Minimal Residual Disease Monitoring with Next-Generation Sequencing Methodologies in Hematological Malignancies

Ultra-deep next-generation sequencing has emerged in recent years as an important diagnostic tool for the detection and follow-up of tumor burden in most of the known hematopoietic malignancies. Meticulous and high-throughput methods for the lowest possible quantified disease are needed to address the deficiencies of more classical techniques. Precision-based approaches will allow us to correctly stratify each patient based on the minimal residual disease (MRD) after a treatment cycle. In this review, we consider the most prominent ways to approach next-generation sequencing methodologies to follow-up MRD in hematological neoplasms.This work received no specific external funding; our research group was supported by the Fundación Cris contra el cáncer.S

Ocena minimalnej choroby resztkowej w szpiczaku plazmocytowym

Minimal residual disease (MRD) in a patient with multiple myeloma (MM) is defined as the mini-mum levels of pathological plasma cells remaining after treatment when a patient is in complete response (CR). The ultimate aim of studying MRD is to identify patients with different prognosis and to tailor treatment for individual patients. MRD studies in MM should be recommended in young patients in CR after autologous hematopoietic stem cells transplantation and in older patients in CR after regimens including proteasome inhibitors. Bone marrow is the only recommend location to assess MRD in MM. The recommended methods of MRD testing include next generation sequencing of immunoglobulin genes or multiparametric flow cytometry (MFC), depending on the experience of each center and the possibility of study samples being available in the first 24 hours for MFC analysis. MRD should be considered as a therapeutic objective. However, there is not enough evidence for taking clinical decisions based on MRD status alone, and for this reason we encourage the design of new clinical studies to address these questions.Minimalną chorobę resztkową (MRD) u pacjenta z rozpoznaniem szpiczaka plazmocytowego defi-niuje się jako populację nowotworowych komórek plazmatycznych, która pozostała w organizmie chorego po osiągnięciu odpowiedzi całkowitej (CR). Ostatecznym celem badań MRD jest dążenie do identyfikacji chorych o odmiennym rokowaniu i indywidualizacji leczenia na tej podstawie. Ocenę MRD u chorych na szpiczaka plazmocytowego zaleca się u młodszych pacjentów, którzy osiągną CR po przeszczepieniu autologicznych krwiotwórczych komórek macierzystych oraz u chorych starszych osiągających CR po chemioterapii opartej na inhibitorach proteasomu. Powinno się oznaczać MRD wyłącznie w szpiku kostnym. Do rekomendowanych metod oceny MRD w szpiczaku plazmocytowym zalicza się sekwencjonowanie następnej generacji genów immunoglobulinowych oraz wieloparametryczną cytometrię przepływową, przy czym wybór jednej z tych metod powinien zależeć od doświadczenia danego ośrodka oraz możliwości wykonania badania cytometrycznego w czasie 24 godzin od pobrania próbki szpiku. Eradykację MRD powinna się obecnie uważać za istotny cel terapii szpiczaka plazmocytowego. Jednak, ze względu na brak wystarczających danych do podejmowania decyzji klinicznych wyłącznie na podstawie wyniku badania MRD, istnieje po¬trzeba dalszych, dobrze zaprojektowanych badań klinicznych w tym zakresie

Novel saliva biomarkers for stress and infection in pigs : Changes in oxytocin and procalcitonin in pigs with tail-biting lesions

There is a need for feasible and reliable measures to improve and evaluate production animal health and welfare. Oxytocin is a promising novel stress-related biomarker and procalcitonin may be a measure of sepsis. Both have potential for use in pigs and can be measured from saliva, which allows on-farm sampling with minimal impact on the animals. The current study sought to further validate these measures using a spontaneous situation that causes both stress and an increased risk for infections in pigs, namely a tail-biting outbreak. Grower pigs on a commercial farm belonging to three different phenotype groups were selected: control pigs from control pens (CC, N = 30), control pigs (CTB, N = 10), and pigs with tail lesions from pens with a tail-biting outbreak (LTB, N = 27). A single sample of saliva was collected from each pig and analysed for a range of biomarkers related to stress, infection, inflammation, and immune activation. Oxytocin tended to be higher in CC pigs than in LTB pigs, while cortisol was higher in CTB than CC pigs. Procalcitonin tended to be higher, and haptoglobin was higher in LTB than in CC pigs. Adenosine-deaminase levels were similar between phenotypes. These results provide further evidence for the link between stress and tail biting, and indicate that tail-biting lesions are potential routes for systemic spread of bacteria. Further research into saliva oxytocin as a stress biomarker and saliva procalcitonin as a sepsis biomarker in pigs is warranted.Peer reviewe

Droplet Microfluidics for the ex Vivo Expansion of Human Primary Multiple Myeloma Cells.

