Malignant Blood Research Articles

Identification of autophagy-associated genes and prognostic implications in adults with acute myeloid leukemia by integrated bioinformatics analysis.

Acute myeloid leukemia (AML) is one of the most common malignant blood neoplasma in adults. The prominent disease heterogeneity makes it challenging to foresee patient survival. Autophagy, a highly conserved degradative process, played indispensable and context-dependent roles in AML. However, it remains elusive whether autophagy-associated stratification could accurately predict prognosis of AML patients. Here, we developed a prognostic model based on autophagy-associated genes, and constructed scoring systems that help to predicte the survival of AML patients in both TCGA data and independent AML cohorts. The Nomogram model also confirmed the autophagy-associated model by showing the high concordance between observed and predicted survivals. Additionally, pathway enrichment analysis and protein-protein interaction network unveiled functional signaling pathways that were associated with autophagy. Altogether, we constructed the autophagy-associated prognostic model that might be likely to predict outcome for AML patients, providing insights into the biological risk stratification strategies and potential therapeutic targets.

MMRFVariant: Prioritizing variants in Multiple Myeloma

Background:Multiple Myeloma (MM) is a blood malignancy that occurs in the plasma cells of bone marrow. Due to the drug resistance and frequent relapses, MM remains a key area of therapeutic challenge. Analysis of variants could reveal important prognostic features in MM. A reliable interpretation on how a variant affects a protein remains yet a not trivial task. The usage of appropriate bioinformatic tools for the analysis of variants data, sourcing from the Multiple Myeloma Research Foundation (MMRF) CoMMpass (Clinical Outcomes in MM to Personal Assessment of Genetic Profile) study, a large-scale observation project focusing on MM, offers great opportunities. To the best of our knowledge, a specific tool dealing with variants data sourcing from MMRF-CoMMpass datasets does not exist yet. Material and methods:MMRFVariant is a novel R package able to identify and prioritize the disease-causing variants in MM with the aim of providing valuable insights for the MM variants interpretation. MMRFVariant deals with the datasets (IA15 release) retrieved from the MMRF-CoMMpass Study. The MM variants analysis exploits pathogenicity predictions scores and survival analysis results. Conclusion and Discussion:MMRFVariant provides five functionalities that produce graphical and tabular results: the impact-effect correlation plot, the Kaplan–Meier (KM) survival curves, the heatmap of the occurrence number and the impact table can be computed. The potentiality of the tool is highlighted in a case study in which a set of six genes is selected due to their notoriety in the MM pathogenicity: the results provided by MMRFVariant are confirmed by what is reported in other studies.

Retrospective study of 613 canine cytologically normal bone marrows associated to altered hemograms: description, possible interpretation, and review of the principal physiological hematopoietic mechanisms.

In clinical routine, it can happen that to an abnormal hemogram corresponds an unexpected cytological normal bone marrow examination that can be difficult to interpret and to manage. This cytologically retrospective study wants to evaluate a consistent number of qualitative and quantitative normal bone marrow exams according to the hematological and clinical-pathological data to judge if this normality is by itself a pathologic state. Six hundred and thirteen bone marrow samples were examined. The bone marrow cytological examinations were performed using morphological and numerical criteria together with a complete hemogram, after the identification of clinical or hematological alterations such as multiple lymph nodes enlarged, positive leishmania serological result, staging of neoplasia, cytopenia, increased number of cells, or suspicion of malignant blood disorders. Of the 613 bone marrow samples evaluated, 85 (14%) were classified as normal or without cytological abnormalities; however, only 28 (33%) of those cases had a normal hemogram associated, whereas 55 (65%) had one or more cytopenia and 2 (2%) had increased blood cells count. From this study emerges that cytological bone marrow examinations without any morphological or numerical abnormalities are often associated with altered hematological exams and for this reason, they should not be considered normal and should lead to other deepened investigations.

Prevention of induced nausea and vomiting in patients with malignant blood disorders. Experience of using netupitant/palonosetron (Akynzeo)

Supporting therapy is a necessary component of successful antitumor treatment in patients with both solid tumors and malignant blood disorders. Adherence to administration protocol for antiemetic drugs aimed at prevention of induced gastrointestinal complications (nausea and vomiting), allows to improve patients' quality of life and treatment compliance. Development of a combination antiemetic drug (netupitant/palonosetron) simultaneously affecting 2 main mechanisms of nausea and vomiting as well as its easy administration allowed to significantly improve control of these complications in patients with solid tumors and patients with malignant blood disorders.We present a literature review on the effectiveness of netupitant/palonosetron and describe our own experience of using this antiemetic drug for prevention of chemotherapy-induced nausea and vomiting.

