General Considerations

Klijanienko, Jerzy; Cohen-Gogo, Sarah; Choucair, Marie Louise; Orbach, Daniel; Cellier, Cécile; Brisse, Hervé J.; Cappellesso, Rocco; Fassina, Ambrogio; Theocharis, Stamatios; Berrebi, Patsy Dominique; Peuchmaur, Michel

doi:10.1007/978-3-319-61027-6_1

Abstract

The time when an excisional biopsy was a standard procedure for the cyto/histopatologic diagnosis is over. Very few indications still remain for an excisional biopsy. Such a procedure is necessary for a diagnosis of bone tumors or for low-grade, presumed benign lesions. Small volume biopsy, which is a combination of fine-needle aspiration (FNA), core needle biopsy (CNB), and molecular analyses, offers the new horizons in this specific and complicated field of pathology [1]. It is preferred that specific molecular analyses be performed on aspirates, knowing that histological material obtained by core needle is very precious for standard histological and immunochistochemical techniques. Today, in experienced hands, this combination is an extremely powerful and rapid diagnostic method.

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Introduction and Application of Fine Needle Aspiration Biopsy

Introduction

Keywords

1.1 Introduction

Jerzy Klijanienko M.D., Ph.D., M.I.A.C.

Department of Pathology, Institut Curie, Paris Cedex 05, France
Jerzy Klijanienko M.D., Ph.D., M.I.A.C.

The time when an excisional biopsy was a standard procedure for the cyto/histopatologic diagnosis is over. Very few indications still remain for an excisional biopsy. Such a procedure is necessary for a diagnosis of bone tumors or for low-grade, presumed benign lesions. Small volume biopsy, which is a combination of fine-needle aspiration (FNA), core needle biopsy (CNB), and molecular analyses, offers the new horizons in this specific and complicated field of pathology [1]. It is preferred that specific molecular analyses be performed on aspirates, knowing that histological material obtained by core needle is very precious for standard histological and immunochistochemical techniques. Today, in experienced hands, this combination is an extremely powerful and rapid diagnostic method.

There is no doubt that the use of “scant” cellular material will, in the future, play an important role, since small volume will mean: non-invasive or minimally invasive, repetitive, easy-to-perform, and rapid procedures providing sufficient material that will be destined for standard and ancillary techniques.

In pediatric pathology, there are many reciprocal relationships between clinical, radiological, and pathological aspects. The appearance of tumors in specific age groups is of great clinical importance for determining the prognosis. Some tumors, such as neuroblastoma , may occur in infants in the perinatal period. Detection at a very young age improves the parameter for a favorable prognosis. Moreover, rapidity of evolution and clinical symptomatology are the first important pieces of diagnostic information.

In some patients, the rapidity of tumor evolution and important tumor volume may restrict the use of general anesthesia or invasive diagnostic procedures. In such cases, “urgent” fine-needle aspiration is an excellent initial diagnostic method to confirm malignancy, sarcoma, or lymphoma.

Radiological evaluation is one of the capital elements of diagnosis [2]. Radiology informs the initial diagnosis and tumor “geography.” Moreover, radiology helps define the optimal technique and anatomical position before taking a tumor sample. Sampling should be based on the collaboration between the radiologist and pathologist. Sampling by “technique of four hands” is, in our opinion, optimal. The radiologist takes samples while the pathologist evaluates specimen quality by macroscopic examination of core biopsies and microscopic examination of cellular smears. The pathologist is essential also for distributing cellular material for various uses: material to be smeared; material to be preserved in liquid media for cell-bloc preparation or immunocytochemistry; material to be embedded in paraffin; and material to be sent for molecular techniques. Furthermore, “rapid on site evaluation” (ROSE) may be performed for immediate diagnosis and for the decision on the ideal diagnostic material (smears versus core needle biopsy versus molecular studies, etc).

Fine-needle aspiration is one of the most challenging techniques in the pediatric age group [3,4,5]. Difficulty resides in the fact that a large spectrum of tumors is possible in each anatomical location. Additionally, numerous tumors may have overlapping cytological patterns. Nodular fasciitis and low-grade spindle-cell sarcoma are the typical examples. In solid malignancies, cytology diagnosis becomes much easier compared to spindle-cell tumors. The differential diagnosis between blastemal tumors is mainly based on tumor localization. Similarly, in round-cell sarcomas, rhabdomyoblastic, rhabdoid, or rosette-like differentiations are easily detectable on smears. However, in the case of a superficial low-grade spindle-cell tumor , the technique of “let it run its course” is indicated and the final diagnosis may be made through prepared core needle biopsy or surgical excision.

Core needle biopsy is minimally invasive diagnostic technique, but when deeply located tumors are sampled, a general anesthesia is indicated. The quality of core needle biopsy depends on the radiologist’s experience. This technique is judged to be safe and accurate but, surprisingly, the cellular material is less representative than the material obtained by fine-needle aspiration. This is particularly true in round-cell and blastemal tumors and less true in spindle-cell tumors.

Molecular diagnosis is one of the newcomers in the diagnosis in pediatric malignancies. Many tumors exhibit specific molecular alterations which may be used in the diagnosis. After an enthusiastic period in the development of molecular diagnosis, many alterations were shown to be “not so specific” and present in several types of tumors. Additionally, the constantly growing number of alterations requires a specialized technical team and systematic updating.

All these techniques may be successfully applied in children if the hospital or oncology center has a well-trained team. A multidisciplinary discussion informs the final diagnosis and appropriate medical or surgical treatment.

1.2 Clinical Aspects of Semi-Malignant and Malignant Tumors in Children and Adolescents

Sarah Cohen-Gogo M.D., Ph.D.,
Marie Louise Choucair M.D. &
Daniel Orbach M.D.

Institut Curie, 26 Rue d’Ulm, Paris, 75005, France
Sarah Cohen-Gogo M.D., Ph.D., Marie Louise Choucair M.D. & Daniel Orbach M.D.

Many parameters should be taken into account when forming hypotheses about a tumor in a child: age of occurrence, tumor site, natural history, and of course the tumor’s clinical and radiological characteristics. Clinical syndromes predisposing pediatric tumors are listed in Table 1.1. Epidemiology of pediatric tumors is shown in Table 1.2.

Table 1.1 Clinical syndromes predisposing pediatric tumors

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Table 1.2 Epidemiology of pediatric tumors according to the Slovenian cancer registry

Full size table

Before any aspiration or biopsy, pediatricians, radiologists, and pathologists should always, as a group, discuss and define the best methods of sampling and the fate of the tumor samples. This first step is important because some tumors should not be biopsied at diagnosis due to their potential to spread, in case of rupture: adrenal carcinoma, pseudo-papillar pancreatic tumor, gonadal germ cell tumors, and sex stromal tumors, for instance, fall into this category. In this case, diagnosis should be based on the clinical and radiological presentation and confirmed by histological analysis of the resected tumor. Figure 1.1 displays the main possible malignancies according to the primary tumor site —the reader will then find detailed clinical information below. Figure 1.2 displays the main possible malignancies correlated to the age at diagnosis.

1.2.1 Cervical Nodes

Enlarged cervical nodes are a frequent clinical finding in children and may arise from a wide variety of benign or malignant disorders. Clinical history, physical examination, and laboratory and radiological investigations may give some important clues for differential diagnosis (Fig. 1.3). In 90% of cases, the lymphadenopathies (LAPs) are benign and might arise from viral or bacterial infections (EBV, CMV, HIV, cat scratch disease, etc), tuberculosis, and autoimmune diseases [6]. Some symptoms can be suggestive of a malignant origin, such as unexplained fever, unintentional weight loss, night sweats, pruritus, dyspnea, and poor general condition. Physical examination should be complete and systematic. Each lymph node should be evaluated for its location (localized or generalized; supra-clavicular location is always highly suspicious for malignancy); size (LAP >3 cm are highly suspicious); and consistency, tenderness or skin inflammatory reaction. Further investigations are then recommended with complete blood count (CBC), erythrocyte sedimentation rate (ESR), lactate dehydrogenase concentration, and simple radiological exams, with a chest X-ray and an ultrasound examination of the affected site. Fine-needle aspiration (FNA) of the lymph node and/or excisional biopsy can then be scheduled if considered appropriate.

Leukemia is the most common childhood cancer; in leukemia, generalized lymphadenopathies can be a prominent feature. Acute lymphocytic leukemia accounts for about one-third of all pediatric malignancies. Treatment consists mainly of chemotherapy, and prognosis may vary depending on molecular characteristics. Lymphomas can present either with generalized or localized lymphadenopathy. They are divided into Hodgkin’s lymphomas (HL) and non-Hodgkin’s lymphomas (NHL) . HL has a bimodal distribution with a peak in adolescence and adulthood. About 80% of HL patients present with asymptomatic cervical adenopathy [7]. NHL is a heterogeneous group of lymphoid malignancies. In the pediatric setting, the tumor is often a Burkitt’s lymphoma or large B cell NHL, but anaplastic large-cell lymphoma (ALCL) and T-lymphoblastic lymphoma can also occur. Treatments of lymphoma have improved a lot over the last decades and consist in short but intensive chemotherapy sometimes associated with immunotherapy. More detailed information on lymphomas will be available in the following sections about thoracic and abdominal tumors [8].

Finally, malignancy can be due to nodal metastasis of solid tumors such as cervical neuroblastoma, nasopharyngeal rhabdomyosarcoma, or undifferentiated carcinoma of nasopharyngeal type (UCNT) . UCNT is rare in Europe and the USA and represents 1% of all childhood cancer. It mainly concerns adolescents and young adults. These tumors are usually revealed by their cervical nodal involvement and also by nasal obstruction, epistaxis, trismus, and headache. The nasopharyngeal mass can be discovered during an ear, nose, and throat examination and confirmed with medical imaging investigations. UCNT has a high chemo- and radio-sensibility in children. The global prognosis is satisfactory with an overall survival approaching 90% after treatment with a chemo-radiotherapy association [9].

1.2.2 Thoracic Tumors

The discovery of a thoracic lesion in a child can lead to a wide possibility of benign or malign lesions, but can also correspond to pseudo tumoral images secondary to infectious or malformative diseases. The physician will need more data to give a more precise diagnosis: age, clinical presentation, genetic predisposition context, anatomical location of the lesion as defined by imaging, and, eventually, specific cytology or histology samples.

Chest radiograph and most often CT scan will be mandatory to assess where exactly the thoracic mass is located. The majority of intra-thoracic malignant tumors will be found in the anterior or middle mediastinum and will correspond to hematopathies. Diagnosis and treatment are then an emergency. Classical radiological presentation is a mediastinal enlargement initially diagnosed on a chest radiograph performed for a number of different reasons: cough, dyspnea, chest pain, or other symptoms such as cervical adenopathies or and abdominal mass. Pathology assessment has to be done quickly and treatment will rest upon steroids and polychemotherapy.

