
Tumors arising from the sympathoadrenal lineage of neural crest-derived tissues are collectively known as peripheral neuroblastic tumors (pNTs) and include Neuroblastoma, Ganglioneuroblastoma and Ganglioneuroma. pNTs are the third most common childhood neoplasm after leukemias and brain tumors, the most common neoplasms during the first year of life, and the most common extra-cranial solid tumors in the first 2 years of life. pNTs are composed of biologically different groups and demonstrate a wide spectrum of clinical courses to include spontaneous regression, tumor maturation, and aggressive progression refractory to therapy. These clinical behaviors are closely associated with defects in various molecular mechanisms that regulate sympathetic nervous system development [1]. Recent advances in tumor biology and normal embryology have increased understanding of the functions of the molecular markers and enhanced efforts to find actionable targets for precision medicine.
Historically, various prognostic factors predicting clinical behavior of pNTs are reported. Those are clinically, histopathologically, and genetically/molecularly defined factors. In order to develop efficient clinical management protocols, risk-grouping systems have been established to stratify affected patients by combinations of the risk factors (please see Risk grouping section). These important prognostic factors are presented below.
Clinical stage and Age at diagnosis are well known prognostic factors. Historically, they have been accepted and included in the risk grouping systems.
(1) Clinical stageThere are two widely used clinical staging systems: They are INSS (International Neuroblastoma Staging System) [2] and INRGSS (International Neuroblastoma Risk Group Staging System) [3]. The INSS is based on the post-surgical evaluation of disease extent, and distinguishes localized disease (Stage 1, 2 and 3), metastatic disease (Stage 4) and special metastatic disease with favorable clinical outcome (Stage 4S). The INRGSS is based on presurgical determination of disease extent, and localized disease is distinguished into two Stages based on presence or absence of Image-Defined Risk Factors (IDRFs): they are L1 (localized disease without IDRFs) and L2 (localized disease with IDRFs). INRGSS also include Stage M (metastatic disease) and Stage MS (special metastatic disease). By definition, disease status of Stage 4S is the same as that of MS, but age at diagnosis of the former is less than one year (365 days) and the latter is less than 1.5 years (548 days). More than 50% of pNT patients have metastatic disease at the time of diagnosis. It is reported that the event-free survival (EFS) rate and overall survival (OS) rate for the patients with stage 4/M disease are 35% and 42%, respectively. In contrast those for the patients with localized and special metastatic disease are 83% and 91%, respectively [4].
(2) Age at diagnosisIn pNTs, the patient’s age at diagnosis has been used as one of the prognostic indicators. Historically, one year of age was used as the cut-off for distinguishing a better prognostic group (<365 days) from a worse prognostic group (≥365 days). However, in 2005, London et al. reported that (1) the prognostic contribution of age to the clinical outcome is continuous in nature: Whichever age cut-off was adopted, survival rate of younger patients was always better than that of older patients, and (2) there was statistical evidence of an age cut-off greater than one year for risk stratification [5]. Since then, national and international clinical studies have been moving the age cut-off for prognostic distinction from one year (365 days) to 18 months (548 days). Age should be regarded as a surrogate for other risk factors. For example, the International Neuroblastoma Pathology Classification (INPC, see below) already has a built-in age cut- off of 18 months for distinguishing prognostic groups. Furthermore, Sano et al. demonstrated that the INPC was able to add independent prognostic information beyond the contribution of age [6]. In other words, the INPC clearly distinguishes two prognostic groups (Favorable Histology Group and Unfavorable Histology Group, the former identifying significantly better prognosis than the latter) in various age groups, such as < vs. ≥12 months (365 days), < vs. ≥18 months (548 days), and < vs. ≥24 months (730 days) of age on diagnosis (Fig. 1).
Histopathology has also been included in the risk grouping systems. It is interesting to note that pNTs can offer one of the best models to investigate biologically significant relationship between genomic/molecular alterations and morphologic manifestations (please see Subgrouping of Unfavorable Histology Neuroblastoma and Precision Medicine section).
