Neutropenia in Pediatric Oncological Patients

Abstract

In 2020, more than 275,000 children and adolescents from 0 to 19 years of age were diagnosed with cancer in the world. Acute myeloid leukemia or acute lymphoblastic leukemia are the most frequent types of cancer. Leukemia is a serious condition that is fatal in many cases. Since tumor cells are present in both, bone marrow and circulating blood, very aggressive therapeutic treatments are required to eliminate tumor cells. Neutrophils are white blood cells that first respond against microbial pathogens and are produced in the bone marrow. Several drugs used in leukemia cancer treatment can reduce the total neutrophil number causing neutropenia. In this chapter we will briefly describe neutrophil maturation and functions as well as the different types of neutropenia. We will also focus on neutropenia consequences and some clinical approaches for treating neutropenia in pediatric patients.

1. Introduction

Neutrophils are the most abundant white blood cells (leucocytes) that are firstly recruited from the circulation into tissues with infection and/or inflammation [1]. Neutrophils derive from the bone marrow, where they mature in response to (G-CSF). Myeloid stem cells differentiate into granulocyte-monocyte progenitors, which in turn, differentiate into neutrophils by intermediate stages of promyelocytes, myelocytes, metamyelocytes, band cells, and segmented polymorphonuclear cells (Figure 1). The size of mature neutrophils varies from 8 to 11 μm [3].

Figure 1.

Neutrophil differentiation in the bone marrow and stages affected by neutropenia. Neutrophils derive from myeloid stem cells. The maturation stages include myeloblast, promyelocyte, myelocyte, metamyelocyte, neutrophil in band, and mature neutrophil. Between the stages of promyelocyte and myelocyte, many factors affect neutrophil maturation and release from the blood marrow causing neutropenia. Modified from [2].

After maturation, neutrophils migrate from the bone marrow into the blood. From the circulation, neutrophils migrate into affected tissues through a process known as the leukocyte adhesion cascade. In tissues, neutrophils destroy microorganisms by several cellular mechanisms such as phagocytosis, degranulation (releasing of antimicrobial substances), production of reactive oxygen species (ROS), and production of neutrophil extracellular traps (NET) (Figure 2) [4, 5].

Figure 2.

Neutrophil release, migration, and functions. a) Neutrophils mature and are released from the bone marrow into the bloodstream. b) Neutrophils circulate in the bloodstream until they are recruited to tissues. c) Neutrophil functions in tissues. Phagocytosis is the engulfment and degradation of a pathogen, degranulation is the release of granule content including enzymes, proinflammatory substances and reactive oxygen species, and NETosis is the release of neutrophil extracellular traps to capture and kill pathogens [4, 5].

1.1 Phagocytosis

Neutrophils are professional phagocytes. Phagocytosis is a cellular process for ingesting and eliminating particles larger than 0.5 μm in diameter, including apoptotic cells, foreign substances, and microorganisms. The process of phagocytosis involves several steps that include (a) recognition of the particle to be ingested, (b) activation of the internalization process, (c) phagosome formation, and (d) maturation of the phagosome into a phagolysosome (Figure 2) [6].

1.2 Degranulation

In the bone marrow, immature neutrophils synthesize proteins that are stored in different granules. Neutrophils are also called granulocytes because they have many granules. The granules are classified into three different types based on their content: azurophilic granules, specific granules, and gelatinase granules [7]. Neutrophils also form secretory vesicles at the last step of their differentiation process in the bone marrow [5]. Degranulation is the secretion of proinflammatory substances that are derived from intracellular stored granules (Figure 2). Neutrophils also release reactive oxygen species to kill extracellular bacteria [3].

1.3 Neutrophil extracellular traps

When microorganisms are larger than 0.5 μm in diameter, neutrophils cannot phagocyte them. Instead, they release neutrophil extracellular traps (NET). These extracellular traps are DNA fibers adorned with histones and several neutrophil granule proteins (Figure 2). The process of NET formation is called NETosis, and it requires myeloperoxidase and neutrophil elastase release into the cytosol and NADPH oxidase activation for reactive oxygen species production. These enzymes and ROS cooperate to degrade the nuclear membrane and decondense the chromatin so it can be released from the cell. Since, neutrophils die in this process, NETosis has been described as a special form of programmed cell death [3, 8].