The impact of donor-to-recipient gender compatibility on outcomes of haploid hematopoietic stem cell transplantation in patients with hematological malignancies

Objective: To investigate how gender differences between the donor and the recipient affect the effectiveness of antithymocyte globulin (ATG) and pure peripheral blood stem cell (PBSC) hematopoietic stem cell transplantation (haplo-HSCT) in the treatment of malignant hematological diseases. Methods: From February 2015 to September 2020, 648 hematological malignancies patients underwent myeloablative condition regimen haplo-HSCT treatment at the Bone Marrow Transplant Center of the First Affiliated Hospital of Zhejiang University. The median age was 32 (14-62) years, with 363 males (56.0% ) and 285 females (44.0% ) present. 242 cases of acute lymphoblastic leukemia (ALL) (37.3% ) , 293 cases of acute myeloid leukemia (AML) (45.2% ) , 56 cases of myelodysplastic syndrome (MDS) (8.7% ) , 27 cases of non-Hodgkin's lymphoma (NHL) (4.2% ) , and 30 cases of other hematological malignancies (4.6% ) . Results: ① The 3-year overall survival (OS) , DFS, the incidence of Ⅱ-Ⅳ grade acute graft-versus-host disease (aGVHD) , the incidence of Ⅲ-Ⅳ grade aGVHD, the 3-year incidence of moderate & severe chronic GVHD (cGVHD) , severe cGVHD, the 3-year incidence of relapse, and NRM of the whole group were (73.10±1.90) % , (70.80±1.90) % , (33.96±1.87) % , (13.08±1.33) % , (35.10±2.14) % , (10.66±1.38) % , (19.43±1.67) % , and (9.80±1.24) % , respectively. ②There was no statistically significant difference between the donor-recipient gender match and donor-recipient gender mismatch groups in the 28-day cumulative neutrophil engraftment rate, 28-day cumulative platelet engraftment rate, the incidence of Ⅱ-Ⅳ grade aGVHD, the incidence of Ⅲ-Ⅳ grade aGVHD, 3-year OS, 3-year DFS, the cumulative incidence of relapse, NRM, and incidence of moderate & severe cGVHD, severe cGVHD. ③The 28-day cumulative neutrophil engraftment rate did not differ statistically between the male-female, female-female, male-male, and female-male groups (P=0.148) . The incidence of Ⅱ-Ⅳ grade aGVHD, the incidence of Ⅲ-Ⅳ grade aGVHD, 3-year OS, 3-year DFS, cumulative relapse rate, and NRM, and the incidence of cGVHD were not statistically different among the four groups (P>0.05) . The 28-day cumulative platelet engraftment rate of the female-male group was significantly lower than male-female group, and the female-female group [ (91.45±2.63) % vs. (94.77±1.75) % , P=0.004; (91.45±2.63) % vs. (95.54±2.05) % , P=0.005]. No significant difference existed in the 28-day cumulative platelet engraftment rate between the female-male group and the male-male group [ (91.45±2.63) % vs. (95.08±1.41) % , P=0.284]. ④Among patients ≤35 years old, the 3-year incidence of severe cGVHD patients receiving sister donors and sibling donors were (26.71±5.90) % and (10.33±4.43) % , respectively (P=0.054) . Patients accepting daughter donors and son donors had a 3-year incidence of moderate and severe cGVHD that was 40.07% vs. 27.41% , respectively, among those over 35 (40.07±6.65) % vs. (27.41±4.54) % (P=0.084) . ⑤Female donors to male recipients had a significantly lower 28-day cumulative platelet engraftment rate compared to the other groups [ (91.45±2.63) % vs. (95.08±0.95) % , P=0.037]. ⑥ Female donors to male recipients had a significantly lower 28-day cumulative platelet engraftment rate than the other groups in the ATG-Fresenius (ATG-F) 10 mg/kg group [ (89.29±4.29) % vs. (94.49±1.45) % , P=0.037]. But when compared to the other groups in the Rabbit Antihuman Thymocyte Immunoglobulin (rATG-T) 6 mg/kg group, the 28-day cumulative platelet implantation rate between female donors and male recipients was not significantly different [ (93.44±3.38) % vs. (95.62±1.26) % , P=0.404]. Conclusion: The main clinical outcomes of patients with malignant blood diseases following transplantation are unaffected by the gender combination of the donor and patient in the haplo-HSCT mode based on ATG and PBSC sources. Female donors to male recipients have a lower 28-day cumulative platelet engraftment rate and longer platelet engraftment times.

