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Keywords:

  • multidisciplinary approach;
  • paediatric tumours;
  • fine needle biopsy;
  • cytology;
  • rapid on-site evaluation;
  • ancillary techniques

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. A multidisciplinary approach to diagnosis
  5. A practical approach and the role of ancillary techniques
  6. Summary
  7. Acknowledgments
  8. References

Fine needle biopsy (FNB) with cytology has long been regarded as an excellent technique as the first choice for diagnosing adult tumours. Being an inexpensive minimally invasive technique with high accuracy and diagnostic immediacy through rapid on-site evaluation, it is also ideal for implementation in the paediatric setting, particularly in developing countries. Furthermore, it allows complementary and advanced procedures such as flow cytometry, polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH), among others, which enhances the diagnostic capacity of this technique and gives it a key role in risk stratification and therapeutic decision-making for several tumours. The advantages of FNB are optimized in the setting of a multidisciplinary team where cytologist, clinician and radiologist play leading roles. Paediatric tumours are rare and most ancillary techniques are cost-effective but complex to be implemented in small centres with limited experience in paediatric pathology. Therefore reference centres are essential, in order to establish teams with extensive experience and expertise. Hence, any child with a suspected malignancy should be directly referred to a paediatric oncology unit. Focusing on a practical approach to the assessment of paediatric lymphadenopathies and non-central nervous system solid tumours we review the effectiveness of FNB as applied concurrently with ancillary techniques in a multidisciplinary approach to the diagnosis, prognosis and therapeutic decisions of paediatric tumours and tumour-like lesions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. A multidisciplinary approach to diagnosis
  5. A practical approach and the role of ancillary techniques
  6. Summary
  7. Acknowledgments
  8. References

In the last few years we have witnessed major steps forward in the diagnosis and therapy of paediatric cancer. The improvement in survival rates of children with cancer is a remarkably successful achievement in the whole oncology field. Several factors have contributed to this progress: (1) a multidisciplinary team approach; (2) treatment stratification based on the results of international cooperative protocols; (3) combining and optimizing chemotherapeutic regimens; and (4) continuous development in the understanding of cancer biology.

Fine needle biopsy (FNB) with cytology is a widely accepted technique as the first line to the diagnosis of paediatric masses. Its most attractive qualities are its diagnostic immediacy and high diagnostic accuracy with minimal invasiveness.

In an initial approach to diagnosis, the main goals of FNB are to separate benign from malignant clinical settings, and to proceed for prompt therapy avoiding needless surgery, longer recovery times and lengthy hospitalization. Paediatric cancer is a psychological emergency for the child and their family. No other disease carries the serious psychological burden of paediatric cancer. Paediatric cancers differ from adult cancers in many ways. They are in general characterized by a high proliferative rate and invasiveness, reaching large sizes and compressing vital structures. Consequently, paediatric cancer does not stand the delaying times that may be acceptable in adults. Decisions should be quick and effective. This is one of the major reasons for the use of FNB in paediatrics.

Cytogenetic and molecular studies provide pivotal biological and clinical insights into paediatric pathology. An understanding of the potential role of tumour biology led to a huge improvement in therapies, identifying therapeutic risk groups and achieving better outcomes while reducing unwarranted late effects and morbidity. The use of a rapid, safe and accurate approach, allowing the collection of enough material for genetic testing, underlines the importance of FNB in the initial assessment of paediatric tumours. Furthermore, most treatment schemes in paediatric solid tumours use pre-operative chemotherapy for tumours not amenable to immediate resection.

Paediatric tumours are rare and most ancillary techniques are cost-effective but too sophisticated to be implemented in small centres with limited experience in paediatric pathology so, in order to establish a team with enough overall experience, centralization is essential.

In this work, we will review the usefulness of a multidisciplinary approach, as well as the role of FNB in the paediatric setting when combined with ancillary techniques. We will concentrate on the role of FNB in the assessment of paediatric lymphadenopathies and solid non-central nervous system tumours, with the aim of addressing the challenge raised by both conditions, in accordance with their relative frequency and usual presentations. We will also comment on the advantages and disadvantages of FNB compared with core biopsy, and the situations in which the latter may be indicated.

Incidence of cancer in childhood

Although cancer is rare among those younger than 20 years of age, the presence of a lump or a mass in a child is always a cause of concern and should be subjected to clinical evaluation. In Europe about 15 000 children under 14 years are diagnosed with cancer each year.[1] The Advanced Composites Centre for Innovation and Science (ACCIS) project has reported an increase in the incidence for all types of cancer both in children and adolescents in the last decades of about 1–1.5% per year.[2]

Types of cancers in childhood according to age group

Cancer in children encompasses a spectrum of different malignancies varying with the histology type, age, location, race and gender (Figures 1 and 2). In children younger than 14 years, leukaemia and central nervous system tumours represent the two most frequent paediatric cancers. In infancy, malignant tumours correspond to about 10% of all tumours and although teratomas are the most common neoplasm in this age group, neuroblastomas (NB) seem to represent the most frequent malignant tumour, whereas leukaemia, central nervous system tumours, retinoblastoma, nephroblastoma, germ cell tumours and soft tissue sarcomas represent the next most frequent malignancies in this age group.[3, 4] Some tumours are essentially diagnosed among infants, such as mesoblastic nephroma, congenital fibrosarcoma, rhabdoid tumour of the kidney and hepatoblastomas, whereas others, such as clear cell sarcoma of the kidney, nephroblastomas, NBs and rhabdomyosarcomas (RMS) may affect a broader range of ages in spite of predominance in the first decade of life. Finally other tumours such as bone tumours, carcinomas, Hodgkin lymphoma and germ cell tumours mainly affect teenagers (Figure 2).

image

Figure 1. Topographic distribution of non-central nervous system sporadic paediatric tumours.