In addition to hematopathies, many other tumors can be found in the thorax. A monocentric study conducted in 2005 included 205 children presenting with thoracic mass; 38% of the subject had, in fact, chest wall tumors, and 62% intra-thoracic tumors. The most frequent diagnoses were neuroblastoma (41%), Ewing sarcoma family of tumors (17%), rhabdomyosarcoma (RMS) (9%), malignant germ cell tumors (8%), thymomas (4%), and Langerhans cell histiocytosis (4%) [10]. Other tumors with intermediate malignancy, such as pulmonary pneumoblastoma or inflammatory myofibroblastic tumors, are rarely found. Tumors arising from the lung or the pleura are exceptional in childhood [11]. Neuroblastomas are located mainly in the posterior mediastinum. These tumors can have two extreme clinical presentations: either strictly asymptomatic or responsible for medullar compression due to paravertebral endocanalar extension (dumb-bell tumors). In case of severe initial paraplegia, immediate chemotherapy should be discussed in emergency before any tumor sampling. In this case, tumor biopsy will be planned after reduction of the spinal cord compression. Chest wall sarcomas (Ewing’s or rhabdomyosarcoma) may be revealed by pain and swelling.

Secondary malignant pulmonary tumors can be seen in pediatric oncology, but are rarely the initial symptom. They arise from solid tumors that are different than those found in adults: Wilms’ tumor, bone sarcomas, or soft tissue sarcomas. Pulmonary involvement is also rare in Hodgkin’s lymphoma [12].

1.2.3 Mesenteric and Peritoneal Tumors

The discovery of an abdominal mass revealed by abdominal pain, a mass, or an intussusception is relatively frequent in pediatric oncology. This situation is frequent for non-Hodgkin’s lymphomas (NHL) , which represents about 10% of all childhood cancers. NHLs usually occur in previously healthy children, although some might appear within rare immunodeficiency disorders such as AIDS, ataxia telangiectasia, Wiscott-Aldrich syndrome, or after organ transplants. Abdominal lymphomas account for approximately 40% of all NHL. The median age of onset is 7–8 years. In this situation, the tumor is a highly malignant B cell proliferation, corresponding to a clonal proliferation of immature lymphoid precursors. The Epstein Barr virus often has a role in malignant transformation, even in immuno-compromised children.Tumor proliferation is centered on the ileocaecal area, Peyer’s patches, and mesenteric lymph nodes, explaining the frequent symptoms of secondary intussusception. Abdominal NHLs are frequently associated with poor general condition. Ultrasound and abdominal CT often find a large intraperitoneal tumor mass combined with thickened bowel loops, mesenteric lymphadenopathies, or ascites. It is essential to avoid extensive initial surgery. In the absence of major gastrointestinal symptoms due to intestinal perforation, medical care can quickly lever the intestinal compression and avoid extensive surgery. Diagnosis is confirmed by FNA of the affected sites, usually by transcutaneous way.

B-NHL may be life-threatening and need an urgent diagnosis. Prognosis of these lymphomas is primarily related to lactate dehydrogenase (LDH) level, initial disease extension, response to induction chemotherapy, and the possibility to reach complete remission at the end of treatment. Currently, overall survival is between 70 and 90% with multichemotherapy regimen ± rituximab [12].

Other peritoneal tumors are very rare and include desmoplastic small round-cell tumors (DSRCT) or peritoneal mesotheliomas . DSRCTs are rare tumors that occur mainly in adolescents and young adults. The diagnosis is suggested by the presence of a mass located primarily on the peritoneum, most often associated with liver metastases. Tumor biopsies are usually performed during an exploratory laparoscopy or by radiological trans-peritoneal route. Despite treatment using prolonged poly-chemotherapy, an extensive peritoneal surgery, sometimes in association with whole abdomen radiotherapy, the prognosis remains very severe with a survival rate of 20% after 5 years [13].

1.2.4 Hepatic Tumors

Malignant hepatic tumors are rare and represent 2% of childhood cancers. Two-thirds of pediatric liver tumors are malignant. The two most common malignant tumors are hepatoblastoma (HB) and hepatocellular carcinoma (HCC) , which together represent 90% of all hepatic malignant tumors. HB is seen in younger children and HCC in older ones. Other malignant liver tumors are quite rare and include hepatic rhabVd tumor, embryonal undifferentiated sarcoma, and biliary rhabdomyosarcoma. Ultrasonography is the first line of examination. Once the hepatic origin of the mass is confirmed, the main aim is to assess disease extension according to the PRETEXT classification (PRE-Treatment-EXTension of the disease). Eighty percent of hepatoblastoma cases occur before 2 years of age and the median age at diagnosis is 18 months. Many risk factors have been identified: Beckwith-Wiedemann syndrome, familial adenomatous polyposis, very low birth weight (<1000 g) and prematurity (<33 weeks). HB mostly presents as an asymptomatic abdominal mass. Laboratory investigations usually show normal liver tests and a very high blood alphafoetoprotein (AFP) level (AFP > 10⁴ to 10⁷ ng/mL). If the hepatic mass looks malignant but there is no AFP elevation, then, depending on the clinical and radiological findings, several differential diagnoses must be discussed relative to the child’s age. In infants, differential diagnosis would be a rare form of HB without AFP secretion or a hepatic rhabdoid tumor, both of which have a very poor prognosis. Molecular analysis, with testing for loss of SMARCB1/INI1 expression, will help to rule out rhabdoid tumor diagnosis. In older children, it could be a HCC or an embryonal undifferentiated sarcoma. Of note, HCC is more frequent in children with an underlying liver condition such as chronic B hepatitis, tyrosinemia, type 1 glycogen storage disease, and biliary atresia. A biopsy should always be performed, except in case of tumoral rupture, to differentiate between HB and HCC. In Europe, treatment consists in neoadjuvant chemotherapy followed by surgery and adjuvant chemotherapy. The intensity of the treatment depends on initial AFP level, the PRETEXT classification, and the presence of metastases [14,15,16,17].

1.2.5 Pancreatic Tumors

Pancreatic tumors are very rare in pediatrics, the most frequent one being the pseudopapillary and solid tumor of the pancreas. Further diagnoses are nevertheless possible: pancreatoblastoma in young children and neuro-endocrine tumors in adolescents. The diagnosis is mainly evoked by the discovery of a mass on CT or MRI localized in the retroperitoneum in the pancreas. Pseudopapillary and solid tumor of the pancreas or Frantz tumor occurs mainly in young women (sex ratio of 1:9), at an average age of 22 years. Ultrasound and abdominal CT show a heterogeneous mass, solid and cystic, well-encapsulated, sometimes with calcifications. Biopsy should be avoided because it might be a risk factor for relapse. Complete surgical resection can be performed when clinical and radiological orientation is strong. The long-term prognosis is excellent (survival >95%) [18].

Pancreatoblastoma (PB) is an extremely rare pancreatic tumor seen in young children, with a male predominance. PB may arise in the context of Wiedemann-Beckwith syndrome . The telltale sign is usually the discovery of an abdominal mass sometimes associated with abdominal pain, asthenia, or jaundice. PPB is a solid mass, rather well-encapsulated, round, of soft consistency, and often large, exceeding 10 cm in major axis and extending beyond the limits of the pancreas. It can show necrosis, hemorrhage, or cystic changes. Metastases are rare. Diagnosis of PB is strongly suspected after detection of elevated serum AFP levels. Final diagnosis is confirmed at the time of tumor resection, or a biopsy performed if resection is not immediately possible. Treatment of PB is primarily surgical. Neoadjuvant chemotherapy is sometimes given with the objective of reducing the tumor volume to allow complete surgical excision and perform prophylaxis of metastasis. Survival depends on the initial spread of the disease. Relapse-free survival at 5 years is 59% and overall survival 79%. The only known prognostic factor, besides initial extension, appears to be the possibility of complete resection with or without chemotherapy [19].

Neuro-endocrine tumors of the pancreas are very rare in children, and are more frequent after puberty. Insulinomas and gastrinomas are the most frequent and may occur in association with multiple endocrine neoplasia type I or II.

1.2.6 Adrenal Tumors

In childhood, neuroblastoma accounts for more than 90% of adrenal tumors, while adrenal cortical tumors account for 6% of adrenal cancers in children. Even if adrenal adenomas and carcinomas occur also in childhood, these tumors are indistinguishable on imaging from neuroblastoma. Usually, radiologic criteria for the diagnosis of adrenal carcinoma include size larger than 5 cm, a tendency to invade the inferior vena cava and to metastasize, but none of them are specific to adrenal carcinoma and are frequently seen in adrenal neuroblastoma as well. Furthermore, chromaffin-cell proliferation contributes to pediatric neoplastic processes in the form of an adrenal pheochromocytoma [20]. Neuroblastoma (NBL) , along with ganglioneuroblastoma and ganglioneuroma, constitute a group of ganglion cell-origin tumors that originate from primordial neural crest cells, which are the precursors of the sympathetic nervous system [21]. Neuroblastoma accounts for 8–10% of childhood cancers. The most undifferentiated and aggressive NBL presents in young children (median age ≤2 years). The more mature tumor type is ganglioneuroma, which affects older age groups. Approximately 50% of NBL occurring in children older than 18 months of age are metastatic at diagnosis. The main prognostic factors in NBL are age, stage of disease at presentation, and molecular abnormalities such as MYC-N amplification or segmental chromosome alterations [22]. Localized neuroblastoma and those arising in infants have a 90% survival rate except in cases with myc-N amplification, where survival is below 30% [18, 19]. Risk-stratified therapy has facilitated the reduction of therapy for children with low-risk and intermediate-risk disease. Advances in therapy for patients with high-risk disease include intensive induction and myeloablative chemotherapies, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy; these have improved 5‑year overall survival to 50% [23].

Adrenocortical tumors (ACT) are very rare in children, with a worldwide annual incidence of 0.3 per million children below the age of 15 years [24]. This tumor is frequently associated with p53 germline mutations [25]. The incidence is higher in young girls, with a female/male ratio of 2:1, whereas in adolescence the sex ratio is equal. Virilization, with early onset of pubic hair, hypertrophy of the clitoris or penis, accelerated growth, gynaecomastia or acne, is the most common presentation. The second most common manifestation is with hypercortisolism (Cushing’s syndrome ), while presentation with a palpable abdominal mass is unusual. Diagnosis should be evoked on the clinic-biologic-radiological presentation. In order to avoid the tumor spreading and therefore deteriorating outcome, this tumor should not be biopsied at diagnosis. Immediate surgery is the gold standard for localized tumors: surgical resection is the mainstay of treatment. The role of radiotherapy is uncertain. Similarly, the role of perioperative chemotherapy in association with mitotane (O’PDDD) is limited, as in adult ACTs, and its efficacy in children has not been well-studied prospectively [26].

Pheochromocytomas arise from the adrenal medulla. However, up to one-third of pheochromocytomas may occur outside of the adrenal gland. They are generally sporadic in childhood, usually occurring in adolescence. High blood pressure is often associated and should be controlled before any type of tumor sampling. Pheochromocytoma could also be associated with multiple endocrine neoplasia syndromes (mostly type 2), von Hippel–Lindau syndrome, or neurofibromatosis. Biopsy or tumor sampling should be avoided. Immediate surgery is required in treating this tumor.

