Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia

Several hemolytic markers are available to guide the differential diagnosis and to monitor treatment of hemolytic conditions. They include increased reticulocytes, an indicator of marrow compensatory response, elevated lactate dehydrogenase, a marker of intravascular hemolysis, reduced haptoglobin, and unconjugated hyperbilirubinemia. The direct antiglobulin test is the cornerstone of autoimmune forms, and blood smear examination is fundamental in the diagnosis of congenital membrane defects and thrombotic microangiopathies. Marked increase of lactate dehydrogenase and hemosiderinuria are typical of intravascular hemolysis, as observed in paroxysmal nocturnal hemoglobinuria, and hyperferritinemia is associated with chronic hemolysis. Prosthetic valve replacement and stenting are also associated with intravascular and chronic hemolysis. Compensatory reticulocytosis may be inadequate/absent in case of marrow involvement, iron/vitamin deficiency, infections, or autoimmune reaction against bone marrow-precursors. Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases and is a poor prognostic factor. Increased reticulocytes, lactate dehydrogenase, and bilirubin, as well as reduced haptoglobin, are observed in conditions other than hemolysis that may confound the clinical picture. Hemoglobin defines the clinical severity of hemolysis, and thrombocytopenia suggests a possible thrombotic microangiopathy or Evans' syndrome. A comprehensive clinical and laboratory evaluation is advisable for a correct diagnostic and therapeutic workup of the different hemolytic conditions

Autoimmune Cytopenias in Chronic Lymphocytic Leukemia: Focus on Molecular Aspects

Autoimmune cytopenias, particularly autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP), complicate up to 25% of chronic lymphocytic leukemia (CLL) cases. Their occurrence correlates with a more aggressive disease with unmutated VHIG status and unfavorable cytogenetics (17p and 11q deletions). CLL lymphocytes are thought to be responsible of a number of pathogenic mechanisms, including aberrant antigen presentation and cytokine production. Moreover, pathogenic B-cell lymphocytes may induce T-cell subsets imbalance that favors the emergence of autoreactive B-cells producing anti-red blood cells and anti-platelets autoantibodies. In the last 15 years, molecular insights into the pathogenesis of both primary and secondary AIHA/ITP has shown that autoreactive B-cells often display stereotyped B-cell receptor and that the autoantibodies themselves have restricted phenotypes. Moreover, a skewed T-cell repertoire and clonal T cells (mainly CD8+) may be present. In addition, an imbalance of T regulatory-/T helper 17-cells ratio has been involved in AIHA and ITP development, and correlates with various cytokine genes polymorphisms. Finally, altered miRNA and lnRNA profiles have been found in autoimmune cytopenias and seem to correlate with disease phase. Genomic studies are limited in these forms, except for recurrent mutations of KMT2D and CARD11 in cold agglutinin disease, which is considered a clonal B-cell lymphoproliferative disorder resulting in AIHA. In this manuscript, we review the most recent literature on AIHA and ITP secondary to CLL, focusing on available molecular evidences of pathogenic, clinical, and prognostic relevance

The Changing Landscape of Autoimmune Hemolytic Anemia

Autoimmune hemolytic anemia (AIHA) is a greatly heterogeneous disease due to autoantibodies directed against erythrocytes, with or without complement activation. The clinical picture ranges from mild/compensated to life-threatening anemia, depending on the antibody's thermal amplitude, isotype and ability to fix complement, as well as on bone marrow compensation. Since few years ago, steroids, immunesuppressants and splenectomy have been the mainstay of treatment. More recently, several target therapies are increasingly used in the clinical practice or are under development in clinical trials. This has led to the accumulation of refractory/relapsed cases that often represent a clinical challenge. Moreover, the availability of several drugs acting on the different pathophysiologic mechanisms of the disease pinpoints the need to harness therapy. In particular, it is advisable to define the best choice, sequence and/or combination of drugs during the different phases of the disease. In particular relapsed/refractory cases may resemble pre-myelodysplastic or bone marrow failure syndromes, suggesting a careful use of immunosuppressants, and vice versa advising bone marrow immunomodulating/stimulating agents. A peculiar setting is AIHA after autologous and allogeneic hematopoietic stem cell transplantation, which is increasingly reported. These cases are generally severe and refractory to standard therapy, and have high mortality. AIHAs may be primary/idiopathic or secondary to infections, autoimmune diseases, malignancies, particularly lymphoproliferative disorders, and drugs, further complicating their clinical picture and management. Regarding new drugs, the false positivity of the Coombs test (direct antiglobulin test, DAT) following daratumumab adds to the list of difficult diagnosis, together with the passenger lymphocyte syndrome after solid organ transplants. Diagnosis of DAT-negative AIHAs and evaluation of disease-related risk factors for relapse and mortality, notwithstanding improvement in diagnostic approach, are still an unmet need. Finally, AIHA is increasingly described following therapy of solid cancers with inhibitors of immune checkpoint molecules. On the whole, the double-edged sword of new pathogenetic insights and therapies has changed the landscape of AIHA, both providing enthusiastic knowledge and complicating the clinical management of this disease

