108 CHAPTER 2 Red cell disorders Table 2.16 Clinical features of Fe overload (homozygous haemochromatosis) Skin pigmentation \200 Hepatic dysfunction Slate gray or bronze discolouration \200 Hepatomegaly, chronic hepatitis, fi brosis, \200 cirrhosis, hepatocellular carcinoma (20 \200\22330%) \200 Retinopathy, nephropathy, neuropathy, vascular Diabetes mellitus complications Gonadal dysfunction \200 Other endocrine dysfunction \200 Abdominal pain \200 Cardiac dysfunction Hypogonadism, impotence \200 Hypothyroidism, hypoparathyroidism, adrenal insuffi ciency Unknown etiology (25%) \200 Cardiomyopathy, heart failure, dysrhythmias (10 \200\22315%) \200 Arthropathy Chondrocalcinosis The % transferrin saturation provides confi rmatory evidence but no measure of the extent o f Fe overload. Useful screening test. If >60% in \231\202 or 45 \200\22350% in \231\200 r epeat and investigate further. MRI useful tool for assessment of Fe overload in heart (T2*) and liver (T2*/R2). Useful for monitoring response to treatment (e.g., venesection). Liver biopsy provides a direct albeit invasive measure of Fe stores (% Fe concentration by weight) and visual assessment of Fe distribution, and the extent of tissue damage. Only ind icated if histological investigation also planned, e.g., staging of cirrhosis. MRI T2* useful tool for assessment of Fe overload in heart, liver and other sites. SQUID (superconducting quantum interference device) uses high- power magnetic fi eld and de tectors that measure the interference of Fe within the fi eld. Linear correlations have bee n demonstrated between SQUID measurements and liver biopsy Fe levels but restricted availab ility. Diagnosis May be diffi cult to diff erentiate haemochromatosis from Fe overload 2 to othe r causes, particularly that associated with chronic liver disease. Recent identifi cation o f HFE gene will provide a tool for more defi nitive diagnosis and screening of relatives (p reviously performed by serum fer- ritin estimation). Management \200 Aim to reduce ferritin to <50mcg/L and prevent complications of overload. Achieved by regular venesection (500mL blood = 200 \200\223250mg Fe) on weekly basis until Fe defi ciency develops (may take many months). 109 IRON OVERLOAD Hb should be measured prior to each venesection and response to therapy can be monitored by intermittent measurement of the serum ferritin. R2 liver (some individuals also require T2* heart) 6 \200\22312-monthly. \200 Once Fe def i ciency develops a maintenance regimen can be commenced with venesection every 3 \200\2234 months. Avoid alcohol, exogenous Fe. \200 Liver cirrhosis and hepatoma risk are not reversible. Natural history Cirrhosis and hepatocellular carcinoma are the most common causes of death in patients with haemochromatosis and are due to hepatic Fe accumulation. Cirrhosis does no t usually develop until the hepatic Fe concentration reaches 4000 \200\2235000mcg/g of live r (normal 50 \200\223500mcg/ g). Hepatocellular carcinoma is the cause of death in 20 \200 \22330% but does not occur in the absence of cirrhosis which increases the risk over 200 \227. If venesection can be commenced prior to the development of cir- rhosis and other com plications of haemosiderosis the life expectancy is that of a normal individual. Reduction of Fe overload by venesection has only a small eff ect on symptomatology which has already developed: skin pigmentation diminishes, liver function may improve, cardiac abnor- malitie s may resolve, diabetes and other endocrine abnormalities may improve slightly, arthropathy is unaff ected. Further reading Beutler, E. et al. (2002). Penetrance of 845G >A (C282Y) HFE hereditary ha emochromatosis mutation in the USA. Lancet, 359, 211 \200\22318. Bulaj, Z.J. et al. (2000). Disease-related conditions in relatives of patients with hemochromatosis. N Engl J Med, 34 3, 1529 \200\22335. Olynyk, J.K. et al. (1999). A population-based study of the clinical ex pression of the hemochromatosis gene. N Engl J Med, 341, 718 \200\22324. Sanchez, A.M. et a l. (2001). Prevalence, donation practices, and risk assessment of blood donors with hemochr omatosis. JAMA, 286, 1475 \200\22381. 