1.2.7 Pelvic Tumors

Pelvic tumors can be diagnosed in connection with an abdominal or perineal mass discovered during a routine examination, or by parents. Patients may also present with pain related to a pelvic nerve root compression, vesico-sphincter dysfunction, inguinal lymphadenopathies, or poor general condition. Pelvic masses can be benign, especially when of ovarian origin. Still, many cancer types can be diagnosed in this area, such as germ cell tumors (GCT), sex cord stromal (SCT) tumors, rhabdomyosarcomas, or neuroblastomas. Inflammatory myofibroblastic tumors (IMT) can also be found. Imaging is mandatory and ultrasound should be the first step.

Pediatric GCTs are very diverse. They can be diagnosed from in utero to adolescence, at gonadal and non-gonadal sites, and from the head to the sacro-coccygeal region. Some of them secrete alpha-fetoprotein (AFP) or hCG, which can then be used as a marker for disease. GCTs remain rare and represent approximately 3% of all childhood cancers [27, 28]. The most frequent primaries are gonadal GCTs, as well as sacro-coccygeal teratoma.

Eighty percent of ovarian masses are benign, and 90% of tumors are non-malignant GCT (mature teratoma). Females present with pain, lower abdominal fullness, and, less commonly, acute abdomen caused by torsion or tumor rupture. Early pubic hair and breast enlargement can occur in case of β-hCG or, more frequently, estrogen secretion (granulosa cells tumors or SCT). An ovarian malignant tumor is a germ cell tumor in 85% of the cases. Serologic markers (AFP, β-hCG, HCG) are essential to assess the nature of the tumor. All the histological subtypes of GCTs may be represented in the ovary tumor. The most common malignant entity is the yolk sac tumor (AFP+) but choriocarcinoma (β-hCG, HCG +) also occurs.

Two age peaks are seen for testicular GCTs: (a) children under 3 years old, who may experience mature teratoma or yolk sac tumors; and (b) adolescents and young adults who may also have seminomas or other mixed tumors. Most often, those tumors will present as painless scrotal masses. Differential diagnosis includes para-testicular rhabdomyosarcoma, leukemia, and lymphoma.

Sacro-coccygeal teratoma is the most common extragonadal GCT and occurs in newborn and infants. Generally, this tumor presents with either one of two distinct clinical patterns: (a) large, predominantly external lesions that are detected prenatally or at delivery, rarely malignant, and with a favorable evolution after surgery; or (b) older infants who present with less apparent pelvic tumors with a very high rate of malignancy. In both cases, serial blood AFP levels must be performed.

Surgical resection remains the main step in management of GCTs. No biopsy should be performed in ovarian and testicular tumors and orchidectomy should be performed by inguinal approach. The management of all benign tumors, and of localized and completely resectable malignant tumors, is surgery alone. Chemotherapy is very effective in infants and children with unresectable or metastatic disease and allows a high survival rate (>90%).

Rhabdomyosarcoma can also occur in the pelvis, mostly as bladder/prostate, paratesticular, and vaginal RMS. Inflammatory myofibroblastic tumors (IMT) can also occur in this setting [29]. Those tumors have intermediate aggressiveness, with a very low metastasis rate but a tendency for local recurrence. The pelvic mass can be associated with fever, weight loss, anemia, thrombocytosis, polyclonal hyperglobulinemia, and an elevated erythrocyte sedimentation rate. Transcutaneous or bladder/vagina per-endoscopy biopsy is needed to allow diagnosis.

1.2.8 Renal Tumors

Renal tumors in children are rare and account for 6–7% of all cancers in children. Initial diagnosis is most often made when an abdominal mass is discovered by the parents or the physician. There might be hematuria, hypertension, or abdominal pain. These tumors can also be found during ultrasound follow-up of children with a genetic predisposition (Wiedemann-Beckwith, Drash, WAGR, etc.). The gender ratio is rather balanced. Among children aged 6 months to 5 years, Wilms’ tumor is by far the most common diagnosis [30]. When confronted with a patient who exhibits a renal mass, the current European SIOP (Société Internationale d'Oncologie Pédiatrique) strategy is to assess how probable Wilms’ histology might be. The clinician is helped by clinical, biological, and radiological criteria. When the presentation is atypical, the SIOP recommends confirm diagnosis through a biopsy. Moreover, immediate tumor biopsy (or immediate nephrectomy ) is recommended when (a) the child is older than 5–6 years old or younger than 6 months old: Wilms’ tumors are less frequent and other tumors may be found, such as congenital mesoblastic nephroma, hypercellular renal fibrosarcoma, and aggressive rhabdoid tumors, in the youngest patients, and renal carcinomas or clear cell renal sarcomas in the oldest ones; (b) when urinary tract infection cannot be ruled out easily: pseudotumoral pyelonephritis or abscess might be a differential diagnosis; (c) when abdominal adenopathies can be seen: they are not frequent with Wilms’ histology; and (d) when the tumor is not obviously intra-renal: neuroblastoma can be evoked.

Different techniques can be used to perform diagnosis: cytology through fine-needle aspiration, or histology through core needle biopsy, but always through a posterior retroperitoneal approach, with the help of an ultrasound scan. Surgical biopsies are not recommended. As diagnosis of Wilms’ tumor is mainly presumptive, urinary catecholamines tests should be systematic to help rule out neuroblastoma, which may mimic nephroblastoma.

Prognosis of Wilms’ tumors is now good with survival rates above 90% (regarding all stages altogether) [31]. These results have been reached with two very different initial approaches: (a) in the USA, the National Wilms’ Tumor Study recommends total nephrectomy as a first step, and then adjuvant treatments based on stage and histology; (b) elsewhere, the SIOP recommends neo-adjuvant chemotherapy, then surgery, then adjuvant treatment based on stage and histology [32, 33]. Overall prognosis of renal tumors is linked to histology (high-risk tumors are Wilms’ tumors with anaplastic or blastemal predominant components and clear cell sarcomas), locally spread disease and metastatic presentation at diagnosis.

1.2.9 Soft Tissue Lesions

Sarcomas in children and adolescents are rare diseases and include various histological types that could be classified as soft tissue sarcomas of “pediatric-type” (i.e., rhabdomyosarcoma), “adult type” (i.e., synovial sarcoma, malignant peripheral nerve sheath tumor), specific entities (infantile fibrosarcoma, desmoid tumor, dermatofibrosarcoma protuberans), and bone sarcomas. Clinical presentation frequently associates a rapidly growing mass with signs depending on the primary location (Fig. 1.4). The group of “non rhabdomyosarcoma soft tissue sarcomas” (NR-STS) gathers all soft tissue sarcomas, except rhabdomyosarcoma and Ewing sarcoma, occurring during childhood and adolescence. Median age of patients with RMS at diagnosis is 5 years, versus 9 years for NR-STS. These sarcomas may occur in every part of the body, but some sites are more frequent: head and neck location for RMS and limbs for NR-STS. Sensitivity to medical therapy depends on the disease type, which must be taken into account in the therapeutic strategy. RMS are very chemosensitive tumors [34]. The age of the patient, the tumor extension, and the potential resectability of the primary tumor also play an important role. Survival for most of these sarcomas is favorable, although lower in adolescents than in younger patients and in certain histological types and metastatic presentations that are difficult to cure with current treatments [35, 36].

1.2.10 Bone Tumors

Malignant bone tumors are most often primary in children. Bone metastases can be seen in neuroblastoma, Wilms’ tumors, and in primary bone tumors—but in these cases, clinical context is obvious [37]. This chapter will therefore focus on primary bone tumors only. Primary bone tumors are the sixth most common neoplasm occurring in children and constitute approximately 6% of all childhood malignancies [38]. There is a peak incidence in 15–19-year-old individuals with these lesions being the third most common tumors in adolescents and young adults, exceeded only by leukemia and lymphoma [39]. Osteosarcoma and Ewing sarcoma are the most common malignant primary bone tumors in this age group [40,41,42]. Although the overall incidence of osteosarcoma is higher than Ewing sarcoma in adolescents younger than 20 years, Ewing sarcoma is more common in children younger than 10 years of age. Of note, focal bone Langerhans cell histiocytosis can be a differential diagnosis.

Patients carrying a bone tumor are most often symptomatic with pain, swelling, pathologic fractures, and, sometimes, constitutional symptoms such as fever or weight loss. Osteosarcoma occurs mostly in the extremities such as the knee, femur, and humerus; Ewing sarcoma’s most common sites are pelvis, femur diaphysis, and chest wall. Metastases to the lungs or other bones are not rare, with Ewing sarcoma also leading to possible bone marrow involvement. Local radiological assessment most often comprises local X-rays and MRI.

Formal diagnosis will need a surgical bone tumor biopsy, but cytology or tru-cut biopsy can be of help in case of soft tissue involvement, which is frequent in Ewing sarcoma. Detection of the specific EWS-FLI1 transcript in molecular biology is helpful to confirm Ewing’s sarcoma diagnosis [43]. Treatment consists of a combination of chemotherapy and limb-sparing surgery. Adjuvant radiotherapy is also discussed in Ewing sarcoma. Overall survival can reach 60–70% for patients with a localized disease [39, 42].

1.3 Radiological Diagnostic Approach in Extracerebral Pediatric Tumors

Cécile Cellier M.D. &
Hervé J. Brisse M.D., Ph.D.

Institut Curie, 26 Rue d’Ulm, Paris, 75248, France
Cécile Cellier M.D.
Institut Curie, 26 Rue d’Ulm, Paris, 75005, France
Hervé J. Brisse M.D., Ph.D.

It should always be kept in mind that the aim of radiological examinations is not to define a single diagnosis, but to define a “group of possible diagnoses” and consequently to propose the appropriate management.

The radiological patterns of the various tumor types have been already largely described in review articles [44,45,46,47,48,49] or reference books [50, 51]. Depending on the anatomical localization of the lesion, it is generally agreed that the radiological diagnosis should consist of initial X-ray and ultrasound examination followed by CT scan or MRI. The role of imaging is essential in these cases, either to confirm its benign nature or, on the contrary, to guide the indication for biopsy if the lesion is potentially aggressive or of a nonspecific appearance.

The nature of the sample and its modality should always be previously discussed in a multidisciplinary team. Clinical examination is still the first step of diagnosis. Age, sex, and site of the lesion are useful pieces of information leading to diagnosis [48, 49]. Tumors can occur anywhere, but some sites can help to guide the diagnosis. Genetic-predisposing diseases such as type-1 neurofibromatosis, Beckwith-Wiedemann syndrome, Hereditary retinoblastoma (RB1 gene mutation), and so on must be clinically investigated because they may orientate on tumor type (Table 1.1).

Diagnostic samples may be obtained via simple palpation-guided cytology without aneasthesia, especially in infants presenting superficial lesions. In deep lesions a combination of cytology and core needle biopsy is preferred.

