance Pb, Ge, and Na-doped SnSe samples. InSn and VSn, doping achieved appropriate bandgap (Eg) and energy band degeneracy (NV) from the valence and conduction band, obtaining the highest electrical conductivity of 4726 S m-1 at 773 K. The impurity state controls the carrier transport process below 573 K, while Eg and NV control the process above 573 K. InSn and VSn doping induces quality fluctuation and local strain, which decreases the lattice thermal conductivity. Owing to the higher power factor and low lattice thermal conductivity, the ZT value of the Sn0.985In0.01Se sample was 1.3 at 773 K. Dual regulation of the valence and conduction band provides a new idea for adjusting the transport behavior of semiconductors.

Biogeochemical cycling of Cd, Mn, and Ce in the Eastern Tropical North Pacific oxygen‐deficient zone

AbstractOxygen‐deficient zones (ODZs) play an important role in the distribution and cycling of trace metals in the ocean, as important sources of metals including Fe and Mn, and also as possible sinks of chalcophile elements such as Cd. The Eastern Tropical North Pacific (ETNP) ODZ is one of the three largest ODZs worldwide. Here, we present results from two sectional surveys through the ETNP ODZ conducted in 2018, providing high‐resolution concentrations of several metals, along with complimentary measurements of nutrients and iodine speciation. We show that samples obtained from the ship's regular rosette are clean for Cd, Mn, Ni, and light rare earth elements, while uncontaminated Fe, Zn, Cu, and Pb samples cannot be obtained without a special trace‐metal clean sampling system. Our results did not show evidence of Cd sulfide precipitation, even within the most oxygen‐depleted water mass. High Mn and Ce concentrations and high Ce anomalies were observed in low‐oxygen seawater. These maxima were most pronounced in the upper water column below the oxycline, coincident with the secondary nitrite maxima and the lowest oxygen concentrations, in what is generally considered the most microbially active part of the water column. High Mn and Ce features were also coincident with maxima in excess iodine, a tracer of shelf sediment sources. Mn and Ce maxima were most prominent within the 13°C water mass, spanning a density horizon that enhances isopycnal transport from the shelf sediments, resulting in transport of Mn and Ce at least 2500 km offshore.

Chronic Myelomonocytic Leukemia Diagnosis By Flow Cytometry: An Italian Center Experience

Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by dysplasia, abnormal production of monocytes, and an increased risk of leukemic evolution. According to WHO, CMML patients show a persistent (≥3 months) absolute PB monocytosis (≥1×109/L) and relative monocytosis (≥10% of PB leukocytes), with the exclusion of Chronic Myeloid Leukemia (CML), Myeloproliferative Neoplasms, and a blast cell count <20% in PB and/or BM. In addition, morphological and/or histopathological evidence for diagnostic dysplasia ≥1 of the three major BM cell lineages must be present. If dysplasia is absent or not diagnostic (<10%), the presence of cytogenetic or molecular alterations or flow cytometry (FC) abnormalities may be utilized as co-criteria for the diagnosis of CMML. FC is helpful in confirming the monocyte and blast cell counts. In addition, FC is useful for confirming the presence of distinct monocyte populations. Based on the expression of CD14 and CD16, monocytes are classified into classical (MO1) (CD14+/CD16−), intermediate (MO2) (CD14+/CD16+) and non-classical (MO3) (CD14-/CD16+). FC analysis of PB monocytes was proposed as a quick and efficient tool for distinguishing CMML from reactive monocytosis by highlighting an increase in the MO1 fraction above 94% and a decrease in the percentage of MO3. Using FC, we evaluated 25 patients with a confirmed diagnosis of CMML in BM and PB samples, as well as 11 patients with other malignancies (MPN-monocytosis), and 28 patients with reactive monocytosis only in PB samples. Among these patients, 21/25 (84%) of CMML patients displayed MO1 ≥94%, while in patients with reactive monocytosis, MO1≥94% was observed in 11/28 (39%) and 2/11 (18%) in patients with MPN-monocytosis. Then we analyzed the MO3 fraction, which was <1.13% in 18/25 (72%) CMML patients, only one patient with MO1 lower than 94% resulted in an M3 fraction >1.13%. The evaluation of SLAN positivity in this fraction MO3 was always lower than 1.7%. The patient with MO1<94% and MO3 > 1.13% had an active ureteral carcinoma and, at NGS evaluation, had TET2, JAK2, and ZRSR2 mutations. In BM and PB samples from MPN patients, the MO3 fraction was in 2/11 <1.13%, the same patients with MO1> 94%, the diagnosis was a CML in one patient and a PMF triple negative in the other patient. PB samples from reactive monocytosis MO3 fraction were in 11/28 (39%) <1.13%, the same patients with MO1> 94%. The evaluation of SLAN positivity in this fraction MO3 in MPN and reactive monocytosis was always superior at 1.7% (range 4.9-60%). BM samples from CMML and MPN patients reflected the percentage of MO1 and MO3 of PB. Commonly PB and BM monocytes in CMML display aberrant expression of CD56. Furthermore, it was detectable a fraction of non-maturing neutrophils (CD66b+/CD11b-) in nearly half of CMML patients. In conclusion, our real-life experience of routinely FC study on PB and BM CMML samples is similar to the studies described in the literature. It confirms the comparable percentages of monocyte subpopulations in both samples, suggesting that PB samples can be used for diagnostic FC in these patients.