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device We demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double layered bead composed by an inner layer of alginate and an outer layer of puramatrix constructed using a soft technology without the use of any external force. In this work, we use this micro manipulation technique to build a 3D scaffold as a biomimetic model to recapitulate the niche of patient-derived multiple myeloma cells (MM cell) using a multilayered 3D tissue scaffold constructed in a microfluidic device and cultured in 10% FBS culture medium. In the current study, we included the use of this biomimetic model comprising supporting human Mesenchymal stem cells to show the mid-term survival of MM cells in the proposed structures. We found that the generated microniches were suitable for the maintenance of MM cells with and without supporting cells. Additionally, cultured MM cells in droplets were exposed to both Bortezomib and Lenalidomide to test their toxicity in the cultured patient derived cells. Results indicate that the maintained MM cells were consistently responding to the applied medication, opening a wide field of possibilities to use the presented micro device as an ex vivo platform for drug screening.This research was funded by European Commission H2020 Marie Curie Research Grants Scheme MSCA-IF-GF (705163).S

Durable Response After Tisagenlecleucel in Adults With Relapsed/Refractory Follicular Lymphoma: ELARA Trial Update

Tisagenlecleucel is approved for adults with relapsed/refractory (r/r) follicular lymphoma (FL) in the ≥3rd-line setting. The primary analysis (median follow-up: 17 months) of the Phase II ELARA trial (ClinicalTrials.gov identifier: NCT03568461) reported high response rates and excellent safety profile in extensively pretreated patients with r/r FL. Here we report longer-term efficacy, safety, pharmacokinetic, and exploratory biomarker analyses after a median follow-up of 29 months. As of March 29, 2022, 97 patients with r/r FL (grades 1-3A) after ≥2 lines of therapy or who relapsed after autologous stem cell transplant received tisagenlecleucel infusion (0.6-6×108 CAR+ viable T cells). Bridging chemotherapy was allowed. Baseline clinical factors, tumor microenvironment (TME), blood soluble factors, and circulating blood cells were correlated with clinical response. Cellular kinetics were assessed by quantitative polymerase chain reaction. Median progression-free survival (PFS), duration of response (DOR), and overall survival (OS) were not reached after 29 months median follow-up (IQR, 22.2-37.7). Estimated 24-month PFS, DOR, and OS rates in all patients were 57.4% (95% CI, 46.2-67), 66.4% (95% CI, 54.3-76), and 87.7% (95% CI, 78.3-93.2). Complete response rate and overall response rate were 68.1% (95% CI, 57.7-77.3) and 86.2% (95% CI, 77.5-92.4), respectively. No new safety signals or treatment-related deaths were reported. Low levels of tumor-infiltrating LAG3+CD3+ exhausted T-cells and higher baseline levels of naïve CD8+ T-cells were associated with improved outcomes. Tisagenlecleucel continued to demonstrate highly durable efficacy and a favorable safety profile in this extended follow-up of 29 months in patients with r/r FL enrolled in ELARA

Reflexiones latinoamericanas en bioética

La reflexión sobre bioética está enraizada en la situación actual. Los países desarrollados comienzan el tercer milenio en condiciones de bienestar generalizadas, superiores a las que tenían hace treinta años, pero la calidad de vida requiere algo distinto del simple crecimiento, la situación personal se ha deteriorado. Los países pobres están dispuestos a sacrificar lo que añoran los países desarrollados por disfrutar del crecimiento mecanicista. La producción requiere cada día menos personas de las que hoy están ocupadas. La generación de desechos aumenta. Los hombres encuentran dificultades para relacionarse. Por ello, la situación presente es vista como un desafío que supera a los retos tradicionales, donde no sólo los principios clásicos que rigen la bioética necesitan ser pensados.En este momento de desequilibrio social, cuando la corrupción y la impunidad son males sociales que necesitan ser extinguidos, parece que toda la sociedad habla de ética. El problema no radica en la frecuencia con la que se exige ética, rama de la filosofía, sino en la situación por la que –de acuerdo con lo que decía Jaspers al respecto de la filosofía– todos se sienten competentes para hablar del tema, parecería que la sola condición humana es suficiente para hacerlos capaces. Así, se escuchan todo tipo de argumentos, la mayoría insostenibles, pero no todos los discursos son vacíos; este libro es un ejemplo de la actitud opuesta, quienes lo escriben se han dedicado a la investigación sobre el tema. Los especialistas en bioética aquí recogidos tienen una larga trayectoria dentro de la misma. En el curso de sus vidas profesionales y personales han encontrado algunas explicaciones acerca del ejercicio de la práctica del cuidado de la salud y han tenido claro que la ética toma en cuenta elementos del deber ser. Aunque en dicha práctica la escucha del inconsciente y la manifestación del deseo ofrecen una apertura distinta hacia el conocimiento del ser humano