1.3.1 Imaging Techniques

Conventional radiography is of diagnostic value as first-line investigation, particularly for limb lesions. Added to plain films, ultrasound is part of the simple first-line examination for cases clinically and radiologically suggestive; the radiography-ultrasound establishes the diagnosis of some pseudotumors (adenitis, abscess), benign tumors (lipomas, infantile hemangioma, fibromatosis colli) or malformations (cystic lymhangioma, venous malformation). Doppler analysis confirms the avascular nature of cystic lesions or, conversely, assesses the type of blood supply of solid lesions.

MRI technique is currently the gold standard for evaluation of soft tissue and bone tumors due to its excellent tissue contrast [47, 49, 52,53,54,55,56] and is mandatory before biopsy. If paravertebral neuroblastoma is suspected, MRI is a mandatory technique to detect an endocanalar extension, but in other localizations, CT scan may be sufficient [57, 58]. All other thoraco-abdominal tumors are diagnosed using MRI or CT scan if MRI is not available [59,60,61].

Figure 1.5 illustrates clinic- radiologic strategy to obtain pathologic samples in pediatric tumors. Figures 1.6, 1.7, 1.8, 1.9 and 1.10 provide examples of radiological evaluation of pediatric masses.

1.3.2 Tumor Biopsy

Deep-sited lesions should be sampled under radiological guidance. Superficial tumors may be sampled under either radiological or palpatory guidance.

1.3.2.1 Surgical and Core Needle Biopsies

In the absence of definite signs of benign lesion, a biopsy should always be performed. Consultation with the radiologist, the surgeon, and the pathologist allows them to define the biopsy tract using compartmental anatomy definitions [62, 63], the biopsy site (especially the most suspicious portion), and appropriate processing of biopsy specimens (tissue preservation for genetic studies). A surgical excisional biopsy or a percutaneous procedure (core needle biopsy) is decided according to the size and location of the mass. Although large lesions of the limbs can easily be biopsied without image guidance, deep-seated musculoskeletal lesions are difficult to target, and benefit from CT or US guidance. Imaging-guided percutaneous core needle biopsies are performed by trained radiologists, under local or general anesthesia, using CT or US guidance [64,65,66,67,68]. The procedure should ideally be performed by both the radiologist and the pathologist, the latter being the most qualified to evaluate the specimen quality and to separate the tissue for morphological and biological studies. If the specimen cannot be frozen immediately, it must not be fixed directly, but should be temporarily placed in culture medium such as Roswell Park Memorial Institute medium, RPMI.

1.3.2.2 Fine-Needle Aspiration

Fine-needle aspiration (FNA) usually does not replace biopsy but, in our experience, constitutes an excellent first-line and reliable diagnostic procedure, provided that it is performed and examined by trained pathologists. It is an inexpensive technique, almost without morbidity [69], and it can be performed under local anaesthesia. Using fine needles (23 Gauge, 0.6 mm of outer diameter), with ultrasound guidance if necessary, provides highly contributive cell aspirates [70, 71]. In the diagnostic strategy, FNA may be used right after initial imaging when the decision to perform or not perform a biopsy is pending, e.g., in case of clinically and radiologically presumed benign or pseudotumoral lesion, in case of a highly vascularized lesion at risk for a biopsy, or for suspected relapses. FNA material is excellent for ancillary techniques (this material is tumor-cell rich and stroma-cell poor) and allows karyotyping and molecular analyses. Cells should be stored in ethylenediaminetetraacetic acid (EDTA) (Figs. 1.11 and 1.12).

1.4 Ancillary Methods

Rocco Cappellesso M.D. &
Ambrogio Fassina M.D., Ph.D.

Department of Surgical Pathology and Cytopathology Unit, University of Padova, Via 8 Febbraio 1848, 2, Padova, 35122, PD, Italy
Ambrogio Fassina M.D., Ph.D.
Department of Medicine, Cytopathology Unit, University of Padova, Via 8 Febbraio 1848, 2, Padova, 35122, PD, Italy
Rocco Cappellesso M.D.

For decades, open biopsy has been the gold standard for the diagnosis of pediatric tumors. Only after the demonstration of similar diagnostic accuracy, core needle biopsy (CNB) has been accepted as less invasive valuable diagnostic alternative. Fine-needle aspiration (FNA), on the other hand, was not used in routine diagnostics until few years ago, mainly because of the publication of earlier studies showing it achieved lower diagnostic sensitivity and specificity than CNB. A great contribution in the recent success of FNA was related to the possibility of performing ancillary techniques on the aspirates. These tests enormously enhanced the overall diagnostic performances of FNA and nowadays its reliability is almost unanimously accepted. FNA yields material suitable for immunocytochemistry (ICC), flow cytometry (FC), and molecular analyses. Moreover, the amount of collected cells is almost the same as in CNB. Furthermore, FNA material can be more representative than CNB in small or heterogeneous tumors, since the movements of the needle allow for sampling of different areas of the neoplasm. This chapter covers the main issues related to the application of ancillary methods in the cytological diagnosis of pediatric tumors [72,73,74,75,76,77].

1.4.1 An Overview of Molecular Alterations in Pediatric Tumors

The vast majority of pediatric tumors are represented by mesenchymal neoplasms and lymphomas. The diagnosis and classification of both these categories of tumors are based on morphology, immunophenotype, and demonstration of specific molecular alterations in the appropriate clinical and radiological context. Thus, the cytologist who is faced with a tumor likely belonging to one of these groups of malignancies is required to know which molecular modifications must be investigated and how the aspirate must be handled.

From a molecular point of view, these tumors can be divided in two main broad categories:

1.
Neoplasms with stochastic, multiple, and complex molecular alterations
2.
Neoplasms with recurrent simple molecular alterations

The first category encompasses all those tumors harboring random transfer, gain, or loss of large parts of chromosomes or DNA resulting in aneuploidy, composite and nonspecific karyotype, or multiple gene aberrations. These neoplasms are usually characterized also by a low degree of differentiation and marked pleomorphism. Molecular analyses in such cases are applied only to achieve a diagnosis of exclusion. Undifferentiated and pleomorphic sarcomas are clear examples. In the latter group, instead, are included all those tumors strongly related to a frequent definite cytogenetic modification or single gene mutation. The detection of the molecular alteration in such cases is often mandatory to attain a correct diagnosis. For instance, Burkitt lymphoma is characterized by a chromosomal translocation combining the oncogene MYC on chromosome 8 with immunoglobulin locus regulatory elements in chromosomes 2, 14, or 22, and the cytogenetic demonstration of MYC rearrangement is the gold standard for the diagnosis. Other examples are pediatric anaplastic large cell lymphoma and inflammatory myofibroblastic tumor, both harboring a chromosomal rearrangement involving the ALK gene. A cytologist should be aware of the specific molecular abnormalities of each tumor and how these can be identified. There are three levels at which the molecular alteration can be detected:

1.
Chromosome
2.
DNA/RNA
3.
Protein

Each level corresponds to a technique to be applied. The example of Ewing sarcoma (ES)/primitive neuroectodermal tumors (PNET) is useful to clarify this issue (Fig. 1.13). For what concerns the first level, the translocation involving the chromosomal region 22q12 can be readily demonstrated by fluorescence in situ hybridization (FISH). In turn, the chromosomal modification causes the juxtaposing of the EWSR1 gene contained in the translocated material with another gene and this is detectable by amplification through polymerase chain reaction (PCR) of the fused genetic region and sequencing. However, this method is not used in routine diagnostics for technical reasons and it is preferred to identify the transcript by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. Indeed, the resulting combined gene is normally transcribed to RNA as any other gene and then translated into a chimeric protein that, in turn, can be detected by ICC, if a specific antibody is already available. Further examples of pediatric tumors with known chromosomal abnormalities that lead to genetic alteration and aberrant protein product are listed in Table 1.3.

Table 1.3 Examples of pediatric tumors with definite recurrent chromosomal abnormalities resulting in gene alteration and aberrant protein product^a

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1.4.2 Molecular Techniques

This section presents only the most widely used methods applied to FNA in the routine diagnostics. The scope is to highlight the advantages and limitations of each technique in order to provide rational and practical information on how to handle an FNA sample that needs ancillary studies.

1.4.2.1 In Situ Hybridization

Fluorescence in situ hybridization (FISH) is a molecular technique that allows identification of rearrangement, gain, deletion, or amplification of genetic material on chromosomes (Fig. 1.14). Pros and cons are summarized in Table 1.4. This method uses DNA probes labeled with different fluorescent dyes able to specifically hybridize to unique sequences on the DNA. In the pediatric diagnostic context, almost all the probes are of the break-apart type. These probes are designed to hybridize to centromeric and telomeric DNA sequences closely flanking a gene or locus of interest. In nuclei with normally paired chromosomes, each containing the gene or locus of interest, the signals are adjacent or overlying. In nuclei with balanced chromosomal translocation, instead, the signal of the telomeric probe is separated from that of the centromeric one, meaning that part of the genetic material was transferred to another segregated chromosome. This strategy is widely applied in the diagnostics of pediatric tumors to detect gene rearrangements. It has a great limitation, however: the fusion gene partner is unknown. Indeed, the probes highlight only that the gene or locus of interest is transferred, without providing any information about the new chromosomal attachment site. For instance, the demonstration of a chromosomal translocation using a break-apart set of probes specifically designed for the EWSR1 gene in a small round cell tumor is not diagnostic for ES/PNET. Indeed, EWSR1 may have combined with FLI1, ERG, E1AF, FEV, or ETV1 as it happens in ES/PNET or with WT1 as in desmoplastic small round cell tumor (DSRCT). In either case, this characteristic can also be an advantage since with a single set of probes all the spectrum of rearrangements of EWSR1 is covered. FISH is applicable to any type of cytological preparations, such as air-dried/alcohol-fixed smear, liquid-based preparation, and formalin-fixed paraffin-embedded (FFPE) cell-block sections. Of note, in smears and liquid-base preparations the nuclei are intact; the method is thus not affected by loss of nuclear material, which is one of the major technical pitfalls of histologic or cell-block FFPE section. Another application of in situ hybridization is the demonstration of viral infection, as for Epstein-Barr virus (EBV) in lymphomas.

Table 1.4 Advantages and disadvantages of in situ hybridization

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1.4.2.2 Polymerase Chain Reaction , Reverse Transcriptase-Polymerase Chain Reaction , and Sequencing

PCR allows amplification of a target sequence of DNA included between the regions bound by two specifically designed primers. Thus, it is possible to determine the precise order of nucleotides of the amplicons through sequencing. This method is mainly used to detect gene mutations, insertions, duplications, and deletions. An example of application is the search for mutations in KIT and PDGFRA genes in the uncommon pediatric gastrointestinal stromal tumors (GIST). This is crucial not for the diagnosis but for the therapeutic implications. Indeed, patients with mutated GIST could benefit from targeted therapy.

