Large B-cell lymphoma with IRF4 rearrangement: a multi-centric study with focus on potential misleading phenotypes.

Large B-cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a rare lymphoid neoplasm, usually occurring in the pediatric/young-adult age. Despite this, subsets of cases occur in elderly patients and express CD5, possibly entering the differential diagnosis with adult aggressive lymphomas, such as blastoid/pleomorphic mantle cell lymphoma (MCL-B/P). To better characterize the clinical-pathological features and differential diagnosis of LBCL-IRF4, we conducted a multi-centric study on 12 cases, focusing on CD5, Cyclin D1, and SOX11 expression. While most cases had typical presentation, adult-to-elderly age at diagnosis and unusual anatomic locations were reported in 3/12 (25.0%) and 2/12 (16.7%) patients, respectively. Histologically, CD5 was positive in 4/12 (33.3%) cases, Cyclin D1 was invariably negative, and SOX11 was weakly/partially expressed in 1/12 (8.3%) case. In conclusion, LBCL-IRF4 can have unconventional clinical presentations that may challenge its recognition. Although CD5 is frequently expressed, negativity for Cyclin D1 and SOX11 contributes to the differential diagnosis with MCL-B/P.

Genomic Profiling of Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System Suggests Novel Potential Therapeutic Targets.

Primary diffuse large B-cell lymphoma of the primary central nervous system (CNS-DLBCL) is an aggressive disease, with dismal prognosis despite the use of high-dose methotrexate-based polychemotherapy. Our study aimed to expand the biologic profiles of CNS-DLBCL and to correlate them with clinical/imaging findings to gain diagnostic insight and possibly identify new therapeutic targets. We selected 61 CNS-DLBCL whose formalin-fixed paraffin-embedded samples were available at first diagnosis. These were investigated by immunohistochemistry, cMYC rearrangements were explored by fluorescence in situ hybridization, and CNS-DLBCL mutated genes were evaluated by next-generation sequencing. CD10, BCL6, and IRF4 were observed in 16%, 83.6%, and 93% of cases, respectively. As typical of CNS lymphoma, 10 (16.4%) of 61 cases were classified as germinal center (GCB) type and 51 (83.6%) of 61 as non-germinal center (non-GCB) type according to the Hans algorithm. Double-expression status for BCL2 and cMYC was detected in 36 (59%) of 61 cases whereas 25 (41%) of 61 were non-DE. Rearrangement of the cMYC gene was detected in 2 cases, associated with BCL6 translocation only in 1 case MYD88, PIM1, CD79B, and TP53 were mutated in 54.5%, 53.5%, 30.2%, and 18.4% cases, respectively. Novel mutations not previously reported in CNS-DLBCL were found: AIP in 23.1%, PI3KCA in 15%, NOTCH1 in 11.4%, GNAS in 8.1%, CASP8 in 7.9%, EGFR in 6.4%, PTEN in 5.1, and KRAS in 2.6% of cases. Survival was significantly longer for patients with mutated MYD88 (8.7 months vs 1.7 months; log-rank test = 5.43; P = .020) and for patients with mutated CD79B (10.8 months vs 2.5 months; log-rank test = 4.64; P = .031). MYD88 and CD79B predicted a longer survival in patients affected by CNS-DLBCL. Notably, we identified novel mutations that enrich the mutational landscape of CNS-DLBCL, suggest a role of PTEN-PI3K-AKT and receptor tyrosine kinase-RAS-mitogen-activated protein kinase signaling in a subset of CNS-DLBCL, and provide new potential therapeutic targets.

Follicular Lymphoma Microenvironment Traits Associated with Event-Free Survival.

The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease "virtually" incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution (intrafollicular, IF and extrafollicular, EF) of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte (CD8, CD4, PD1) and macrophage (CD68, CD163, MA4A4A)-associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan-Meier estimates were linked with shorter EFS (event-free survival), with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts.

The Medium Obtained from the Culture of Hodgkin Lymphoma Cells Affects the Biophysical Characteristics of a Fibroblast Cell Model.