110 CHAPTER 2 Red cell disorders Transfusion haemosiderosis Fe overload occurs in patients with transfusion dependent anemia , notably thalassaemia major, Diamond \200\223Blackfan syndrome, aplastic anemia, and acqui red refractory anemia. Also increasingly seen in transfused sickle patients. In many of the se conditions Fe overload is aggravated by physiological mechanisms which promote i dietary absorption of Fe in response to ineff ective erythropoiesis. Each unit of blood contains 2 00 \200\223250mg Fe and average transfusion dependent adult receives 6 \200\22310g of Fe/ye ar. Distribution of Fe is similar to haemochro- matosis with primarily liver parenchymal ce ll accumulation followed by pancreas, heart, and other organs. Cardiac deposition occurs in patients who have received 100 units of blood (20g Fe) without chelation, and is followed by damage to the liver, pancreas, and endocrine glands. Pattern of distribution also determ ined by genetic factors. Clinical features of Fe overload in children who require transfusion support for hereditary anemia are listed. Similar problems excluding those related to growth and sexual maturatio n develop in patients who commence a transfusion program for acquired refractory anemia in later life. Features of transfusion haemosiderosis in hereditary anemia \200 Growth retardation in 2n d decade. \200 Hypogonadism \200\224delayed or absent sexual maturation. \200 Skin pigm entation \200\224slate gray or bronze discolouration. \200 Hepatic dysfunction \200\224he patomegaly, chronic hepatitis, fi brosis, cirrhosis, hepatocellular carcinoma. Diabetes mellitus. \200 Other endocrine dysfunction \200\224rarely hypothyroidism, hypoparathyroidism, adrenal insuffi ciency. Cardiac dysfunction \200\224cardiomyopathy, heart failure, dysrhythmias (main cause of death). Death from heart disease in adolescence. Management \200 Fe chelation therapy using currently available chelation agents (desferrioxamine, deferiprone and deferasirox) is the mainstay of treatment for patients wi th transfusion haemosiderosis, who remain anemic. Haemosiderosis due to previous transfusio ns in conditions where Hb now normal, e.g. treated AML, may be venesected to remove Fe. Regular treatment is required in transfusion dependent children if they are to avoid the consequences of Fe overload in the 2nd decade of life. SC administration of desferrioxamine by portable syringe pump over 9 \200\22312h on 5 \200\2237 nights/week is a common regimen. Ascorbic acid supplementation is given at the same time to aid excretion. 111 TRANSFUSION HAEMOSIDEROSIS Deferiprone (Ferriprox) is orally active but needs to be taken 3 times a day. Can also be g iven as combination therapy with desferrioxamine. Contraindicated for use in patients with DBA. Deferasirox (Exjade) is the newest licensed agent. Given as a suspension once daily. Avoid in renal impairment. Chelation therapy is started after a year of regular transfusions or when ferritin > 1000mc g/L. d hepatic Fe and improves hepatic function, promotes growth and sexual development, an d protects against heart disease and early death. Newer chelation therapies are under investigation. Natural history The prognosis of the underlying hematological condition in transfusion- dep endent elderly patients may eliminate the need for Fe chelation. In others with a longer li fe expectancy, a period of venesections (if good Hb) or iron chelation therapy should be co nsidered. Other causes of haemosiderosis Dietary Fe overload may also occur as a result of chronic ov er-ingestion of Fe-containing traditional home-brewed fermented maize beverages peculiar to sub-Saharan Africa, which overwhelms physiological controls on Fe absorption. Fe stores ma y >50g and Fe is initially deposited in both hepatocytes and Kupff er cells but when cirrho sis develops, accumulates in the pancreas, heart, and other organs. Over-ingestion of medic inal Fe may possibly have a similar though less dramatic eff ect but is certainly harmful t o patients with Fe-loading disorders. The excessive Fe absorp- tion seen in patients with c hronic liver disease is associated with accumu- lation in Kupff er cells rather than hepati c parenchyma. Rare congenital defects associated with Fe overload have been reported. This page intentionally left blank 113 Chapter 3 White blood cell abnormalities Neutrophilia 114 Neutropenia 116 Lymphocytosis and lymphopenia 118 Eosinophilia 119 Basophi lia and basopenia 120 Monocytosis and monocytopenia 121 Mononucleosis syndromes 122 114 CHAPTER 3 White blood cell abnormalities Neutrophilia Neutrophils are derived from same precursor as monocytes. Cytoplasm contains g ranules; the nucleus has 3 \200\2234 segments. Functions include chemotaxis \200\224neutrop hils migrate to sites of infl ammation by chemot- actic factors, e.g. complement components (C5a and C3), and cytokines. Cytotoxic activity is via phagocytosis and destruction of par ticles/ invading microorganisms (latter often antibody coated = opsonized). Granules contai n cationic proteins l lyse Gram \210\222ve bacteria, \200\230defensins \200\231, myeloper oxidase \200\224interacts with H2O2 and HCl l hypochlorous acid \210\222) and (HOCl); lyso zyme (hydrolyses bacterial cell walls); superoxide (O2 hydroxyl (OH \210\222) radicals. Neu trophil lifespan is 71 \200\2232d in tissues. Normal neutrophil count 2.0 \200\2237.5 \227 109/L (neonate diff ers from adult; E Normal ranges, p.809). Neutrophilia is defi ned as a n absolute