Introduction
Severe neutropenia (absolute neutrophil count < 500 / ul) results in increased risk of bacterial infection [1] . Most neutropenias result from extrinsic causes that include infection, drugs (including chemotherapy ), autoimmune disorders, and marrow-occupying malignancies such as chronic or acute lymphoid leukemia [2, 3] . Less common ly, severe neutropenia may result from intrinsic defects in neutrophil proliferation, maturation, and survival [4] . Most intrinsic defects in adults are acquired ; this is the case in myelodysplastic syndrome (MDS) and acute myeloid leukemias (AMLs). In contrast, most intrinsic defects in children are congenital or inherited and are usually diagnosed in the newborn and young infants (Table 1). The most common inherited isolated neutropenia is severe congenital neutropenia (SCN), also called Kostmann syndrome. SCN presents with characteristic clinical and pathologic features that are demonstrated in this case presentation.

Clinical History
The patient was delivered by C aesarean section at 41 weeks of gestation and she was discharged following an uneventful 5-day hospital stay. However, a week later, the patient was seen in the emergency department (ED) for fever (101 ° C) and upper respiratory infectious symptoms (cough and rhinorrhea). The latter responded to treatment with a vaporizer. Two days later, the patient returned to the ED for a small cutaneous vesicular lesion on her neck. Two days later, the patient was admitted for fever and cellulitis of the neck. Laboratory studies were significant for an absolute neutrophil count (ANC ) of 0 with normal hemoglobin and increased platelets. The patient was started on antibiotics with resolution of the cellulitis. Serial CBCs over 2 weeks showed persistent absence of neutrophils, ANC of 0. ( Fig. 1) A bone marrow aspirate was performed and the findings were consistent with SCN. The patient was started on G-CSF at 15 ug/kg/day with prompt elevation in the ANC . She was subsequently followed mostly in the outpatient clinic.

The patient is now 14 years old and has received a daily injection of G-CSF that maintains her ANC between 1000 and 3000 / ul. She has had only a few hospitalizations for fevers, herpes stomatitis, dental abscesses, and pneumonia. She suffers from chronic gingivitis, hepatosplenomegaly, severe osteoporosis with vertebral compression fractures, and short stature. She has being doing well in school, receiving honors. Her marrow is examined on an annual basis by morphology and cytogenetics. Bone marrow aspirates and biopsies have not shown definitive evidence of MDS or AML and the karyotypes have been normal 46,XX.

Pathology
The patient's bone marrow has been evaluated many times at our institution over the past14 years. As typical in most pediatric hospitals, most of the marrow examinations have been via bone marrow aspirate smears, although biopsies were performed at ages 2 months, 11 months, 7 years, 8 years, and 12 years. All of the marrow studies showed similar morphology.

The bone marrow aspirate smears showed normocellular to hypercellular marrow



Figure 2a: Bone marrow aspirate smear - 100X magnification, Wright stain.

with granulocytic hypoplasia and maturation arrest , with most of the cells arrested at the promyelocyte to myelocyte stage



Figure 2b: Bone marrow aspirate smear - 500X magnification, Wright stain.



Figure 2c: Bone marrow aspirate smear - 1000X magnification, Wright stain.

The myelocytes showed asynchronous maturation of cytoplasm and nucleus , with the presence of secondary granules without chromatin condensation. The promyelocytes contained decreased numbers of primary granules . The decreased number of mature neutrophils (0 - 8% of marrow cellularity) was strikingly disparate from the increased number of maturing eosinophils (7 – 13%). Monocytes were also increased in number (2 - 9%, normal less than 1%). Erythroid precursor cells were relatively increased in number and showed normal maturation. The megakaryocytes were normal to increased in number and showed normal matur ation. Aspirate smears at younger ages showed increased numbers of lymphocytes and immature-appearing lymphocytes consistent with hematogones. Blasts were not increased in number.

The biopsies generally confirmed the aspirate findings. In addition, there was prominent expansion of the myeloid precursors that was confined to the paratrabecular region



Figure 3a: Bone marrow biopsy - 25X - magnification, hematoxylin and eosin stain.



Figure 3b: Bone marrow biopsy - 100X - magnification, hematoxylin and eosin stain.



Figure 3c: Bone marrow biopsy - 400X - magnification, hematoxylin and eosin stain.