The neoplastic Hodgkin-Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) represent only 1-10% of cells and are surrounded by an inflammatory microenvironment. The HL cytokine network is a key point for the proliferation of HRS cells and for the maintenance of an advantageous microenvironment for HRS survival. In the tumor microenvironment (TME), the fibroblasts are involved in crosstalk with HRS cells. The aim of this work was to study the effect of lymphoma cell conditioned medium on a fibroblast cell population and evaluate modifications of cell morphology and proliferation. Hodgkin lymphoma-derived medium was used to obtain a population of "conditioned" fibroblasts (WS-1 COND). Differences in biophysical parameters were detected by the innovative device Celector®. Fibroblast-HL cells interactions were reproduced in 3D co-culture spheroids. WS-1 COND showed a different cellular morphology with an enlarged cytoplasm and enhanced metabolism. Area and diameter cell values obtained by Celector® measurement were increased. Co-culture spheroids created with WS-1 COND showed a tighter aggregation than those with non-conditioned WS-1. The presence of soluble factors derived from HRS cells in the conditioned medium was adequate for the proliferation of fibroblasts and conditioned fibroblasts in a 3D HL model allowed to develop a representative model of the in vivo TME.

Primary pulmonary T-cell lymphoproliferative disorders with a limited-stage, low proliferative index, and unusual clinical behavior: two cases of a rare occurrence.

Extranodal T-lymphoproliferative disorders or T-cell lymphomas (TLPD) are classified according to the WHO Classification (4th and upcoming 5th editions) (Swerdlow et al., IARC Press 1; Alaggio et al., Leukemia 36(7):1720-1748, 2) and to the International Consensus Classification Update (Campo et al., Blood 140(11):1229-1253, 3) upon several morphologic, phenotypic, and genetic features. None of those at present included has been characterized by primary pulmonary onset. We herein present two such cases which, to the best of our knowledge, have not been previously reported and that might represent another variant of T-cell proliferation at mucosal sites. The two cases share similar histological and phenotypic features, suggesting an origin from CD4 + effector memory T cells with the expression of a CD279/PD-1 antigen. They are both monoclonal, harbor few mutations, and show no disease progression outside the lung. They only differ concerning the local extension of the process and clinical setting. The two cases are examples of so far unreported primary pulmonary TLDP, with limited stage and low proliferative index. A possible relationship with a local yet unknown inflammatory trigger that might have favored the development of the T-cell clone cannot be ruled out.

TOX Expression in Mycosis Fungoides and Sezary Syndrome.

Mycosis fungoides (MF) and Sezary syndrome (SS) are the two most common type of cutaneous T-cell lymphoma (CTCL). Currently, no markers can be clearly related to prognosis or to differential diagnosis between early stages and inflammatory benign diseases (IBD). The thymocyte selection-associated high mobility group box factor (TOX), has been proposed as a possible marker in differential diagnosis between early CTCL stages and IBD. Recently TOX has been related to prognosis. We aimed to investigate whether TOX may be a diagnostic or prognostic marker. MF and SS biopsies between 2010 and 2020 were retrieved. New tissues slides were stained with an anti-TOX antibody, (Clone NAN448B). On each slide, 5 fields were examined at high magnification (400×), to evaluate the percentage of marker-positivity in a quantitative way. Thirty-six patients (12 females and 24 males) and 48 biopsies were collected. Nine patients had multiple biopsies. TOX expression in MF/SS cases showed an increase from early to advanced phases. TOX was not regarded as a prognostic marker due to the absence of significant changes by comparing early MF cases with reactive conditions. TOX statistical significance increased in patients alive with disease and in those dead of disease (p = 0.013 and = 0.0005, respectively) as compared with patients in complete remission. Our results show that TOX should be regarded more as a prognostic than a diagnostic marker.

The role of peritumoral CD8 + /TIA1 + lymphocytes in hepatocellular carcinoma aggressiveness and recurrence after surgical resection.

Hepatocellular carcinoma (HCC) is characterized by a low mutation burden and a relatively low number of tumor-infiltrating lymphocytes (TILs), making still difficult to identify targets for specific therapies. The aim of this study was the identification of the prognostic role of TILs in HCC, focusing on their distribution and status of activation. We retrospectively enrolled 41 patients, undergone to liver resection for HCC. A significant increase of CD8 + intratumoral lymphocytes was observed in HCCs with prevalent solid architecture, but with a higher PD-1/TIA1 ratio, suggesting that HCCs with solid architecture have more peri-tumoral lymphocytes, but with minor functionality. At multivariate and univariate analyses, TIA1/CD8 ratio correlated with tumor recurrence, meaning that HCC with more activated TILs are characterized by a higher tumor aggressiveness. The use of a feasible and cheap immunohistochemical panel can help in post-surgical prognostic stratification, focusing not only in the raw number and density of TILs, but more on their state of activation and morphology.