(1) Categories and subtypes (Fig. 2)“Neuroblastoma” is often used as an omnibus term for all tumors in the pNTs. In 1999, the International Neuroblastoma Pathology Committee defined 4 categories [7]: They are Neuroblastoma (Schwannian stroma-poor; no or limited Schwannian stromal development occupying up to 50% of tumor tissue; Fig. 2A-C), Ganglioneuroblastoma, Intermixed (Schwannian stroma-rich; Schwannian stromal development occupying >50% of tumor tissue; Fig. 2D), Ganglioneuroma (Schwannian stroma-dominant; predominantly Schwannian stroma containing individual or clusters of ganglion; Fig. 2E) and Ganglioneuroblastoma, Nodular (composite, Schwannian stroma-rich/stroma- dominant and stroma-poor; Fig 2E). The first three categories represent tumor maturation prompted by “cross-talk” between neuronal tumor cells and Schwannian stromal cells supported by various signaling pathways, including trkA/NGF signaling and Nrg1/ErbB signaling [8-10]. As described by Willis [11], pNTs are embryonal tumors: It is believed that all Ganglioneuromas were once Neuroblastomas in their early stage of tumor development. The fourth category of Ganglioneuroblastoma, nodular is defined as composite or multi-clonal tumor. Among all the pNTs reviewed and filed at the COG Neuroblastoma Pathology Reference Laboratory, approximately 80% are in the Neuroblastoma category and the rests are in the Ganglioneuroblastoma, Intermixed (9%), Ganglioneuroma (3%), and Ganglioneuroblastoma, Nodular (8%) category (unpublished data).
In the Neuroblastoma category, there are three subtypes depending on the grade of neuroblastic differentiation: They are Undifferentiated (totally undifferentiated tumor without potential of neuroblastic differentiation, immunohistochemical and/or molecular tests required for confirming the diagnosis; 3% in the Neuroblastoma category; Fig. 2A), Poorly differentiated (tumor with or without potential of further neuroblastic differentiation, diagnosis confirmed by identifying neurite formation by tumor cells; 89% in the Neuroblastoma category; Fig. 2B), and Differentiating (tumor with more than 5% of neoplastic cells having an appearance of differentiating neuroblast; 8% in the Neuroblastoma category; Fig. 2C) subtype. In the tumor tissue of the Ganglioneuroblastoma, Intermixed (one step behind of complete maturation towards Ganglioneuroma), neuritic processes produced by the neuroblastic cells are not completely incorporated in the cytoplasm of Schwann cells, so that foci of naked neurites are still identified microscopically (Fig. 2D). While in the tumor tissue of the Ganglioneuroma (final stage of tumor maturation), all neuritic processes produced by ganglion cells are immediately incorporated in the cytoplasm of Schwann cells, so that no naked neuritic processes are detected (Fig. 2E). In the Ganglioneuroma category, two subtypes, maturing and mature, are recorded: Ganglion cells are embedded in the Schwannian stroma of both subtypes, and the latter is composed only of completely mature ganglion cells covered with satellite cells (Fig. 2E, inset).
(2) International Neuroblastoma Pathology Classification (INPC)The INPC was established in 1999 by adopting the original Shimada classification [12] with minor modifications [13] and revised in 2003 [14]. According to the INPC, pNTs are classified into either Favorable Histology (FH) Group or Unfavorable Histology (UH) Group (Please see Fig. 1A). As shown in Table 1, all tumors in the Ganglioneuroblastoma, Intermixed category and in the Ganglioneuroma category are FH with an excellent clinical outcome. While tumors in the Neuroblastoma category and Ganglioneuroblastoma, Nodular (composite tumor of Ganglioneuroblastoma, Intermixed or Ganglioneuroma containing neuroblastoma nodule) category are distinguished into the FH or UH Group based on age-dependent evaluation of histologic indicators of the tumor or neuroblastoma nodule: They are the grade of neuroblastic differentiation (i.e., subtype of Undifferentiated, Poorly differentiated or Differentiating; see Categories and Subtypes above) and the mitotic and karyorrhectic activities [MKI, Mitosis-Karyorrhexis Index; low (<100/ 5,000 cells), intermediate (100-200/5,000 cells) or high (>200/5,000 cells)]. Determination of MKI class is based on averaging the activities by counting mitotic and karyorrhectic cells in multiple representative microscopic fields. In other words, MKI class is not determined by counting the activities from the hottest, i.e. most condensed, fields. Cells with mitosis and nuclear fragmentations (i.e. karyorrhexis) are included in the counting, while simply hyperchromatic (darkly stained) nuclei are excluded.