1.4 Role of neutrophils

Neutrophils are important not only for fighting infectious microorganisms but also for maintaining a homeostatic environment in all tissues in the body. Neutrophils are important for tissue repair [9] and for maintaining a healthy oral cavity [10]. Neutrophils can also fight larger microorganisms such as Entamoeba histolytica, a protozoan parasite that causes amoebiasis and has a high prevalence in developing countries [11] or such as Trychomonas vaginalis, a parasite that causes the most common non-viral sexually transmitted infections worldwide [12]. These larger parasites are controlled by different mechanisms, while amoebas are stopped by NET [13], T. vaginalis are killed by a novel process known as trogocytosis [14]. In addition, in the female reproductive system, vaginal neutrophils also eliminate sperm by trogocytosis [15]. Thus, neutrophils in normal conditions contribute to keep tissue homeostasis. However, in pathological conditions, neutrophil numbers can be seriously altered. For example, in periodontal disease [10] or in obesity [16], overactivation and/or accumulation of neutrophils can cause tissue damage. Also, in serious inflammatory conditions such as acute respiratory distress syndrome, COVID-19, or septic shock [17, 18, 19], accumulation of neutrophils can be lethal. On the contrary, if neutrophil numbers are very low, a condition called neutropenia, life-threatening conditions result from overgrowth of bacteria, virus, or fungi at sites of injury [20].

2. Neutropenia classification

2.1 Neutropenia

Neutropenia is defined as an abnormally low number of neutrophils in the blood. Normal neutrophil counts range from 1500 to 7000 neutrophils/microliter. When neutrophil counts are less than 1500 neutrophils/microliter, the person presents neutropenia [20]. Neutropenia is often diagnosed after a routine blood count revealing a low count of neutrophils but with absolute counts for monocytes, eosinophils, and basophils in normal values. In the absence of active infection or inflammation, the hematocrit/hemoglobin and platelet count are also usually normal or only moderately reduced. In young children, congenital anomalies suggest a genetic cause for neutropenia. In other cases, such as autoimmune neutropenia or chronic idiopathic neutropenia, diagnosis is complicated since a generalized enlargement of lymph nodes, liver, or spleen is not normally found [21]. In addition to the low neutrophil counts if some other characteristics are present in the patient, neutropenia has other names.

2.2 Chronic neutropenia

Neutropenia becomes chronic if it occurs on at least three occasions in a three-month period [22]. Chronic neutropenia is characterized by (a) reduced or ineffective neutrophil production in the bone marrow, (b) increased neutrophil margination, (c) sequestered neutrophils in the spleen, (d) accelerated neutrophil destruction, and (e) mutations of a variety of neutrophil genes, including ELANE, and HAX1 [23, 24, 25]. Chronic benign neutropenia is the most common form of chronic neutropenia in the pediatric age group, occurring in approximately 1/100,000 children/year, with the median age at diagnosis being 7–9 months [26].

2.3 Idiopathic neutropenia

Neutropenia is called idiopathic when the agent causing it, is not clear. In this case, neutropenia cannot be attributed to a drug nor to an autoimmune, genetic, infectious, inflammatory, or malignant origin [22]. Patients with chronic idiopathic neutropenia and autoimmune neutropenia can overlap in this category because it is difficult to precisely detect circulating antibodies directed toward antigens present on the surface of neutrophils [27].

2.4 Chronic idiopathic neutropenia

Chronic idiopathic neutropenia combines the features of chronic and idiopathic neutropenia. It is then, a type of neutropenia that occurs on at least three occasions in a three-month period and is not attributable to drugs nor to a specific autoimmune, genetic, infectious, inflammatory, or malignant origin. About 30% of patients with chronic neutropenia do not have an apparent underlying cause [22, 28].

2.5 Autoimmune neutropenia

Autoimmunity is a disease caused by antibodies produced against substances naturally present in the body and has been recognized as a sign of primary immunodeficiency [29]. Autoimmune neutropenia is characterized by chronic neutropenia and the presence of antibodies against human neutrophil antigens. Chronic idiopathic neutropenia and autoimmune neutropenia are rare conditions, also referred to as “primary” or “isolated” because in some diseases, neutropenia is the primary hematological abnormality, both in children and in adults [30].