neutrophil count >7.5 \227 109/L. Mechanisms \200 i production. \200 Accelerated/early release from marrowlblood. \200 Demargination (marginal poollcirculating pool). Causes \200 Infection (bacterial, viral, fungal, spirochaetal, rickettsial). \200 Infl ammation (trauma, infarction, vasculitis, rheumatoid disease, burns). Chemicals, e.g. drugs, hormones, toxins, haemopoietic growth factors, e.g. G-CSF, GM-CSF, adrenaline, corticosteroids, venoms. \200 Physical agents, e .g. cold, heat, burns, labor, surgery, anesthesia. \200 Hematological, e.g. myeloprolifer ative disease, CML, PPP (1 proliferative polycythaemia), myelofi brosis, chronic neutrophilic leukemia. Other malignancies. \200 Cigarette smoking. \200 Post-splenectomy. \200 Chronic bleed ing. \200 Idiopathic. Investigation History and examination. Ask about cigarette smoking, symptoms sug- gesting o ccult malignancy. Other investigations \200 ESR. \200 CRP. Treatment Usually treatment of underlying disorder is all that is required. 115 NEUTROPHILIA Leukaemoid reaction May resemble leukemia (hence name); see i WBC (myeloblasts and promyelo cytes prominent). Occurs in severe and/or chronic infection, metastatic malignancy. 116 CHAPTER 3 White blood cell abnormalities Neutropenia Defi ned as absolute peripheral blood neutrophil count of <2.0 \227 109/L. Rac ial variation: black and Middle Eastern people may have neutrophil count of <1.5 \227 109/ L normally. Congenital neutropenia syndromes \200 Kostmann syndrome \200\224E Pediatric hematology, p .597. \200 Chediak \200\223Higashi \200\224E Pediatric hematology, p.603. \200 Shwachma n \200\223Diamond syndrome \200\224E Pediatric hematology, p.597. \200 Cyclical neutropen ia \200\2243 \200\2234-week periodicity; often 21d cycle, lasts 3 \200\2236d. Miscellaneous \200\224transcobalamin II defi ciency, reticular dysgenesis, dyskeratosis congenita. Acquired neutropenia Table 3.1 Acquired neutropenia: commonest causes Infection Viral, e.g. infl uenza, HIV, hepatitis, overwhelming bacterial sepsis Drugs Anticonvulsants (e.g. phenytoin) Antithyroid (e.g. carbimazole) Phenothiazines (e.g. chlorpromazine) Antiinfl ammatory agents (e.g. phenylbutazone) Antibacterial agents (e.g. co-trimoxazole) Others (gold, penicillamine, tolbutamide, mianserin, imipramine, cytotoxics) Immune mediated Autoimmune (antineutrophil antibodies) SLE Felty \200\231s syndrome (rheumatoid arthritis + neutropenia + splenomegaly; no correlation between spleen size and degree of neutropenia) As part of pancytopenia Bone marrow failure Leukemia, lymphoma, LGLL, haematinic defi ciency, anorexia Splenomegaly Any cause Clinical features When severe neutropenia: throat/mouth infection, oral ulceration, sep- ti caemia. (See Table 3.1 for commonest causes.) Diagnosis Examine peripheral blood fi lm, check haematinics, autoimmune profi le, antineutr ophil antibodies, haematinics, bone marrow aspirate and tre- phine biopsy if indicated (e.g . severe or prolonged neutropenia, or fea- tures suggestive of infi ltration of marrow fail ure syndrome). 117 NEUTROPENIA Treatment Consists of prompt antibiotic therapy if infection, IVIg and corticoster- oids ma y be helpful but eff ects unpredictable. In seriously ill patients consider use of G-CSF (n eed to exclude underlying leukemia before starting therapy with growth factors). Consider p rophylaxis with low- dose antibiotics (e.g. ciprofl oxacin 250mg bd) and antifungal (e.g. f l ucona- zole 100mg od) agents. Drug-induced neutropenia usually recovers on stopping suspe cted agent (may take 1 \200\2232 weeks). Further reading Bux, J. et al. (1998). Diagnosis and clinical course of autoimmune neutrope nia in infancy: analysis of 240 cases. Blood, 91, 181 \200\2236. Dale, D.C. et al. (2002). Cyclic neutropenia. Semin Hematol, 39, 89 \200\22394. 118 CHAPTER 3 White blood cell abnormalities Lymphocytosis and lymphopenia Lymphocytes are small cells with a high N:C ratio; some (e.g. natural killer cells) have prominent cytoplasmic granules. Two principal types: B and T ly mphocyte. B-cells express monoclonal surface (not cytoplasmic) IgM and often IgD. B-cell st imulation through cross linkage of surface Ig molecules or via eff ector T cells causes the ir diff erentiation into plasma cells. Predominant role is humoral immunity via Ig secretio n. T cells are derived from stem cells that undergo maturation in thymus and express the T-cel l receptor molecule (CD3) on the cell surface. Responsible for cell-mediated immunity, e.g. delayed hypersensitivity, graft rejection, contact allergy, and cytotoxic reactions agains t other cells. Lymphocytosis (peripheral blood lymphocytes >4.5 x 109/L) \200 Leukaemias and lymphomas i ncluding: CLL, NHL, Hodgkin disease, acute lymphoblastic leukemia, hairy cell leukemia, Wal denstr m macroglobulinaemia, heavy chain disease, mycosis fungoides, S