Myeloid nuclear differentiation antigen: an aid in differentiating lymphoplasmacytic lymphoma and splenic marginal zone lymphoma in bone marrow biopsies at presentation.

The differential diagnosis between lymphoplasmacytic lymphoma (LPL) and marginal zone B-cell lymphoma, particularly splenic type (SMZL), can be challenging on onset of bone marrow biopsy (BMB) since morphology and phenotype are not specific and clinical features can overlap or be mildly developed at diagnosis. The LPL-specific L265P mutation in the MYD88 gene is not available in all laboratories, and genetic aberrancies identified in SMZL (del7q, mutations of NOTCH2 and KLF2) are seldom searched in routine practice. The study aim is to investigate the potential role of myeloid nuclear differentiation antigen (MNDA) expression in this specific differential diagnosis. We report MNDA reactivity in 559 patients with small B-cell lymphoma including bone marrow biopsies from 90 LPL and 91 SMZL cases. MYD88 p.Leu265Pro mutation status was assessed and confirmed as positive in 24 of 90 LPL cases, which served as the test set. MNDA staining was negative in 23 of 24 LPL cases in the test set (96%). In the 157 remaining cases (66 LPL, 91 SMZL), which served as the validation set, the MYD88 p.Leu265Pro mutation was unavailable and MNDA was more frequently expressed in SMZL (p < 0.00001). In addition, immunohistochemical features more consistent with SMZL (i.e., presence of CD23+ follicular dendritic cell meshworks, polytypic plasma cells, DBA44 reactivity) were more often present in MNDA-positive cases (statistically significant for 2 such parameters). On the widest case series so far published focusing on LPL and SMZL immunohistochemical diagnosis at onset of BMB, we demonstrated that MNDA expression significantly support the diagnosis of SMZL. This observation may be of particular help in cases where the MYD88 p.Leu265Pro mutational status and/or SMZL-related genetic aberrations are unavailable.

Three-dimensional models: a novel approach for lymphoma research.

PURPOSE: Three dimensional (3D) "in vitro" models are progressively being applied to investigate tumor cell biology and the interaction of cancer cells with tumor microenvironment under conditions more similar and realistic to "in vivo" behavior than standard bidimensional (2D) cultures. METHODS: In the last years, different methods have been developed to create spheroids and organoids and each technique has advantages and limitations also based on individual needs and cell types used. This review offers an overview of methodologies used for 3D systems: scaffold-free and scaffold-based methods up to bioreactors and organ-on-chip models. RESULTS: The principal goal for researchers is to select the 3D system that best suits their needs and that reflects the tumor model they want to study. A large chapter is dedicated to the application of these models to lymphomas' study, a neoplasm still little explored in the 3D field. CONCLUSION: These innovative and advanced models may represent new tools for cancer research and pre-clinical studies of new therapies in the perspective of precision medicine.

Prognostic impact of TP53 mutation in newly diagnosed diffuse large B-cell lymphoma patients treated in the FIL-DLCL04 trial.

The prognostic role of TP53 disruption has been established in diffuse large B-cell lymphoma (DLBCL). Aim of this analysis was to correlate TP53 mutations by Sanger sequencing, cell of origin (COO) profile by Lymph2Cx panel on the NanoString platform and MYC, BCL2 and BCL6 overexpression or re-arrangements by immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH), with outcome in DLBCL patients enrolled into the FIL-DLCL04 trial (NCT00499018). One hundred and twenty-five DLBCL patients with tumour block available were analyzed. TP53 was mutated in 11/125 (9%) cases; 60/125 patients received high-dose chemoimmunotherapy up-front, as for the randomization arm; COO was reported in 88 patients: 48 germinal centre B-cell like, 25 activated B-cell like and 17 unclassified; 26 patients were double expressors in IHC and 11 double hit in FISH. After a median follow-up of 72 months, five-year failure-free survival (FFS) for TP53 mutated versus wild-type was 24% and 72%, and five-year overall survival (OS) was 34% and 83%, respectively. Adjusted hazard ratio (HR) was 2·28 [95% confidence interval (CI) 0·89-5·86, p = 0·086] and 4·05 (95% CI 1·37-11·97, p = 0·011) for FFS and OS, respectively. In this series of young DLBCL patients, TP53 gene mutation identified a poor prognosis subgroup, regardless of treatment and other biological markers.