2.6 Febrile neutropenia

Patients with suppressed immune systems might present fever as a sign of an underlying infection. When neutropenia is accompanied by fever which is called febrile neutropenia. Fever, in this case, is defined as a temperature higher than 37.8°C for at least 1 h or two measurements within 24 h, or a temperature higher than 38°C in a single measurement [31]. Febrile neutropenia which usually lasts 7 days, is a common complication of myelosuppressive chemotherapy in oncological children and one of the most important causes of morbidity and mortality in these patients [32, 33, 34]. Febrile neutropenia is common in children who have received chemotherapy as treatment for acute myeloid leukemia and acute lymphoblastic leukemia. Other conditions associated with febrile neutropenia and prolonged neutropenia include Ewing’s sarcoma, malignant brain tumors, and myeloablative conditioning for autologous and allogeneic hematopoietic stem cell transplantation [34, 35].

Even though febrile neutropenia affects both adult and pediatric patients, children with febrile neutropenia have a higher risk than adults of infections of unknown origin [36]. Patients with a high-risk of presenting febrile neutropenia also present some of the following factors: (a) C-reactive protein (CRP)  > 90 mg/L, (b) hypotension, (c) platelet count below 50,000 platelets/microliter, (d) relapsed leukemia, or (e) the elapsed time between the end of chemotherapy and the beginning of fever being less than 7 days [36, 37].

2.7 Severe neutropenia

If neutrophil count is less than 500 neutrophils/microliter, the patient has severe neutropenia [2]. Severe congenital neutropenia is a genetically heterogeneous syndrome associated with mutations in (a) ELANE gene 2 which encodes neutrophil elastase, (b) HAX1 gene which encodes HS-1-associated protein X-1 (HAX-1), a protein involved in the regulation of apoptosis [38], (c) GFI1 gene which encodes the growth factor independence 1 (GFI1) transcriptional repressor protein, that plays an essential role in the differentiation of myeloid and lymphoid progenitors [39], (d) WAS gene encoding the Wiskott–Aldrich syndrome protein, and (e) CSF3R gene, encoding the granulocyte colony-stimulating factor (G-SCF) receptor [24]. Mutated ELANE gene 2 is present in half of the people with severe congenital neutropenia [40]. Severe neutropenia is a risk factor for vulnerability to bacterial infections, which puts people at high-risk for infection with significant morbidity and mortality [41]. In addition, an important clinical feature of severe congenital neutropenia is the risk for disease progression to myelodysplasia and/or acute myeloid leukemia [42]. Progression to leukemia is strongly associated with acquired mutations of the gene CSF3R [43]. Thus, the clinical use of recombinant G-CSF in patients with severe congenital neutropenia to improve granulopoiesis must be considered carefully, since G-CSF may elevate the risk for malignant transformation [44].

2.8 Cyclic neutropenia (cyclic hematopoiesis)

Cyclic neutropenia is a rare idiopathic disorder estimated at one in one million. It is characterized by regular periodic reductions in neutrophil counts. The cause of this type of neutropenia seems to be a mutation in the ELANE gene, resulting in the arrested development of neutrophils at the promyelocyte stage within the bone marrow [40]. The signs and symptoms of cyclic neutropenia appear in uniformly spaced episodes every 21 days. Patients typically complain of recurrent episodes of fever, anorexia, cervical lymphadenopathy, malaise, pharyngitis, and oral mucosal ulcerations. Other gastrointestinal mucosal areas, including the colon, rectum, and anus, may be affected by recurrent ulcerations. Oral ulcerations develop on any oral mucosal surface that is exposed to even minor trauma, particularly the lips, tongue, and oropharynx. Symptoms usually begin in childhood. When the neutrophil count is at its lowest point, the patient experiences problems with infections. As the neutrophil count rises toward normal, the signs and symptoms decline. Very low neutrophil counts usually are present for 3–6 days, and blood monocyte and eosinophil levels are typically increased when the neutrophil count is depressed. Even when the neutrophil count is at its peak, the levels are often less than normal [45]. Cyclic neutropenia should be diagnosed after sequential complete neutrophil blood counts (three times per week for 6–8 weeks), in which neutrophil numbers are less than 500 neutrophils/microliter for 3–5 days during each of at least three successive cycles [46].