zary syndrome, lar ge granular lymphocyte leukemia, ATLL. Infections, e.g. EBV, CMV, Toxoplasma gondii, rickettsial infection, Bordetella pertussis, mumps, varicella, coxsackievirus, rubella, hepatitis virus, adenoviru s. \200\230Stress \200\231, e.g. myocardial infarction, sickle crisis. \200 Trauma. \200 Rheumatoid disease (occasionally). \200 Adrenaline. \200 Vigorous exercise. \200 Post -splenectomy. \200 thalassaemia intermedia. Lymphopenia (peripheral blood lymphocytes <1.5 x 109/L) \200 Malignant disease, e.g. Hodg kin disease, some NHL, non- haematopoietic cancers, angioimmunoblastic lymphadenopathy. MDS. \200 Collagen vascular disease, e.g. rheumatoid, SLE, GvHD. \200 Infections, e.g. HIV. \200 Chemotherapy. \200 Surgery. \200 Burns. \200 Liver failure. \200 Renal failure (acute and chronic). \200 Anorexia nervosa. \200 Fe defi ciency (uncommon). \200 Aplastic anemia. \200 Cushing \200\231s disease. \200 Sarcoidosis. \200 Congeni tal disorders (rare) such as SCID, reticular dysgenesis, agammaglobulinaemia (Swiss type), thymic aplasia (DiGeorge \200\231s syndrome), ataxia telangiectasia. 119 EOSINOPHILIA Eosinophilia Diff erential diagnosis Common \200 Drugs (huge list, e.g. gold, sulfonamides, penicillin ); erythema multiforme (Stevens \200\223Johnson syndrome). Parasitic infections: hookworm, Ascaris, tapeworms, fi lariasis, amoebiasis, schistosomiasis. Allergic syndromes \200\224asthma, eczema, urticaria. Less common \200 Pemphigus. \200 Dermatitis herpetiformis (DH). \200 Polyarteritis no dosa (PAN). \200 Sarcoid. \200 Tumors esp. Hodgkin. \200 Irradiation. Rare \200 Hypereosinophilic (Loeffl er \200\231s) syndrome. \200 Eosinophilic leukemia. \200 AML with eosinophilia esp. M4Eo (E see Table 4.1, p.127). Discriminating clinical features \200 Drugs \200\224history of exposure, time course of e osinophilia with resolution on cessation of drug. Allergic conditions \200\224history of eczema, urticaria, or typical rashes. Symptoms and signs of asthma. Parasites \200\224history of exposure from foreign travel, symptoms and signs of Fe defi ci ency anemia (hookworm is commonest cause worldwide). Blood fi lm may show fi lariasis. Stoo l microscopy and culture for ova, cysts, and parasites for amoebiasis, Ascaris, Taenia, sch istosomiasis. Skin diseases \200\224typical appearances confi rmed by biopsy, e.g. dermatitis herpetiformis and pemphigus. PAN \200\224renal failure, neuropathy, angiography and ANCA positivity. \200 Sarcoid \200 \224multisystem features with non-caseating granulomata in biopsy of aff ected tissue or on BM biopsy; high serum ACE. Hodgkin \200\224lymphadenopathy, hepatosplenomegaly \200\224BM or node biopsy. Hypereosinophilic syndrome \200\224history of allergy, cough, fever, and pulmonary infi ltrates on CXR, may be cardiac involvement. Eosinophils on blood fi lm have normal morphology and granulation. Diagnosis on exclusion of similar causes. Eosinophilic leukemia \200\224eosinophils on blood fi lm have abnormal morphology with hyperlobular and hypergranular forms. BM heavily infi ltrated with same abn ormal cells. Other signs of myeloproliferative disease may be present. AML M4Eo \200\224blasts with myelomonoblastic features on BM and blood fi lm (E see Table 4.1, p.127). 120 CHAPTER 3 White blood cell abnormalities Basophilia and basopenia Basophils are found in peripheral blood and marrow ( \211 mast ce lls in tissues). Short lifespan (1 \200\2232d), cannot replicate. Degranulation results in hypersensitivity reactions (IgE Fc receptors trigger), fl ushing, etc. Basophilia (peripheral blood basophils >0.1 x 109/L) \200 Myeloproliferative disorders: CGL. \200 Other chronic myeloid leukaemias. \200 PRV. \200 Myelofi brosis. \200 Ess ential thrombocythaemia. \200 Basophilic leukemia. AML (rare). \200 Hypothyroidism. \200 IgE-mediated hypersensitivity reactions. \200 I nfl ammatory disorders, e.g. rheumatoid disease, ulcerative colitis. \200 Drugs, e.g. oes trogens. \200 Infection, e.g. viral. \200 Irradiation. \200 Hyperlipidaemia. Basopenia (peripheral blood basophils <0.1 x 109/L) \200 As part of generalized leucocyto sis, e.g. infection, infl ammation. \200 Thyrotoxicosis. \200 Hemorrhage. \200 Cushin g \200\231s syndrome. \200 Allergic reaction. \200 Drugs, e.g. progesterone. 