Cellular and Molecular Effects of Eribulin in Preclinical Models of Hematologic Neoplasms.

Despite the advances in understanding the biology of hematologic neoplasms which has resulted in the approval of new drugs, the therapeutic options are still scarce for relapsed/refractory patients. Eribulin is a unique microtubule inhibitor that is currently being used in the therapy for metastatic breast cancer and soft tissue tumors. Here, we uncover eribulin's cellular and molecular effects in a molecularly heterogeneous panel of hematologic neoplasms. Eribulin reduced cell viability and clonogenicity and promoted apoptosis and cell cycle arrest. The minimal effects of eribulin observed in the normal leukocytes suggested selectivity for malignant blood cells. In the molecular scenario, eribulin induces DNA damage and apoptosis markers. The ABCB1, ABCC1, p-AKT, p-NFκB, and NFκB levels were associated with responsiveness to eribulin in blood cancer cells, and a resistance eribulin-related target score was constructed. Combining eribulin with elacridar (a P-glycoprotein inhibitor), but not with PDTC (an NFkB inhibitor), increases eribulin-induced apoptosis in leukemia cells. In conclusion, our data indicate that eribulin leads to mitotic catastrophe and cell death in blood cancer cells. The expression and activation of MDR1, PI3K/AKT, and the NFκB-related targets may be biomarkers of the eribulin response, and the combined treatment of eribulin and elacridar may overcome drug resistance in these diseases.

The Mutually Mediated Chloride Intracellular Channel Protein 1 (CLIC1) Relationship between Malignant Cells and Tumor Blood Vessel Endothelium Exhibits a Significant Impact on Tumor Angiogenesis, Progression, and Metastasis in Clear Cell Renal Cell Carcinoma (ccRCC).

Background: Overexpression of chloride intracellular channel protein 1 (CLIC1) in tumor cells has been confirmed, but it has received less attention in the tumor blood vessel endothelium. Aim: The assessment of CLIC1 expression in ccRCC tumor blood vessels and its relationship with TNM parameters and tumor cell CLIC1 expression. Methods: CLIC1 immunostaining in ccRCC was evaluated in 50 cases in both malignant cells and tumor blood vessels (CLIC1 microvessel density-CLIC1-MVD) and was correlated with TNM staging parameters. Results: CLIC1-MVD was observed in approximately 65% of cases, and CLIC1 co-localization in both tumor and endothelial cells was observed in 59% of cases. ccRCC was classified into four groups (Classes 0−3) based on the percentage of positive tumor cells, with each group including sub-groups defined by CLIC1 expression in the endothelium. Class 3 (60−100% positive tumor cells) had the highest CLIC1-MVD, with an impact on T and M parameters (p value = 0.007 for T, and p value = 0.006 for M). For cases with CLIC1 intracellular translocation, there was a strong correlation between CLIC1-MVD and M (p value < 0.001). Conclusions: Co-expression of ccRCC tumor and endothelial cells promotes tumor progression and metastasis and should be investigated further as a potential therapeutic target for ccRCC and other human malignancies.

Identification of the CD16low CD56low CD38pos Natural Killer Cell as a Key Subset in Patients with Multiple Myeloma.

Natural killer (NK) cells are classified as innate immune cells which can directly recognize and kill tumor cells without antigen sensitization. NK cell-based adoptive immunotherapy for blood malignancies has attracted more attention in recent years. To analyze different NK cell subsets in the peripheral blood and bone marrow (BM) of patients with multiple myeloma (MM). Using flow cytometry we analyzed: (i) the distribution of distinct NK cell subpopulations (i.e. CD16low CD56low, CD16pos CD56high, CD16neg CD56high, CD16high CD56low, CD16neg CD56low, CD16low CD56low CD38pos) in the BM from MM patients at distinct disease stages. (ii) the expression of NKG2D, DNAM-1 and NKp30, and (iii) the expression of CD107a in CD16low CD56low CD38pos and CD16low CD56low CD38neg NK cells subsets. CD16low CD56low CD38pos was the dominant subset in BM from patients with MM at the CR stage with a decreased expression of NKp30. CD16low CD56low CD38pos subset showed a higher proportion of CD107a expression compared to CD16low CD56low CD38neg cells. In vitro experiments indicated that the CD16low CD56low CD38pos NK cell subset possesses more cytotoxicity than CD16low CD56low CD38neg NK cells. Our data suggest that CD16low CD56low CD38pos NK cells may reflect as an effector population with the potential therapeutic target in patients with MM. This group of cells may be useful for adoptive immunotherapy in MM in the future.