Alterations of Human Bone Marrow Niche in Clonal Hematopoiesis and AML

Acute myeloid leukemia (AML) is a heterogeneous disease, predominantly caused by acquisition of mutations in the hematopoietic stem and progenitor cells. Some of these mutations are acquired in a preleukemic state termed clonal hematopoiesis of indeterminate potential (CHIP). Recent studies show that alterations in the bone marrow niche can support progression and therapy resistance of myeloid neoplasia. In our study, we aim to elucidate alterations in the bone marrow niche, in particular in mesenchymal stem cells (MSC), during the evolution from healthy to CHIP and further to AML. We collected primary human bone marrow (BM) and peripheral blood (pB) samples from hematologically healthy individuals who underwent hip replacement surgery at the LMU University Hospital as well from AML patients treated at our institution (LMU University Hospital Munich). Individuals were assigned to non-CHIP or CHIP cohorts based on targeted mutation analyses on pB samples. So far, we have included 105 healthy individuals and 92 have undergone targeted sequencing, revealing 27 individuals (29%) with CHIP. While there was no difference between the gender of the non-CHIP and CHIP groups, individuals with CHIP tended to be of older age (median, 64 y non-CHIP vs. 73.5 y CHIP). We detected DTA gene mutations as the most common CHIP alteration and 1.4 mutations per individual were found on average. Variant allele frequencies varied between 1% and 17%. The amount of mutations per individual increased with age. In the non-CHIP and CHIP individuals, we analyzed various blood parameters such as hemoglobin, leukocyte counts, mean corpuscular volume and others, as well as various comorbidities such as myocardial infarction, hypertesia, previously treated cancer, etc. We found a trend for more frequent myocardial infarction in the CHIP individuals. We did not find any significant differences between CHIP and non-CHIP for the blood parameters. MSC from donor BM samples were isolated via plastic adherence over 10-14 days of in vitro culture. MSC were characterized regarding their surface marker expression (FACS), differentiation capacity (osteogenic, adipogenic, chondrogenic) and metabolic activity (Seahorse XF). Analysis of MSCs from non-CHIP (n=5) and CHIP (n=5) individuals revealed no difference in surface marker expression over 10 passages. Also, differentiation assays of MSCs (non-CHIP=4, CHIP= 2) showed no differences in adipogenic and chondrogenic differentiation after 21 days. For osteogenesis, we noticed a trend towards an increased differentiation potential in the MSCs of CHIP individuals. This experiment is ongoing with the goal to increase the sample number up to 10 per condition, as well as including the AML controls. The data will be shown at the ASH meeting. To gain deeper insights into the BM niche we sorted 4 non-hematopoietic and 6 hematopoietic cell populations from non-CHIP (n=3), CHIP (n=4) and AML (n=4) individuals and performed bulk-RNA sequencing on each population. So far, changes in the composition of the bone marrow niche were only detected in AML samples. Currently, in a series of pilot experiments, we established primary MSC and HSPC cross co-cultures from non-CHIP, CHIP and AML samples. A preliminary experiment with one non-CHIP and one CHIP sample showed that both non-CHIP and CHIP HSC produced less hematopoietic cells and less colony forming units (CFUs) after co-culturing with CHIP MSCs, than with non-CHIP MSCs. The results of the main experiment with 3 individuals per condition and including the AML samples will be shown at the conference.

Dissecting MRD Kinetics By Automated Computational Analysis to Improve Outcome Prediction in Mantle Cell Lymphoma: A Bioinformatic Substudy from the Fondazione Italiana Linfomi (FIL) MCL0208 Clinical Trial