121 MONOCYTOSIS AND MONOCYTOPENIA Monocytosis and monocytopenia Bone marrow monocytes give rise to blood monocytes and tissue mac- rophages. Part of RES. Other components of RES: lung alveolar mac- rophages; pleural and peritoneal macrophages; Kupff er cells in liver; histiocytes; renal mesangial cells; ma crophages in lymph node, spleen and marrow. Contain 2 sets of granules: (1) lysosomal (acid phosphatase, arylsul- phatase and peroxidas e), and (2) function of second set unknown. Monocytosis (peripheral blood monocytes >0.8 x 109/L) Common \200 Malaria, trypanosomiasi s, typhoid (commonest worldwide causes). \200 Post-chemotherapy or stem cell transplant e sp. if GM-CSF used. \200 Tuberculosis. \200 Myelodysplasia (MDS). Less common \200 Infective endocarditis. \200 Brucellosis. \200 Hodgkin lymphoma. \200 AML (M4 or M5). Discriminating clinical features \200 Malaria \200\224identifi cation of parasites on thi ck and thin blood fi lms. \200 Trypanosomiasis \200\224parasites seen on blood fi lm, lym ph node biopsy, or blood cultures. Typhoid \200\224blood culture, fecal and urine culture, and BM culture. \200 Infective en docarditis \200\224cardiac signs and blood cultures. \200 Tuberculosis \200\224AFB seen a nd cultured in sputum, EMU, blood, or BM, tuberculin positivity on intradermal challenge, c aseating granulomata on biopsy of aff ected tissue or BM. Brucellosis \200\224blood cultures and serology. \200 Hodgkin \200\224lymphadenopathy, he patosplenomegaly, eosinophilia, biopsy of node or BM. MDS \200\224typical dysplastic features on blood fi lm or BM (see E Myelodysplastic syndromes, p.228). AML (M4 or M5) \200\224monoblasts on blood fi lm and BM biopsy. Skin and gum infi ltration common (see E Acute myeloblastic leukemia, p.124). Monocytopenia (peripheral blood monocytes <0.2 x 109/L) \200 Autoimmune disorders, e.g. S LE. \200 Hairy cell leukemia. \200 Drugs, e.g. glucocorticoids, chemotherapy. 122 CHAPTER 3 White blood cell abnormalities Mononucleosis syndromes De \201 nition Constitutional illness associated with atypical lymphocytes in the blood. Clinical features Peak incidence in adolescence: may be subclinical or acute presenta- tion consisting of fever, lethargy, sweats, anorexia, pharyngitis, lym- phadenopathy (cervical > axillary > inguinal), tender splenomegaly hepatomegaly, palatal petechiae, maculopapul ar rash especially if given ampicillin. Rarely also pericarditis, myocarditis, encephalitis . Usually self- limiting illness but complications include lethargy persisting for months o r years (chronic fatigue syndrome), depression, autoimmune haemo- lytic anemia, thrombocyto penia, 2 infection, and splenic rupture. Causes EBV, CMV, Toxoplasma, Brucella, Coxsackie and adenoviruses, HIV sero- conversion ill ness. Pathophysiology In EBV-related illness, EBV infection of B lymphocytes results in immortal- ization and generates a T-cell response (the atypical lymphocytes) which controls EBV prol iferation. In severe immunodefi ciency following prolonged use of ciclosporin, oligoclonal EBV-related lymphoma may develop which usually regresses with reduction of immunosuppressiv e therapy but may evolve to a monoclonal and aggressive lymphoma, e.g. after MUD stem cell transplant. In malarial Africa, EBV infection is associ- ated with an aggressive lymphoma \200\224Burkitt lymphoma (E Burkitt lym- phoma, p.200). Diagnosis: hematological features \200 Atypical lymphocytes on blood fi lm (recognized by the dark blue cytoplasmic edge to cells and invagination (scalloping) around RBCs). Usually lymphocytosis with mild neutropenia. \200 Occasionally anemia due to cold antibod y mediated haemolysis (anti-i) \200\224identify with cold haemagglutinin titre. Paul \200\223Bunnell/monospot test for presence of heterophile antibody +ve when cause is EBV but only in the fi rst few weeks. False +ves can occur in lymphoma. i bilirubin and abnormal LFTs. \200 Serological testing should include EBV capsid Ag, CMV IgM, Toxoplasma titre, Brucella titre, HIV-1 and -2 Ag and Ab. Immunophenotype of peripheral blood B lymphocytes shows polyclonality (distinguishes from lymphoma and other lymphoproliferative disorders). Treatment Rest and symptom relief are mandatory. No other specifi c treatment has been show n to infl uence outcome. 123 Chapter 4 Leukemia Acute myeloblastic leukemia (AML) 124 Acute lymphoblastic leukemia (ALL) 138 Chronic myeloi d leukemia (CML) 148 Chronic lymphocytic leukemia (B-CLL) 158 Prolymphocytic leukemia (PLL) 168 Hairy cell leukemia and variant 170 Large granular lymphocyte leukemia (LGLL) 174 Adul t T-cell leukemia-lymphoma (ATLL) 178 124 CHAPTER 4 Leukemia Acute myeloblastic leukemia (AML) Malignant tumor of haemopoietic precursor cells of non-ly mphoid lineage arising in the BM. Incidence Most common acute leukemia in adults; 3 per 100,000 annually. i fre- quency with age (median 64 years; incidence 35/100,000 at age 90). Infrequent in children <15 years. 