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image

Figure 2. Distribution of sporadic paediatric non-central nervous system tumours according to age group. *Kidney; **Liver; ***in developed countries.

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Types of cancers in childhood according to topographic location

Apart from the central nervous system, the three most frequently affected topographic compartments for paediatric masses are the abdomen/retroperitoneum, mediastinum and extremities.[5] Except in the neonatal period, most abdominal masses should be clinically considered as likely to be malignant.[6] The kidney, adrenal and liver are frequently the source of benign and malignant neoplasms. Not infrequently non-Hodgkin lymphomas and germ cell tumours also occur in these locations. Mediastinal masses encompass a heterogeneous group of malignant and benign entities, best characterized by their anterior-posterior situation (Figure 1).[7] Malignant tumours of the extremities are diagnosed most commonly in teenagers and young adults and affect bones [Ewing family of tumours (EFTs) and osteosarcoma] or soft tissues. RMS is the most common soft tissue sarcoma in children and, whenever arising in the extremities, is most frequently of the alveolar subtype.

A multidisciplinary approach to diagnosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. A multidisciplinary approach to diagnosis
  5. A practical approach and the role of ancillary techniques
  6. Summary
  7. Acknowledgments
  8. References

Clinical presentation of childhood cancer

Childhood cancer can present with signs and symptoms shared by other childhood diseases, whose detailed description is beyond the scope of this review. Laboratory testing can provide further clues to diagnosis and assess disease progression. An elevated white blood count can be observed in lymphomas. Thrombocytosis is frequent in hepatoblastomas. Various degrees of pancytopaenia can be detected in extensive bone marrow infiltration by lymphoma, neuroblastoma (NB) or EFTs. NB can present as a systemic disease with pancytopaenia and metastasis to the retro-orbital tissues (‘Racoon eyes’). Some tumour markers are very useful in sorting out the diagnosis: urinary catecholamines in NB, serum alpha-fetoprotein and beta human chorionic gonadotropin (β-HCG) in liver tumours or germ cell tumours. Serum lactate dehydrogenase is usually elevated in dysgerminoma.

Paediatricians should be aware of a personal and family history, and of any physical sign that may warn of the possibility of hereditary cancer syndromes, metabolic diseases or a tumour predisposition syndrome. An infrequent diagnosis in a given age group, an odd location, or the presence of bilateral tumours, should alert to such a possibility. Moreover, the knowledge of a clinical syndrome background should also be taken in consideration as it points to the most probable oncological diagnosis in that context. The pathologist should be informed of all these clinical details.

Besides age and clinical presentation, location and imaging features provide other clues for reducing the possible differential diagnoses (Figures 1 and 2) and a multidisciplinary approach with radiology plays a key role in these settings. The goals of initial tumour imaging include detection, description, differential diagnosis, extent, staging and imaging-guided biopsy of the tumour. In clinical practice various combinations of imaging techniques are used to achieve these aims.

Imaging in the investigation of childhood tumours

Sonography is frequently the initial imaging method in the work-up of abdominal, pelvic and soft tissue masses. It is widely available, does not require sedation and gives valuable information such as localization, morphological features (solid/cystic areas, calcifications, vascular encasement and lymphadenopathy) and the relation to neighbouring organs and structures. After sonography better resolution is generally required with computed tomography (CT) or magnetic resonance imaging (MRI).

CT is commonly employed as a first examination because it requires a short scan time and can be used without or with only minor sedation, and in severely ill patients. It is the favoured method to detect lung metastases. It has the disadvantages of using ionizing radiation and iodinated contrast agents. Consequently, it is not indicated for follow-up studies, which need several repeated examinations to evaluate a response to treatment or a relapse. MRI is the preferred imaging method for paediatric oncology, not only because of the absence of ionizing radiation but owing to the superior soft-tissue contrast resolution. It is also frequently needed in suspicious soft-tissue lesions. However, MRI requires a calm child, and it is also time consuming. There is controversy over whether general anaesthesia or sedation should be used. In our hospital the support for these children is provided by anaesthesiologists. These sophisticated imaging techniques and experienced radiologists are not accessible in peripheral hospitals or health centres, emphazing once again the idea that it is essential that paediatric cancer should be dealt with in reference centres.

Planning and carrying out the fine needle biopsy procedure

A multidisciplinary approach is also crucial in the correct planning of the biopsy procedure in order to sample the areas of the mass most likely to be informative, and to gather precise anatomical details that will allow the most accurate diagnosis. It is critical that the pathologist has the perception of the numerous tissues crossed by the needle, which in spite of the use of mandrel needles are usually dragged on its way and may be seen as contaminants.

It is sometimes important to collect material from the neighbouring areas in order to compare them with the tumour sample; this is particularly helpful in well-differentiated hepatic tumours. FNB can be used in palpable or non-palpable lesions. In our institution deep lesions are usually managed by a radiologist or interventional radiologist, with ultrasound or CT guidance, together with the pathologist, whereas palpable lesions are often biopsied by the pathologist.

According to our experience, FNB should preferably be performed without aspiration, using a 25-gauge needle. Depending on the morphological features of the lesion (such as marked vascularization with the risk of haemorrhage or marked desmoplasia) thinner (27-gauge) or larger (23-gauge) needles should be used. The mandrel needles enable the collected sample to be split for various purposes, controlling the amount of ejected material from the needle. Each pass should collect material for at least two or three smears, and rinse into one or two PBS/RPMI Eppendorf tubes. In spite of these advantages, mandrel needles are longer and more difficult to control.