A variant of PCR followed by sequencing provides an initial step of reverse transcription to obtain complementary DNA (cDNA) molecules from RNA templates. This enables the molecular investigation to move from a long DNA sequence (including both introns and exons of the gene) to its shorter messenger RNA (including only exons of the gene). The advantage is the possibility to overcome (at least partially) the problem of nucleic acids fragmentation and cross-linking due to the use of fixatives (mainly formalin). Indeed, a PCR analysis on fixed cells is reliable only if it is based on amplicons shorter than 200 base pairs, a length too limited to cover both introns and exons of a combined gene (other pros and cons of the technique are reported in Table 1.5). In messenger RNA, instead, amplicons of this length can extend into two juxtaposed exons. This is particularly useful in the analysis of known fusion genes resulting from chromosomal translocations. Indeed, by designing the primers so that each is positioned in one of the supposed combined genes, if the fusion occurred they will be able to drive the amplification (Fig. 1.15); if the fusion did not occur, they will not be able to drive the amplification. Thus, RT-PCR could be a valid alternative to FISH in this setting, since it demonstrates the occurrence of the rearrangement and with which gene. Regarding the example of small round cell tumor, using RT-PCR one can differentiate ES/PNET from DSRCT, being able to recognize the gene combined with EWSR1. However, all the possible fusion genes must be known a priori.

Table 1.5 Advantages and disadvantages of polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and sequencing

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Another major limitation is related to the low sensitivity of classic Sanger sequencing that might bring about false negative results when applied to cytological samples. Indeed, the relative amount of tumor cells harboring the molecular alteration to be investigated could be reduced by an excess of inflammatory or non-tumoral cells in the sample. A neoplastic component higher than 25% of the cells is required for an adequate analysis. Thus, tumor cells enrichment through micro-dissection of the neoplastic cells must be performed in some cases. Obviously, the method suffers also when facing a low absolute amount of neoplastic cells. The result in these cases should be interpreted with caution. Where available, it is advisable to use a more sensitive technique. As for FISH, this method is feasible in any type of cytological sample. However, the best performances are achieved with fresh or frozen specimens. In any case, personnel must be aware of the risk of contamination and take all the necessary precautions. PCR and RT-PCR can be applied also for the detection of oncogenic viral DNA or RNA [78,79,80].

1.4.2.3 The Future: Next-Generation Sequencing

The current molecular biology workflow applied to FNA is destined to radically change in the coming years. Indeed, next-generation sequencing (NGS) will probably replace Sanger sequencing and FISH. NGS is a technique with an extraordinary sensitivity which, by parallelizing the sequencing process, is able to produce up to millions of sequences at the same time. This allows for performance of multiple gene analysis using a minimum of nucleic acids, a crucial aspect for aspirates. Moreover, NGS can cover all the range of genomic alterations, such as base substitutions, short insertions and deletions, amplifications, homozygous deletions, and gene rearrangements. Thus, in the same aspirate it will be possible to analyze concurrently several molecular abnormalities identifying a diagnostic molecular alteration, even if present in less than 1% of the cells.

1.4.3 Other Ancillary Techniques

1.4.3.1 Immunocytochemistry

ICC is a simple, valuable, and cost-effective method to reveal the lineage of differentiation of tumors and is available in almost all pathology laboratories worldwide. ICC is also used to analyze therapeutic targets on neoplastic cells and can be applied for the identification of molecular alteration [81, 82] since it can detect chimeric protein resulting from chromosomal translocation (pros and cons summarized in Table 1.6). In this latter context, ICC is effective in highlighting the presence of the protein product of a fusion transcript (Fig. 1.16). However, the antibodies usually bind to a small part of the protein (epitope) belonging to only one of the combined genes, and therefore does not provide direct information about the fusion gene partner. This is a limitation if the recognized epitope is in a protein that can combine with several others. For instance, a positive immunoreaction to ALK in inflammatory myofibroblastic tumors just means that a translocation involving the ALK gene happened. It is impossible to know if it concerned TPM3, TPM4, CLTC, RANBP2, ATIC, CARS, or SEC31A. However, indirect approximate clues about the translocation can be derived from the subcellular localization of the immunostaining, since the chimeric protein can have nuclear, cytoplasmic or membranous expression depending on the combined gene. ICC can be performed on air-dried/alcohol-fixed smears, liquid-based preparations, and FFPE cell block sections. However, ICC protocols must be optimized according to the material sampled, the fixative used, the times of each step, and the antibodies selected to guarantee reliability.

Table 1.6 Advantages and disadvantages of immunocytochemistry

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1.4.3.2 Flow Cytometry

FC is a technique that enables concurrent testing of the presence of several antibodies labeled with different fluorochromes directed toward membranous or cytoplasmic antigens, which maximizes the amount of data obtained from a few cells, as in the case of cytological samples. It is used to help diagnose and classify neoplasms, mainly lymphomas (Fig. 1.17), to detect therapeutic molecular targets on the surface of malignant cells, and to monitor disease [83,84,85]. In order to determine the quantity and the immunophenotype of a malignant cell population, FC requires viable cells. Thus, fixation is forbidden. Aspirates must be immediately suspended in sterile saline solution or cell culture medium after sampling and processed as soon as possible to prevent cell loss. Indeed, FC analysis may be affected by nonspecific binding of the antibodies to dead or damaged cells. One should be aware that in such cases a negative search for neoplastic cells could be a false negative. Finally, even small tissue fragments may interfere with FC analysis, thus they must be broken down to allow the release of the cells in suspension into fluid before the analysis. Mechanical rather than enzymatic disaggregation is preferred, since enzymes could modify the epitopes recognized by the antibodies. Pros and cons of FC are summarized in Table 1.7

Table 1.7 Advantages and disadvantages of flow cytometry

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1.4.3.3 Rapid On-Site Evaluation

Rapid On-Site Evaluation (ROSE) [86, 87] is an invaluable procedure that should be mandatorily implemented in each service dealing with FNA, especially in the pediatric context. Indeed, ROSE allows the medical team to perform the following tasks:

1.
To evaluate the morphology of the cells and the background of the smear in order to achieve a timely first diagnosis or to understand which are the possible differential diagnoses to consider.
2.
To assess the adequacy of the cytological sample in terms of cellularity, size of tissue fragments, presence of contaminants (blood, inflammatory cells, necrosis), preservation of the tumor cells, and representativeness of the lesion in order to immediately perform a second needle pass if needed.
3.
To properly collect and process the cytological sample having in mind which could be the necessary ancillary methods to solve the case. As has been shown above, each technique presents specific requirements and an inappropriate management of the material may preclude some analyses.

Cases occur in which the cytopathologist performing ROSE needs a second opinion not attainable soon and is thus not able to decide how to handle the sample. In such circumstances, the cells may be collected and temporarily kept in a non-fixative solution (sterile saline or cell-culture medium) to delay the decision for a few hours. Then, the samples can be appropriately processed.

In services where ROSE is not feasible, part of the aspirates (the needle rinse or a second needle pass) should always be conserved for the preparation of a cell block. Indeed, this type of preservation of the sample is suitable for the application of most of the ancillary techniques.

As regards the quantity of material necessary for the various methods, it depends on the number of analyses that should be performed and on the sensitivity of the techniques. As a general rule, however, a needle pass for each technique is a good compromise, at least until the advent in the routine clinical practice of NGS.

1.4.4 Perspectives of Basic Research in Pediatric Tumors’ Diagnosis, Classification and Treatment

Stamatios Theocharis M.D., Ph.D.

First Department of Pathology, National and Kapodistrian University of Athens, 75, Mikras Asias Street, Goudi, Athens, GR11527, Greece
Stamatios Theocharis M.D., Ph.D.

Pediatric malignant tumors represent a heterogeneous group with distinct morphology and characteristic molecular profiles. Data obtained from sequencing projects suggested that, in general, tumors in childhood share fewer somatic mutations and present less genetically complex than those in adulthood [88]. Prognostic molecular features as predictive factors are currently used in some of them, mainly in pediatric leukemia, neuroblastoma, and Wilms’ tumor. Additionally, an increasing number of clinical trials regarding targeted therapies in pediatric cancer patients have been performed. Although the majority of the molecular targeted therapies have been applied on unselected populations, pediatric trials that included genomic biomarkers or distinct molecular cohorts have demonstrated powerful results [89, 90]. Hematologic malignancies represent 40% of all of the pediatric cancers, and targeted therapies have dramatically improved patients’ outcomes. The challenge still remains to better treat refractory and relapsed disease, and advances in pediatric leukemia genomics identify new therapeutic targets as BCR-ABL1 translocation, NOTCH pathway members, altered genes expression regulating Ras signaling and histone modification [89]. Increased progress has been reported for the genomic landscape of pediatric brain tumors, in relation mainly to diagnosis and subclassification as also to prognosis and therapy. ACVR1 mutations in high-grade diffuse (infiltrating) gliomas and the presence of KIAA1549-BRAF fusions and BRAF^V600E mutations in low-grade gliomas represent examples for future therapeutic interventions. In medulloblastoma, molecular studies established four distinct genomic subgroups, presenting novel genes’ mutations or specific translocations related to recurrence, giving information not only for tumor formation but also serving as specific subgroup prognosticators [89]. MYCN amplification was initially associated with aggressive disease state and poor outcome for neuroblastoma patients [91], and MYCN was recently confirmed as a central signaling mediator important for therapeutic target discovery [92]. On the other hand, ALK alterations in both familial and sporadic neuroblastoma cases have been reported [93, 94], suggesting also a treatment option with crizotinib, an ALK inhibitor already used in other malignancies [95, 96]. In rhabdomyosarcoma, comprehensive genome and transcriptome sequencing documented sporadic genomic alterations provided convincing evidence that fusion-positive and fusion-negative tumors consist of biologically distinct subgroups with benefits for directed therapy [97]. Recent studies on the genetic landscape of the Ewing sarcoma family of tumors confirmed that although tumors presenting the EWSR1 fusion exert low mutational burden, they frequently contain cohesin complex subunit STAG2 loss-of-function mutations, CDKN2A deletions and TP53 alterations [89, 98]. An additional issue in different pediatric tumors remains the molecular heterogeneity within the same sample as also from the time of diagnosis to relapse, or within the primary and/or metastatic sites [99]. Detectable molecular alterations could have prognostic and predictive value and should modify treatment options, as recently shown for ALK mutations in neuroblastoma [100] and expansion of loss- of function STAG2 mutations in Ewing sarcoma at relapse [101].

International collaborations on larger patient populations are further encouraged using novel research platforms, such as whole transcriptome sequencing for the discovery of novel genetic alterations, to establish biologic markers and to highlight new therapeutic targets for pediatric tumors [102].

1.5 Specific Considerations Using Small Volume Material: Diagnostic and Sampling Modalities

Jerzy Klijanienko M.D., Ph.D., M.I.A.C.

Department of Pathology, Institut Curie, Paris Cedex 05, France
Jerzy Klijanienko M.D., Ph.D., M.I.A.C.