Literature review: Kaposi’s sarcoma pathogenesis, symptoms, diagnostic and therapeutic innovations

Paulina Petraitytė1 1 Vilnius University, Faculty of Medicine, Vilnius, Lithuania. Abstract Introduction. Kaposi’s sarcoma is a complex multifactorial angioproliferative disease caused by a virus and damage to the immune system. Clinically malignant blood or lymphatic vascular disease of many vascular nodules in the skin and internal organs. Four subtypes of the disease are distinguished, ranging from slow, only with skin lesions to lightning, which is characterized by severe visceral damage and complications. Materials and methods. Sources were colected using the PubMed database. Mainly 4 filters were used in the search: “Kaposi sarcoma”, “HHV-8”, “Kaposi sarcoma treatment”, “Kaposi sarcoma HIV”. Additionally we applied language, type, publication access, and time filters. Used sources were published in the last 10 years. Innovations in treatment and diagnostics are reviewed using sources published in 2019-2021. A total of 21 bibliographic records were used in the review. After a thorough review of the titles, abstracts, and full text, 16 publications were selected and an additional 5 publications were selected manually. Aim. To review the latest data on the clinical, diagnostic, and therapeutic features of Kaposi’s sarcoma. Results and Conclusions. Kaposi’s sarcoma is a significant diagnostic challenge in a physician’s professional practice. The main goal of treatment of Kaposi’s sarcoma is to inhibit the progression of the disease and to improve the patient’s general and physical well-being. In order to do that and prevent further complications this article comments on newest local, systemic and combined treatments. Key words: Kaposi’s sarcoma, rash, AIDS, HHV-8.

Oral Candida colonization and anti-fungal susceptibility pattern in patients with hematological malignancy.

Candidiasis is regarded as one of the most important fungal infections and a cause of disease and mortality in patients with hematological malignancy. Accordingly, antifungal prophylaxis is of significant importance in this regard. This study aimed to identify the epidemiology of Candida colonization and evaluate its antifungal susceptibility pattern in patients with hematological malignancy. In this study, the samples were collected from the oral cavity of 100 patients, and Candida colonization was confirmed by fungal culture. Candida strains were also identified by ITS-PCR. In vitro antifungal susceptibility tests against fluconazole, amphotericin B, and caspofungin were performed according to CLSI M60. Demographic characteristics, comorbidities, distribution of Candida species (spp.), and antifungal susceptibility were analyzed in this study. The study participants included 100 patients with a mean age of 15.48%±48.74 years (age range: 17-84 years). Regarding gender distribution, the majority (64%) of the patients were male. In terms of the distribution of underlying hematologic malignancy, 27% of the cases had lymphoma. The most commonly isolated species among patients were C. albicans complex (49%; n=49), C. glabrata (39%; n=39), and co-colonization of C. albicans complex and C. with C. glabrata (10%; n=10). The overall resistance of C. albicans complex was 5% to fluconazole (n=5) and 2% to amphotericin B (n=2). Furthermore, C. glabrata showed 11% (n=11) resistance to fluconazole and was susceptible to amphotericin B. All Candida spp. isolated from patients who were susceptible to caspofungin. The high rate of colonization of Candida spp., especially the significant increase in the frequency of C. glabrata in patients with blood malignancies and the gradual increase in resistance to fluconazole, necessitate a change in the use of antifungal drugs for the prevention and experimental treatment of hematological malignancy.

