Tailored Digital PCR Follow-Up of Rare Fusion Transcripts after Initial Detection through RNA Sequencing in Hematological Malignancies.

The management of hematologic malignancies has entered a new era in which minimal residual disease (MRD) monitoring plays a pivotal role. Well-established molecular targets, such as PML::RARA, CBFB::MYH11, or RUNX1::RUNX1T1, are conventionally tracked by quantitative RT-PCR. Recently, a broader landscape of fusion transcripts has been unveiled through transcriptomic analysis. These newly discovered fusion transcripts may emerge as novel molecular markers for MRD quantification. In this study, we compared a targeted RNA-sequencing (RNA-seq) approach (FusionPlex) with a whole-transcriptomic strategy (Advanta RNA-Seq XT) for fusion detection in a training set of 21 samples. We evidenced a concordance of 100% for the detection of known fusions, and showed a good correlation for gene expression quantification between the two techniques (Spearman r = 0.77). Additionally, we prospectively evaluated the identification of fusions by targeted RNA-seq in a real-life series of 126 patients with hematological malignancy. At least one fusion transcript was detected for 60 patients (48%). We designed tailored digital PCR assays for 11 rare fusions, and validated this technique for MRD quantification with a limit of detection of <0.01%. The combination of RNA-seq and tailored digital PCR may become a new standard for MRD evaluation in patients lacking conventional molecular targets.

A modern view of LGL leukemia.

Large granular lymphocytic (LGL) leukemia is a rare lymphoproliferative chronic disorder characterized by expansion of either T- or NK- cytotoxic cells. Contrary to EBV-induced aggressive NK-LGL leukemia, chronic T- and NK-LGL leukemia are indolent diseases affecting elderly patients with a median age of 66.5 years old. LGL leukemia is frequently associated with autoimmune disorders, most frequently rheumatoid arthritis. An auto/allo antigen is tentatively implicated in disease initiation. LGLs expansion is then triggered by proinflammatory cytokines such as interleukin (IL) IL-15, MIP-1, and RANTES. This proinflammatory environment contributes to deregulation of proliferative and apoptotic pathways. Following the initial description of the JAK-STAT pathway signaling activation in the majority of patients, recurrent STAT3 gain of function mutations have been reported. The JAK-STAT pathway plays a key role in LGL pathogenesis by promoting survival, proliferation and cytotoxicity. Several recent advances have been made towards understanding the molecular landscapes of T and NK LGL leukemia, identifying multiple recurrent mutations affecting the epigenome, such as TET2 or KMT2D, and crosstalk with the immune microenvironment, such as CCL22. Despite an indolent course, published series suggest that the majority of patients will eventually need treatment. However, it is noteworthy that many patients may have a long-term observation period without ever requiring therapy. Treatments rely upon immunosuppressive drugs, namely cyclophosphamide, methotrexate and cyclosporine. Recent advances have led to the development of targeted approaches, including JAK-STAT inhibitors, cytokine targeting and hypomethylating agents, opening new developments in a still-incurable disease.

[Incidence, characteristics and survival of patients with pneumocystis pneumonia in solid oncology]. / Incidence, caractéristiques et survie des patients présentant une pneumocystose en oncologie solide.

INTRODUCTION: Pulmonary pneumocystis causes interstitial lung disease, particularly in patients with solid cancers. The aim of this study is to clarify its incidence, which remains poorly understood, and to identify patients at risk and prognostic factors. METHODS: Data on patients with solid tumors and pulmonary pneumocystis were retrospectively collected from January 1, 2014 to December 31, 2019 in two hospitals in Rennes. Incidence was estimated via the Poisson model. Survival data were estimated using Kaplan-Meier method and Log-rank test. A multivariate Cox model was performed to identify risk factors for death. RESULTS: The incidences of pulmonary pneumocystis in metastatic cancer patients receiving parenteral systemic therapy are 198 and 349 cases per 100,000 patients per year in these two centers, respectively. Most patients were being treated with corticosteroids and chemotherapy at the time of pulmonary pneumocystis. The mortality rate for patients with pulmonary pneumocystis is 38%. Median overall survival was 2,7 months. Risk factors for death are corticotherapy greater than 20mg, prednisone equivalent, daily and chemotherapy. DISCUSSION: Pulmonary pneumocystis pneumonia is rare but not exceptional and has a poor prognosis in solid oncology. It frequently occurs in patients treated with long-term corticosteroids. Oncologists need to be better informed to discuss prophylaxis whenever corticosteroids are prescribed for several weeks.