Despite its morbidity and elevated price, excisional surgical biopsy is still a standard diagnostic procedure in many centres and countries. In the last two decades less invasive biopsy techniques have also demonstrated a highly accurate rate of diagnosis. Fine-needle and core needle biopsies are today optimal and noninvasive diagnostic methods for pediatric tumors [5]. Specific biopsy technique should be used in appropriate clinical and radiological settings. Histological material obtained using core needle biopsy is sufficient to elaborate a diagnosis which is reproducible among pathologists. However, it is not always necessary for pathologists to reach a final diagnosis, since many pediatric tumors have specific molecular abnormalities, allowing highly specific and definitive diagnosis. This specific diagnostic molecular signature is well defined in many tumors such as Ewing’s sarcoma, alveolar rhadbomyosarcoma, synovial sarcoma, desmoplastic small round cell tumor, etc. In such instances, we can speculate that the cytological diagnosis of “round cell sarcoma ,” showing specific abnormality of alveolar rhabdomyosarcoma, is largely sufficient [103, 104]. Similarly, initial cytological diagnosis of “benign connective tissue tumors” or “low-grade connective tissue tumor” (examples: Langerhans cell histiocytosis, lipoblastomas, desmoids, etc.) is sufficient since a final diagnosis will be performed on “non-urgent and representative surgical specimen.” However, several studies have shown that in the hands of a well-trained team with good collaboration between pediatrician, radiologist, and cytopathologist, FNA is a highly accurate approach for quickly rendering a preliminary or final diagnosis [69, 105, 106].

Samples that serve diagnosis are: smears for cytological diagnosis, core needle biopsy, cell block for immunohistochemistry, cell pooling for immunocytochemistry and molecular techniques, and cryopreservation for future biological analyses (Figs. 1.18–1.25) [1, 2].

Part of the FNA material is always used to preapare smears which should be representative. We recommend making two to three slides, depending on the amount of material obtained. Cytologic material is usually paucicellular in fibrous connective tumors (e.g., fibromatosis), or extremely cellular in round cell tumors (e.g., round cell sarcomas, blastemal tumors, lymphomas) [107].

In cases when ancillary techniques are required, we propose to preserve cells in EDTA or in DeLaunay solution for immunocytochemistry and molecular analyses. Once in the laboratory, cells are counted and the quality is evaluated. Cytospins or cell blocks are prepared from material in these preservation media. Extra cellular material may also be frozen for cell banking.

Routine cytological stains are May-Grünwald Giemsa (MGG) and Papanicolaou stainings , but Diff-Quik is also useful. Some centres use hematoxylin-and-eosin safran (HES) . The choice of the staining depends on the tradition of a particular laboratory and on the experience of pathologists.We suggest using MGG stain, which is well suited for examination of cytoplasms, nuclei, and surrounding connective tissue (Figs. 1.26–1.28).

Core needle biopsy is usually performed with thin needles. The material obtained is precious and scant. Its preservation for definitive routine-morphologic diagnosis supported by immunohistochemical studies should be the first intention of the pediatric pathologist [108]. Fixation in Formol for biopsies and double fixation in Formol and Acid-Formol-Alcohol of larges fragments is recommended.

Paraffin-embedded cell blocks being an ancillary cytologic material should be used with corresponding cytological smears (Fig. 1.29). Cell blocks, which are comparable in quality to the histological sections, are extremely useful for immunohistochemical studies. As already mentioned, immunocytochemical and molecular analyses may also be performed on direct or liquid-based cytospins.

The combination of all these samples is used differently in particular clinical settings. Our diagnostic modalities vary in palpable and non-palpable lesions. In palpable lesions children are given local anaesthesia in the form of a cream, which is applied over the area to be sampled 30–60 min before the procedure. Only babies are held down in order to ensure that they will not move during the procedure. With older children who recognize the doctor and injections, we ask them for cooperation and the patients usually do so perfectly. If they are unable to cooperate, the sample is taken under sedation or light general anaesthesia. In both cases it is recommended that the cytopathologist perform the procedure.

In non-palpable lesions (see Sect. 1.3), sample acquisition is guided by imaging techniques (usually ultrasound) and performed either by the cytopathologist and/or the radiologist. The needles we use are 0.6–0.7 mm in diameter. It is important to emphasize that the presence of the cytopathologist is crucial for checking sample adequacy and ensuring proper handling of the specimen for smearing, fixation, and triage for ancillary techniques.

Rapid on-site evaluation (ROSE) , using 30 s Diff-Quik staining , is performed whenever possible regardless of palpable or non-palpable lesions. In addition, to ensure sample adequacy and preliminary diagnosis, this may greatly help in identification of hemopathy, blastemal tumor, or round cell sarcoma. Use of FNA combined with core needle technique, and using molecular examinations, allows us to introduce a “one-day diagnostic procedure,” which is an optimal method used with pediatric patients. This fast diagnostic procedure has been practised for 30 years at the Institut Curie, and was made possibly by the creation of a highly specialized diagnostic multidisciplinary team.

1.6 General Cytological Morphological Aspects

Jerzy Klijanienko M.D., Ph.D., M.I.A.C.

Department of Pathology, Institut Curie, Paris Cedex 05, France
Jerzy Klijanienko M.D., Ph.D., M.I.A.C.

Cytological and histological differential diagnosis modalities are similar, and based on the same parameters. A large variety of cellular and stromal components may be present in a sample, whether this is a cytological smear or paraffin sections of biopsy/cell block. In tumoral pathology cells are epithelial, epitheliod, blastemal, round, and spindle-shaped. They may be aggregated or single. If aggregated, fascicles, clusters, tubes, or rosettes may be present. Stromal elements consist of either nonspecific connective tissue or are composed of fibrous, myxoid, adipocytic, cartilaginous (chondroid), or osseous components. Necrosis, calcifications, inflammatory cells, and different secretions are precious in the diagnosis. The quality, quantity, and proportions of these elements are often indicative and diagnostic.

Epithelial cells are seen in many varieties of tumors. Epithelial neoplasms like hepatocellular carcinomas, thyroid gland tumors, and salivary tumors in children do not differ morphologically from those of adults. However, epithelial cells are also present in some blastemal tumors like nephroblastoma and hepatoblastoma [109]. They are easy to detect when clustering is present. Epithelial component is particularly detectable when Papanicolaou stain is used. Epithelial cells are observed in malignant germinal tumors including teratomas [110, 111]. Usually they show mature tissues which may be regarded as non-representative. The anucleated squamous cell is the most frequently encountered cell type in mature teratomas. Dysgerminomas/seminomas do not look like epithelial cell tumors. Cells are dissociated, medium-to-large size with huge nuclei, prominent nucleoli, and scant cytoplasm in a tigroid background. However, they may show epithelial differentiation which can be demonstrated with positivity for cytokeratins. Yolk sac tumors and embryonal carcinomas, on the other hand, are not epithelial neoplasms; they are, however, cytologically similar to adenocarcinomas with well-formed, three-dimensional groups sometimes in the form of papillae. Groups of epithelial cells can be present in the biphasic type of synovial sarcoma (Figs. 1.30–1.32) [112].

Cells with epithelioid or plasmacytoid features are characterized with eccentric nuclei and abundant, well-delineated cytoplasm. They are observed in many benign conditions as well as in some malignancies. Tumors and tumor-like conditions are rarely composed entirely of epithelioid cells, but they do predominate in some tumors, such as rhabdoid tumor [113, 114] and malignant melanoma. Some myofibroblasts also have epithelioid morphology and can predominate in cases of nodular fasciitis and fibromatosis. Many epithelioid cells may be present in rhabdomyosarcoma, while a few plasmacytoid cells are seen also in some round cell sarcomas such as Ewing’s sarcoma [115]. Epithelioid cells of various tumors differ somewhat in their morphology regarding cell size and the presence and size of nucleoli. Classical rhabdoid tumor , for example, has a population of large polygonal cells with very prominent nucleoli and dense, periodic acid-Schiff-positive globoid cytoplasmic inclusions (Figs. 1.33 and 1.34) that correspond, as seen by electron microscopy and immunochistochemical analysis, to accumulations of perinuclear intermediate filaments. However, morphology alone cannot always differentiate tumors with epithelioid features, and ancillary techniques are mandatory.

Blastemal cells are present in all blastemal tumors, but their morphology varies somewhat depending on the cell of putative origin. Nevertheless, they are small cells with high nuclear/cytoplasmatic ratio. Nephroblastoma shows roundish and oval blastemal cells with regular nuclei and clearly visible nucleoli. Cytoplasm is grayish and may contain some microvacuolizations (Figs. 1.35 and 1.36) [116]. Blastemal cells in neuroblastoma are characterized by excentred, round, kidney-shaped, or V-shaped nuclei with numerous mitotic figures (Figs. 1.37 and 1.38) [117, 118].

Hepatoblastomas are rarely composed of a single cell type [109]. Fetal cell type has monomorphic round-to-spindle-shaped cells, resembling hepatocytes, while embryonal type cells are pleomorphic, hyperchromatic, and exhibit an elevated nuclear/cytoplasmic ratio. Finally, retinoblastomas are composed of undifferentiated cells as well as of more differentiated cells with cytoplasmic extensions. The morphological differences in blastemal cells are usually not sufficient to arrive at the correct diagnosis. Cellular arrangement, mesenchymal components, necrosis, and calcifications have to be considered.

Round cells are seen in lymphomas and in round cell sarcomas [119,120,122]. Some sarcomas are predominantly composed of round cells like alveolar rhabdomyosarcoma, Ewing’s sarcoma and desmoplastic small round cell tumors (Figs. 1.39 and 1.40), and mesenchymal chondrosarcoma [115, 123,124,125]. Cells in rhabdomyosarcoma show excentred nuclei and are occasionally spindle or binucleated. Cells in Ewing’s sarcoma frequently show rosettes and double cell population: smaller, darker, and larger and clearer cells (Figs. 1.41 and 1.42). Cells in desmoplastic round cell tumors are small and without specific patterns. Dense connective tissue is found in smears and histological sections (Figs. 1.43 and 1.44). In some sarcomas round cells predominate only in the so-called round cell variants as in synovial and osteosarcomas. Finally, there are sarcomas such as malignant rhabdoid tumor in which round cells are a minor component.

Spindle-shaped cells may exhibit nonspecific or clearly malignant morphology. In low-grade/benign spindle-cell tumors such as desmoids, nodular fasciitis, schwannomas, congenital fibrosarcomas, or dermatofibrosarcoma protuberans, cells are spindle and regular (Figs. 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53 and 1.54) [119, 126, 127]. Nuclei are well-formed and chromatin is dusty. In spindle cell sarcomas such as synovial sarcoma or high-grade malignant peripheral nerve sheath tumors, cyto-nuclear atypia may be prominent, nuclei may be irregular, and signs of malignancy are immediately found [128].