66 % are >60 years of age. Etiology Unclear \200\224association with pre-existing myelodysplasia, prior cytotoxic chem otherapy (particularly alkylating agents and epipodophyllotoxins), ionizing radiation, benz ene exposure, constitutional chromosomal abnormalities (e.g. Down (older patients) and Fanc oni syndromes) and smoking. Diagnosis \200 FBC \200\224usually shows leucocytosis, anemia, and thrombocytopenia. Can show pancytopenia Blood fi lm \200\224usually contains blasts. \200 BM aspirate \200\224 \211 20% blasts (s ee Figs. 4.1 \200\2234.5). \200 Trephine biopsy \200\224to exclude fi brosis and multilin eage dysplasia. \200 Immunophenotyping to diff erentiate AML from ALL: CD3, CD7, CD13, CD14, CD33, CD34, CD64, CD117, cytoplasmic myeloperoxidase (MPO). Cytochemistry \200\224MPO or Sudan Black (SB), combined esterase. \200 Cytogenetic analys is \200\224to identify prognostic group. \200 Molecular analysis \200\224RT-PCR and FISH in selected cases. Cytochemistry Formerly the mainstay of leukemia diagnosis \200\224SB, MPO, and esterase (ch loroacetate and non-specifi c esterase (NSE)) stains are +ve in AML and \210\222ve in ALL (<3% blasts +ve). Note: M0 and M7 AML are MPO \210\222ve. NSE is +ve in monocytic cells. Cytochemistry is no t essential if 4-color fl ow cytometry and estimation of cytoplasmic MPO is available. 125 ACUTE MYELOBLASTIC leukemia (AML) Fig. 4.1 BM showing myeloblasts in AML (E see color plate section). Fig. 4.2 BM showing myeloblasts in AML (E see color plate section). Fig. 4.3 AML: myeloblast with large Auer rod (left) (E see color plate section). 126 CHAPTER 4 Leukemia Fig. 4.4 AML: myeloblast with large Auer rod (top of cell) (E see color plate section). Fig. 4.5 AML: Sudan Black stain (dark granules clearly seen in myeloblasts) (E see color pl ate section). 127 ACUTE MYELOBLASTIC leukemia (AML) Morphological classi \201 cation The WHO classifi cation (Table 4.2) is used and has super seded the French \200\223American \200\223British (FAB) system. The FAB classifi cation, ba sed on predominant diff erentiation pathway (Table 4.1), remains useful for pre- liminary c lassifi cation of a newly diagnosed patient before the cytoge- netics result is known. For this reason it remains included here. Table 4.1 FAB morphological classifi cation of AML1,2,3 M0 AML with minimal diff erentiation (SB and MPO cytochemistry \210\222ve but myeloid immunop henotyping; may also express CD4 and CD7); 3% of cases M1 AML without maturation (<10% promyelocytes/myelocytes or monocytes; may have Auer rods) 20% of cases M2 AML with maturation ( \211 10% promyelocytes/myelocytes; < 20% monocytes; may have Auer rod s; t(8;21)) commonest subtype: 30% of cases M3 Acute promyelocytic leukemia (APL; >30% promyelocytes; multiple Auer rods (faggot cells); t (15;17)) 10% of cases M3v Microgranular variant of APL (high WBC count; minimal granulation; Auer rods rare; t(15;17) ) M4 Acute myelomonocytic leukemia (mixed myeloid (>20% blasts and promyelocytes) and monocytic ( \211 20%) maturation; monocytic cells are NSE +ve; may have Auer rods) 20% of cases M4Eo M4 variant with 5 \200\22330% eosinophils; associated with inv(16) chromosome abnormality. 5% of cases M5a Acute monoblastic leukemia (poorly diff erentiated subtype with \211 80% monocytoid cells of which \211 80% are monoblasts; Auer rods unusual) 10 \200\22315% cases are M5a or M5b M5b Acute monocytic leukemia (diff erentiated subtype with \211 80% monocytoid cells including NSE-+ve cells with typical monocytic appearance; Auer rods rare) M6 Acute erythroleukaemia (myeloblasts sometimes with Auer rods plus \211 50% bizarre often m ultinucleated erythroblasts; erythroblasts often PAS-positive) 3 \200\2235% of cases but 10 \200\22320% of 2 leukaemias M7 Acute megakaryoblastic leukemia (diffi cult to diagnose morphologically; often dry tap due to fi brosis; requires immunophenotyping with antiplatelet antibodies or electron microscop e analysis of platelet peroxidase) rare 128 CHAPTER 4 Leukemia WHO classi \201 cation of acute myeloid leukemia Correlates morphological, genetic, and cl inical features to categorize cases of AML4 into unique clinical and biological subgroups. The WHO system is recommended for the defi nitive diagnosis and classifi cation of AML. \200 The blast threshold for the diagnosis of AML is reduced from 30% to 20% BM blast compared to older classifi cation systems. Patients with clonal recurring abnormalities t(8;21)(q22;q22), inv(16) (q13q22), t(16;16)(p13;q22), or t(15;17)(q22;q12) should be considered to have AML regardle ss of the blast percentage. Table 4.2 2008 WHO classifi cation of acute myeloid leukemia Acute myeloid leukemia with recurrent genetic abnormalities AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 APL with t(15;17)(q22;q12); PML-RARA AML with t(9;11)(p22;q23); MLLT3-MLL AML w ith t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 A ML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 AML with mutated NPM1, provisional entry AML with mutated CEBPA, provisional entry Acute myeloid leukemia with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukemia, not otherwise speci \201 ed AML with minimal diff erentiation (FAB M0) AML without maturation (M1) AML with maturation (M2) Acute myelomonocytic