In the non-paratrabecular regions, there was normal maturation of erythroid precursors in small collections (erythrons), scattered megakaryocytes, and maturing eosinophils



Figure 3b: Bone marrow biopsy - 100X - magnification, hematoxylin and eosin stain.



Figure 3d: Bone marrow biopsy - 400X - magnification, hematoxylin and eosin stain.

Mature neutrophils were rare to absent throughout the marrow, suggesting that the mature neutrophils seen on the aspirate smears represented peripheral blood components. The thickened paratrabecular regions that contained the myeloid precursor cells were sharply delineated from the intertrabecular regions that contained erythroid elements, eosinophils, and megakaryocytes.

Discussion
Definition: SCN is an inherited disorder presenting in the newborn with severe chronic neutropenia (ANC less than 500 / ul) and little to moderate anemia/thrombocytopenia. Often the ANC is less than 200 / ul even shortly after birth. SCN should be distinguished from cyclic neutropenia, another intrinsic defect in granulopoiesis that shares many similarities but does not progress to MDS or AML.

Synonyms: Congenital agranulocytosis, infantile congenital agranulocytosis, Kostmann's syndrome, Kostmann syndrome, congenital neutropenia, congenital severe chronic neutropenia. Some authors distinguish SCN from Kostmann syndrome, reserving the latter for the first described family or to those cases with documented autosomal recessive inheritance pattern. Others, including this author, use these terms interchangeably.

Epidemiology: SCN is a rare disease with approximately 1-2 cases / million population [5] . The first cases were reported in 1950 and 1956 by Rolf Kostmann, who designated the disease as infantile genetic agranulocytosis [6] . These cases were transmitted in an autosomal recessive pattern of inheritance. Since then, other cases of SCN have been described, some with autosomal dominant [7] or sporadic pattern of inheritance. Similar to SCN, cyclic neutropenia is a rare disease with less than 1 case / million population. The inheritance pattern of cyclic neutropenia is autosomal dominant or sporadic [8] .

Clinical features: The patients have neutropenia at birth that deteriorates to agranulocytosis with recurrent infections, typically bacterial sepsis or pneumonia (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa). Prior to 1994, most patients were treated with prophylactic antibiotics [9] and rarely, bone marrow transplant [10] . The prognosis in these patients was poor with median age of death as low as 0.8 - 2 years [11, 12] . In addition, there was a 2% incidence of acute leukemia.

In the late 1980s and early 1990s, multiple studies demonstrated the clinical effectiveness of G-CSF in the treatment of SCN [9, 13] . In 1994, G-CSF became widely available and dramatically changed the clinical course of this disease. Also, an international registry (Severe Chronic Neutropenia International Registry, SCNIR, http://depts.washington.edu/registry) was established permitting close clinical follow up of this and other diseases with severe chronic neutropenia. As of 2003, 853 patients have been registered, of which 348 are SCN [5] . Over 90% of the patients respond to G-CSF injections (0.15-60 ug / kg/ day) with the ANC increasing to 1,500 - 10,000 / ml. The median life expectancy is 17.3 years and increasing. Death from infection still occurs (35% of deaths) but is more commonly due to leukemia and its complications (37% of deaths). Even more worrisome is the increasing incidence of MDS / AML, 1% / year for the first four years; this accelerates to approximately 13% cumulative risk after 8 years on G-CSF treatment. The accelerating rate suggests that the incidence and percentage of deaths from MDS/AML will continue to increase. In addition to leukemia, other medical complications are becoming evident with increasing age. Many of the patients exhibit hepatomegaly (20-35% of the patients), osteopenia / osteoporosis (59%), and growth retardation (27-51% were less than the tenth percentile). Less frequent are vasculitis (4%) and glomerulonephritis.

Similar to SCN, cyclic neutropenia may present with severe neutropenia. However, cyclic neutropenia is characterized by a 21 day oscillating neutrophil count with ANC ranging from 0 to 1.5K/ul. Severe infections can occur at the nadir of the neutrophil count. G-CSF treatment has also ameliorated the clinical course of this disease. However, approximately 10% of these patients die, exclusively secondary to infection [5] . Unlike SCN, cyclic neutropenia has not been associated with MDS / AML. As of 2003, 145 patients with cyclic neutropenia have been registered in the SCNIR.