Incidence of Secondary Hematological Malignancies in Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: An Analysis of SEER Database

Background: Patients with Chronic lymphocytic leukemia/ Small lymphocytic lymphoma (CLL/SLL) are at risk of experiencing transformation to aggressive hematological malignancies and can develop second primary malignancies. With a dramatic change in the treatment landscape of CLL/SLL, there is paucity of literature on real-world incidence of second hematological malignancies (SHM) in the era of targeted therapy. We analyzed data obtained from Surveillance, Epidemiology, and End Results (SEER) database to estimate the incidence of SHM in patients with CLL/SLL. Method We used SEER 17 registry to identify adult patients with an index diagnosis of CLL/SLL between January 2000- December 2019, and subsequent diagnosis (>2months after index diagnosis) of any SHM. International Classification of Disease code for Oncology (ICD-O-3) was used to identify codes assigned to CLL/SLL (9823/3) and 11 types of SHM [9650/3: Classical Hodgkin lymphoma(HL), 9673/3: Mantle cell lymphoma(MCL), 9680/3: Diffuse large B-cell lymphoma (DLBCL), 9687/3: Burkitt lymphoma (BL), 9689/3: Splenic marginal zone lymphoma (SMZL), Acute lymphoblastic leukemia/lymphoma (ALL: consisting of 9811/3: B lymphoblastic leukemia/lymphoma, 9728/3: Precursor B-lymphoblastic lymphoma, 9836/3: Precursor B-cell lymphoblastic leukemia and 9837/3: T lymphoblastic leukemia/lymphoma), 9833/3: B-cell prolymphocytic leukemia(B-PLL), 9834/3: T-cell prolymphocytic leukemia(T-PLL), 9861/3: Acute myeloid leukemia (AML), 9863/3: Chronic myeloid leukemia (CML), 9989/3: Myelodysplastic syndrome (MDS)]. We considered SHM as any blood malignancy with diagnosis after treated or untreated CLL/SLL. Patients with CLL/SLL who had unknown survival time or a simultaneous diagnosis of another hematological malignancy were excluded. A data cut-off time point (December, 2019) was used to estimate survival duration (in months) and vital status (dead or alive). Additional data including demographics and treatment details were extracted using SEER Stat v 8.4.0.1 software. Statistical analyses were performed using IBM-SPSS Statistics v 28.0.1.1(15). The reverse Kaplan Meier method was used to calculate median follow-up duration. Chi-squared test was used to calculate difference between categorical variables. Kaplan Meier method and log rank test were used to estimate overall survival (OS). Result 1.8% of adult patients with CLL/SLL (1390 of 78,943) had SHM during the study period. 1255 patients met eligibility criteria (CLL/SLL patients with unknown survival duration (n=3), simultaneous diagnosis of other hematological malignancy (n=3) and a gap of <2months between SHM diagnosis and index diagnosis (n=129) were excluded). Of the 1255 patients, 63.7% (799) were males and 90% (1130) had White race. Median ages at the time of diagnosis of CLL/SLL and SHM were 65 and 70 years respectively. DLBCL (57.2% [n=718]), MCL (10.8% [n=136]), and MDS (8.69% [n= 109]) were the most frequently reported SHMs. Median (95% Confidence Interval) follow-up duration after CLL/SLL and SHM diagnosis were 153 (145.0-160.9) and 58 (53.4-62.6) months respectively. Gender association with SHM was not statistically significant (p =0.146). 394 (31.4%) patients received chemotherapy (CT) for CLL/SLL, while 861 (68.6%) patients either did not receive treatment or status was unknown. OS subsequent to any SHM diagnosis was 12 (95%CI 10.1-13.9) months. OS was lowest for AML [3(1.3-4.6)], and highest for SMZL [113(82.7-143.3)]. Patients who did not receive chemotherapy (for CLL) or details were unknown lived longer subsequent to SHM diagnosis compared to patients who received chemotherapy for CLL/SLL (Median survival months (95%CI): 13(10-16) vs 10(8-12), p =0.034, hazard ratio = 1.159 (95% CI: 1.007-1.334) (Figure). Conclusion SHM were seen in approximately 2% of CLL/SLL patients, predominantly among males and white race. Overall survival after the diagnosis of SHM is around 1 year. Improvement in overall survival with newer targeted therapies for CLL/SLL necessitates robust strategies for surveillance of SHM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of Consensus Guidelines for Hematology Fellow Competency in Peripheral Blood Smear Review Utilizing Nominal Group Technique