Background and Aims. Minimal residual disease (MRD) detection is a validated outcome predictor in mantle cell lymphoma (MCL). Recently, the clinical relevance of repeated MRD monitoring has been described in a prospective clinical trial [Ferrero, Blood 2022]. Nonetheless, complex patterns of MRD kinetics over time and availability of results obtained by heterogeneous tissues (bone marrow, BM, vs peripheral blood, PB) might generate substantial interpretation issues and hamper an easy-to-use application of this predictive biomarker. To overcome the limitation of empirical analysis we tested CONNECTOR, a novel automated computational framework to facilitate the interpretation of MRD kinetics and stratify patients in risk classes based on a solid, algorithm-derived, classification. Patients and methods. ASO RQ-PCR MRD data already generated from BM and PB samples in the FIL MCL0208 trial [Ferrero, Blood 2022], offering first-line high dose chemoimmunotherapy and autologous transplantation (ASCT) to youngers MCL patients, were employed. CONNECTOR (https://qbioturin.github.io/connector/) is a data-driven framework based on Functional Data Analysis for analyzing longitudinal high-dimensional data in a straightforward and revealing way. CONNECTOR allows the aggregation of time-series data through an unsupervised approach in informative clusters. Moreover, we developed a classification approach to predict the risk class for new MRD data. The new MRD curves were allocated to existing risk clusters defined on a training set of MRD data. Results. Without knowledge of any clinical or outcome data, CONNECTOR was run on 117 patients characterized by at least 3 MRD timepoints in BM (median 6, 3-9) and separately on 95 patients with at least 4 MRD timepoints in PB (median 7, 4-9). Starting from BM MRD data, CONNECTOR identified four patient risk Clusters (CrC), based on MRD kinetics, Figure1A. CrC_A comprised 73 patients with an overall quick and stable MRD negativization; CrC_B accounted for 14 patients characterized by later MRD negativization, alternating MRD results, or low-level MRD persistence; the 14 patients in CrC_C were always characterized by early MRD negativization followed by late MRD reappearance; CrC_D was composed of 16 patients with stable MRD positivity or only transient MRD negativization followed by early MRD reappearance. The different trends of MRD kinetics dissected by CONNECTOR fairly predicted patients’ outcomes: median TTP was not reached for CrC_A and B, 36 months for CrC_C and 27 months for CrC_D, respectively (p<0.0001), Figure1B. Similar data were generated when CONNECTOR was run on MRD results from PB samples: CrC allocation of BM vs PB was concordant for 89% of patients and survival analysis on PB results confirmed patients stratification in two groups: favorable MRD kinetics (CrC_A and B, median TTP not reached) vs unfavorable MRD kinetics (CrC_C and D, median TTP 35 months, p<0.0001), as depicted in Figure1B. Finally, we tested the performance of pooling together BM and PB results, to increase the number of evaluable patients (irrespective of eventual missing BM samples). Only the BM point at 12 months after ASCT was considered, together with the PB MRD results of all the available time points. CONNECTOR was used to verify if this mixed MRD pattern was able to correctly reclassify patients in the same CrC. Actually, 89 out of 95 patients (94%) were correctly reclassified (p_mean=0.96, p_sd= 0.07). Discussion. CONNECTOR allowed unsupervised identification of patients clusters with distinct MRD kinetics and highly significant different clinical outcome. Such clusterization proved effective using BM, PB and mixed tissues, as well. Validation of CONNECTOR performance in large, independent prospective trials is currently ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Multi-Omics of Peripheral Blood Reveals Tiding and Evolution of Responsive and Resistant Clones upon Daratumumab-Based Treatments in Multiple Myeloma and Plasma Cell Leukemia

Background: Treatment with anti-CD38 antibodies is a standard of care in newly diagnosed and relapsed/refractory multiple myeloma (MM). Clonal tiding and evolution as well as genomic heterogeneity are major drivers towards drug-resistant disease. However, modes of resistance to anti-CD38 directed therapies are currently unknown and might include loss of antigen expression and/or exhaustion of the immune system. We performed longitudinal single-cell multi-omic analyses of circulating tumor cells (CTC) and non-malignant cells to decipher clonal heterogeneity and tiding upon response and resistance to daratumumab (Dara)-based therapies in MM and plasma cell leukemia (PCL). Methods: Peripheral blood mononuclear cells (MNCs) were isolated from 7 patients (PCL n=5, MM n=2) before and after exposure to Dara. Samples were collected at four different time points: 1. before administration of Dara (CD38-naïve). 2. Four days after first application of Dara (CD38-exposed). 3. After relapse from Dara (CD38-refractory). 4. After an additional line of non-CD38 targeted therapy following relapse from Dara. In total, we analyzed 17 PB samples. To demonstrate that results from PB are comparable to bone marrow (BM), four paired PB and BM samples were included (21 samples in total). MNCs were subjected to single cell RNA (scRNAseq), B-cell (scBCRseq), and T-cell receptor sequencing (scTCRseq). Surface protein expression was measured using a custom panel of 57 DNA-barcoded antibodies. Sequencing files were processed with CellRanger (10x Genomics) and data were analyzed with the Seurat toolkit for single cell sequencing. Cell types were annotated with SingleR based on the Human Primary Cell Atlas built-in references. ScRepertoire was used for longitudinal scTCRseq and scBCRseq data. Copy number variations (CNVs) in CTCs were detected with inferCNV, and cellular interactions were investigated with CellPhoneDB. Results: In total, we sequenced 130.038 MNCs (PB: 108.236, range 414-8700/patient; BM: 21802, range 3375-8516/patient, Figure 1A). Malignant plasma cells were identified using scBCRseq and restricted light chain expression from scRNAseq. Even in MM patients without morphological evidence for CTC, we could identify and characterize single malignant plasma cells in PB. By inferring genome-wide CNVs from scRNAseq, we were able to characterize intra-patient genomic heterogeneity. Longitudinal tracking of CTC revealed tiding of clones characterized by distinct CNV profiles and transcriptomes upon Dara-exposure and resistance. Early transcriptional changes in CTCs upon Dara-exposure included inflammatory signaling (e.g. upregulation of IFI6, IFI35, IFIT1, IFITM1). Analysis of CD38 protein surface expression using DNA-barcoded antibodies showed significantly decreased levels after Dara exposure. To rule out that this might have resulted from competitive binding of the diagnostic antibody and Dara, we repeated analyses after discontinuing Dara and after application of a further line of therapy. In contrast to CD38 RNA expression, surface protein expression decreased significantly upon exposure and relapse, but recovered after an additional line of therapy. Expression of other druggable antigens (BCMA, SLAMF7, GPRCD5, FcRL5, Figure 1B) was not affected by treatment with Dara. Next, we investigated co-evolution of non-malignant cells. Longitudinal scTCRseq showed concomitant expansion and stagnation of TCRs. Receptor-ligand interaction analyses demonstrated that exposure to Dara resulted in significant differences in CD38-PECAM1-mediated interactions between CTCs with non-malignant B-cells and CD8+ T-cells. We detected recurrently increased immunosuppressive receptor-ligand interactions between malignant plasma cells and CD4+, CD8+, and regulatory T-cells via CD86-CTLA4 and NECTIN3-TIGIT axis upon Dara exposure. Conclusion: We demonstrate that single-cell multi-omic analyses of PB provide a minimal invasive approach to study genomic heterogeneity and evolution of malignant plasma cells and non-malignant cells of the immune system. Longitudinal analyses reveal different patterns of response and resistance to targeted myeloma therapies and might affect future therapeutic decision-making. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased Levels of Circulating Plasma Cells in Patients with Newly Diagnosed Multiple Myeloma Are Independently Associated with Poor Prognosis