Performing FNB in children is no different from similar procedures in adults; however, several points should be considered: (1) in paediatric oncology the patient–physician relationship is a ‘four person dialogue’: one has to deal not only with the child but also with the parents; (2) children reflect the parent's attitude: calm and informed parents will reflect into the child; and (3) children are in general averse to needles and consequently are less cooperative than adults. One can encourage a child to perform an initial biopsy, but one will have to take into account it being difficult to obtain a sample of good quality on a second attempt. Therefore one should try to get in the first attempt enough material for cytological examination and other laboratory tests. Minimizing the discomfort, with the use of some basic rules, will prevent some of these difficulties:

  1. FNB should be carried out with time and serenity;
  2. Tranquillity should be encouraged in the parents and the child;
  3. Far more frequently than adults, children require sedation for a variety of procedures or imaging tests, usually to access deep or less easily reached locations. When dealing with a probable malignant neoplasm and scheduling for immediate chemotherapy requiring a central venous catheter, or bone marrow biopsy for clinical stage evaluation, FNB should be performed in an operating theatre under general anaesthesia. This procedure allows a number of invasive medical procedures, saving anaesthetic episodes, without major discomfort or distress for the child. The use of topic anaesthetics, (lidocaine/prilocaine cream) or even superficial sedation in extremely anxious children/parents should be considered;
  4. Rapid on-site evaluation (ROSE) should be used whenever possible. A preliminary interpretation of the sample using a quick Giemsa stain will allow, in situations where malignancy is suspected and in which carrying out the FNB in the operating theatre under general anaesthesia is an option, a multidisciplinary team approach where the surgeon, the clinician and the haematologist will have supporting roles in achieving an adequate procedure. ROSE also has an important role in assessing sample adequacy and allows the collection of additional samples for ancillary studies. Needle rinse samples from lymphoid lesions can be used for flow cytometry (FC) in evaluating potential lymphoproliferative lesions (Table 1). When enough material is obtained, a cell block or cytospins can be performed later in the laboratory for histochemical, immunohistochemical and molecular studies. In paediatric oncology, pathologists are challenged, in addition to providing an accurate diagnosis, with predicting the tumour behaviour and frequently contributing to a therapeutic decision Although gene targeting therapy is not yet a current practice in paediatric tumours, the knowledge of the molecular features of the tumour is essential to decide the best therapeutic strategy. Good results with these ancillary techniques are attained both directly in cytological smears, in cytospins, cytoblocs, and in liquid-based thin-layer preparations.[8-10] Microbiological cultures can be performed from lesions that appear to be inflammatory/infectious.
Table 1. Immunophenotype markers in non-Hodgkin lymphomas
AntibodyLBLLTLLPLFLBLDLBCL GCDLBCL non GCAL
BehaviourAggressive with cure Indolent/no cureIndolentAggressive/with cure Aggressive/with cureAggressive/with cure
  1. a

    Adverse prognosis. LBL, lymphoblastic B-cell lymphoma; LTL, lymphoblastic T-cell lymphoma; LPL, lymphoplasmacytic lymphoma; FL, follicular lymphoma; BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; GC, germinal centre; AL, anaplastic lumphoma.

sIg ++−/+weak+++ 
Igs   + +++ 
CD79a++/− + ++ 
CD19++++++ 
CD34+  
Tdt++
CD20+/−++++++ 
CD23  +/−    
CD43+      
CD10+/−+/−+++++ 
FMC7 +++ + 
CD138  +    
CD38  ++++   
CD30+
EMA     +
CD3+    +/−
ALK   +/−
BCL2 +++a  
BCL6 ++ ++/− 
Cyclin- D1 
PAX5+++ ++ 
MUM1   +/− + 

Indications for core biopsy in addition to or instead of FNB cytology

In lesions hard to puncture, mainly owing to fibrosis/sclerosis, as in Hodgkin lymphoma, ganglioneuroblastoma and in some soft tissue tumours with dense stroma, the sample collected is scarce not allowing a reliable diagnosis; ROSE may play an important role helping in the decision for other prompt methods. In these situations the choice to perform a core biopsy or even a surgical biopsy can be crucial. Most protocols also advocate the need for a core biopsy based on the requirement to save tissue for tumour banks. The decision to perform FNB instead of a core or incisional biopsy should be carefully evaluated and tailored individually, and is dependent on the department experience and on the available technology.[11, 12]

Despite the aforementioned benefits of a core needle biopsy, in our experience morphology is best preserved in cytological smears. Our claim is mainly relevant in small round cell tumours and lymphomas where the technical artefacts, namely crushing artefacts that are as a result of the procedure, can change the normal morphology and cytology features or even lead to a complete inadequacy of the specimen. Another major drawback of the core biospy is based on the difficulty in monitoring the representativeness of the collected material for additional ancillary techniques. Nevertheless, whenever architecture is required for diagnosis or even an extensive panel of ancillary techniques is necessary, a core needle biopsy or surgical biopsy undoubtedly has advantages.

A practical approach and the role of ancillary techniques

  1. Top of page
  2. Abstract
  3. Introduction
  4. A multidisciplinary approach to diagnosis
  5. A practical approach and the role of ancillary techniques
  6. Summary
  7. Acknowledgments
  8. References

In spite of the urgency of the clinical situation, there are not many emergencies in paediatrics thus allowing time in most situations to plan the best approach to treatment only after the tumour has been accurately diagnosed and the extent of disease precisely defined. When the probability of a neoplasm is clinically established, the next decision is the selection of the quickest and most reliable method of establishing the pathological diagnosis. The paediatric oncologist should discuss with a paediatric surgeon, an interventional radiologist and a pathologist the optimal site to biopsy, the amount of tissue needed, and the specimens to be collected. The two most common clinical situations in which FNB is required are the assessment of lymphadenopathies and of solid lumps or masses.