A three-gene signature based on MYC, BCL-2 and NFKBIA improves risk stratification in diffuse large B-cell lymphoma.

Recent randomized trials focused on gene expression-based determination of the cell of origin in diffuse large B-cell lymphoma could not show significant improvements by adding novel agents to standard chemoimmunotherapy. The aim of this study was the identification of a gene signature able to refine current prognostication algorithms and applicable to clinical practice. Here we used a targeted gene expression profiling panel combining the Lymph2Cx signature for cell of origin classification with additional targets including MYC, BCL-2 and NFKBIA, in 186 patients from 2 randomized trials (discovery cohort) (NCT00355199 and NCT00499018). Data were validated in 3 independent series (2 large public datasets and a real-life cohort). By integrating the cell of origin, MYC/BCL-2 double expressor status and NFKBIA expression, we defined a 3-gene signature combining MYC, BCL-2 and NFKBIA (MBN-signature), which outperformed the MYC/BCL-2 double expressor status in multivariate analysis, and allowed further risk stratification within the germinal center B-cell/unclassified subset. The high-risk (MBN Sig-high) subgroup identified the vast majority of double hit cases and a significant fraction of Activated B-Cell-derived diffuse large B-cell lymphomas. These results were validated in 3 independent series including a cohort from the REMoDL-B trial, where, in an exploratory ad hoc analysis, the addition of bortezomib in the MBN Sig-high subgroup provided a progression free survival advantage compared with standard chemoimmunotherapy. These data indicate that a simple 3-gene signature based on MYC, BCL-2 and NFKBIA could refine the prognostic stratification in diffuse large B-cell lymphoma, and might be the basis for future precision-therapy approaches.

Histology of the spleen in immune thrombocytopenia: clinical-pathological characterization and prognostic implications.

OBJECTIVE: Immune thrombocytopenia (ITP) is an acquired disorder, characterized by immune-mediated platelet destruction. The spleen plays a key pathogenic role in ITP and splenectomy is a valuable second-line therapy for this disease. Little is known on ITP spleen histology and response to splenectomy is unpredictable. This study aims to characterize ITP spleen histology and assess possible predictors of splenectomy outcome. METHODS: A series of 23 ITP spleens were retrospectively assessed for the following histological parameters: density of lymphoid follicles (LFs), marginal zones (MZs), T helper and cytotoxic T cells; presence of reactive germinal centers (GCs); width of perivascular T cell sheaths; and red pulp features. Clinical and histological data were matched with postsplenectomy platelet counts to assess their prognostic relevance. RESULTS: Three histological patterns were documented: a hyperplastic white pulp pattern, a non-activated white pulp pattern (lacking GCs), and a white pulp-depleted pattern. Poor surgical responses were associated with presplenectomy high-dose steroid administration, autoimmune comorbidities and low T follicular helper cell density. The combination of such parameters stratified patients into different splenectomy response groups. The removal of accessory spleens was also associated with better outcome. CONCLUSION: ITP spleens are histologically heterogeneous and clinical-pathological parameters may help predict the splenectomy outcome.

Whole exome sequencing reveals mutations in FAT1 tumor suppressor gene clinically impacting on peripheral T-cell lymphoma not otherwise specified.