Mesenchymal Stem Cells in Aplastic Anemia and Myelodysplastic Syndromes: The "Seed and Soil" Crosstalk

There is growing interest in the contribution of the marrow niche to the pathogenesis of bone marrow failure syndromes, i.e., aplastic anemia (AA) and myelodysplastic syndromes (MDSs). In particular, mesenchymal stem cells (MSCs) are multipotent cells that contribute to the organization and function of the hematopoietic niche through their repopulating and supporting abilities, as well as immunomodulatory properties. The latter are of great interest in MDSs and, particularly, AA, where an immune attack against hematopoietic stem cells is the key pathogenic player. We, therefore, conducted Medline research, including all available evidence from the last 10 years concerning the role of MSCs in these two diseases. The data presented show that MSCs display morphologic, functional, and genetic alterations in AA and MDSs and contribute to immune imbalance, ineffective hematopoiesis, and leukemic evolution. Importantly, adoptive MSC infusion from healthy donors can be exploited to heal the "sick" niche, with even better outcomes if cotransplanted with allogeneic hematopoietic stem cells. Finally, future studies on MSCs and the whole microenvironment will further elucidate AA and MDS pathogenesis and possibly improve treatment

Management of refractory autoimmune hemolytic anemia after allogeneic hematopoietic stem cell transplantation : current perspectives

Autoimmune hemolytic anemia (AIHA) is increasingly observed after allogeneic hematopoietic stem cell transplantation (allo-HSCT), with a reported incidence between 4% and 6%. The disease is generally severe and refractory to standard therapy, with high mortality, and there are neither defined therapies, nor prospective clinical trials addressing the best treatment. Most of the knowledge on the therapy of AIHAs derives from primary forms, which are highly heterogeneous as well, further complicating the management of post-allo-HSCT forms. The review addresses the risk factors associated with post-allo-AIHA, including unrelated donor, the development of chronic extensive graft-versus-host disease, CMV reactivation, nonmalignant diagnosis pre-HSCT, and alemtuzumab use in conditioning regimens. Regarding therapy, we describe standard treatments, such as corticosteroids, intravenous immunoglobulin, splenectomy, rituximab, cyclophosphamide, and plasma exchange, which have lower response rates than those reported in primary forms. New therapeutic options, including sirolimus, bortezomib, abatacept, daratumumab and complement inhibitors, are promising tools for this detrimental complication occurring after allo-HSCT

The Physiopathology of T- Cell Acute Lymphoblastic Leukemia: Focus on Molecular Aspects

T-cell acute lymphoblastic leukemia/lymphoma is an aggressive hematological neoplasm whose classification is still based on immunophenotypic findings. Frontline treatment encompass high intensity combination chemotherapy with good overall survival; however, relapsing/refractory patients have very limited options. In the last years, the understanding of molecular physiopathology of this disease, lead to the identification of a subset of patients with peculiar genetic profile, namely \u201cearly T-cell precursors\u201d lymphoblastic leukemia, characterized by dismal outcome and indication to frontline allogeneic bone marrow transplant. In general, the most common mutations occur in the NOTCH1/FBXW7 pathway (60% of adult patients), with a positive prognostic impact. Other pathogenic steps encompass transcriptional deregulation of oncogenes/oncosuppressors, cell cycle deregulation, kinase signaling (including IL7R-JAK-STAT pathway, PI3K/AKT/mTOR pathway, RAS/MAPK signaling pathway, ABL1 signaling pathway), epigenetic deregulation, ribosomal dysfunction, and altered expression of oncogenic miRNAs or long non-coding RNA. The insight in the genomic landscape of the disease paves the way to the use of novel targeted drugs that might improve the outcome, particularly in relapse/refractory patients. In this review, we analyse available literature on T-ALL pathogenesis, focusing on molecular aspects of clinical, prognostic, and therapeutic significance

Small Paroxysmal Nocturnal Hemoglobinuria Clones in Autoimmune Hemolytic Anemia: Clinical Implications and Different Cytokine Patterns in Positive and Negative Patients