Efficacy of ruxolitinib in the treatment of relapsed/refractory large granular lymphocytic leukaemia.

Large granular lymphocytic (LGL) leukaemia is a rare chronic lymphoproliferative disorder characterized by an expansion of cytotoxic T or NK cells. Despite a usually indolent evolution, most patients will require a treatment over the course of the disease because of cytopenia or symptomatic associated autoimmune disorders. First-line treatment is based on immunosuppressive agents, namely cyclophosphamide, methotrexate and ciclosporin. However, relapses are frequent, and there is no consensus on the management of relapsed/refractory patients. The implication of the JAK/STAT pathway in the pathogenesis of this disease has prompted our group to propose treatment with ruxolitinib. A series of 21 patients who received this regimen is reported here. Ten patients (47.6%) were refractory to the three main immunosuppressive drugs at the time of ruxolitinib initiation. Ruxolitinib yielded an overall response rate of 86% (n = 18/21), including 3 complete responses and 15 partial responses. With a median follow-up of 9 months, the median response duration was 4 months. One-year event-free survival and 1-year overall survival were 57% and 83% respectively. Mild side effects were observed. Biological parameters, notably neutropenia and anaemia, improved significantly, and complete molecular responses were evidenced. This study supports ruxolitinib as a valid option for the treatment of relapsed/refractory LGL leukaemia.

The complex relationship between large granular lymphocyte leukemia and rheumatic disease.

INTRODUCTION: Large granular lymphocytic (LGL) leukemia is a rare lymphoproliferative disorder characterized by an expansion of clonal T or NK lymphocytes. Neutropenia-related infections represent the main clinical manifestation. Even if the disease follows an indolent course, most patients will ultimately need treatment in their lifetime. Interestingly, LGL leukemia is characterized by a high frequency of autoimmune disorders with rheumatoid arthritis being the most frequent. AREAS COVERED: This review covers the pathophysiology, clinic-biological features and the advances made in the treatment of LGL leukemia. A special focus will be made on the similarities in the pathophysiology of LGL leukemia and the frequently associated rheumatic disorders. EXPERT OPINION: Recent advances in the phenotypic and molecular characterization of LGL clones have uncovered the key role of JAK-STAT signaling in the pathophysiology linking leukemic cells expansion and autoimmunity. The description of the molecular landscape of T- and NK-LGL leukemia and the improved understanding of the associated rheumatic disorders open the way to the development of new targeted therapies effective on both conditions.

Haematopoietic stem cell numbers are not solely determined by niche availability.

Haematopoietic stem cells (HSCs) reside in specialized microenvironments, also referred to as niches, and it has been widely believed that HSC numbers are determined by the niche size alone 1-5 . However, the vast excess of the number of niche cells over that of HSCs raises questions about this model. We initially established a mathematical model of niche availability and occupancy, which predicted that HSC numbers are restricted at both systemic and local levels. To address this question experimentally, we developed a femoral bone transplantation system, enabling us to increase the number of available HSC niches. We found that the addition of niches does not alter total HSC numbers in the body, regardless of whether the endogenous (host) niche is intact or defective, suggesting that HSC numbers are limited at the systemic level. Additionally, HSC numbers in transplanted wild-type femurs did not increase beyond physiological levels when HSCs were mobilized from defective endogenous niches to the periphery, indicating that HSC numbers are also constrained at the local level. Our study demonstrates that HSC numbers are not solely determined by niche availability, thereby rewriting the long-standing model for the regulation of HSC numbers.