The presence of circulating tumor cells (CTCs) has long been suggested as a valuable prognostic biomarker in Multiple Myeloma (MM). Recently, increased baseline CTC levels in patients with newly-diagnosed MM (NDMM) have been associated with adverse outcomes in the GEM2012MENOS65/GEM2014MAIN and FORTE trials. Since January 2017, we have been evaluating the presence of CTCs utilizing Next-generation Flow cytometry (NGF) with a median limit of detection (LOD) reaching 2x10-6. NGF assessment has been performed according to the EuroFlow guidelines for sample preparation, antibodies, cytometer settings and analysis of data. Currently, we have analyzed 525 matched BM and PB samples from NDMM patients and have found clonal plasma cells in 100% of BM and 89.1% (468/525) of PB samples respectively. Patients presented with ISS III/R-ISS III, high-risk cytogenetics, serum β2 microglobulin above median and hemoglobin levels ≤ 8.5 g/dL had increased CTC levels. Inversely, the absence of CTCs or their low prevalence (<10-5) was associated with an increased normal plasma cell compartment within the BM (R2=0.82, P < .0001). No association was detected between the CTC number and the therapeutic response to induction treatment in accordance with previous reports. Nevertheless, over a median follow-up monitoring of 42 months (range: 3-66 months), a running log-rank test with a step increase of 0.1% CTCs, highlighted an optimal cut-off point for PFS risk assessment at the level of 2x10-4. In the multivariable analysis, NDMM patients with CTCs above this cut-off showed a 2.2-fold higher risk of progression which was independent from ISS, induction scheme and baseline cytogenetics (HR: 2.2, 95% CI: 1.24 - 3.28, P< .001). Our large number of matched samples allowed for a phenotypic comparison between BM clonal cells and CTCs. The majority of patients with detectable CTCs (86%) showed a matched phenotypic profile of aberrant plasma cell in the two sites. However, 66 patients showed phenotypic discrepancies based on one of the following patterns: i) all phenotypic subsets were present in both BM and PB but with significantly altered ratios (≥20% of relative prevalence) for at least two concomitant subsets; ii) presence of ≥ 1 phenotypic subsets (with a minimum relative prevalence of 20% of all clonal cells) only in the BM; and iii) presence of ≥ 1 phenotypic subsets only in the PB. Remarkably, patients with phenotypic discrepancies had significantly higher CTC levels than those with a phenotypic agreement on the two sites (P < .001), together with signs of a more diffuse disease pattern, as evidenced by the imaging results. It is likely that these patients may share a patchy BM infiltration (and/or even extramedullary disease) and accumulate circulating clonal cells from various sites (thus leading to increased tumor burden) with a spatial clonal heterogeneity, which, as a total, may differ from a specific phenotype derived from a particular BM site. The biological differences among patients with differential baseline CTCs expand beyond the tumor cells. Performing a deep phenotypic approach for the identification of 32 distinct immune subsets, we analyzed the immune profile of both ΒΜ and PB in NDMM patients differing to their CTC levels by two log decimals. Patients with CTCs ≥ 5x10-3 had substantially increased levels of total T cells (especially CD8+), NK cells, tumor associated macrophages (TAMs) and an elevated ratio of memory to naïve B cells (P < .0001 for all subsets) in their BM microenvironment than those with less than 5x10-5 CTCs. The same differences, though with weaker statistical significance, applied also in PB, where, we could also identify a clear immune signature with characteristics of tumor suppression and T cell exhaustion for NDMM with the high CTC burden. Altogether, our results confirm the negative and independent prognostic impact of increased CTC levels in the NDMM and further imply its relevance in the real-world setting to establish improved risk-stratification models. Moreover, since the liquid biopsy is a better representative of the entire tumor load than a tissue biopsy sample, the analysis of CTCs may serve as the new hallmark for the real-time evaluation of a patient's disease and/or immune status.