Autoimmune hemolytic anemia (AIHA) is characterized by immune mediated erythrocytes destruction by autoantibodies with or without complement activation. Additional pathologic mechanisms include cellular cytotoxicity, cytokline dysregulation, and inadequate bone marrow compensation with fibrosis/dyserythropoiesis. The latter resembles that of bone marrow failures, namely aplastic anemia and myelodysplastic syndromes. Paroxysmal nocturnal hemoglobinuria (PNH) clones are increasingly recognized in bone marrow failure syndromes, and their selection and expansion are thought to be mediated by immune mechanisms. In this study, we aimed to evaluate the prevalence of PNH clones in 99 patients with primary AIHA, and their correlations with disease features and outcomes. Moreover, in the attempt to disclose the physiopathology of PNH positivity in AIHA, serum levels of several immunomodulatory cytokines were tested. A PNH clone was found in 37 AIHA patients (37,4%), with a median size of 0.2% on granulocytes (range 0.03\u201385). Two patients showed a large clone (16 and 85%) and were therefore considered as AIHA/PNH association and not included in further analysis. Compared to PNH negative, PNH positive cases displayed a higher hemolytic pattern with adequate bone marrow compensation. AIHA type, response to therapy, complications and outcome were comparable between the two groups. Regarding cytokine levels, IFN-\u3b3 and IL-17 were lower in PNH positive vs. PNH negative AIHAs (0.3 \ub1 0.2 vs. 1.33 \ub1 2.5; 0.15 \ub1 0.3 vs. 3,7 \ub1 9.1, respectively, p = 0.07 for both). In PNH positive AIHAs, IFN-\u3b3 positively correlated with reticulocytes (r = 0.52, p = 0.01) and with the bone marrow responsiveness index (r = 0.69, p = 0.002). Conversely, IL-6 and IL-10 showed the same pattern in PNH positive and PNH negative AIHAs. IL-6 levels and TGF-\u3b2 positively correlated with clone size (r = 0.35, p = 0.007, and r = 0.38, p = 0.05, respectively), as well as with LDH values (r = 0.69, p = 0.0003, and r = 0.34, p = 0.07, respectively). These data suggest testing PNH clones in AIHA since their prevalence is not negligible, and may correlate with a prominent hemolytic pattern, a higher thrombotic risk, and a different therapy indication. PNH testing is particularly advisable in complex cases with inadequate response to AIHA-specific therapy. Cytokine patterns of PNH positive and negative AIHAs may give hints about the pathogenesis of highly hemolytic AIHA

Mesenchymal Stem Cells in Myeloid Malignancies: A Focus on Immune Escaping and Therapeutic Implications

The importance of the bone marrow microenvironment forming the so-called niche in physiologic hemopoiesis is largely known, and recent evidences support the presence of stromal alterations from the molecular to the cytoarchitectural level in hematologic malignancies. Various alterations in cell adhesion, metabolism, cytokine signaling, autophagy, and methylation patterns of tumor-derived mesenchymal stem cells have been demonstrated, contributing to the genesis of a leukemic permissive niche. This niche allows both the ineffective haematopoiesis typical of myelodysplastic syndromes and the differentiation arrest, proliferation advantage, and clone selection which is the hallmark of acute myeloid leukemia. Furthermore, the immune system, both adaptive and innate, encompassing mesenchymal-derived cells, has been shown to take part to the leukemic niche. Here, we critically review the state of art about mesenchymal stem cell role in myelodysplastic syndromes and acute myeloid leukemia, focusing on immune escaping mechanisms as a target for available and future anticancer therapies

Multifactorial neutropenia in a patient with acute promyelocytic leukemia and associated large granular lymphocyte expansion: A case report

Neutropenia in the setting of acute hematological malignancies may impact disease prognosis, thus affecting therapy dose intensity. This is often due to chemotherapy\u2011induced aplasia as well as to the disease itself. However, chronic neutropenia deserves further investigation, as the management of reversible concomitant causes may avoid treatment delay. The present study describes a case of an acute promyelocytic leukemia patient with chronic severe neutropenia of multifactorial origin, including acute leukemia itself, chemotherapy, autoimmune activation with anti\u2011platelets and anti\u2011neutrophil antibodies positivity, and the rare association of large granular lymphocyte (LGL) expansion. As neutropenia may challenge the diagnosis and treatment of acute malignancies, clinicians and hematopathologists must discuss the differential diagnosis in order to avoid misdiagnosing and undertreating concomitant diseases. In particular, LGL chronic expansion and autoimmunity should be considered