leukemia (M4) Acute monoblastic/monocytic leukemia (M5a/5b) Acute erythroid leukemia (pure erythroid leukemia & erythroleukaemia, erythroid/myeloid) (M6) Ac ute megakaryoblastic leukemia (M7) Acute basophilic leukemia Acute panmyelosis with myelofi brosis Myeloid sarcoma Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm 129 ACUTE MYELOBLASTIC leukemia (AML) Immunophenotyping Monoclonal antibodies to cell surface antigens reliably diff erentiate AM L from ALL and confi rm the diagnosis of M0, M6, and M7 (see Table 4.3). Table 4.3 Immunophenotypic diagnosis of acute leukemia5 Panel of monoclonal antibodies to diff erentiate AML and ALL Myeloid B lymphoid Anti-MPO; CD13; CD33; CD45; CDw65; CD117 CD19; cytoplasmic CD22; cytoplasmic CD79a; CD10 T lymphoid Cytoplasmic CD3; CD2; CD7 Immunophenotypic patterns in AML subtypes Undiff erentiated (M0) Anti-MPO; CD13; CD33; CD34; CDw65; CD117; negative cytochemistry; lymphoid markers Myelomonocytic (M1 \200\223M5) anti MPO; CD13; CD33; CDw65; CD117 Monocytic (M4 & M5) Stronger expression of CD11b & CD14 Erythroid (M6) Anti-glycophorin A Megakaryocytic (M7) CD41; CD61 Biphenotypic leukaemias A minority of acute leukaemias have 2 distinct leukaemic cell popul a- tions on phenotyping and are characterized as biphenotypic leukaemias. Most commonly the se cell populations express B-lymphoid and myeloid markers and are associated with a high f requency of t(9;22)(q34;q11), the Ph chromosome. These patients have variable response rate s. Some may display \200\230lymphoid \200\231 features such as marked lymphadenopathy and high blast counts. Up to 50% of myeloid leukaemias may be positive for lymphoid antigens, m ost commonly CD2 (34%) and CD7 (42%) and this does not appear to have prognostic signifi ca nce. Cytogenetic analysis Should be performed in all cases of acute leukemia. It detects translo - cations and deletions that provide independent prognostic information in AML. Revised MRC prognostic classifi cation based on multivariable analyzes. Favorable risk cytogenetics \200 t(15;17)(q22;q21): 15% adults <45 years, rare older; fus ion gene PML- RAR ( \200\230FAB M3 \200\231). t(8;21)(q22;q22) Irrespective of additional cytogenetic abnormalities. 5 \200\2238% adults <55 years, rare older; fusion gene AML1/ETO. ( \200\230FAB M2 \200\231). inv(16)(p13q22)/t(16;16)(p13;q22). 10% adults <45 years, rare older; fusion gene CBF /MYH11. ( \200\230FAB M4Eo \200\231). Intermediate risk cytogenetics, \200\230standard risk \200\231 Entities not classifi ed as favorable or adverse. 130 CHAPTER 4 Leukemia Adverse cytogenetics, \200\230poor risk \200\231 \200 abn(3q), excluding t(3;5)(q21725;q 31 \200\22335). \200 inv(3)(q21q26)/t(3;3)(q21;q26). \200 add(5q), del(5q), \210\2225. \200 \210\2227, add(7q)/del(7q), excluding favorable karyotype cases. \200 t(6;11)(q2 7;q23). \200 t(10;11)(p11713;q23). \200 t(11q23), excluding t(9;11)(p21722;q23) and t(1 1;19)(q23;p13). \200 t(9;22)(q34;q11). \200 \210\22217/abn(17p). \200 Complex ( \211 4 unrelated abnormalities). Molecular analysis FISH and RT-PCR methods add sensitivity and precision to the detection o f translocations, deletions, and aneuploidy in cases where conventional cytogenetics fails or gives normal results. RT-PCR detects minimal residual disease overlooked by conventional methods and monitoring b y quantifi cation of PML-RARA transcript numbers in APL can identify patients at higher ris k of relapse. This may also be useful in patients with t(8;21) or inv(16). Presence of an activating mutation of the FLT3 gene with internal tandem repeats (25 \200 \22330% of all AML patients) is predictive for poor outcome in all cytogenetic subgroups. Mutations of the nucleophosmin (NPM) gene which normally produces a protein with diverse fu nctions including regulation of the p53 pathway, are found in many cases with a \200\230no rmal \200\231 karyotype and are associated with a favorable response to induction therapy. Abnormal localization of NPM in the cytoplasm can be detected by immunohistochemistry (NPMc +).6 Clinical features \200 Acute presentation usual; often critically ill due to eff ects of BM failure. Symptoms of anemia: weakness, lethargy, breathlessness, lightheadedness, and palpitations. Infection: particularly chest, mouth, perianal, skin (Staphylococcus, Pseudomonas, HSV, Candida). Fever, malaise, sweats. \200 Hemorrhage (especially APL due to DIC): purpura, menorrhagia and epistaxis, bleeding gums, rectal, retina. Gum hypertrophy and skin infi ltration (monocytic leukaemias (M4, M5)) (see Fig. 4.6). Signs of leucostasis, e.g. hypoxia, retinal hemorrhage, confusion, or diff use pulmonary shadowing. Hepatomegaly occurs in 20%, splenomegaly in 24%; the latter should raise the question of transformed CML; lymphadenopathy is infrequent (17%). CNS involvement at presentation is rare in