PD-1 Inhibitors Can be the First-Line Therapy for Myelodysplastic Syndrome (MDS) --Study on the Bone Marrow Immune Microenvironment of MDS By Multispectral Imaging of Multiplex Immunofluorescence

Objective: Myelodysplastic Syndrome (MDS) is a special myeloid neoplasm with still limited treatment choices and poor survival. Although current therapies including HMAs have been shown to prolong survival, only 40-50% of patients respond to therapy, and responses are universally transient, with loss of response within 2 years. With the increasing understanding of the pathogenesis of MDS in recent years, MDS was found closely associated with the bone marrow microenvironment, including immune microenvironment (IME). More and more evidences show that the immune microenvironment of MDS is related with its disease characteristics and prognosis. Knowing more of the IME of MDS may facilitate the diagnose and treatment on MDS. However, till now little was known about the bone marrow immune microenvironment of MDS. Previous studies mostly applied multiparameter flow cytometry and based on the PB samples or frozen bone marrow fluid samples, less was known about the immune landscape of MDS bone marrow biopsies. Methods: We use multispectral imaging of multiplex immunofluorescence (mIF) to analyze the bone marrow biopsy slides and explore the bone marrow immune microenvironment of MDS patients. A total of 20 de novo MDS patients, 20 post-treatment MDS patients and 20 normal controls were included in this study. In HMAs treated group, 10 patients were response to HMA and another 10 patients were not response. HMAs were either decitabine with a dose at 20mg/m2 d1-5 or 5-Azacytidine with a dose at 75mg/m2 d1-7. The multiplex staining panel could differentiate the distinct cell subpopulations into CD34+CD117+ cells, CD8+ cells, PD1+ cells, CTLA4+ cells, CD8+PD1+ cells and CD8+CTLA4+ cells, CD8-PD1+ cells and CD8-CTLA4+ cells, respectively. We analyzed the proportion of these cells in MDS patients and normal controls, and analyzed the proportion of these cells in MDS patients with different IPSS risk stratifications, and we also assessed the degree of myelofibrosis in MDS patients and analyzed the relationship between myelofibrosis and immune cells. Results: We found that compared with the normal control, the proportion of CD8+ cells in de novo MDS patients increased, and CD8+PD1+ T cells increased too, but the CD8+CTLA4+ T cells decreased. Compared with the lower-risk groups, CD8+ T cells as well as CD8+PD1+ T cells increased in higher-risk groups, while CD8+CTLA4+ T cells decreased. We also found a higher CD8+ T cells as well as CD8+/PD1+ T cells in MDS-F patients, especially in MDS-F patients with higher IPSS risk levels. We also found that compared with the normal control, the proportion of CD8+ T cells per CD34+CD117+ cell reduced, and was lower in IPSS int-2 risk/high-risk group than that in low-risk/int-1 risk group. But the proportion of CD8+PD1+/CD34+CD117+ increased compared with normal control. To analyze the bone marrow immune microenvironment after HMAs treatment, CD8+PD1+ T cells decreased in patients who are effective to HMAs, but still remain high in patients who had no response to HMAs, so did the proportion of CD8+PD1+/CD8+ and CD8+PD1+/CD34+CD117+. However, such findings were not found on CD8+/CTLA4+ T cells. Conclusions: Our research findings provide a new insight on the bone marrow IME of MDS. The results confirmed that the immune checkpoint molecule PD-1 positive CD8+ T cells are abundant in MDS IME and associated with the risk. However, CTLA4 may not be a major molecule interfere with the IME of MDS. Our result also suggest that PD-1 inhibitor combines with HMAs might be used as a first-line therapy for de novo MDS patients to cover those patients who may not have response to HMAs. Also, PD-1 inhibitor may cover higher risk MDS and MDS-F, the latter is a high-risk subtype of MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MRD4 Eradication at 6 Months and Early Clearance of MRD with Combination of Ibrutinib Plus Venetoclax Results in Sustained Clinical and MRD Responses: Exploratory Analysis of the Blood Cancer UK TAP Clarity Study

