CP Case ConferenceAplastic Anemia

1/27/12Laura Walters

Clinical Presentation33 yo man with PMH of HTN and 1 month h/o headaches and 20 lb weight loss

3 day h/o worsening headache, dizziness, nausea, vomiting, anorexia, DOE

Laboratory DataMCV 85.2RDW 21.1Auto DiffNeut 0.5Lymph 1.3Mono 0.1Eos 0.0Baso 0.0BP 129/65, P 85, T 37.1, RR 16, 100% on RAHaptoglobin 55Tbili 1.0LDH 114

Lymphocytes73.0Neutrophils 18.0Metamyelocytes 0.0Myelocytes 0.0Promyelocytes 0.0Blasts 0.0Monocytes 9.0Eosinophils 0.0Basophils 0.0

Blasts 0.0Promyelocytes 1.0 LGran Precursors 21.5 LEryth Precursors 27.5 HLymphocytes45.5 HEosinophils 0.5 L Basophils 0.0Monocytes 0.5 Plasma cells 3.5 HM:E Ratio 0.8 L

Ancillary StudiesCytogenetics46, XYFlow CytometryProtocol: Acute Leukemia ProfileMarkers: CD2, CD3, CD5, CD7, CD10, CD11c, CD13, CD14, CD19, CD20, CD22, CD33, CD34, CD38, CD45, CD56, CD117Interpretation: No increased/aberrant blasts or acute leukemia.

Differential DiagnosisAplastic anemiaHypoplastic myelodysplastic syndromeParoxysmal nocturnal hemoglobinuriaHypocellular leukemia

Clinical HistoryAplastic anemia with normal genetics at OSH 10/2001.Treated with ATG and cyclosporine partial response. Cellcept added. Refuses bone marrow transplant.Multiple rounds of immunosuppression and weaning followed by relapse.

Hypoplastic MDSClonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis with bone marrow cellularity 10%)Increase in myeloid-lineage blasts

Hypocellular AML5-12% of all AML casesAML with bone marrow cellularity of

Paroxysmal Nocturnal HemoglobinuriaAcquired somatic mutation in PIG-A geneLoss of GPI-anchored cell membrane proteins (e.g. CD55, CD59)Hemolytic anemia, thrombosis, and/or bone marrow failurePeripheral blood and bone marrow findings variableFlow cytometry: CD55 and CD59 neg

Aplastic Anemia (AA)Pancytopenia due to marrow hypoplasiaTwo new cases per million people60 yrs

Biopsy findings in AAPeripheral bloodPancytopeniaGranulocytes and platelets morphologically unremarkableNo immature myeloid cellsErythrocytes macrocytic/normocyticReticulocytopeniaBone MarrowHypocellular Spicules consist of fatty tissueScant cellularity consists of lymphocytes, plasma cells, histiocytes, other stromal elementsHot pockets of hematopoiesisNo dysplasia

Ancillary studies in AAImmunohistochemistryCD34+ mononuclear cells very rare and scatteredFlow cytometryNormal phenotypeCytogeneticsNormal karyotype

AA PathophysiologyAutoimmune Inverted CD4/CD8Oligoclonal expansion of cytotoxic T cells Direct cell-mediated killing of stem cells or cytokine-transduced inhibition/apoptosisShort TelomeresSekeres et al. (2007) Clinical Malignant HematologyEnvironmental precipitant + Host genetic background + Immune response = AA

Follow-upViral w/u negative RBC & platelet transfusionsAntimicrobial prophylaxis Exjade for iron overloadATG & cyclosporineG-CSFDischarged after 12-day stay

*Mycophenolate - immunosuppressant*Diagnosis:bone marrow cellularity 10% of the cells within the same lineage Erythroids megaloblastic changes, irregularly-shaped nuclei or karyorrhexis of erythroblastsMegas micromegakaryocytes, hypolobated or binucleated megakaryocytesGranocytic pseudo-Pelger Huet anomaly, hypogranulation of granulocytes**PIG-A gene = X-linked gene whose protein product is required for the synthesis of GPD anchors