Can we predict the influence of inflammation on voriconazole exposure? An overview.

Voriconazole is a triazole antifungal indicated for invasive fungal infections that exhibits a high degree of inter-individual and intra-individual pharmacokinetic variability. Voriconazole pharmacokinetics is non-linear, making dosage adjustments more difficult. Therapeutic drug monitoring is recommended by measurement of minimum plasma concentrations. Several factors are responsible for the high pharmacokinetic variability of voriconazole: age, feeding (which decreases absorption), liver function, genetic polymorphism of the CYP2C19 gene, drug interactions and inflammation. Invasive fungal infections are indeed very frequently associated with inflammation, which engenders a risk of voriconazole overexposure. Many studies have reviewed this topic in both the adult and paediatric populations, but few studies have focused on the specific point of the prediction, to evaluate the influence of inflammation on voriconazole pharmacokinetics. Predicting the impact of inflammation on voriconazole pharmacokinetics could help optimize antifungal therapy and improve patient management. This review summarizes the existing data on the influence of inflammation on voriconazole pharmacokinetics in adult populations. We also evaluate the role of C-reactive protein, the impact of inflammation on patient metabolic phenotypes, and the tools that can be used to predict the effect of inflammation on voriconazole pharmacokinetics.

Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation.

B-cell acute lymphoblastic leukemia (B-ALL) reflects the malignant counterpart of developing B cells in the bone marrow (BM). Despite tremendous progress in B-ALL treatment, the overall survival of adults at diagnosis and patients at all ages after relapse remains poor. Galectin-1 (GAL1) expressed by BM supportive niches delivers proliferation signals to normal pre-B cells through interaction with the pre-B cell receptor (pre-BCR). Here, we asked whether GAL1 gives non-cell autonomous signals to pre-BCR+ pre-B ALL, in addition to cell-autonomous signals linked to genetic alterations. In syngeneic and patient-derived xenograft (PDX) murine models, murine and human pre-B ALL development is influenced by GAL1 produced by BM niches through pre-BCR-dependent signals, similarly to normal pre-B cells. Furthermore, targeting pre-BCR signaling together with cell-autonomous oncogenic pathways in pre-B ALL PDX improved treatment response. Our results show that non-cell autonomous signals transmitted by BM niches represent promising targets to improve B-ALL patient survival.

Outcome of patients with newly diagnosed AML admitted to the ICU, including preemptive admission - a multi-center study.

Acute myeloid leukemia (AML) can lead to life-threatening complications that may require intensive care unit (ICU) management. It has been advocated that early preemptive (ePE) ICU admission, before the onset of organ failure, could benefit some high-risk patients such as those with hyperleukocytosis. The aim of this study was to retrospectively analyze the outcome of newly diagnosed AML patients who required ICU admission in five academic centers with a special focus on patients with an ePE admission strategy, i.e., those transferred to the ICU without any organ failure (modified SOFA score ≤ 2 [omitting thrombocytopenia] and no life-sustaining intervention in the first 24 h following ICU admission) before the start of induction therapy. Between January 2017 and December 2019, 428 patients were included among which 101 were admitted to the ICU. Among patients requiring life-sustaining interventions (n = 83), 18 (22%) died while in the ICU but ICU survivors had the same survival as those not admitted to the ICU. Patients with an ePE admission (n = 18) had more comorbidities and high-risk disease features such as hyperleukocytosis but required no life-sustaining interventions while in the ICU. In a subgroup analysis of patients with hyperleukocytosis ≥ 50 G/l at diagnosis (n = 85), patients not admitted to the ICU and those admitted with an ePE strategy had similar outcomes. This study provides encouraging results about ICU outcome in AML patients during induction therapy but the potential benefit of an ePE strategy must be confirmed prospectively.