Lymphadenopathies

A suspicious or persistent lymphadenopathy in children is a frequent situation that the paediatrician has to cope with routinely. The challenge is to reassure the parents' and the physician's fears of malignancy and to do so in a safe, timely and cost-effective manner. In spite of this, FNB should preferably be restricted to cases with a persistent enlarged lymph node or a strong clinical suspicion of a specific infection or neoplasia. Performing an adequate clinical and physical examination is extremely important. Most children have palpable small cervical, axillary and inguinal nodes, but in some locations these are considered definitely abnormal, namely lymphadenopathies in the posterior auricular, epitrochlear and supraclavicular areas.

Reactive lymphadenopathy

Most lymphadenopathies in children are benign and self-limited, and are frequently as a result of viral or bacterial (Staphylococcus aureus and β-haemolytic Streptococcus) or mycobacterium infections. Other less common causes include fungal infection (Figure 3a) and primary or metastatic malignancies. Mycobacteria, fungi or a virus can be identified in cytological preparations with haematoxylin and eosin staining (Figure 3b) or with the assistance of histochemical techniques such as Ziehl Neelsen, Grocott, (Figure 3c), Gram, Gomori's methenamine silver (GMS), periodic acid-Schiff (PAS), alcian blue and even more specific immunostains. Fine needle samples can also be rinsed into a specific culture medium and sent to a microbiology laboratory for bacterial cultures or polymerase chain reaction (PCR).

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Figure 3. (a) Cervical nodules in a 12-year-old African boy. (b) Cytological preparation shows numerous spherical to oval fungal cells (haematoxylin and eosin ×600). (c) A thick double wall on Grocott staining permits the diagnosis of Histoplasma Capsulatum (Duboisii) (Grocott ×600).

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Lymphomas and leukaemia

The primary malignant tumours that involve the head and neck in children are most often lymphomas. The child's age conveys important diagnostic information evaluating enlarged nodes. In children younger than 6 years of age, the most common malignancies are non-Hodgkin lymphoma and leukaemia. From 7 to 13 years of age, Hodgkin lymphoma and non-Hodgkin lymphoma are the most common and over 13 years of age, the former predominates. Nevertheless, although unusual, solid tumours such as neuroblastoma and RMS can present as a lymph node metastasis.

FNB is an excellent technique for addressing lymphomas; however, to perform a diagnosis of lymphoma, nowadays it is no longer acceptable to carry out FNB without using ancillary techniques, namely flow cytometry. The combination of flow cytometry and classic cytological features represent a powerful combination of methods in the accurate diagnosis and subclassification of lymphomas.[13] According to WHO, lymphoma subtyping is based on morphology, immunophenotype and molecular characterization of the lymphoid cells.[14] Most non-Hodgkin lymphomas can be characterized with this multidisciplinary approach. Immunophenotype characterization of lymphocytes can be accomplished by immunocytochemistry (ICC) (preferably in cytospins or cell blocks) or by flow cytometry (Table 1). Immunostains on cell blocks or cytospins require a sample of additional quantity and quality, without blood contamination; a condition that is not always feasible when dealing with children. FNB samples can also be used to perform molecular studies, such as PCR or fluorescence in situ hybridization (FISH), in order to detect translocations, deletions and gene amplifications in the DNA, which are specific of several subtypes of lymphomas (Table 2).[15]

Table 2. Most common molecular alterations found in frequent paediatric lymphomas
LymphomaMolecular alteration
Burkitt lymphomat(14;18)(q32;q21)
t(8;14)(q24;q32)
t(2;8)(p12;q24)
t(8;22)(q24;q11)
Anaplastic large cell lymphomat(2;5)(p23;q25)
t(1;2)(q25;p23)
t(2;3)(p23;q21)
t(2;17)(p23;q23)
t(2;X)(p23;q11-12)
t(2;19)(p23;p13)
t(2;22)(p23;q11)
t(2;17)(p23;q25)
Lymphoblastic T-cell lymphomat(1;14)(p32;q11)
t(10;14)(q24;q11)

Sporadic Burkitt lymphoma frequently presents as a large and rapidly growing abdominal mass with spontaneous tumour lysis syndrome, creating a therapeutic emergency. In Europe and North America, Burkitt lymphoma represents around half of all malignant non-Hodgkin lymphoma in children. In spite of its characteristic round-to-oval nuclei, multiple small nucleoli, speckled chromatin (Figure 4a) and tiny cytoplasmic lipid vacuoles (Figure 4b), other entities such as lymphoblastic lymphoma, diffuse large B-cell lymphoma (DLBCL) and plasmablastic lymphoma can mimic this cytological pattern. Burkitt lymphoma can exhibit an atypical morphological appearance, raising some doubts in the differential diagnosis even with other small round cell tumours. Evaluation of the tumour immunophenotype is crucial in the differential diagnosis with other entities (Figure 4c and Table 1). However, an unusual immunophenotype is not uncommon in Burkitt lymphoma and detection of the typical t(8;14), c-myc translocation is sometimes a determining factor for the correct diagnosis (Figure 4d). A more superficial idea of all these techniques may give us the misconception that they represent a solution for the diagnosis. MYC translocation for instance, although a consistent feature of Burkitt lymphoma, is not specific and may be shared with DLBCL. Once again we emphasize the importance of the multidisciplinary approach in the evaluation of the appropriate techniques.