Peripheral T-cell lymphoma not otherwise specified represents a diagnostic category comprising clinically, histologically, and molecularly heterogeneous neoplasms that are poorly understood. The genetic landscape of peripheral T-cell lymphoma not otherwise specified remains largely undefined, only a few sequencing studies having been conducted so far. In order to improve our understanding of the genetics of this neoplasm, we performed whole exome sequencing along with RNA-sequencing in a discovery set of 21 cases. According to whole exome sequencing results and mutations previously reported in other peripheral T-cell lymphomas, 137 genes were sequenced by a targeted deep approach in 71 tumor samples. In addition to epigenetic modifiers implicated in all subtypes of T-cell neoplasm (TET2, DNMT3A, KMT2D, KMT2C, SETD2), recurrent mutations of the FAT1 tumor suppressor gene were for the first time recorded in 39% of cases. Mutations of the tumor suppressor genes LATS1, STK3, ATM, TP53, and TP63 were also observed, although at a lower frequency. Patients with FAT1 mutations showed inferior overall survival compared to those with wild-type FAT1. Although peripheral T-cell lymphoma not otherwise specified remains a broad category also on molecular grounds, the present study highlights that FAT1 mutations occur in a significant proportion of cases, being provided with both pathogenetic and prognostic impact.

Correction: Whole exome sequencing reveals mutations in FAT1 tumor suppressor gene clinically impacting on peripheral T-cell lymphoma not otherwise specified.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel and Rare Fusion Transcripts Involving Transcription Factors and Tumor Suppressor Genes in Acute Myeloid Leukemia.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.

Interferon gamma inducible protein 16 (IFI16) expression is reduced in mantle cell lymphoma.

IFI16, member of the IFN-inducible PYHIN-200 gene family, modulates proliferation, survival and differentiation of different cell lineages. In particular, IFI16 expression, which is regulated during the differentiation of B cells, was recently studied in B-CLL as well. Here, we compared IFI16 expression in several lymphomas including Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma and mantle cell lymphoma with respect to normal cell counterparts. We observed that IFI16 expression was significantly deregulated only in mantle cell lymphoma (p < 0.05). Notably, IFI16 was associated with the expression of genes involved in interferon response, cell cycle, cell death and proliferation and, interestingly, lipid and glucose metabolism, suggesting that IFI16 deregulation might be associated with relevant changes in cell biology. In our group of mantle cell lymphoma samples a correlation between patient survival and IFI16 expression was not detected even though mantle cell lymphoma prognosis is known to be associated with cell proliferation. Altogether, these results suggest a complex relationship between IFI16 expression and MCL which needs to be analyzed in further studies.

Cortactin expression in non-Hodgkin B-cell lymphomas: a new marker for the differential diagnosis between chronic lymphocytic leukemia and mantle cell lymphoma.

Cortactin is a cytoskeletal-remodeling adaptor protein, playing an oncogenic role in solid tumors. Little is known on cortactin expression in non-Hodgkin B-cell lymphomas (B-NHLs). The present study aimed to characterize cortactin expression in B-NHLs and to assess its role in the differential diagnosis of such entities. Cortactin protein expression was first assessed by immunohistochemistry in a series of 131 B-NHLs, including B-cell chronic lymphocytic leukemia (CLL; n = 17), mantle cell lymphoma (MCL; n = 16), follicular lymphoma (FL; n = 25), marginal zone lymphoma (MZL; n = 30), hairy cell leukemia (HCL; n = 10), splenic diffuse red pulp small B-cell lymphomas (SDRPBL; n = 3), and diffuse large B-cell lymphoma (DLBCL; n = 30) cases. Cortactin was expressed in 14 of 17 CLLs, 10 of 10 HCLs, and 22 of 30 DLBCLs. MCLs, SDRPBLs, most FLs, and MZLs were cortactin negative. The immunohistochemical results were in keeping with in silico gene expression data. In CLL, cortactin positivity did correlate with LEF1 and CD200 expression, and the combined positivity for ≥2 markers strongly predicted CLL diagnosis. Such preliminary data suggested a role for cortactin in the differential diagnosis between CLL and MCL. This hypothesis was confirmed in a large validation set of 112 CLLs (n = 55) and MCLs (n = 57), which also disclosed rare cortactin-expressing MCLs. The immunohistochemical and gene expression results were sustained by flow cytometry and Western blot analysis on CLL and MCL cell lines. In conclusion, cortactin is mainly expressed in subsets of CLL and DLBCL and in HCL. Cortactin may represent a novel marker for the differential diagnosis between CLL and MCL.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia.

BACKGROUND: Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. METHODS: The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. RESULTS: We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. CONCLUSIONS: Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.