2.9 Benign ethnic neutropenia

Benign ethnic neutropenia, the most common form of neutropenia worldwide, is also diagnosed when neutrophil counts are less than 1500 neutrophils/microliter. However, people with this condition do not seem to have a higher risk of infections. This condition appears in some individuals from African, Middle Eastern, and West Indian descent. In these individuals, neutrophil numbers as low as 800–1000 neutrophils/microliter are considered normal [47]. Ethnic benign neutropenia has been associated with a single nucleotide polymorphism in the ACKR1/DARC gene, the same variation that also confers the Duffy-null trait [48]. ACKR1 is the atypical chemokine receptor 1 (Duffy blood group). Duffy antigen/chemokine receptor (DARC), also known as Fy glycoprotein (FY) or CD234, is a protein that in humans is encoded by the ACKR1 gene. Duffy antigen is a glycosylated membrane protein and a non-specific receptor for several chemokines, located on the surface of red blood cells. The pathophysiology of ethnic benign neutropenia is not completely understood [49]. Many studies suggest that this condition results from a defect in the release of mature granulocytes from the bone marrow; however, newer studies favor an increase in the egress and migration of neutrophils into the organs and tissues as the cause [49, 50].

2.10 Neutropenia in premature infants

This type of neutropenia is strongly associated with maternal complications during pregnancy e.g., hypertension and preeclampsia. Other complications are placental blood flow or intrauterine growth restrictions, severe asphyxia, and infections. It has been suggested that because of these complications, neutrophil production in the bone marrow of neonates is reduced [22, 51, 52]. Neutrophil function is less strong in preterm neonates than in adults and might also contribute to the increase in propensity to infections which lead to neutropenia caused by sepsis. Therefore, neonatal intensive care units hold low birth weight neonates with neutropenia during their first week of life. Supportive management is helpful, typically neonates get well, and the condition follows a benign progression.

3. Causes of neutropenia

The origins of neutropenia are not completely understood. In many cases, the association of certain gene mutations with this condition suggests a genetic cause for neutropenia. The same may be true for autoimmune neutropenia or chronic idiopathic neutropenia, however, in these cases, no clear genetic associations have been reported. Therefore, neutropenia may also be caused by other yet unidentified factors. The known genetic causes of neutropenia are described next.

3.1 Mutations in the ACKR1/DARC gene

As mentioned before, benign neutropenia is an intrinsic condition of some individuals in certain ethnic groups. This condition is not associated with negative clinical consequences due to decline in innate immunity. African ancestry people have shown a strong association between familial neutropenia and a single nucleotide polymorphism in the promoter region of the atypical chemokine receptor 1 gene (ACKR1), also termed Duffy antigen/chemokine receptor (DARC), which is part of the Duffy blood group system [48, 53]. Neutropenia is associated with the null Duffy genotype (Fy-/Fy-), but not with the heterozygote (Fy-/Fy+) and wild-homozygote (Fy+/Fy+) genotypes, suggesting an autosomal recessive inheritance for the condition [54].

3.2 Mutations in the neutrophil elastase gene 2

In contrast to the benign neutropenia, whose genetic influence results in nonthreatening variations in neutrophil numbers among certain individuals in the general population, there are other neutropenic disorders with a strong genetic component. This so-called “Mendelian” or hereditary neutropenia includes primarily two types. The first is cyclic neutropenia, in which neutrophil numbers oscillate with approximately 21-day periodicity, changing between almost normal levels to undetectable levels that last for several days [55]. Nearly all cases of cyclic neutropenia are associated with autosomal dominant mutations in the ELANE gene 2, which encodes neutrophil elastase [56]. The second is the so-called Kostmann syndrome or non-cyclical “infantile agranulocytosis”, in which non-cyclical severe neutropenia is observed. This neutropenia is characterized by an arrest of granulocytic differentiation at the promyelocyte stage [40]. Nowadays, the disorder is most often referred to as severe congenital neutropenia and it is the result of allelic, heterozygous mutations in the ELANE gene 2 [40]. Although some ELANE mutations overlap with the mutations observed in cyclic neutropenia [56], it is now recognized that severe congenital neutropenia represents a genetically heterogeneous group of disorders, in which multiple mutated genes participate, including those encoding for the HAX1, G6PC3, WAS, GFI1, STK4, and tafazzin proteins [25, 57, 58].

The ELANE gene 2, also known as ELA2, HLE, HNE, NE, and SCN1, encodes neutrophil elastase. ELANE gene 2 consists of five exons and six introns and is located on chromosome 19 (19p13.3). Neutrophil elastase is a protein of 267 amino acids synthesized as an inactive form of pro-pre-enzyme (zymogen) [40]. Neutrophil elastase is a serine protease stored in the azurophil granules of neutrophils that degrades extracellular matrix proteins, destroys microorganisms, and regulates inflammation by degrading soluble proteins such as immunoglobulins, cytokines, coagulation factors, and protease inhibitors [59]. Mutations in the ELANE gene lead to the production of a mutant protein. However, no general biochemical malfunction, including effects on proteolysis, has been identified. Therefore, it is not clear how mutations in neutrophil elastase are responsible for neutropenia. Two non-mutually exclusive theories have been proposed to explain how elastase mutations might cause neutropenia. One theory declares that mutations within elastase elicit elastase accumulation in inappropriate neutrophil compartments. Another theory proposes that mutations cause misfolding of the protein, thereby inducing the stress response pathway within the endoplasmic reticulum. In both cases, neutrophil precursor cells will arrest their development resulting in neutrophil maturation arrest and in consequence smaller number of mature cells in circulation.