Background: Treatment of chronic lymphocytic leukemia (CLL) has been revolutionized by targeting the B-cell receptor pathway with inhibitors such as ibrutinib (IBR), and molecules that reactivate apoptosis such as venetoclax (VEN). The Blood Cancer UK TAP CLARITY trial combined IBR with VEN to eradicate detectable CLL with the intention of stopping therapy. Methods: CLARITY is a Phase II trial combining IBR with VEN in 50 patients with relapsed/refractory (R/R) CLL. After 2 months of IBR monotherapy, VEN was added at a daily dose of 10mg/day or 20mg/day, escalating weekly to a final daily dose of 400mg/day. The primary outcome was the proportion of patients eradicating MRD (<0.01%) in the bone marrow (BM) at Month 14. Secondary outcomes included the proportion of patients eradicating MRD at Months 8, 26 and 38 and IWCLL response at Months 14, 26 and 38, progression-free and overall survival. CLL MRD was quantified using >6 color ERIC-standard flow cytometry (detection limit 10-5/0.001%). Paired PB and BM samples were assessed at Months 8, 14, 26 and longitudinal PB samples were taken at additional intermediate time points. Therapy was discontinued upon confirmation of a CR according to the IWCLL2008 criteria and MRD reduction to <10-4 CLL cells in PB and BM (MRD4). MRD4 at Month 8 indicated to stop IBR and VEN at Month 14; MRD4 in PB and BM at Months 14 and/or 26 indicated to stop IBR and VEN at Month 26; while detectable MRD at Month 26 indicated to stop VEN and to continue IBR until progression or toxicity. The original trial protocol limited venetoclax to 24 months of treatment, but this was later amended in January 2019 to allow patients, who had not obtained an MRD4 at month 26, to have an extra 12 months of VEN in addition to IBR. The aim of this exploratory analysis is to ascertain whether 2 log reduction in MRD at 2 months of combination or achievement of MRD4 at 6 months of combination results in persistence of MRD and clinical responses with prolonged follow-up. Results: Fifty-four patients were recruited from May 2016 to November 2017. The median number of prior therapies was 1 (range: 1-6). 20% patients had del(17p); 25% del(11q); 75% had unmutated IGVH. Four patients that discontinued treatment due to ibrutinib-related adverse events in the first 8 weeks before starting VEN were replaced so that, in total, 50 patients received the combination of IBR and VEN. Of the 50 evaluable participants recruited to the study, 14 patients achieved MRD-negativity at a level of <0.01% in PB and BM at 6 months of combination of IBR and VEN. At month 38 of follow-up, one patient has relapsed but not needed treatment. 10/14 patients continue at MRD4 levels and 3/14 have a missing MRD sample at Month 38 but all three patients were MRD-negative at Month 26. There were no clinical progression in the 13 patients to date. Of the 25 patients achieving 2 log reduction in MRD levels at 2 months of combination of IBR and VEN, 15/25 patients were still MRD-negative at levels of <0.01% at month 38; 4/25 patients were MRD-positive at Month 38. 6/25 patients had missing samples at month 38; 5 patients were still MRD-negative at month 26 with only one patient being MRD-positive at month 26. There were two clinical progressions to date at month 41 and 42 respectively. Conclusions: Early MRD eradication with IBR+VEN results in sustained MRD and clinical responses in R/R CLL. Achievement of MRD <0.01% with 6 months of combination or 2 log reduction in MRD levels at 2 months of combination co-related with sustained MRD and clinical responses at 3 years follow-up suggesting that this approach may help to ascertain the best responders to this combination.

Enabling the Implementation of Next-Generation Sequencing into Clinical Diagnosis: Nemhesys Project

Training for the use and analysis of Next-Generation Sequencing (NGS) is uncommon, as shown by the lack of specialized health professionals in this area. This delays NGS implementation into the healthcare system, with the result that NGS solutions hardly ever translated into real and precise experiences. . NEMHESYS (NGS-Establishment in Multidisciplinary HEalthcare SYStem) is an Erasmus Plus Knowledge Alliance programme with the aim of improving the translation of NGS into clinical practice for hematological diagnosis and prognosis by providing clinical and biomedical staff with suitable training in NGS NEMHESYS was initiated in March 2020 with partners from five Academic Institutions (University of Salamanca, Queen's University Belfast, University of Helsinki, Central European Institute of Technology (CEITEC) at Masaryk University and Charité University Medicine Berlin) and three small/medium enterprises (SME) (Artelnics, Mnemotix and IDimás). The project developed an online Masterclass for participants to complete over a 12-16-week period. The course, via the Moodle Platform hosted at the University of Helsinki, included a pre-module and five modules of which, in order to successfully pass the course, modules A & B were mandatory and at least one of the optional modules C, D or E. Regarding the contents, Module A was an introduction to all clinical and laboratory essentials for NGS in the clinic, whilst Module B was focused on all aspects of the analysis of NGS data. Module C looked at the barriers and risks around the implementation of NGS in health systems, Module D explored the role of artificial intelligence, linked data and knowledge graph, and Module E examined innovative programs and intra-entrepreneurship for NGS in the clinic. Three cohorts of the NGS Masterclass were run (March 2021, October 2021, and February 2022). Registration was high for each of the cohorts (161, 253, and 172 participants, respectively). Around 75% of the participants were from Europe but a significant number were from USA, Brazil, Argentina, Egypt, Australia as well as from Africa and Middle Eastern countries. The background of the participants was reported to be medics, biomedical and clinical scientists, graduate students, and a small proportion of bioinformaticians. As expected, there was a large number who didn't participate further than registration, but overall, at least 180 participants across the three cohorts successfully completed the Masterclass program. Additionally, participants who belonged to one of the five partner academic institutions, and had passed the online NGS training course, were entitled to apply for a learning mobility opportunity. Pre-covid this had been planned to for periods of 2 -12 weeks, but this was modified to enable more participants to be involved when considering covid related travel and other restrictions. Therefore, five Mobility Workshops were planned of one week duration at each of the academic centres, taking place from April to October (2022), and covering fundamental and of interest aspects of the application of NGS methodologies in the clinical practice and research. At the end, twenty-eight (28) participants were involved in the Mobility. The Brno hands-on workshop "Basic Skills in NGS: from PB sample to the Report" covered the clinical and laboratory essentials of NGS. The Salamanca workshop further explored novel technologies and trends in Real World NGS, whilst a focus on Precision Medicine and blood cancer diagnosis was covered by the Belfast workshop. Then, with more innovative methodologies, Charité covered the 4th generation Nanopore Technology, and the Helsinki workshop focused on Single Cell Sequencing Analytics. Overall, the NEMHESYS Masterclass and mobility program achieved the aim of giving online and practical training to a significant number of clinical and biomedical participants around the globe in the use of NGS for hematological disorders. This will enable the delivery of higher quality clinical services. PROJECT REF.: 612639-EPP-1-2019-1-ES-EPPKA2-KA. Disclaimer: The European Commission support for the production of this publication does not constitute an endorsement of the contents that reflects the views only of the authors, and the Commission cannot be held responsible for any use that may be made of the information contained therein. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Study of Betulin Particles Containing Hydrogels Prepared by Antisolvent Precipitation