Stroma is also an important element of diagnosis. Stroma in neuroblastoma appears in two different patterns: the neuropil and schwannian fragments (Fig. 1.55). Neuropil presents as a delicate and fibrillary pink matrix on MGG staining, and as fine microlinear material on Papanicolaou staining. Neuropil is present both inside the rosettes and as background. The schwannian tissue fragments in neuroblastoma resemble those in schwannoma or malignant peripheral sheath tumor. Connective fragments and vascular structures are easily identified in alveolar rhabdomyosarcoma. Myxoid stroma, seen in nodular fasciitis, may be misleading.

Osseous and cartilaginous components may be present in osteosarcoma [129], mesenchymal chondrosarcoma,; hepatoblastoma, and teratoma (Figs. 1.56–1.58). Cytological examples of pleuropulmonary blastoma showing epithelial cells, spindle-shaped cells and cartilage were also reported [130].

Calcifications are usually present in Ewing sarcoma, retinoblastoma, and neuroblastoma. Calcifications are usually associated with necrotic areas (Fig. 1.59).

Necrosis may be prominent in retinoblastoma, Ewing sarcoma, and neuroblastoma. Necrotic areas may occasionally be seen in nephroblastoma and other round cell sarcomas.

The combination of cellular and stromal components may vary in different types of tumors. Table 1.8 presents some particular associations that may be helpful in the preliminary cytological diagnosis. Proposition of cytological classification of soft tissue tumors arising in children and in adults was previously reported [119].

Table 1.8 Cellular and stromal components useful for the preliminary diagnosis

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1.7 Histopathologic Evaluation of Pediatric Tumors Containing Round Cells

Patsy Dominique Berrebi M.D., Ph.D. &
Michel Peuchmaur M.D., Ph.D.

Hôpital Robert Debré, Paris, 75019, France
Patsy Dominique Berrebi M.D., Ph.D. & Michel Peuchmaur M.D., Ph.D.

Histopathological examination is performed on core needle biopsies, surgical biopsies, and surgical specimens. Fresh tumoral material should be partially fixed and partially frozen for tissue banking. Additional material may be a subject of molecular studies.

Pediatric tumors are benign or malignant. Benign lesions are usually composed of small spindle cells. Malignant lesions are usually composed of spindle or round cells. Tumors containing round cells may be of various putative origins like connective, blastemal, or hematopoietic tissues, and constitue the subject of this paragraph.

Malignant small round cell tumors (MSRCT) is a term used for tumors composed of malignant round cells that are slightly larger or double the size of red blood cells. This group of neoplasms mainly occurs in children and is characterized by rapidly growing, relatively small, round, and undifferentiated/primitive cells with round to oval nuclei, no nucleoli, and pale chromatin [131]. Classification distinguishes several groups in function of site and histological type [132]. The “4 main tumors” between 0 and 16 years include lymphoma/leukemia, neuroblastoma, rhabdomyosarcoma, and Ewing’s sarcoma (EWS) (Table 1.9). But, interestingly, the percentage of solid MSRCT (except central nervous system tumors) before the age of 5 years is also indicated, showing that neuroblastoma and nephroblastoma are the two most frequent solid tumors before the age of 5 years.

Table 1.9 Classification of pediatric tumors and percentage before the age of 5 years

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Some of them are small cell tumors or organ-specific blastomas: nephroblastoma (or Wilms’tumor), hepatoblastoma, pancreatoblastoma, sialoblastoma, or pleuropulmonary blastoma. Others occur in specific sites: desmoplastic small round cell tumor (DSRCT) in peritoneal cavities; germ cell tumors in ovary or testis; nuclear protein in testis (NUT); translocation carcinoma in the head, neck, and mediastinum. Still others, such as synovial sarcoma and rhabdoid tumor, occur in diverse sites.

Differential diagnosis of small round cell tumors is particularly difficult for pathologists due to their undifferentiated or primitive characters and requires extensive immunohistochemistry (IHC) and molecular studies [132]. Indeed, age, sites, cooperation between surgeons, clinicians, radiologists, and the use of a large panel of antibodies and molecular studies are mandatory to differentiate this group of neoplasms (Table 1.10) from malignant hemopathies (Fig. 1.60a) and new entities such as “Ewing-like tumors” with non-EWS fusions: CIC-DUX4 (Fig. 1.60b) or BCOR-CCNB3 sarcomas. In addition, these SRCT can mimic each other, notably DSRCT without desmoplasia on fine-needle biopsy (Fig. 1.60c), which can show a highly variable immunohistochemistry pattern but a characteristic translocation t (11;22)(p13;q12).

Table 1.10 Main useful antibodies for immunohistochemistry to differentiate MSRCT

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1.7.1 Peripheral Neuroblastic Tumors

Peripheral neuroblastic tumors represent one of the most frequent groups of solid tumors in children (Table 1.9). A considerable amount of publications describe these enigmatic tumors, which encompass a very large spectrum of clinical behaviors, morphologic characteristics, and genomic abnormalities [133,134,135]. One of the most important contributions has been the establishment of a risk grouping to obtain a fine-tuning for tailoring the treatment. International cooperation allowed the definition of different risk group, the International Neuroblastoma Risk Group (INRG), including the staging (with the characterization of image defined risk factors, or IDRF), the pathology, and the genomic abnormalities [96, 136,137,138]. Five robust criteria are taken into account for the risk grouping :

1.
Age (less or more than 18 month old)
2.
Stage (localized without IDRFs: L1, localized with IDRFs: L2, metastatic: M, metastatic special: Ms)
3.
Pathology (Favorable or Unfavorable) (Tables 1.11a and 1.11b),
4.
Nmyc status (not amplified or amplified)
5.
Genomic profile obtained by CGHarray (numeric or segmental chromosome alterations) [139]

Table 1.11a The INPC (International Neuroblastoma Pathology Classification ) recognizes four categories of Peripheral Neuroblastic Tumors: ganglioneuroma, ganglioneuroblastoma intermixed, ganglioneuroblastoma nodular, neuroblastoma. The analysis relies on the presence or not of a Schwannian (i.e, “stromal”) component and on the cytological characteristics of tumor cells, mainly the differentiation (differentiating D, poorly differentiated PD or undifferentiated U) and the Mitotic Karyorrhectic Index (MKI). These two major cytological characteristics must be described both in neuroblastoma categories and in the nodule of the ganglioneuroblastoma, nodular, categories. In the INPC, the histoprognostic depends on these cytoarchitectural characteristics combined with the age of the patient (F—favourable; UF—unfavorable)

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Table 1.11b Histoprognostic of peripheral neuroblastic tumors according to INPC

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To focus on the pathology, the age-dependent classification of the peripheral neuroblastic tumors was established by the International Neuroblastoma Pathology Committee (INPC) [140,141,142]. INPC recognizes four categories of peripheral neuroblastic tumors (Table 1.11a):

1.
Ganglioneuroma (GN) (Fig. 1.61a)
2.
Ganglioneuroblastoma intermixed (GNBi) (Fig. 1.61b)
3.
Ganglioneuroblastoma nodular (GNBnod) (Fig. 1.61c)
4.
Neuroblastoma (NB) (Fig. 1.61 d–f)

Peripheral neuroblastic tumors are tumors derived from the peripheral sympathetic nervous system and are basically constituted by a tumor component made of neuroblasts. This neuroblastic tumor cells component could be associated with a schwannian cell component . The precise classification of a peripheral neuroblastic tumor relies on the analysis of the presence or not of a schwannian cell component, the relation between this schwannian cell component and the tumor cell component (for example, in the GNBnod category, the interface between the schwannian cell component and the tumor cell component is defined by the presence of a pushing border), and the cytological characteristics of the tumor cells. Two major characters of the neuroblasts must be defined (Fig. 1.61g): (1) the grade of differentiation (differentiating, poorly differentiated, and undifferentiated); and (2) the MKI (low: <2%; intermediate: 2–4%; high: more than 4%). The analysis of these cyto-architectural characteristics, along with the age of the patient, allowed performing the diagnostic and the histoprognostic as Favorable or Unfavorable, taking into account in the neuroblastoma tumor cell component of the grade of differentiation, the mitosis karyorrhexis index MKI, and the age of the patient (Table 1.11b) [143]. By definition, the International Neuroblastoma Pathology Classification relies on the study of pre-chemotherapy surgical specimens. But the complexity of clinical presentation in neuroblastic tumors, namely the stage L2, M, or MS, justifies that most often the pathologist is faced with a core biopsy specimen, or, in some countries, with a surgical biopsy specimen [144]. Actually, no data is available to be sure that the biopsy (either core or surgical) is representative of the whole tumor. Nevertheless, several international protocols (e.g., the Localized and Intermediate Risk Neuroblastomas European Study: LINES protocol under the care of the SIOPEN, Cf. http://www.siopen.org/siopen-studies/current/lines), indicate that not only the diagnosis, but also the histoprognostic according to INPC recommendations, have to be performed on a core biopsy. To be representative, the biopsy must contain more than 5000 tumor cells. Moreover, the biopsy should allow one toto rule out a ganglioneuroblastoma nodular subtype in case of a predominance of schwannian component. This underlines that the application of INPC on biopsy must be made with caution, all the more because recent data describe tumor heterogeneity in peripheral neuroblastic tumors. Nevertheless, in a majority of cases, the application of INPC when core biopsy is indicated could be proposed (Fig. 1.61h). The use of immunohistochemistry is relevant in cases of undifferentiated neuroblastomas (Fig. 1.61f) and/or in cases of poorly differentiated neuroblastoma if the neurofibrillary background is difficult to find out. As usual, a panel of antibodies must be used to rule out other malignant small round cell tumors (MSRCT) of children. Two antibodies are very useful to recognize the neuroblast cells: anti-PHOX2B (Fig. 1.61f) and anti-tyrosine hydroxylase (Table 1.10) [145]. Besides the cytoarchitectural characteristics defined in the INPC, two other cytological characteristics with unfavorable signification must be pointed out when analyzing tumor cell morphology: the presence of a large, often pinky nucleoli, which could be correlated with NMyc amplification (Fig. 1.61i) [146], and the presence of nuclear polymorphism, which could be linked with segmental chromosome alterations (Fig. 1.61j). After chemotherapy, there is at this date no international recommendation describing the way to manage the surgical specimen, but several publications argued in favor of an extensive analysis of the surgical specimen, particularly to detect alterations in morphology (Fig. 1.61j) and/or in genomic abnormalities in persistent tumor cell components compared with those found in the initial pre-chemotherapy specimen. The emergence of these new genomic alterations could represent targets for future treatment [94, 100, 147, 148].