3.3 Autoimmunity

Autoimmunity responses are thought to be responsible for eliminating neutrophils. However, little is known about the cellular and molecular mechanisms involved in the autoimmunity of granulocytic disorders. Currently existing explanations for autoimmunity against neutrophils, include a deficit in the elimination of apoptotic cells, deficiency in regulatory cells, hyperactivation of inflammatory cytokines, repeated infections, or a loss of tolerance to neutrophil autoantigens. Although no clear explanations are known, excessive cytokine activation might explain defective neutrophils and more importantly the loss of these leukocytes by apoptosis or other mechanisms [60]. Thus, in conditions of strong chronic neutrophil activation, such as those found in several autoimmune conditions, neutropenia can develop [29]. However, in autoimmune diseases where autoantibodies against neutrophils are present neutropenia can more clearly develop. In a study where blood from 402 children with neutropenia was analyzed, it was found that 302 (75%) of them had anti-neutrophil antibodies. These children also had a significantly lower absolute neutrophil count and a 2-times greater risk of hospitalization than patients without anti-neutrophil antibodies [61]. Thus, in several autoimmune conditions particularly when anti-neutrophil antibodies develop, neutropenia can be a negative result from autoimmunity.

3.4 Cancer treatment in pediatric patients

In 2020, more than 275,000 children and adolescents from 0 to 19 years of age were diagnosed with cancer in the world [62]. The most common types of cancer in this population are leukemias (80,500 cases), brain and central nervous system tumors (30,750 cases), lymphomas (40,000 cases), kidney tumors (14,500 cases), thyroid cancer (10,000 cases), and gonadal tumors (testicular and ovarian; 10,000 cases) (Table 1) [63].

Age (years)	Cancer type
0–14	Central nervous system tumors, lymphoma, neuroblastoma, kidney tumors, and malignant bone tumors
0–19	Brain and central nervous system tumors, leukemia, and lymphoma.
15–19	Brain, central nervous system, lymphoma, leukemia, thyroid, gonadal germ cell tumors, and malignant bone tumors

Table 1.

Most common types of cancer according to childhood age group [63].

Among all these types of cancer, acute myeloid leukemia or acute lymphoblastic leukemia are the most frequent. Leukemia then becomes a serious condition that is fatal in many cases. Since tumor cells are present both in the bone marrow and in circulating blood, very aggressive therapeutic treatments are required to eliminate tumor cells. One of the most used treatments is chemotherapy with high doses of drugs that directly damage the bone marrow. This type of treatment can eliminate malignant cells but also, in many cases, damages leukocyte progenitor cells in the bone marrow leading to loss of mature leukocytes. Therefore, chemotherapy of pediatric leukemia patients frequently leads to the development of neutropenia.

3.4.1 Therapeutic drugs for leukemia cancer treatment

Several drugs used in leukemia cancer treatment can also lead to neutropenia. Methotrexate is a potent therapeutic agent administered at high doses for the treatment of acute lymphoblastic leukemia [64], osteosarcoma [65], and lymphoma [66] in both pediatric and adult patients. This drug is transported from the blood into the liver, where it is metabolized so that it can be cleared from the body. The organic anion transporting polypeptide 1B1 (OATP1B1) is a transporter protein on liver cells that promotes methotrexate uptake. Defects or low expression of the OATP1B1 transporter, in some individuals, can affect methotrexate clearance. This would cause an accumulation of methotrexate in circulation leading to deleterious effects on neutrophil precursors and resulting in neutropenia [67]. Several myeloid leukemia patients present mutations in the onco-genic tyrosine kinase FMS-related tyrosine kinase 3 (FLT3). Patients with these mutations can be treated with tyrosine kinase inhibitors such as midostaurin and gilteritinib. A large proportion of patients treated with gilteritinib developed febrile neutropenia [68, 69]. Other negative side effects were also anemia (20–40.7%), and thrombocytopenia (13–22.8%) [68, 69]. Patients with acute myeloid leukemia who are not eligible for aggressive chemotherapy can be treated with venetoclax in combination with low doses of hypomethylating agents. Unfortunately, in most of these patients (75%) serious adverse events also occurred. Febrile neutropenia (44%) and pneumonia (13%) were the most commonly detected [70].