The aim of the study was to investigate the effect of process parameters on the production of hydrogels with antisolvent precipitation using unpurified (UB) and purified (PB) birch outer bark betulin powder samples. Experimental activities are focused on the production of hydrogels using concentration of colloidal dispersions with filtration of UB or PB sample particles obtained by antisolvent precipitation method varying the dilution of saturated (at boiling temperature) ethanol solutions with different water content. During the study the maximum solubility in ethanol at boiling point of PB (22.0 g/L) and UB (55.0 g/L) was determined. For obtaining gel with the highest liquid content (absorption capacity 37.45 g per 1 g of dry matter) from PB by antisolvent precipitation the optimal saturated ethanol solution dilution with water was 12.5 vol%. In the case of UB hydrogel, by dilution of ethanol from 25 to 10 vol%, a gradual decrease of hydrogel dry matter yield and a simultaneous increase of the filtrate dry matter yield can be observed, which is related with leaching of the particles through the filter paper together with a solution and form a colloidal suspension of particles. For obtaining gels with high liquid content from UB the optimal saturated ethanol solution dilution with water was in the range from 20.0 to 12.5 vol% (absorption capacity in average 18 g per 1 g of dry matter). Comparing the chemical compositions of the raw materials and dry matter of hydrogels in the case of PB composition remains quite similar while in UB dry matter composition the betulin content increased from 52 to even 69 wt%. PB at the optimal dilution of 12.5 vol% of ethanol managed to achieve average particle size - 231.7 nm and with UB in the optimal dilution range from 20 to 12.5 vol% the range of average particle size was from 304.7 to 189.8 nm.

Individualized Antigen Expression in Precursor T-Cell Acute Lymphoblastic Leukemia

BACKGROUND: In T-acute lymphoblastic leukemia (T-ALL), multi-parametric flow cytometry can serve to detect minimal residual disease (MRD) by using immature or aberrant antigens expression as well as the altered expression of T-cell antigens. The latter approach has been specifically introduced to overcome the absence of leukemia-associated antigens. However, there is no agreed-upon method for the use of T-cell antigens in T-ALL MRD testing. AIMS AND OBJECTIVES: To compare the expression of classic T-cell antigens on T-lymphoblasts and T-lymphocytes to establish a protocol for their use in MRD analysis. MATERIALS AND METHODS: Flow cytometric data of PB or BM samples from 63 adults with T-ALL were collected. We assessed the frequency and degree of brightness or dimness of each T-cell marker, in addition to studying the uniformity of the events scatter of a total of 287 follow-up BM samples from 50 patients. RESULTS: Significant differences in expression intensity of T-cell markers were found between T-lymphoblasts and T-lymphocytes; they were reasonably stable on blasts in follow up samples. This detailed study has nominated the conjoint use sCD3neg/dim and CD5dim/neg in the identification of residual cells, to be supported by other T-cell markers. CONCLUSION: The suggested gating sequence showed an acceptable level of accuracy in detecting residual leukemia, supporting their use in T-ALL MRD especially when other distinguishing markers might be absent in the diagnosis sample, or susceptible to be lost with induction therapy.

P1402: ALTERATIONS OF HUMAN BONE MARROW NICHE IN CLONAL HEMATOPOIESIS AND AML

Background: Acute myeloid leukemia (AML) is a heterogeneous disease, predominantly caused by acquisition of mutations in the hematopoietic stem and progenitor cells. Some of these mutations are acquired in a preleukemic state termed clonal hematopoiesis of indeterminate potential (CHIP). Recent studies show that alterations in the bone marrow niche can support progression and therapy resistance of myeloid neoplasia. To date, it is unclear when these niche alterations occur and how they contribute to disease progression. In our study, we aim to elucidate alterations in the bone marrow niche, in particular in mesenchymal stem cells (MSC), during the evolution from healthy to CHIP and further to AML. Aims: Primary goal of this project is to examine how hematopoietic and niche cells from different health status (healthy, CHIP and AML) interact with each other and to what extent the presence of clonal hematopoiesis and leukemia have an impact on function and structure of the bone marrow niche. Methods: We collected primary human bone marrow (BM) and peripheral blood (pB) from hematologically healthy individuals who underwent hip replacement surgery at the LMU University Hospital. BM samples from AML patients are provided by the Laboratory for Leukemia Diagnostics, LMU University Hospital. Individuals are assigned to non-CHIP or CHIP cohorts based on targeted mutation analyses on pB samples. To study transcriptional changes in the bone marrow compartments, non-CHIP, CHIP and AML BM samples were FACS-sorted for 10 hematopoietic and non-hematopoietic cell types, and further processed for bulkRNA sequencing. MSC from donor BM samples were isolated via plastic adherence over 10-14 days of in vitro culture. MSC were characterized regarding their surface marker expression (FACS), differentiation (osteogenic, adipogenic, chondrogenic) potential and metabolic activity (Seahorse XF). Results: So far, we have included 98 healthy individuals and 64 have undergone targeted sequencing, revealing 19 individuals (30%) with CHIP. While there was no difference between the gender of the non-CHIP and CHIP groups, individuals with CHIP tended to be of older age (median, 64 y non-CHIP vs. 73.5 y CHIP). Most common mutations were DNMT3A (10/19), TET2 (7/19), ASXL1 (2/19), with variant allele frequencies varying between 1.0% and 16.8%, and 1.42 mutations per individual on average. The amount of mutations per individual increased with age. Analysis of MSCs from non-CHIP (n=5) and CHIP (n=5) individuals revealed no difference in surface marker expression over 10 passages. Differentiation assays of MSCs (non-CHIP=4, CHIP= 2) showed no difference in adipogenic and chondrogenic differentiation after 21 days. For osteogenesis, we noticed a trend towards an increased differentiation potential in the MSCs of CHIP individuals. To gain deeper insights into the BM niche we sorted 4 non-hematopoietic and 6 hematopoietic cell populations from non-CHIP (n=3), CHIP (n=4) and AML (n=4) individuals and performed bulkRNA sequencing on each population. So far changes in the composition of the bone marrow niche were only detected in the AML samples. Summary/Conclusion: Currently, we are analyzing the bulkRNA sequencing data and performing further experiments on cultured MSC to increase group sizes, as well as MSC and HSPC co-cultures in vitro to gain more insight into the bone marrow niche in non-CHIP, CHIP and AML. Additionally, we are continuously recruiting new individuals into our study. A comprehensive update on our cohort, sequencing data and MSC characterization will be shown at the conference.