INTRODUCTION: Graduate medical education (GME) training in hematology in the United States is accredited by the Accreditation Council for Graduate Medical Education (ACGME). Trainees provide patient care that requires manual review and interpretation of peripheral blood smears (PBS). The ACGME milestones were developed with significant input from the American Society of Hematology (ASH) and mandate fellow competency in the interpretation of peripheral blood smears. However, there are currently no publications that define competency or propose consensus recommendations (CRs) for PBS review. The primary aim of this study was to establish CRs on PBS review with a focus at the GME level. METHODS: Nominal group technique (NGT) and constructivist grounded theory (CGT) were selected given the lack of existing published theory and consensus on the methodology of PBS review or other analogous visuospatial skills within GME. A convenience sample of hematologists with expertise in training and assessing fellows was recruited through the ASH Medical Educators Institute. Goal accrual was 5-10 hematologists. Participants were required to regularly review PBSs as part of their clinical practice and attend two 1-hour virtual focus group sessions. The facilitator guide for session 1 was developed a priori to prompt open discussion on PBS review. Dialogue was transcribed and organized into thematic concepts through inductive coding. Data from session 1 were analyzed using a mixed methods approach and were used to develop a facilitator guide for session 2. Draft CRs were created using CGT. Weak and strong consensus were pre-specified as 50 - 69%, and >70% agreement, respectively. CRs underwent iterative review which would continue for a pre-specified minimum of 2, but no more than 3 rounds. RESULTS: Eight academic hematologists (5 men, 3 women) enrolled in the focus group (Table 1). Duration of clinical practice ranged from 5 to 33 years. Clinical focus spanned classical and malignant hematologic conditions. Half had formally instructed trainees in course work on PBS morphology. All routinely worked with fellows. CRs were generated through CGT. Three rounds of review occurred. Participants provided a statement of agree vs. disagree and offered qualitative suggestions for each statement in which they disagreed. Response rates per round were 87.5%, 87.5%, and 100%. The most commonly identified themes included recognition of morphology suggestive of thrombotic microangiopathies and distinguishing between malignant vs. benign white blood cells (WBC), specifically as it pertains to acute leukemias. Strong consensus was reached on multiple aspects of PBS education, including curricular considerations, method of review, and morphology (Table 2). All agreed that trainees should learn PBS review through a systematic approach, although some agreed that this systematic approach is not always used in clinical practice. The group agreed that trainees should be educated through PBS review of patients under their clinical care as well as through formal slide "libraries". Many discussed the changing landscape of PBS education with the advent of digital microscopy, and formed strong consensus that trainees should understand the benefits and limitations of digital microscopy. Group discussion largely focused on disorders of WBCs and red blood cells (RBC) (43% and 48% of all coded statements respectively). Most discussion focused on disorders in which prompt recognition was imperative to avert significant patient morbidity or mortality. However, all agreed that trainees must also be competent in the identification of common conditions, regardless of the degree of clinical importance, including normal peripheral blood smear morphology. The group was unable to reach consensus on how to balance these competing philosophies. CONCLUSIONS: A formal theory of PBS education was systematically developed using CGT. Experts were able to form strong CRs on almost every aspect of PBS review at the GME level. FUTURE DIRECTIONS: We propose that these guidelines serve as a template for future research and curricular development for both PBS review and other visuospatial tasks in medical education. This framework can also be used by organizations, such as ACGME and ASH, when formally reviewing and codifying guidelines on PBS review. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of Mannose Receptor C-Type 2 (MRC2) in Leukemic Stem Cell Maintenance in AML