Complement-mediated lysis of RBCs and complement-mediated activation of platelets

One-third of PNH patients carry an AA diagnosis before developing symptoms of PNHPNH-AA overlap syndrome = PNH patients who meet the criteria for severe AA

1:100,000Median age 40 yrs

Hemolysis picture: polychromasia, increased retics, hypercellular bone marrow with erythroid predominance (may see dyspoietic changes only in the erythroid series)Bone marrow failure: macrocytic anemia, lack polychromasia, hypocellular bone marrow

*IPOX: Increased CD34+ blasts or clusters of CD34+ cells h-MDS

FLOW:Abnormal CD34+ populations h-MDSCD55- and CD59- population PNH

Clonal cytogenic abnormalities h-MDS

Not part of the diagnostic criteria

A cytogenic abnormality does not necessarily exclude the diagnosis of AA. Our techniques to identify a low level clone are becoming increasingly sensitive phenotypically (flow) and cytogenetically (FISH), which creates a problem with disease classification.

H-MDS and PNH not infrequently are diagnosed in patients that were previously diagnosed with AA.

Expansion of clones in repeated relapses and with high Neupogen use

**Immune mechanisms best evidence is its responsiveness to immunosuppression

Immune reaction to viral infection that, through molecular mimicry, lead to the breach of tolerance toward antigens residing on hematopoietic stem cellsCross-reactive antigens generated by chemical modification or conjugation with drugsNeoantigens caused by transcription of mutated fused genes

Inverted CD4/CD8 ratio, activated cytotoxic lymphocytes (HLA-DR and CD25 pos) and skewing of the variable beta-chain of the TCR -=-> c/w expansion of autoimmune T-cell clones Th1 polarizationDamage to stem cells = direct cell-mediated killing by CTL or cytokine-transduced inhibition

In support of this hypothesis, we also have found that hematopoietic progenitor cells of aplastic anemia patients with short telomeres display increased chromosomal abnormalities in vitro, such as chromosomal breakage, aneuploidy, and end-to-end fusions.26 These cells display chromosomal instability and are probably more likely to evolve to a clonal disorder. Additionally, other healthy hematopoietic stem and progenitor cells with short telomeres that effectively activate cell signaling via p53 and p21 undergo proliferation arrest and senescence. The result is the selection of abnormal, chromosomally unstable stem and progenitor cells in the bone marrow.

Nucleotide repeats at ends of chromosomesPrevent erosion of DNA during cell division

Telomeres short in 1/3 of aplastic anemia patients but mutations only in about 10% of cases

*Exjade iron chelator

Antilymphocyte globulins are immunosuppressive. ATGs contain a heterogeneous mix of antibody specificities for lymphocytes, including reactivity to antigens such as CD2, CD3, CD4, CD8, CD25 (the receptor for IL-2), and HLA-DR.[294296] ATGs fix human complement efficiently, and all preparations are cytotoxic to T cells in vitro. Lot-to-lot differences have been difficult to demonstrate in the laboratory.[295] Commercial rabbit ATG is more potent than is horse ATG on a weight basis, and a few studies in renal transplant recipients have suggested that it might also be more effective clinically.[297,][298] In vitro, antilymphocyte globulins efficiently inhibit T-cell proliferation and block IL-2 and IFN- production and IL-2 receptor expression[299]; ATG induced Fas-mediated apoptosis in T cells, especially after activation.[300] Monkey experiments have suggested that persisting specific antibodies are responsible for the induced chronic anergy and tolerance.[301] In patients, ATG results in rapid reduction in the number of circulating lymphocytes and lymphocytopenia persists for several days after discontinuing the last infusion. When lymphocyte numbers have returned to pretreatment values at 3 months, activated lymphocyte numbers are reduced.