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Figure 4. Burkitt lymphoma fine needle biopsy. (a) A monotonous population of intermediate-sized cells: nuclei have a characteristic speckled chromatin (haematoxylin and eosin ×600). (b) Deep blue cytoplasm with tiny lipid vacuoles can be seen on Giemsa staining (×600); note the close resemblance to rhabdomyosarcoma in Figure 5c. (c) Demonstration on flow cytometry of a monoclonal (K expression) B-cell population with intense staining with CD19; CD10 and CD38, in the absence of leukocyte-associated IG-like receptor (LAIR) expression, point to the diagnosis of Burkitt lymphoma. (d) Fluorescence in situ hybridization analysis using Vysis LSI IGH/MYC, CEP 8 Tri-color, Dual Fusion Translocation Probe shows a classic MYC/IgH rearrangement pattern with two fused signals (IGH-MYC fusions on der(8), t(8;14) and der(14), t(8;14)), one red signal (normal MYC allele), one green signal (normal IGH allele) and two blue signals for the two centromeric probes for chromosome 8.

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Hodgkin lymphoma comprises about 6% of childhood cancers according to the National Cancer Institute (NCI) data. An accurate cytological diagnosis of Hodgkin lymphoma relies on a combination of morphological findings: Reed–Sternberg (RS) cells in a background of small mature T cells, (CD57-positive) and immunostains (CD30 and CD15 immunophenotype in RS cells). To date, flow cytometry has not been considered useful to confirm this diagnosis. In most situations, RS cells represent less than 1% of the total cells present in the sample, and fall outside the area that is usually used for recognition of individual (non-aggregated) cells, or remain located at the very edge or beyond the high end of the measurable range of forward scatter parameters set of analysis. Besides this, the rosette complex, between RSC and T-cell lymphocytes, which are generally produced in Hodgkin lymphoma, display a composite immunophenotype combining these two cell immunophenotypes, thus effacing the CD30 and CD15 that typically characterizes RS cells.[16, 17] Recently, modern cytometers have increased sensitivity, as well as the capacity to identify these minor populations with increased confidence.[17]

Hodgkin lymphoma should also be differentiated from other non-Hodgkin lymphomas that may involve the mediastinum of children, namely mediastinal large B-cell lymphoma (MLBCL) and anaplastic large cell lymphoma, mainly the large cell subtype (ALCL). In both these lymphomas cytological features can be similar; however, both in MLBCL and in ALCL neoplastic cells are CD45-positive, which differentiates them from RS cells, and in ALCL neoplastic cells can express T-cell markers (CD2 and CD4). In a great majority of the cases neoplastic lymphocytes of ALCL also express ALK protein and in FISH analysis a t(2;5) or t (1;2) can be detected (Table 2). The differentiation between Hodgkin and anaplastic lymphoma is critical, owing to the fact that they have a different prognosis and anaplastic lymphoma implies a different and more intensive treatment. Sometimes in reactive lymph nodes, sustentacular reticular or endothelial cells assume atypical shapes, similar to popcorn or RS cells. The differential diagnosis lays in the careful observation of the remaining lymphoid population which is polymorphic as well as the presence of macrophages with tangible bodies, which are more frequent in a reactive lymph node.

Solid tumours

Small round cell tumours

These tumours share a similar light microscopic morphology. An experienced cytopathologist will detect subtle details that will allow a more accurate diagnosis in the majority of routine cases (Table 3). Most of these tumours are associated with highly specific recurrent chromosomal translocations and are easily detected with basic molecular tools (Table 4).[15] FISH performed with break-apart probes (with split signals reflecting presence of gene fusion) or dual fusion translocation probes (with fusion of signals indicating presence of gene fusion) can identify essentially all known breakpoints of a given translocation, allowing increased sensitivity. Besides the advantages of evaluating multiple breakpoints, FISH is also helpful in situations in which one translocation partner is constant but the second translocation partner changes. The high sensitivity contrasts with the lower diagnostic specificity of the FISH technique which ought to be addressed by a multidisciplinary team.

Table 3. Cytological evaluation of small round cell tumours
 Diagnostically important featuresDiagnostic ancillary techniques
  1. NB, neuroblastoma; EFT, Ewings family of tumours; ARMS, alveolar rhabdomyosarcoma; DSRCT, desmoplastic round cell sarcoma; EMCS, extraskeletal myxoid chondrosarcoma; WT, Wilms tumour/nephroblastoma; ICC, immunocytochemistry.

NB

Neuropil background

Dispersed cells and clusters

Rosette formation

Anisokaryosis

Cells in different maturation stages

Salt and pepper chromatin

ICC:

 Positive: NB84, synaptophysin; CD56;chromogranin; Leu 7

 Negative: vimentin; CD99; CK; myogenin

EFT

Tigroid background

Dispersed or poorly aggregated cells

Occasional rosettes

Double cell population : large light and small

dark cells

Cytoplasmic glycogen vacuoles

Bland Monotonous nuclear morphology

Inconspicuous nucleoli

ICC:

 Positive: synaptophysin; chromogranin; CD99; FLI 1; CAV; EMA; CK; vimentin

 Negative: CD56; myogenin; CK20

Molecular: t(11;22)(q24;q12)- 85–90%

ARMS

Cellular smears

Apoptotic bodies

Tigroid background

Loosely cohesive aggregates

Uniform cells

Little or no specific differentiation

Rhabdoid phenotype can be seen

Multinucleated neoplastic giant cells with

eosinophilic cytoplasm

Fine chromatin

Inconspicuous nucleoli

Cytoplasmic glycogen vacuoles

ICC:

 Positive: vimentin; desmin; Myo-D1; myogenin; CD99; synaptophysin; CD56

 Negative: chromogranin; CD45

Molecular: t(2;13)(q35;q14); t(1;13)(p36;q14)

DSRCT

Poorly cellular sample

Occasionally stromal fragments

Dispersed cells and loosely cohesive clusters

Nuclear moulding

Fine chromatin

Inconspicuous nucleoli

Scant cytoplasm

ICC:

 Positive: WT1;CD99; AE1/AE3; desmin; vimentin; NSE

 Negative: myogenin; Myo-D1; chromogranin

Molecular: t(11;22)(p13;q12)

Synovial sarcoma

Hypercellular smears

Tight tissue fragments with irregular borders

Vascular network

Dispersed round cells

Bland cells with inconspicuous nucleoli

Scant to abundant cytoplasm

Stripped nuclei

Mast cells

ICC:

 Positive: BCL2; CD99; EMA; vimentin

 Negative: desmin; CD56; CD34; WT1

Molecular: t(X;18)(p11;q11); t(X;18)(p11;q13); t(X;20)(p11;q13)

EMCS

Aspirate with abundant whitish fluid

Metachromatic myxoid/chondroid matrix

Round to oval cells

Nuclei with fine granular chromatin

Nuclear grooves can be present

Small prominent nucleoli

Pale blue cytoplasm

Multinucleated cells

ICC:

 Positive: S100;vimentin; lysozyme; synaptophysin

 Negative: EMA; CK

Molecular: t(9;22)(q22;q12); t(9;17)(q22;q11); t(9;15)(q22;q21); t(9;22)(q22;q15)

 

WT (blastema)

Isolated cells, diffuse sheets or pseudo rosettes

Nuclear moulding

Monotonous pale nuclei

Very fine chromatin

Frequent in smears owing to poor cohesiveness

ICC:

 Positive: vimentin; WT1; BCL2; CK; NSE; synaptphysin; NB84; CD56; CD99

Table 4. Paediatric tumours sharing the same or similar molecular alterations
Shared GeneMolecular alterationGene fusionTumour
EWSR1t(11;22)(q24;q12)EWSR1-FLI1Ewing family tumours
t(21;22)(q22;q12)EWSR1-ERG
t(7;22)(p22;q12)EWSR1-ATV1
t(2;22)(q33;q12)EWSR1-ETV4
t(11;22)(p13;q12)EWSR1-WT1Desmoplastic small round cell tumour
t(21;22)(q22;q12)EWSR1-ERG
CREB1t(12;22)(q13;q12)EWSR1- ATF1Clear cell sarcoma
t(2;22)(q33;q12)EWSR1-CREB1
CREB1t(12;22)(q13;q12)EWSR1-ATF1Angiomatoid fibrous histiocytoma
t(2;22)(q33;q12)EWSR1-CREB1
t(9;22)(Q22;q129EWSR1-NR4A3Extraskeletal myxoid chondrosarcoma
t(12;22)(q13;q12)EWSR1-ATF1Myxoid/round cell liposarcoma
 EWSR1Neuroendocrine carcinomas
 EWSR1Soft tissue myoepithelioma
ETV6t(12;15)(p13;q25)ETV6-NTRK3Congenital mesoblastic nephroma
NTRK3t(12;15)(p13;q25)ETV6-NTRK3Congenital fibrosarcoma
ASPSLt(X;17(p11;q25)ASPSL-TFE3Alveolar soft part sarcoma
TFE3t(X;17(p11;q25)ASPSL-TFE3Renal cell carcinoma
Neuroblastoma

NB is one of the most common malignant tumours in paediatrics. Approximately two-thirds arise in the abdomen/retroperitoneum. Neuroblastoma embodies the classic example in which cytology and genetics work together in defining the clinical behaviour and treatment stratification. The great majority are not associated with any inherited condition or syndrome. Iodine-123 metaiodobenzylguanidine (MIBG) scintigraphy is mandatory for diagnosis and staging. Currently, treatment stratification requires the evaluation of DNA content (ploidy), genetic testing for MYC amplification and segmental chromosomal anomalies.[18] Neuroblastomas are rapidly growing tumours and may present as an oncologic emergency, owing to local mass effect, massive hepatomegaly, spinal cord compression, paraneoplastic syndrome or even catecholamine release.[19, 20] A good prognosis is associated with ages younger than 18 months, low disease stage (Stage 1, 2, or 4S), DNA triploidy, the absence of segmental chromosomal alterations and a lack of N-myc amplification. Cases with these features are expected to have a 3-year survival rate of 95% with no or little therapy.[21] In contrast to this, patients whose tumours lack all these features have a poor prognosis, even with strengthened therapy.[21]

At the time of diagnosis, material should be kept to evaluate DNA content, and also to perform FISH or Southern blotting analysis to detect N-myc amplification. Although in the current International Neuroblastoma Risk Group (INRG) histology is required for evaluation, (core needle or surgical biopsy), in our experience we have discovered that FNB material is excellent and accurate for diagnosis as well as for the evaluation of ploidy and in the assessment of other prognostic factors (e.g. N-myc status, the detection of possible chromosomal alterations, del 1p, dupl 17q21); and, above all, in only a few hours after admission.[22, 23]