Acute myeloid leukemia is a blood malignancy organized as a hierarchy with leukemic stem cells (LSC) at its summit. LSCs are well known and described for their role in initiating the disease as well as relapse after apparent remission. Over the last decades, efforts have been made to specifically and selectively target LSCs and as a result, many have demonstrated the heterogeneous composition of this AML sub-population. In a previously reported transcriptomic data set comparing LSCs with non-LSCs in 10 patients with molecularly heterogeneous AML, we noted a not yet appreciated enrichment of mannose receptor C-type 2 (MRC2) mRNA in LSCs. MRC2 is a molecule interacting with extracellular matrix (ECM) components such as collagen and described as a pro-migratory and -invasive receptor. By flow cytometry screening, we noted a diversified distribution of MRC2 expression among healthy and n=42 primary AML samples. Hematopoietic stem cells (HSCs) display the highest percentage of MRC2 (88 to 97%) while AML samples show a very variable MRC2 expression (with 0 to 90% positive cells). Interestingly, MRC2-positive cells often represent a distinct cellular subpopulation that phenotypically resemble LSCs as they are mostly CD34 positive, NKG2DL negative and show enhanced expression of CD123 and GPR56. Functionally, when compared to MRC2-negative cells sorted from the same patient, MRC2-positive cells from n=12 AML cases showed increased clonogenicity in in vitro CFU assays. Importantly, MRC2-positive cells also showed higher in vivo leukemia induction capacity (as compared to their MRC2-negative counterpart) in transplantation assays performed in xenograft mouse models (n=2 analyzed AML cases, n=6 mice analyzed for MRC2-negative and MRC2-positive cells, respectively). These results indicate that MRC2 expression might be a feature of LSCs. To further investigate its functional role in AML, we knocked-out (KO) MRC2 in AML cell lines and observed a decreased capacity of KO cells to migrate, invade and interact with ECM proteins. The most striking impact of our KO models is that, compared to wild-type cells, KO cells are unable to uptake FITC-labelled degraded collagen (gelatin), the most described function of MRC2 in the literature. We could also demonstrate that hypoxia impairs the ability of AML cells to uptake degraded collagen by reducing the level of MRC2 expression. On the other hand, TGF-ß1 treatment increased the potential to uptake degraded collagen, which was directly correlated to increased MRC2 expression upon treatment. In line with this, blocking of TGF-ß1 signaling significantly affected the uptake of gelatin and the level of MRC2 expression. We could also note such similar effects with TGF-ß1-treated primary AML samples, and conclude that TGF-ß1 is a potent up-regulator of MRC2 expression. Altogether, our data propose MRC2 as a new LSC marker that potently regulates the interaction of LSCs with their microenvironment. Mechanisms by which MRC2 expression is modulated are likely to be responsible for AML heterogeneity and plasticity and has new implications for LSC identification.

Preclinical Evaluation of CRISPR-Edited CAR-NK-92 Cells for Off-the-Shelf Treatment of AML and B-ALL.

Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines' cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.

Atención al adolescente con cáncer. ¿Qué ha cambiado en nuestro país en una década?

Introduction and objectiveIn 2012, the Adolescents with Cancer working group published the results of a survey on care delivery for the adolescent population in Spain as a starting point for future intervention. The aim of this nationwide survey was to outline the current situation and assess whether the implemented strategies have resulted in changes in care delivery. Material and methodsSurvey consisting of the same items analysed and published in 2012. The questionnaire was structured into sections devoted to epidemiology, psychosocial care, infrastructure, treatment and follow-up of adolescents with cancer. It was submitted to all hospitals in Spain with a paediatric haematology and oncology unit. We conducted a descriptive analysis of the results. ResultsThe percentage of patients aged up to 18 years managed in paediatric units has increased from 35.9% to 77.5% in the past decade. The proportion of malignant blood tumours treated in paediatric units increased from 31% to 52%, and the proportion of solid tumours from 49% to 85%. In 2012, 30 units (out of 39) reported that new cases in adolescents amounted to up to 10% of the total. At present, only 14 (out of 40) continue to report this percentage. A decade ago, there were no specific adolescent cancer units in Spain. Now, 7 centres (out of 40) have specific multidisciplinary units. There has been little change in psychological support services for adolescents. The follow-up of survivors is carried out by paediatric specialists in 82.5% of the hospitals. ConclusionsThe efforts made to centralise the care of adolescents with cancer in specific multidisciplinary adolescent units or, failing that, paediatric units, is reflected in the changes in care delivery in Spain in the past decade. Much remains to be done in key components of the management of adolescents with cancer.

Laser Micromachining of Bone as a Tool for Studying Bone Marrow Biology.

The bone marrow (BM) has traditionally been a difficult tissue to access because it is embedded deep within the bone matrix. It is home to the hematopoietic stem cells (HSCs) that give rise to all blood cells in the body. It is also the site of origin for malignant blood cells such as leukemia and multiple myeloma, as well as a frequent site of metastasis for many solid tumors including prostate and breast cancer. The following chapter describes how laser micromachining of bone can be used to improve both optical and physical access to the BM. For example, laser thinning of the overlying bone can improve optical access, enabling deeper imaging into the BM as well as enhancing optical resolution by reducing scattering and aberration. Laser micromachining can also be used to provide physical access into the BM by creating access ports for micropipette insertion and delivery of cells to precise locations in the BM, as well as for the extraction of BM cells and interstitial fluid, all under image guidance. This chapter provides a detailed protocol for installing a laser-micromachining capability for users with an existing multiphoton microscope. Additionally, we briefly outline how such a system improves the optical resolution during imaging as well as its potential useto study injury response.