Morphology: The aspirate smear findings in this case are typical of SCN cases . The typical SCN bone marrow aspirate shows a maturation arrest of the myeloid precursors at the promyelocyte/myelocyte stage of differentiation with increased numbers of eosinophils and monocytes. The promyelocytes have normal [14, 15] to abnormally lucent primary granules [16] with paucity of secondary granules in the more mature myeloid cells. The cytoplasm and nucleus show asynchronous maturation [16, 17] . Occasional myeloid precursors showed cytoplasmic vacuolation [14, 16] . In one published case, the promyelocytes and myelocytes were enlarged and multinucleated but this appears to be the exception to the rule [18] . All of these changes, including left shift in myeloid maturation, enlarged myeloid cells, hypogranulation, nuclear to cytoplasmic maturation asynchrony, cytoplasmic vacuolation , eosinophilia, and monocytosis, have been associated with G-CSF treatment [19, 20] and thus, it is unclear how much chronic G-CSF treatment contributes to these morphologic changes. However, such changes are seen in SCN without G-CSF treatment, indicating that they may be intrinsic to SCN. Finally, prominent and increased azurophilic granulation, typical of G-CSF effect, is not seen.

The bone marrow biopsies have been generally described as showing a left shift in myeloid maturation but detailed descriptions are lacking. For example, increased numbers of eosinophils and monocytes have not been noted in the biopsies but given the aspirate findings, one would expect to find that they are also increased in the biopsy. Our case details specific findings that have not been previously described for bone marrow biopsies of patients with SCN. The marrow biopsies showed thickened paratrabecular region comprised exclusively of immature myeloid cells. The non-paratrabecular regions showed normal erythroid precursors, normal to increased megakaryocytes, increased numbers of maturing eosinophils, and the near absence of mature neutrophils. The last point is particularly interesting because normal numbers of mature neutrophils are found in the peripheral blood with G-CSF treatment, suggesting that segmented neutrophils are immediately released from the bone marrow upon maturation or that maturation to neutrophils occurs outside the marrow from circulating immature cells. This would suggest that the mature neutrophils seen in the bone marrow aspirate smears [21] represent peripheral blood contamination. We have seen virtually identical biopsy findings in another case of SCN (data not shown), suggesting that these findings are defining features of a subset of SCN on chronic G-CSF.

The differential diagnosis is limited for the expanded myeloid precursors in the paratrabecular regions with zonation of the myeloids from the erythroid, eosinophils, and megakaryocytes. Possible diagnoses include acute response to G-CSF therapy, early regeneration of the myeloid cells from a toxic event, and MDS. Clinical history excludes the first two possibilities. In theory, autoimmune neutropenia could present with similar morphology if the antibodies react against myelocytes and metamyelocytes. In practice, autoimmune neutropenia typically results in compensatory myeloid hyperplasia in the marrow and occasional neutrophil phagocytosis [2, 22] . The paratrabecular myeloid expansion is unlikely to be a manifestation of MDS because this feature was present for 14 years without any evidence of significant anemia, thrombocytopenia, increased blasts, or cytogenetic abnormalities. Furthermore, MDS often shows disorganization of erythroid precursors from their usual erythrons and immature myeloid cells in the interparatrabecular areas (atypical localization of immature precursors). Finally, the paratrabecular myeloid expansion is unlikely to result solely from chronic G-CSF therapy . While G-CSF therapy can initially cause paratrabecular expansion, prolonged treatment (> 1 month) leads to normalization of the myeloid maturation and localization [19].

Bone marrow findings reported in cyclic neutropenia have been based mostly on bone marrow aspirate smears. The marrow findings range from those similar to SCN at the nadir of the neutrophil count to those similar to normal marrow at normal neutrophil counts. The two disease s can be distinguished by serial CBCs over several weeks [23] .

Immunophenotype: Very few studies of the immunophenotype of bone marrow cells from SCN patients have been reported. Flow cytometry analysis of SCN bone marrow without G-CSF demonstrates normal numbers of the very early CD34+, CD38- hematopoietic progenitor cells but decreased numbers of the slightly more committed CD34+, CD38+ hematopoietic progenitor cells [24] . Apoptotic CD34+ progenitor cells and CD33+ or CD15+ more committed myeloid cells are increased in number in cultured SCN bone marrow but not in freshly isolated marrow cells [25] . Peripheral neutrophils in SCN on G-CSF appear to have increased expression of CD64, CD32, CD14, HLA-DR, decreased expression of CD16, and constant expression of CD11b/CD18, CD11a/CD18 [26] . However, later studies indicate that most if not all changes are secondary to G-CSF effect [27, 28] . Currently, there is no consensus immunophenotype that characterizes SCN.