4. Neutropenia consequences

Neutropenia is a severe clinical condition that leads to many complications due to the lack of neutrophil defensive functions. Neutrophils play a central role in innate immune defense against many microorganisms, particularly bacteria and fungi [3]. Bacteria, including Staphylococci, Streptococci, and Escherichia coli, among others and fungi, including Candida albicans cause recurrent infections in patients whose neutrophil counts become very low. This situation is similar to the one seen in patients with chronic granulomatous disease. In these individuals, although neutrophils are present, they fail to produce reactive oxygen species, so that neutrophils can perform phagocytosis of microorganisms, but they are unable to kill them [71]. In neutropenia, many recurrent infections are a serious problem that can be life-threatening, particularly when they are on top of other clinical conditions. This is the situation most observed among pediatric cancer patients. With these children, chemotherapy treatments try to control cancer, but they provoke more complications after inducing neutropenia.

4.1 Infections in pediatric oncological patients

Infectious diseases are associated with high morbidity and mortality rates among pediatric cancer patients undergoing neutropenia after cancer treatment [72]. Among these patients, those experiencing prolonged periods of neutropenia are at a higher risk of acquiring bacterial, viral, and fungal infections (Table 2) [74].

Microorganism	Percentage	Species
Gram + bacteria	> 40%:	Streptococcus pneumoniae, Staphylococcus aureus, Nocardia spp., Mycoplasma spp.
Gram – bacteria	> 30%	Pseudomonas spp., Klebsiella spp., Escherichia coli, Group D enterococcus, Chlamydia trachomatis.
Fungi	Around 5%	Aspergillus spp., Candida spp., Fusarium spp., Pneumocystis jirovecii, Lomentospora prolificans.
Virus	3%	RhV, RSV, Influenza A, PIV, HBoV, HMOV, CMV, HHV-6/7/8, Adenovirus, Sars-CoV-2
Non classified	13%

Table 2.

Main opportunists in pediatric oncological patients affected by febrile neutropenia [72, 73].

Abbreviations. RhV: Rhinovirus; RSV: Respiratory Syncytial Virus; PIV: Parainfluenza; HBoV: Human Bocavirus; HMOV: Human Metapneumovirus; CMV: Cytomegalovirus; HHV-6/7/8: Human Herpesvirus types 6,7,8; SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus type 2.

4.1.1 Bacterial infections

One-fifth of pediatric patients with acute leukemia develop an infection. Ninety percent of these infections are caused by bacteria [72]. One critical infection associated with the cytotoxic effects of chemotherapy agents used for cancer treatment is neutropenic enterocolitis [75]. Patients with neutropenic enterocolitis present fever, abdominal symptoms, and radiologic bowel wall thickening. These symptoms are frequently associated with severe and life-threatening clinical conditions such as sepsis, perforations, and gastrointestinal bleeding [76]. Another serious condition in pediatric cancer patients with febrile neutropenia is bacterial sepsis. Despite international guidelines on sterile insertion and appropriate maintenance and use of central venous catheters, Gram-positive bacterial infections remain a common complication caused by contaminated tunneled long-term central venous catheters, and totally implanted devices or ports which are frequently used in cancer treatment [77, 78].

4.1.2 Fungi infections

Invasive fungal infection is a significant problem in neutropenic individuals [79]. The most frequent causes of infection are Aspergillus and Candida spp., although a growing number of other fungi (including species of Fusarium and Lomentospora) have been more recently implicated [80, 81]. The prevalence of invasive fungal infections has been estimated at around 23%, with a mortality rate of 9.45% [82]. The clinical manifestations of Candida infections involve more frequently cutaneous lesions, manifested as diffuse erythematous papules that usually do not develop central necrosis or eschar [83]. Unfortunately, recognizing the cutaneous manifestations of fungal infections in neutropenic patients is often delayed, resulting in more severe cases of not initiating a proper treatment sooner. For this reason, prophylactic treatment with amphotericin B and the triazole compounds itraconazole and fluconazole, is recommended soon in pediatric neutropenic patients [83, 84]. However, this course of action has led to the appearance of some resistant strains. Though the frequency of resistant strains is still low in neutropenic cancer patients, and mostly limited to Candida glabrata and Candida krusei, drug resistance in Candida albicans and Candida tropicalis has also been reported [81, 85]. Lomentospora prolificans, is a rare but highly virulent filamentous fungus with intrinsic resistance to antifungals. This microorganism has also been associated with a diversity of infections with high mortality in neutropenic patients. Indeed around 50% of patients with neutropenia develop this type of infection [86].