adults with AML. 131 ACUTE MYELOBLASTIC leukemia (AML) Fig. 4.6 Gum hypertrophy in AML (E see color plate section). Investigations and diagnosis \200 FBC and blood fi lm. \200 EBV, CMV and HIV serology. \200 BM aspirate biopsy. BM infi ltrated with blasts ( \211 20%). \200 BM cytogeneti cs and molecular analysis. \200 Immunophenotyping of blood or marrow aspirate. \200 WBC usually i with blasts on fi lm \200\224but WBC may be low. \200 Hb, neutrophils, and pla telets usually d. Diff erential diagnosis ALL, blast crisis of CML, infectious mononucleosis (in younger pati ents), Megaloblastic anemia. Emergency treatment \200 Seek expert help immediately and refer to AML treatment center i f appropriate. Intensive cardiovascular and respiratory resuscitation may be needed for septic shock or massive hemorrhage. Immediate empirical broad-spectrum antibiotic treatment for neutropenic sepsis. Leucapheresis if peripheral blast count high or signs of leucostasis (clinical, diff use pulmonary shadowing on CXR, or hypoxia). Note: not in APL. Intensive hydration to prevent acute tumor lysis syndrome in patients with a high peripheral blast cell count Urgent chemotherapy; can usually be delayed until necessary clinical and laboratory assessments available. The exception is APL. 132 CHAPTER 4 Leukemia Supportive treatment \200 Explain diagnosis and off er counseling \200\224the word \200 \230leukemia \200\231 and prospect of prolonged chemotherapy are distressing. RBC and platelet transfusion support will continue through treatment: \200 CMV \210\222v e products? Most countries have switched to use of CMV unselected products due to leucodepletion at source. Irradiated products for patients treated with purine analogs. \200 Platelet transfusion t o maintain count >10 \227 109/L, unless septic, haemorrhagic or other haemostatic abnormal ity (>20 \227 109/L); in bleeding APL >50 \227 109/L. Start neutropenic infection prophylaxis regimen. Prompt antibiotic treatment might be required if febrile/septic. Start hydration aiming for urine output >100mL/h throughout induction therapy. Start allopurinol or consider rasburicase (200mcg/kg for up to 7d) to prevent hyperuricaemia/tumor lysis syndrome. Insert tunneled central venous catheter. \200 Routine use of growth factors is not recomm ended. Chemotherapy Where available patients should be entered into clinical trials. Initial aim o f chemotherapy7 is to eliminate leukaemic cells and achieve complete hematological remissio n (CR), defi ned as normal BM cellu- larity (blast cells <5% and normal representation of t rilineage haemat- opoiesis), normalization of peripheral blood count with no blast cells, n eutrophils \211 1.5 \227 109/L, platelets \211 100 \227 109/L and Hb >10g/dL. Leukemia is undetectable by conventional morphology but may be demonstrated by more sensitive molecu lar techniques (when available). CR is not synonymous with cure. Standard treatment is with 4 cycles of intensive combination chemotherapy, each cycle followed by a 2 \200\2233- week period of profound myelosuppression, during which good sup- portive therapy is essential. AML treatment consists of two phases: 1. Remission induction to achieve CR: usually 2 cours es of anthracycline- containing combination chemotherapy; assess BM response after 3 \200 \2234 weeks. \200 DA: daunorubicin 35 \200\22360mg/m2 IV \227 3d plus cytarabine 100mg/ m2/d as 12h IV infusion or divided dose bd bolus \227 8 \200\22310d. 2. Consolidation therapy: essential to reduce risk of relapse; optimum number unknown and p art of ongoing trials. Standard 2 cycles, i.e. HD cytarabine. \200 Enter patient into nat ional or international multicentre clinical trial if possible. Randomized studies are based on large patient numbers and compare increm ental experimental therapy with best treatment arm from previous trials. Allows access to n ovel therapies. Treatment protocols are generally age related; patients >60 \200\22370 years will only tolerate less intensive treatments and rarely transplantation. 133 ACUTE MYELOBLASTIC leukemia (AML) Risk stratifi cation using prognostic factors may determine consolidation therapy in some clinical trials or indeed for patients off -study. Patients who have resistant disease with >5% BM blasts after course 1 or adverse risk cytog enetic or molecular features should be considered for alternative treatment for high-risk A ML preferably within study. This would include intensifi cation of treatment with an aim to move to allo-SCT. Major complications are infective episodes which may be bacterial (Gram +ve and Gram \210 \222ve) or fungal (Candida and Aspergillus); less commonly viral (esp. HSV, HZV) and hemorr hage. No role for maintenance treatment. \200 Eff ectiveness of therapy assessed by CR rate, re lapse-free or disease-free survival, and overall survival.