P596: CLINICAL IMPACT OF TP53 DISRUPTION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS TREATED WITH A BCR INHIBITOR. A CAMPUS CLL EXPERIENCE

Background: In chronic lymphocytic leukemia (CLL), the presence of TP53 aberration (mutations and/or deletion) predicts an increased risk of relapse and death after chemo-immunotherapy (CIT). Although TP53 deletion and mutations mostly co-occur and are considered equal prognosticators, the clinical impact of isolated or concomitant mutations and deletions remains unclear in the context of targeted agents. Aims: To investigate the clinical relevance of isolated or concomitant TP53 aberrations in a large cohort of CLL patients treated with BCR inhibitors. Methods: In the framework of an institutional Italian multicenter working group on CLL (Campus CLL), a retrospective analysis of 229 CLL patients (51 treatment naïve, and 178 relapsed/refractory) treated with ibrutinib was carried out. All patients referred to a single institution for del17p analyses by FISH (167-kb 17p13 orange probe, MetaSystems) and TP53 mutations by NGS, both analyses carried out on CD19-purified (>85% pure) CLL samples within 6 months prior the start of ibrutinib treatment. The median follow-up from ibrutinib treatment was 36.3 months (95% CI 29.5-41.5 months). Overall survival (OS) and progression free survival (PFS) were computed from the start of ibrutinib treatment. Results: In the CLL cohort, 76 patients with del17p showed a trend for inferior OS respect to wt cases (P=0.0662, Fig.1A), while a significant correlation was found with PFS (P=0.0162). With regard to TP53 mutations, 296 TP53 mutations were found in 126 patients (range of mutations/patients 1-11). As with CIT, TP53-mutated patients, irrespective of VAF (Fig.1B), experienced a significantly worse OS and PFS than wt cases also by univariate analyses (P=0.0160, and P=0.0378, respectively). The combination of del17p with TP53 mutation data identified 95 cases with no TP53 aberrations, 8 cases del17p only, 58 cases TP53-mutated only, and 68 cases bearing both del17p deletion and TP53 mutation (Fig.1CD). Only patients with concomitant TP53 mutations and del17p experienced significantly shorter OS and PFS compared to TP53wt cases (P=0.0122, and P=0.0076, respectively; Fig.1CD). Conversely, patients presenting a single aberration showed no significant differences compared to wt patients (Fig.1CD). The simultaneous presence of TP53 mutations and del17p remained an independent predictor factor both for OS and PFS by multivariate analysis, together with the previous therapy lines (0-1 vs. >1), and anemia. The evolution of TP53-mutated clones was assessed by longitudinal NGS analysis of sequential PB samples collected from 38 patients (16 relapsed, and 22 on treatment) corresponding to 127 TP53 mutations. Among relapsed cases, 7 showed a prominent expansion of the TP53-mutated clone, 8 remained stable, and the remaining case displayed an evident decrease (Fig.1E). In non-relapsed patients, 3 cases presented an increase of TP53 mutations, 13 remained stable, and 6 showed a reduction (Fig.1F), with no significant difference compared to relapsed cases (P=0.0623, c2 test). BTK and PLCG2 mutations were found in 9/16 (56%) relapsed cases and in 3/22 (14%) patients still on ibrutinib (P=0.0492, c2 test). Of note, only 3/7 relapsed cases that presented a positive selection for TP53 mutations showed the presence of BTK mutations at the time of relapse. Image:Summary/Conclusion: This retrospective study indicates that only the concomitant presence of TP53 mutations and deletion is an independent negative prognostic factor for OS and PFS in patients with CLL on ibrutinib treatment; the simultaneous investigation of del17p and TP53 mutations may lead to a more precise risk assessment.