1.7.2 Nephroblastoma

Wilms’ tumor (nephroblastoma), an embryonal type of renal cancer, is one of the most common solid malignant neoplasms in children. It accounts for approximately 90% of all paediatric tumors of the kidney. More than 80% of children who are diagnosed with Wilms’ tumor are diagnosed before the age of 5 years, and the median age at diagnosis is 3.5 years. There are three main types of tumor cells: blastema, resembling the undifferentiated embryonic metanephric mesenchyme, together with epithelium and stroma, both thought to have differentiated from the blastema [149,150,151]. These cell types are distinguished histologically and currently there are no good markers to specifically identify blastema. Several distinctive patterns occur in blastemal cells, including the diffuse, serpentine, nodular, and basaloid patterns. In the SIOP (Société Internationnale d’Oncologie Pédiatrique) classification, where histology is assessed after chemotherapy, Wilms’ tumors are sub-classified and risk-stratified based on the percentage of each of these types of cells. Fine-needle aspiration biopsy can be used to address a correct diagnosis of nephroblastoma if age and/or radiological findings are atypical (Fig. 1.62a). Both survival of a high proportion of blastemal cells after chemotherapy and/or the presence of diffuse anaplasia, which is defined morphologically, are considered high risk. The histological criteria for making a diagnosis of anaplastic nephroblastoma are the presence of all three criteria for anaplasia: (1) the presence of atypical tri/multipolar mitotic figures; (2) marked nuclear enlargement, with diameters at least three times those of adjacent cells; and (3) the presence of hyperchromatic tumour cell nuclei. The main differential diagnosis on fine-needle aspiration biopsy is the newly discovered entity named anaplastic sarcoma (Fig. 1.62b) [152].

Histological subtyping and risk-grouping of renal tumors in children according to SIOP initial treatment approach is shown in Table 1.12.

Table 1.12 Renal tumor classification after chemotherapy according to SIOP

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1.7.3 Ewing Family (ESF) of Tumors

This family includes Ewing sarcoma of bone and soft tissues and peripheral primitive neuroectodermal tumor. They arise in bone (mostly diaphyseal), less often in soft tissue, and rarely in organs such as kidney. ESF harbors characteristic genetic fusions of EWS gene with ETS group of genes (FLI1/ t(11;22) in about 85% and ERG/t(21;22) in about 10–15%. ESF is slightly more common in boys, mostly adolescents and young adults. Histologically, we distinguish classical Ewing sarcoma, atypical Ewing sarcoma, and peripheral neuroectodermal tumor (PNET). Classical ES show small nuclear size, regular nuclear contours, no rosette, and inconspicuous nucleoli, as illustrated in Fig. 1.62c, whereas atypical/PNET show opposite features. Positivity of CD99 (membranous) (Fig. 1.62c) and FLI-1 by immunohistochemistry is characteristic although nonspecific, but the combined use of CD99 and a new marker NKX2.2 (transcription factor for neuronal development and the target gene product of EWS-FLI1 fusion protein) considerably improved specificity. Histological characteristics of Ewing-like sarcoma with non-EWS fusions are indicated in Table 1.13 [153,154,155,156].

Table 1.13 CIC-DUX4 or BCOR-CCNB3 Sarcomas

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1.7.4 Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most frequent of soft tissue tumor. It is rare before the age of 1 year, and the median age is 7 years. The three main sites are extremities, head and neck, and bladder and genital area. The International Classification of Rhabdomyosarcoma [157, 158] classifies childhood rhabdomyosarcoma into the following categories:

1.
Botryoid rhabdomyosarcoma is a favorable prognosis subtype of embryonal RMS. It occurs in sites adjacent to an epithelial surface, particularly bladder, vagina, nasal cavity and sinuses, and biliary tract. Diagnosis of the botryoid variant requires a cambium layer (condensed layer of rhabdomyoblasts) underlying an intact epithelium (Fig. 1.63a).
Fig. 1.63
Bladder, and pelvic illustrating rhabdomyosarcoma, others renal tumors (rhabdoid tumor). (a), Bladder rhabdomyosarcoma, botryoid subtype (macroscopy, left; HES, middle and right). The tumor cells are arranged in a cambium layer beneath an intact urothelium. (b), Bladder rhabdomyosarcoma, embryonal subtype (HES, left and middle). The rhabdomyoblast tumor cells expressed cytoplasmic desmin and a portion of them nuclear myogenin (immunoperoxidase on formalin-fixed embedded paraffin sections with anti-desmin antibody, Gx25, middle, and anti-myogenin antibody, right). (c), Pelvic rhabdomyosarcoma, alveolar subtype (HES, left and reticulin, right). The tumor cells were discohesive and lined up along fibrous septae. This tumor presented with the PAX3-FKHR fusion gene. (d), Core needle biopsy of a solid renal mass (macroscopy, left) in a 6-month-old boy with hypercalcemia showing a rhabdoid tumor (HES, Gx40, middle), with loss of INI1 expression (immunoperoxidase on formalin-fixed embedded paraffin section with anti-INI 1 antibody, right)
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2.
Embryonal rhabdomyosarcoma , not otherwise specified, is composed of mesenchymal cells that show variable degrees of cytoplasmic skeletal muscle differentiation. They are typically moderately cellular but may contain both hypo- and hypercellular areas with a loose, myxoid stroma (Fig. 1.63b).
3.
Alveolar habdomyosarcoma is a poor prognosis subtype composed of malignant small rounded cells that are typically discohesive with a tendency to attach to and line up along thin fibrous septae. The differential diagnosis of alveolar RMS includes the panoply of other MSRCT, but a strong nuclear expression for myogenin and desmin is always present. RT-PCR for PAX3- and PAX7-FKHR fusion gene products occur in approximately 85% of alveolar RMS cases (Fig. 1.63c).
4.
Spindle cell rhabdomyosarcoma is considered a subtype of embryonal RMS, composed almost exclusively of elongated spindle cells, and one-third of spindle cell RMS are located in the paratesticular region.
5.
Malignant rhabdoid tumor (MRT) is a rare neoplasm originally described in the kidney (Fig. 1.4d), but is also found in soft tissues and the central nervous system. It occurs in infants and young children with about 80% of patients younger than 2 years, while it is extremely rare after 5 years of age [159]. Two characteristic associations of MRT are hypercalcaemia and the development of synchronous or metachronous primary brain tumors. The classic rhabdoid tumor cell appears large with an eccentrically placed nucleus with vesicular chromatin and a prominent eosinophilic nucleolus (Fig. 1.63d). These cells characteristically have abundant eosinophilic cytoplasm with a globular pink cytoplasmic inclusion. Molecular genetic investigations of malignant rhabdoid tumors have identified a characteristic loss or mutation of the INI1 (BAF47) gene in chromosome band 22q11.2. Deletion and/or mutation of both copies of the INI1 gene results in loss of INI1 expression at the protein level, which can be detected using immunohistochemistry with an anti-INI1 antibody (Fig. 1.63d). Recent data extend the spectrum of INI1-deficient tumors, and several malignant tumors other than MRT showed INI1 abnormalities with loss of INI1 protein expression such as malignant peripheral nerve sheat tumor (MPNST) or epithelioid sarcoma [160].

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Department of Pathology, Institut Curie, Paris, France
Jerzy Klijanienko M.D., Ph.D., M.I.A.C.
Departments of Pediatric Oncology and Radiology, Institut Curie, Paris, France
Sarah Cohen-Gogo M.D., Ph.D., Marie Louise Choucair M.D., Daniel Orbach M.D., Cécile Cellier M.D. & Hervé J. Brisse M.D., Ph.D.
Department of Medicine, Cytopathology Unit, University of Padova, Via 8 Febbraio 1848, 2, Padova, 35122, PD, Italy
Rocco Cappellesso M.D.
Department of Surgical Pathology and Cytopathology Unit, University of Padova, Via 8 Febbraio 1848, 2, Padova, 35122, PD, Italy
Ambrogio Fassina M.D., Ph.D.
First Department of Pathology, National and Kapodistrian University of Athens, 75, Mikras Asias Street, Goudi, Athens, GR11527, Greece
Stamatios Theocharis M.D., Ph.D.
Hôpital Robert Debré, Paris, 75019, France
Patsy Dominique Berrebi M.D., Ph.D. & Michel Peuchmaur M.D., Ph.D.

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Jerzy Klijanienko M.D., Ph.D., M.I.A.C.
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Correspondence to Jerzy Klijanienko M.D., Ph.D., M.I.A.C. , Jerzy Klijanienko M.D., Ph.D., M.I.A.C. , Sarah Cohen-Gogo M.D., Ph.D. , Cécile Cellier M.D. , Rocco Cappellesso M.D. , Stamatios Theocharis M.D., Ph.D. , Jerzy Klijanienko M.D., Ph.D., M.I.A.C. , Jerzy Klijanienko M.D., Ph.D., M.I.A.C. or Patsy Dominique Berrebi M.D., Ph.D. .

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Department of Pathology, Institut Curie, Paris Cedex, France
Jerzy Klijanienko
Department of Cytopathology, Institute of Oncology, Ljubljana, Slovenia
Živa Pohar Marinšek
Department of Pathology, Skåne University Hospital, Lund, Sweden
Henryk A Domanski

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Klijanienko, J. et al. (2018). General Considerations. In: Klijanienko, J., Pohar Marinšek, Ž., Domanski, H. (eds) Small Volume Biopsy in Pediatric Tumors. Springer, Cham. https://doi.org/10.1007/978-3-319-61027-6_1

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DOI: https://doi.org/10.1007/978-3-319-61027-6_1
Published: 17 July 2017
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General Considerations

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Introduction and Application of Fine Needle Aspiration Biopsy

Introduction

Introduction

Keywords

1.1 Introduction

1.2 Clinical Aspects of Semi-Malignant and Malignant Tumors in Children and Adolescents

1.2.1 Cervical Nodes

1.2.2 Thoracic Tumors

1.2.3 Mesenteric and Peritoneal Tumors

1.2.4 Hepatic Tumors

1.2.5 Pancreatic Tumors

1.2.6 Adrenal Tumors

1.2.7 Pelvic Tumors

1.2.8 Renal Tumors

1.2.9 Soft Tissue Lesions

1.2.10 Bone Tumors

1.3 Radiological Diagnostic Approach in Extracerebral Pediatric Tumors

1.3.1 Imaging Techniques

1.3.2 Tumor Biopsy

1.3.2.1 Surgical and Core Needle Biopsies

1.3.2.2 Fine-Needle Aspiration

1.4 Ancillary Methods

1.4.1 An Overview of Molecular Alterations in Pediatric Tumors

1.4.2 Molecular Techniques

1.4.2.1 In Situ Hybridization

1.4.2.2 Polymerase Chain Reaction , Reverse Transcriptase-Polymerase Chain Reaction , and Sequencing

1.4.2.3 The Future: Next-Generation Sequencing

1.4.3 Other Ancillary Techniques

1.4.3.1 Immunocytochemistry

1.4.3.2 Flow Cytometry

1.4.3.3 Rapid On-Site Evaluation

1.4.4 Perspectives of Basic Research in Pediatric Tumors’ Diagnosis, Classification and Treatment

1.5 Specific Considerations Using Small Volume Material: Diagnostic and Sampling Modalities

1.6 General Cytological Morphological Aspects

1.7 Histopathologic Evaluation of Pediatric Tumors Containing Round Cells

1.7.1 Peripheral Neuroblastic Tumors

1.7.2 Nephroblastoma

1.7.3 Ewing Family (ESF) of Tumors

1.7.4 Rhabdomyosarcoma

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