Periosteal skeletal stem cells can migrate into the bone marrow and support hematopoiesis after injury.

Functional stromal cells are known to support bone marrow regeneration after chemotherapy or radiation-induced injury to prevent prolonged myelosuppression. However, it is not known how stromal cells within the bone marrow are regenerated after injury. We have utilized a whole bone transplantation model that mimics the initial bone marrow necrosis and fatty infiltration that is seen after bone marrow injury and subsequent recovery. We demonstrate that periosteal skeletal stem cells (P-SSCs) can migrate into the bone marrow and contribute to stromal regeneration and hematopoietic recovery. Once in the bone marrow, P-SSCs are phenotypically and functionally reprogrammed into bone marrow mesenchymal stem cells (BM-MSCs), expressing high levels of hematopoietic stem cell (HSC) niche factors, such as Cxcl12 and Kitl. Additionally, our results further indicate that P-SSCs are more resistant to acute stress than BM-MSCs. Here, we report a new function of P-SSCs, highlighting their major plasticity and the role of the periosteum as a potential source of BM-MSCs following acute bone marrow injury.

Gilteritinib activity in refractory or relapsed FLT3-mutated acute myeloid leukemia patients previously treated by intensive chemotherapy and midostaurin: a study from the French AML Intergroup ALFA/FILO.

The real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients. Among them, 140 received gilteritinib as single agent (cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (cohort C). The main differences in patient characteristics in this study compared to the ADMIRAL trial were ECOG ≥ 2 (83.6% vs. 16.6%), FLT3-TKD mutation (21.0% vs. 8.5%), primary induction failure (15.0% vs. 40.0%) and line of treatment (beyond 2nd in 37.1% vs. 0.0%). The rates of composite complete remission, excluding those that occurred after hematopoietic stem cell transplantation (HSCT), were similar at respectively 25.4% and 27.5% in cohorts B and C. Median overall survival (OS) for these two groups was also similar at respectively 6.4 and 7.8 months. Multivariate analyses for prognostic factors associated with OS identified female gender (HR 1.61), adverse cytogenetic risk (HR 2.52), and allogenic HSCT after gilteritinib (HR 0.13). Although these patients were more heavily pretreated, these real-world data reproduce the results of ADMIRAL and provide new insights into the course of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment who can benefit from treatment in an outpatient setting.

Comparison of scoring systems evaluating suitability for intensive chemotherapy in adults with acute myeloid leukemia-a Grand Ouest Against Leukemia (GOAL) study.

Several scoring systems have been developed to assess suitability of individual patients for intensive acute myeloid leukemia (AML) therapy. We sought to compare the performance of these scores in a cohort of 428 consecutive adults with AML who received conventional induction chemotherapy in five academic centers in France. All scoring systems identified a subset of patients with increased 28 and 56-day mortality although the prediction accuracy was overall limited with C-statistics of ranging from 0.61 to 0.71 Overall survival (OS) prediction was more limited and restricted to scoring systems that include AML-related parameters. The outcome of 104 patients (24%) considered unsuitable for intensive chemotherapy based on criteria used in recent randomized trials was similar to that of the other 324 patients (28-day mortality, odds ratio [OR] = 1.88, P = 0.2; 56-day mortality, OR = 1.71, P = 0.21; event-free survival, hazard ratio [HR] = 1.08, P = 0.6; OS, HR = 1.25, P = 0.14) with low discrimination (C-statistic: 0.57, 0.56, 0.50, and 0.52 for 28-day, 56-day mortality, EFS, and OS, respectively). Together, our findings indicate that the accuracy of currently available approaches to identify patients at increased risk of early mortality and shortened survival after intensive AML therapy is relatively limited. Caution regarding the use of available scoring systems should be warranted in clinical decision-making.