Advances and Hurdles in CAR T Cell Immune Therapy for Solid Tumors.

Simple SummaryChimeric antigen receptor (CAR) T cells are genetically engineered T cells that recognize markers present on tumor cells and drive the degradation of the tumor itself. CAR T immunotherapy has obtained remarkable success in targeting a number of blood malignancies; however, its outcome is typically modest when applied to solid tumors, because of specific structural, biological, and metabolic aspects of the solid tumor environment. This article offers an overview of the interactions between CAR T cells and the solid tumor microenvironment, highlighting the main strategies that have been attempted to overcome CAR T suppression, both in preclinical models and in clinical trials.Chimeric antigen receptor (CAR) T cells in solid tumors have so far yielded limited results, in terms of therapeutic effects, as compared to the dramatic results observed for hematological malignancies. Many factors involve both the tumor cells and the microenvironment. The lack of specific target antigens and severe, potentially fatal, toxicities caused by on-target off-tumor toxicities constitute major hurdles. Furthermore, the tumor microenvironment is usually characterized by chronic inflammation, the presence of immunosuppressive molecules, and immune cells that can reduce CAR T cell efficacy and facilitate antigen escape. Nonetheless, solid tumors are under investigation as possible targets despite their complexity, which represents a significant challenge. In preclinical mouse models, CAR T cells are able to efficiently recognize and kill several tumor xenografts. Overall, in the next few years, there will be intensive research into optimizing novel cell therapies to improve their effector functions and keep untoward effects in check. In this review, we provide an update on the state-of-the-art CAR T cell therapies in solid tumors, focusing on the preclinical studies and preliminary clinical findings aimed at developing optimal strategies to reduce toxicity and improve efficacy.

Evaluation of two new highly multiplexed PCR assays as an alternative to next-generation sequencing for IDH1/2 mutation detection.

IDH1 and IDH2 somatic mutations have been identified in solid tumors and blood malignancies. The development of inhibitors of mutant IDH1 and IDH2 in the past few years has prompted the development of a fast and sensitive assay to detect IDH1R132 , IDH2R140 and IDH2R172 mutations to identify patients eligible for these targeted therapies. This study aimed to compare two new multiplexed PCR assays - an automated quantitative PCR (qPCR) on the PGX platform and a droplet digital PCR (ddPCR) with next-generation sequencing (NGS) for IDH1/2 mutation detection. These assays were evaluated on 102 DNA extracted from patient peripheral blood, bone marrow and formalin-fixed paraffin-embedded tissue samples with mutation allelic frequency ranging from 0.6% to 45.6%. The ddPCR assay had better analytical performances than the PGX assay with 100% specificity, 100% sensitivity and a detection limit down to 0.5% on IDH1R132 , IDH2R140 and IDH2R172 codons, and a high correlation with NGS results. Therefore, the new highly multiplexed ddPCR is a fast and cost-effective assay that meets most clinical needs to identify and follow cancer patients in the era of anti-IDH1/2-targeted therapies.

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.

HOXA9 has the hallmarks of a biological switch with implications in blood cancers

Blood malignancies arise from the dysregulation of haematopoiesis. The type of blood cell and the specific order of oncogenic events initiating abnormal growth ultimately determine the cancer subtype and subsequent clinical outcome. HOXA9 plays an important role in acute myeloid leukaemia (AML) prognosis by promoting blood cell expansion and altering differentiation; however, the function of HOXA9 in other blood malignancies is still unclear. Here, we highlight the biological switch and prognosis marker properties of HOXA9 in AML and chronic myeloproliferative neoplasms (MPN). First, we establish the ability of HOXA9 to stratify AML patients with distinct cellular and clinical outcomes. Then, through the use of a computational network model of MPN, we show that the self-activation of HOXA9 and its relationship to JAK2 and TET2 can explain the branching progression of JAK2/TET2 mutant MPN patients towards divergent clinical characteristics. Finally, we predict a connection between the RUNX1 and MYB genes and a suppressive role for the NOTCH pathway in MPN diseases.