Rhabdomyosarcoma

RMS is the most common soft tissue sarcoma in childhood.[24] In spite of its age-dependent location, retroperitoneal presentation is not uncommon (Figure 5a). Alveolar RMS (ARMS) usually presents as a small round cell tumour with occasional rhabdomyoblasts or multinucleated giant cells and, besides the presence of glycogen cytoplasmic vacuoles (Figure 5b) and of a tigroid background that can guide an experienced cytologist to the diagnosis, it is essential to differentiate it from other tumours sharing a similar cytological pattern namely neuroblastoma, DSRCT, EFTs, synovial sarcoma (SS), rhabdoid tumour or even lymphoma (Burkitt lymphoma).[18] A broad panel of immunostains can help in the correct diagnosis such as myogenin and MyoD1 (muscle-related antibodies), which are characteristic of RMS (Figure 5c). Two specific translocations involving the FKHR gene are found in ARMS, detectable in FNB material (Figure 5d). The translocation (2;13)(q35;q14) is identified in nearly 60% of cases, and t(1;13)(p36;q14) is found in approximately 20% of cases.[25, 26]

image

Figure 5. Rhabdomyosarcoma. (a) Magnetic resonance imaging (MRI) from an adolescent female displaying a huge retroperitoneal mass that involves the bowel. (b) Fine needle biopsy: a small round cell tumour with small glycogen cytoplasmic vacuoles (Giemsa ×400). (c) Cytospins: intense staining to myogenin (immunocytochemistry ×100). (d) Fluorescence in situ hybridization: three interphase nuclei studied with the FKHR Break Apart Rearrangement probe. The lower nuclei exhibits a pattern typical of a FKHR rearrangement, with separation of the centromere (green) and telomere (red) probes to the FKHR locus.

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Ewing family tumour

EFT is the second most common primary soft/bone tissue malignancy in childhood. Recently, numerous variants (adamantinoma-like, large cell, spindle cell, sclerosing, clear cell and vascular-like) as well as cases displaying atypical morphological differentiation (e.g., epithelial differentiation) and infrequent immunostaining features (e.g. epithelial markers) have been described in EFTs.[27-29] In spite of this morphological dissimilarity, all variants share an identical genetic profile.[19, 30-32] In its classic pattern, EFT typically shows a characteristic cytological poorly differentiated round cell pattern. CD99, FLI1, CD57 (HNK-1) and CAV1 are positive markers that characterize this tumour. The absence of CD56/NCAM expression, and of myogenin, allows the exclusion of other small round cell such as neuroblastoma and RMS, and neuroblastoma is rarely positive for CD99 (Table 5).[33] FLI1 although initially regarded as a more promising marker than CD99 is regarded by some authors as a marker of variable sensitivity and specificity.[9, 31, 34-37] In 20% of EFTs, cytokeratin (AE1AE3) may be expressed, as well as synaptophysin and EMA, hindering distinction from the undifferentiated small cell variant of SS or even a neuroendocrine carcinoma.[38] However, cytokeratin expression is focal in most cases. Recently, we had a case of an EFT with intense and diffuse expression of high-molecular-weight cytokeratins (cytokeratin 5) and p63, and focal expression of chromogranin (Barroca et al., [39]).[39]

Table 5. Small round cell tumours: CD99 positive tumours
Tumours with CD99 immunoexpressionCrucial immunostains in differential diagnosis
  1. a

    Variable expression.

Ewing family tumourPositive: FLI-1, CAV, vimentin
Negative: CD56, chromogranin
Synovial sarcoma (small cell undifferentiated)Positive: EMAa, BCL2, CKa
Desmoplastic small round cell tumourPositive: desmin (dot), NSE, CKa, WT1 (C-terminal)
Rhabdoid tumourNegative: INI-1
RhabdomyosarcomaPositive: myogenin
Mesenchymal chondrosarcomaPositive: S100, NSEa, desmina
Osteosarcoma (small cell)Negative: desmin, S100
NephroblastomaPositive: CD56 (NCAM), WT1 (N- terminal)
Merkel cell carcinomaPositive: CK20, chromogranin
Lymphoblastic lymphomaPositive: TdT, CD3, CD79a, CD43

The two most common types of translocation found in the EFTs are the EWS-FLI1 gene fusion [t(11:22) (q24;q12)] and the EWS-ERG gene fusion [t(21;22)(q22;q12)]. Both translocations are diagnostic for EFT and can be detected in FNB material, most commonly by FISH or RT-PCR. Nevertheless, other chimerical genes, including EWS-ETV1 [t(7:22)], EWSE1AF, [t(17;22)] and EWS-FEV [t(2;22)], have been described, as well as several breakpoints of the FLI-1 gene (Table 4). One advantage unique to RT-PCR is its ability to define which of two prognostically relevant transcript structures is present: a type 1 fusion transcript (EWSR1-FLI1) usually carries a good prognosis compared with the alternative fusions.[40] Another advantage of RT-PCR concerns its ability to detect minimal residual disease after therapy.[41]

The EWSR gene is also rearranged in a panoply of other tumours, namely in desmoplastic round cell tumours, in extraskeletal myxoid chondrosarcoma and in clear cell sarcoma among others[38] (Table 4).