4.2 Oral disease related to neutropenia in pediatric oncological patients

In addition to recurrent infections of the skin, respiratory and urinary tracts, and bacterial sepsis, several lesions in the mouth area are also frequently observed in patients with neutropenia. Clinical symptoms include mouth ulceration in the palatal region, and in the posterior lateral region of the tongue, chronic gingivitis, and even periodontitis despite standard medical and dental care [87]. Premature bone loss can be observed in mixed dentition, in the inter-root area of the mandibular deciduous molars [45]. This is not too surprising since neutrophils are known to actively participate in controlling the oral microbiota and maintaining periodontal homeostasis [88]. Recently, it was reported that an increase in periodontal inflamed surface area (PISA), a new periodontal disease parameter, was strongly associated with cancer patients undergoing chemotherapy and having neutropenia, but not with cancer patients without neutropenia [89]. This association was independent of the types of blood cancer or treatment with human G-CSF [89]. Therefore, these reports strongly suggest that periodontitis treatment is recommended before starting cancer treatment as supportive care for preventing the onset of neutropenia during chemotherapy and later periodontal disease [87].

5. Neutropenia treatment in pediatric oncological patients

5.1 Prophylaxis

Pediatric patients with neutropenia are at the highest risk for infection. Infection prophylaxes have a focus on both pharmacologic and supplementary interventions. Bacterial and fungal prophylaxis decreases the risk of infection in certain high-risk groups. Consider utilizing bacterial and fungal prophylaxis in patients with acute myeloid leukemia or relapsed acute lymphoblastic leukemia. Adolescent and young adult Down syndrome patients may benefit from additional supportive care measures and protocol modifications [90].

5.1.1 Pharmacologic prophylaxis for bacterial infections

After acute lymphoblastic leukemia chemotherapy in children and adolescents, bacterial infections remain the principal cause of morbidity and mortality [91]. Even though systemic antibacterial prophylaxis is a well-established practice for adult patients [92], antibacterial prophylaxis in pediatric patients is still a matter of controversy [93, 94].

Bacteriemia can be highly reduced with the use of levofloxacin, and moderately reduced with sulfamethoxazole-trimethoprim, ciprofloxacin, fluoroquinolones, cefepime, vancomycin plus cefepime, and vancomycin plus ciprofloxacin [95]. The early detection of a bacteremia and the rapid therapeutic intervention are crucial to improve the outcome [32] of pediatric patients with neutropenia. Prompt empiric broad-spectrum antibiotic administration is collectively considered the best therapeutic approach [72]. Patients with acute leukemia are usually treated with empirical broad-spectrum antibiotics third- and fourth-generation cephalosporins and antipseudomonal penicillin [72].

Antimicrobial prevention strategies decrease bacterial infections caused from contaminated tunneled long-term central venous catheters and totally implanted devices or ports [77]. Unfortunately, administering antibiotics before the insertion of these catheters do not prevent Gram-positive related infections. Flushing or locking these catheters with an antibiotic solution tend to reduce Gram-positive infections but may increase microbial antibiotic resistance. Therefore, the use of antibiotics should depend on the risk of the patient to develop bacterial infections [77, 90].

5.1.2 Pharmacologic prophylaxis for fungal infections

Invasive fungal diseases are decisive causes of morbidity and mortality among febrile neutropenic patients after intensive chemotherapy. Significantly less fungal infections related with mortality are seen when using antifungal agents than when no antifungal treatment is used. Antifungal prophylaxis agents with broad-spectrum activity include itraconazole, amphotericin B, isavuconazole, rezafungin, olorofim, and manogepix or Posaconazole [96, 97].

Unfortunately, patients with prolonged neutropenia may still develop mucormycosis even under the prophylaxis of antifungals. If patients are refractory to monotherapy, a combined antifungal therapy is recommended. Also, when a patient is intolerant to amphotericin B the usual treatment is isavuconazole or posaconazole [97].