Results from HARMONY: an open-label, multicentre, 2-arm, phase 1b, dose-finding study assessing the safety and efficacy of the oral combination of ruxolitinib and buparlisib in patients with myelofibrosis

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Ruxolitinib in combination with prednisone and nilotinib exhibit synergistic effects in human cells lines and primary cells from myeloproliferative neoplasms

Ruxolitinib is the front-line non-palliative treatment for myelofibrosis (MF). However, a significant number of patients lose or present suboptimal response, are resistant or have unacceptable toxicity. In an attempt to improve response and avoid the adverse effects of this drug, we evaluated the combination of 17 drugs with ruxolitinib in ex vivo models of peripheral blood mononuclear cells from MF patients and cell lines. We found that the combination ruxolitinib and nilotinib had a synergistic effect against MF cells (ΔEC50 nilotinib, -21.6%). Moreover, the addition of prednisone to combined ruxolitinib/nilotinib improved the synergistic effect in all MF samples studied. We evaluated the molecular mechanisms of combined ruxolitinib/nilotinib/prednisone and observed inhibition of JAK/STAT (STAT5, 69.2+11.8% inhibition) and MAPK (ERK, 29.4+4.5% inhibition) signaling pathways. Furthermore, we found that the triple therapy combination inhibited collagen protein and COL1A1 gene expression in human bone marrow mesenchymal cells. Taken together, we provide evidence that combined ruxolitinib/nilotinib/prednisone is a potential therapy for MF, possibly through the anti-fibrotic effect of nilotinib, the immunomodulatory effect of ruxolitinib and prednisone, and the anti-proliferative effect of ruxolitinib. This combination will be further investigated in a phase Ib/II clinical trial in MF.This study was supported by the Subdireccion General de Investigacion Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. M.L. holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409).S

Risk factors for one-year mortality in hospitalized adults with severe covid-19 comment

As the body''s immunity declines with age, elderly-hospitalized patients due to COVID-19 might be at higher mortality risk. Therefore, the aim of this prospective study was to examine the possible risk factors (demographic, social or comorbidities) most associated with mortality one-year after diagnosis of COVID-19. Routine data were collected from a cohort of hospitalized adults with severe COVID-19. The primary endpoint was mortality at one-year after diagnosis of COVID-19. We used a Cox proportional hazard model to estimate the hazard ratios (HRs) for both all-cause and specific cardiorespiratory mortality. A fully adjusted model included sex, socioeconomic status, institutionalization status, disability, smoking habit, and comorbidities as confounders. A total of 368 severe cases hospitalized on average 67.3 +/- 15.9 years old were included. Participants aged >= 71 years had significantly higher HRs for all-cause mortality (adjusted HRs = 2.86, 95%CI: 2.01-4.07) and cardiorespiratory mortality (adjusted HRs = 2.86, 95%CI: 1.99-4.12). The association between age and mortality after diagnosis of COVID-19 due to both all-causes and cardiorespiratory mortality showed a consistent dose-response fashion. Institutionalization, disability, and socioeconomic status also showed a significant association with mortality. In conclusion, aging itself was the most important risk factor associated with mortality one year after diagnosis of COVID-19. People with disabilities, institutionalized or low socioeconomic status are significantly more likely to die after COVID-19

GMP-Compliant Manufacturing of NKG2D CAR Memory T Cells Using CliniMACS Prodigy

Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3ζ signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA- memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA+ fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 108 CD45RA- cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3ζ lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.This study was funded in part by the National Health Service of Spain, Instituto de Salud Carlos III (ISCIII), FONDOS FEDER grant (FIS) PI18/01301, by the Unoentrecienmil Foundation and by CRIS Cancer Foundation to beat Cancer (http://criscancer.org).LF, AF, IM, and AE are granted by CRIS Cancer Foundation to beat cancer.S