Molecular mechanism: The molecular defect in a subset (57 – 88 %) of SCN cases has been localized to point mutations in the neutrophil elastase 2 gene (ELA-2) [29, 30] , a component of primary granules. A mutation in the transcription factor Gfi1 , which regulates the expression of ELA-2 gene, has been identified in a minor subset of SCN cases without ELA-2 mutations [31] . The identified mutations are heterozygous and cannot explain the autosomal recessive form of SCN [30, 32] . Additional unidentified mutations remain to be identified. The ELA-2 gene is also mutated in 100% of cyclic neutropenia, although most of the point mutations are different from those in SCN (http://archive.uwcm.ac.uk/uwcm/mg/search/118792.html). However, occasional mutations are shared between the two diseases [33] , resulting in the perplexing question of how the same point mutations in ELA-2 cause different diseases.


The identification of ELA-2 using linkage analysis of cyclic neutropenia [34] was a major step in understanding the molecular mechanism s of both cyclic neutropenia and SCN [29] . The obligatory role of ELA-2 was questioned following the identification of a family with two symptomatic children with an ELA-2 mutation and an asymptomatic father with the identical mutation [35] . Later study demonstrated that in such cases the asymptomatic parent was a mosaic for the ELA-2 mutation and the normal ELA-2 genes predominated in the neutrophils, strongly supporting the hypothesis that ELA-2 mutations are sufficient to cause neutropenia [36] .

Despite the identification of the offending gene in the majority of SCN cases and the detection of increased apoptosis in the myeloid precursors [37] , the exact mechanism of disease remains poorly understood. Early models predicted that normal ELA-2 was necessary for normal granulopoiesis and ELA-2 mutations led to loss of the elastase activity of the mutated ELA-2 protein and the normal ELA-2 protein in a dominant negative manner. Most studies appear to be inconsistent with this model. Mouse models that lack ELA-2 [38] or express ectopic mutated ELA-2 [39] have normal neutrophil development. In vitro assay of the mutated ELA-2 proteins demonstrated varying elastase activities including supranormal levels [40] . Finally, mutated Gli1 leads to the overexpression and not suppression of ELA-2 [31] . These findings have led to an alternative model in which abnormal activity or intracellular localization of ELA-2 leads to apoptosis of the neutrophils [41] .

This model is supported by the identification of the molecular defect in a dog model of cyclic neutropenia [42] . This mutated gene encodes an adaptor protein complex b subunit that is important for directing proteins to specific membranes. This model does not explain the mouse studies , but preliminary studies suggest species differences such that mutated ELA-2 alters human but not murine myelopoiesis [43] . Finally, this model also does not define the relationship between abnormal ELA-2 and apoptosis. The relationship may involve the anti-apoptotic protein bcl-2 since bcl-2 is decreased in patients with SCN and is restored to normal levels with G-CSF treatment [44] .

Prognosis and clinical course: The life expectancy in SCN has been greatly increased by the advent of G-CSF. Numerous complications are being identified with increased age of the patients (see clinical course). Of these, the most worrisome is the increasing incidence of MDS / AML that has overtaken infection as the leading cause of mortality. The conversion to AML is associated with the acquisition of G-CSF receptor mutations, loss of chromosome 7, and ras mutations [45] .

Table 1: Partial Differential Diagnosis for Severe Neutropenia

Extrinsic Infection Drug Immune-mediated: immunodeficiency, autoimmune, aplastic anemia Nutritional deficiency: B12, folate Marrow compromise by tumor Pseudo-neutropenia secondary to neutrophil clumping Intrinsic (often seen in newborn / young infant) Severe congenital neutropenia (Kostmann syndrome) Cyclic neutropenia Schwachman-Diamond Syndrome Chediak-Higashi syndrome Dyskeratosis congenita Cartilage hair hypoplasia Griscelli syndrome Myelokathexis (WHIM syndrome) Glycogen storage disease Ib Myelodysplastic syndrome

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