As with bacteria, there is not enough evidence to sustain the prophylactic use of antifungal agents in pediatric patients. It is recommended to perform trustworthy analysis for the diagnosis and follow-up of mucormycosis with CT scans, cultures, PCR tests, and histology. Additionally, the use of high-efficiency particulate air (HEPA) filters and neutropenic diets is needed to prevent fungal infections [97, 98].

5.2 Growth factor therapies such as granulocyte colony-stimulating factor (G-CSF)

There is a guideline based on evidence that recommends prophylactic treatment of G-CSFs to reduce febrile neutropenia incidence while improving chemotherapy dose delivery [35]. This treatment is effective to increase blood neutrophils in almost all cases [99]. G-CSF treatment several times weekly seems to correct the lack of production of neutrophils. Indeed, this treatment improves the clinical course of the disease because it decreases neutropenia from five days to one day. In patients with cyclic febrile neutropenia, inflammatory symptoms, and infections the treatment is reserved. Fortunately, the severity of symptoms related to cyclic neutropenia seems to diminish after the second decade of life, even though the cycling of the neutrophils continues. Optimal oral hygiene should be maintained to reduce the number and severity of oral infections [100].

6. How to proceed with pediatric patients with neutropenia

Neutropenia can be a common finding in pediatric patients. It is often benign but also it can be a life threat. There are mainly four causes for pediatric neutropenia: (1) There is a decrease in neutrophil production, (2) There is an inability to transfer mature neutrophils from bone marrow to peripheral blood, (3) There is an increase in margination and sequestration of neutrophils, also called pseudoneutropenia, (4) There is an increase in neutrophil destruction and clearance.

As we discussed throughout the chapter there are some risk factors that can help to diagnose and treat different types of pediatric neutropenia [51].

6.1 Risk factors to include in the clinic history

1. Ethnicity

2. Infections

   • Age of beginning of the infection

   • Site of infection. Does the infection reappear in the same site?

   • Severity of the infection. Does it become more sever with the frequency?

   • Cause of the infection. Are there any organisms isolated from these infections? Any viral infection?

   • Frequency of infection. Does the infection have a cyclical pattern?

3. Genetic and syndromic features

   • Family history

   • Has someone in the family presented neutropenia or any of the following conditions?

     • Glycogen storage disease 1b (GSD1b) mutation

     • Shwachman-Diamond syndrome

     • Wiskott-Aldrich syndrome

     • Barth syndrome

4. Treatment

   • What were the drugs used to treat neutropenia? In case there were previous episodes

   • Did there any other treatment for neutropenia? In case there were previous episodes

   • Cancer treatments

5. Pregnancy complications.

   • Hypertension

   • Intrauterine growth restriction

   • Placental blood flow restriction

   • Preeclampsia

   • Premature

   • Prevention

6.2 Diagnose

1. Neutropenia characteristics

   • Acute (less than one month)

   • Chronic (more than three months)

   • Congenital

   • Acquired

   • Associated with infections

2. Genetic test for mutations in ELANE and other genes.

3. Last full blood count

   • Frequency

4. Signs of chronic infection.

   • Common sites of infection in pediatric neutropenia are the membranes, mouth, mucus, and skin.

   • Presence of mouth ulcers or gingivitis

   • Presence of abscess/purulent exudate

   • Pneumonia

   • Septicemia

6.3 Treatment

1. Take full blood counts frequently until neutrophil number recovers and discard benign neutropenia.

2. If the patient is at increased risk of infection

   • Start antibiotic treatment

   • Start granulocyte stimulating factor treatment

3. Is there a serious underlying disorder causing neutropenia?

   • Look for maternal neutrophil antigen

   • A bone marrow biopsy can be useful in cases with prolonged neutropenia

7. Conclusion

Neutrophils are very important to prevent infections. Because of chemotherapy, most patients develop some class of neutropenia. The treatment for preventing bacterial and fungal infections in pediatric patients is still under study. If antibiotic prophylactic treatments are used in pediatric patients undergoing chemotherapy, a close monitoring of antibiotic resistance should be addressed. Also, hospital or clinic infrastructure to diagnose and monitor patients is ideal. Finally, personal environment and hygiene are also factors to consider.

Acknowledgments

Authors thank Israel Alvarado Oaxaca for helping with references edition. Research in authors’ laboratories is supported in part by grant PAPIIT IN222120 (to EU-Q) and PAPIIT IN205523 (to CR) from Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México, and grant CF-2023-I-610 (to CR) from Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAHCyT), Mexico.