Toward a Better Classification System for NK-LGL Disorders.

Large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by a clonal expansion of T-lineage lymphocyte or natural killer (NK) cells in 85 and 15% of cases respectively. T and NK large granular leukemia share common pathophysiology, clinical and biological presentation. The disease is characterized by cytopenia and a frequent association with autoimmune manifestations. Despite an indolent course allowing a watch and wait attitude in the majority of patients at diagnosis, two third of the patient will eventually need a treatment during the course of the disease. Unlike T lymphocyte, NK cells do not express T cell receptor making the proof of clonality difficult. Indeed, the distinction between clonal and reactive NK-cell expansion observed in several situations such as autoimmune diseases and viral infections is challenging. Advances in our understanding of the pathogenesis with the recent identification of recurrent mutations provide new tools to prove the clonality. In this review, we will discuss the pathophysiology of NK large granular leukemia, the recent advances in the diagnosis and therapeutic strategies.

Leukemic Stem Cells: From Leukemic Niche Biology to Treatment Opportunities.

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults. While complete remission can be obtained with intensive chemotherapy in young and fit patients, relapse is frequent and prognosis remains poor. Leukemic cells are thought to arise from a pool of leukemic stem cells (LSCs) which sit at the top of the hierarchy. Since their discovery, more than 30 years ago, LSCs have been a topic of intense research and their identification paved the way for cancer stem cell research. LSCs are defined by their ability to self-renew, to engraft into recipient mice and to give rise to leukemia. Compared to healthy hematopoietic stem cells (HSCs), LSCs display specific mutations, epigenetic modifications, and a specific metabolic profile. LSCs are usually considered resistant to chemotherapy and are therefore the drivers of relapse. Similar to their HSC counterpart, LSCs reside in a highly specialized microenvironment referred to as the "niche". Bidirectional interactions between leukemic cells and the microenvironment favor leukemic progression at the expense of healthy hematopoiesis. Within the niche, LSCs are thought to be protected from genotoxic insults. Improvement in our understanding of LSC gene expression profile and phenotype has led to the development of prognosis signatures and the identification of potential therapeutic targets. In this review, we will discuss LSC biology in the context of their specific microenvironment and how a better understanding of LSC niche biology could pave the way for new therapies that target AML.

Impact of High-Dose Methotrexate on the Outcome of Patients with Diffuse Large B-Cell Lymphoma and Skeletal Involvement.

Diffuse large B-cell lymphoma (DLBCL) with extra nodal skeletal involvement is rare. It is currently unclear whether these lymphomas should be treated in the same manner as those without skeletal involvement. We retrospectively analyzed the impact of combining high-dose methotrexate (HD-MTX) with an anthracycline-based regimen and rituximab as first-line treatment in a cohort of 93 patients with DLBCL and skeletal involvement with long follow-up. Fifty patients (54%) received upfront HD-MTX for prophylaxis of CNS recurrence (high IPI score and/or epidural involvement) or because of skeletal involvement. After adjusting for age, ECOG, high LDH levels, and type of skeletal involvement, HD-MTX was associated with an improved PFS and OS (HR: 0.2, 95% CI: 0.1-0.3, p < 0.001 and HR: 0.1, 95% CI: 0.04-0.3, p < 0.001, respectively). Patients who received HD-MTX had significantly better 5-year PFS and OS (77% vs. 39%, p <0.001 and 83 vs. 58%, p < 0.001). Radiotherapy was associated with an improved 5-year PFS (74 vs. 48%, p = 0.02), whereas 5-year OS was not significantly different (79% vs. 66%, p = 0.09). A landmark analysis showed that autologous stem cell transplantation was not associated with improved PFS or OS. The combination of high-dose methotrexate and an anthracycline-based immunochemotherapy is associated with an improved outcome in patients with DLBCL and skeletal involvement and should be confirmed in prospective trials.