Undifferentiated SS with a primitive, small, round cell pattern occurs in 15% of the cases and represents a challenge in the differential cytological diagnosis with all the other small round cell tumours. These tumours share with EFTs expression of CD99 (Table 5) and EMA. The detection through RT-PCR of a reciprocal translocation t(X;18)(p11.2;q11.2) or the detection of a SS translocation-chromosome 18 (SYT) gene rearrangement by FISH is observed in >90% of the cases, and is essential for achieving a correct diagnosis.[42]

Kidney tumours

These are also frequent in children and can be so large that it is difficult to make accurate assessment of the site of origin by ultrasound; however, a CT scan with intravenous contrast enhancement is usually quite accurate for this purpose. As previously mentioned, the nature of renal tumours is closely linked to the age group in which they appear (Figure 2). In general, renal tumours in childhood do not cause major problems in cytology differential diagnosis and are characterized by typical and distinct cytological patterns. Wilms' tumour/nephroblastoma (WT) is by far the most common renal tumour in childhood. This makes the diagnosis of a WT the most probable whenever an abdominal mass is detected in the kidney of a child, leading many institutions to start a chemotherapeutic treatment directed to WT, even without a pathological confirmation (SIOP 2001). Cytological diagnosis of WT is easily performed whenever epithelial, stromal and blastema components are all, or at least in part, present in the smears. One must be aware that blastema and epithelial components are most easily sampled, whereas stroma and mesenchyme components are more cohesive and difficult to sample. It is not uncommon to obtain a predominant blastematous smear or otherwise predominantly epithelial smears. At least three needle biopsies should be performed from different tumour areas in order to obtain a more representative sample. When one of these components predominates or is unique, a differential diagnosis with other less frequent tumours of the kidney must considered. Blastema cells should be differentiated from small round cell tumours (NB, EFTs, DSRCT, SS and lymphoma). Immature epithelial tubules of WT should raise the differential diagnosis with metanephric adenoma, and a mesenchymal component of a WT should be set apart from mesoblastic nephroma or rhabdomyosarcoma whenever rhabdomyoblasts features are detected. Immunostains and molecular studies are required in this difficult task.[43]

In this age range and location one should not expect a diagnosis of carcinoma or pheochromocytoma. Whenever such a diagnosis is suspected, the possibility of a hereditary syndrome should be considered. Except for the recently described translocation associated renal cell carcinomas, these are infrequently diagnosed in children and can be associated with a tuberous sclerosis setting. A diagnosis of carcinoma or pheochromocytoma/paraganglioma should also indicate the possibility of syndromes such as neurofibromatosis, von Hippel-Lindau, MEN1, MEN2 or a pheochromocytomas/paraganglioma syndrome (PGL) (mutation of succinate dehydrogenase gene).[44, 45] More recently, mutations in the FP/TMEM127 gene were identified in patients with familial and sporadic pheochromocytoma, but not paraganglioma, and mutations in the MAX gene were identified in patients with familial pheochromocytoma.[46]

Germ cell tumours

These are frequent paediatric tumours which may occur in the abdominal cavity and the retroperitoneal space, as well as the mediastinum. Teratomas are the most common germ line tumour in newborns whereas malignant germ cell tumours affect mainly post-puberty children. Yolk sac tumours appear most frequently around the age of 3 years, and embryonal carcinomas and choriocarcinomas occur in adolescence. Most of these tumours are easily diagnosed by sonography/CT-guided FNB and immunostaining. Most ovarian tumours are cystic and benign and are identified as such by ultrasound. Teratomas may appear echogenic or echolucent but frequently have mixed characteristics with solid and with cystic components or calcifications that may suggest the correct diagnosis. FNB samples are mostly non-informative, owing to hypocelularity, and may require multiple passes. When suspicion of a teratomatous lesion is raised in a child, sedation is essential. Multiple passes will be needed to collect a representative sample and to allow an accurate diagnosis. In a representative sample of a teratoma, multiple types of mature tissues, such as squamous and glandular epithelia, cylindrical cells sometimes with a brush border, goblet cells, mesenchymal stroma, cartilage, adipose cells, skeletal and smooth muscle, bone, glial tissue, among others may be identified. Occasionally, a component of undifferentiated small round cells is identified as being most of the times neuroblastic or blastema tissue. This component should induce the cytologist to consider the possibility of immature teratoma.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. A multidisciplinary approach to diagnosis
  5. A practical approach and the role of ancillary techniques
  6. Summary
  7. Acknowledgments
  8. References

This review highlights the role of FNB in association with ancillary techniques framed in the daily multidisciplinary management to provide accurate diagnosis, prognosis and adequate therapeutic stratification of paediatric tumours. Its application in paediatrics is not substantially different from the procedure in adults, yet some particularities, distinct from the adult pathology setting and inherent to the fact one is dealing with children support the uniqueness of FNB in paediatrics.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. A multidisciplinary approach to diagnosis
  5. A practical approach and the role of ancillary techniques
  6. Summary
  7. Acknowledgments
  8. References

We thank the courtesy of C. Barroca for the drawing in Figure 1; to L. Guedes Vaz MD, Serviço de Pediatria, Centro Hospitalar S João, for providing the picture in Figure 3a; to C. Marques MD, Serviço de Patologia Clinica-Sector de Citometria de Fluxo, Centro Hospitalar S João, for providing the flow cytometry photograph presented in Figure 4c; to MJ Soares MD, Serviço de Hematologia Clínica, Centro Hospitalar S João, for providing the FISH photograph in Figure 4d; to Castedo S. (PhD MD) Genetica Medica e Diagnostico Pre′-Natal, for providing the FISH photograph presented in Figure 5d; to Sobrinho Simões PhD MD and JM Lopes (PhD MD) for their support and for the review of this paper. We also thank the reviewers for their excellent work and patient review.

Corrections made on 30 December 2013 to the online publication: Figure 2, ‘Epithelioid sarcoma’ (not ‘tumour’). Table 1, CD19 is + for LPL and BL; CD23 is − for LPL; BCL2 is − for BL; ALK is +/− for AL. Figure 4, 4b resembles 5b (not 5c).

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  2. Abstract
  3. Introduction
  4. A multidisciplinary approach to diagnosis
  5. A practical approach and the role of ancillary techniques
  6. Summary
  7. Acknowledgments
  8. References
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