Table 1 . International Neuroblastoma Pathology Classification [7,13].
Favorable histology | Unfavorable histology | |
---|---|---|
Neuroblastoma (Schwannian stroma-poor) | ||
Undifferentiated subtype | All Tumors (Any MKI, Any Age) | |
Poorly differentiated subtype | Low or Intermediate MKI, <548 days | Low or Intermediate MKI, ≥548 days |
High MKI, Any Age | ||
Differentiating subtype | Low MKI, <5 years | Any MKI, ≥5 years |
Intermediate MKI, ≥548 days | ||
High MKI, Any Age | ||
Ganglioneuroblastoma, intermixed (Schwannian stroma-rich) | All Tumors | |
Ganglioneuroma (Schwannian stroma-dominant) | ||
Maturing subtype | All Tumors | |
Mature subtype | All Tumors | |
Ganglioneuroblastoma, Nodular (composite, Schwannian stroma-rich/ Stroma-dominant and stroma-poor) | Favorable histology vs. unfavorable histology distinction: based on age-linked evaluation of Grade of neuroblastic differentiation (subtype) and MKI of the neuro-blastoma nodule |
MKI, Mitosis Karyorrhexis Index; Low, (<100/5,000 cells); Intermediate, (100-200/5,000 cells); High, (>200/5,000 cells).
FH tumors fit into an age-appropriate framework and display differentiation/maturation from Poorly differentiated subtype to Differentiating subtype of Neuroblastoma, then to Ganglioneuroblastoma, Intermixed and finally to Ganglioneuroma. In order to observe tumor differentiation/maturation, however, a certain period of time, i.e.,
UH Group includes tumors of the (1) Neuroblastoma, Undifferentiated subtype in any age-group, tumors of the Neuroblastoma, (2) Poorly differentiated subtype in patients over 18 months of age, and (3) tumors of the Neuroblastoma, Differentiating subtype in patients over 60 months of age. Those tumors are considered to have limited or no differentiation/maturation potential and they are outside the age-appropriate framework. As for MKI classes, (1) High MKI NB tumors in any age group, (2) Intermediate MKI tumors ≥18 months of age at diagnosis, and (3) Low MKI tumors ≥60 months of age at diagnosis are also outside the framework. A significant correlation between
According to the INPC, survival rate of patients in the FH group is estimated to be approximately 90%, whereas that of the UH group has remained approximately 50% for years [6]. In summary, clinically unfavorable behaviors, such as treatment-resistance and aggressive progression, are observed in patients having UH tumors in the Neuroblastoma category and UH neuroblastoma nodule in the Ganglioneuroblastoma, Nodular category. It should be noted that the INPC is applicable only to the tumor specimens obtained before starting chemotherapy/irradiation therapy [7,13]. Post-chemotherapy morphologic changes representing “acute chemotherapy effects” do not reliably predict clinical outcome. Based on our experience, those changes, such as neuroblastic differentiation and even focal Schwannian stromal development, observed in UH tumors after chemotherapy are often misleading. Large areas of necrosis and extensive hemosiderin deposition are frequently seen in the UH tumors but are rarely found in post-therapy FH tumors. We should, however, conduct further study on recurrent tumors after chemo/irradiation therapy, since they could demonstrate different genetic/molecular characteristics from those detected at the time of initial diagnosis due possibly to clonal selection/evolution or therapy-induced events during the individual clinical courses.
Genomic/molecular alterations closely associated with clinically unfavorable tumor behaviors are listed in this portion.
(1)Amplification of the
Determination of DNA index by flow cytometric analysis has been used for predicting clinical outcome of patients with Neuroblastoma tumors. Tumors presenting with numeric whole-chromosomal copy number gains without structural abnormalities (hyperdiploid, DNA index >1) are associated with an excellent prognosis. In contrast, diploid tumors (DNA index=1) are associated with a poor clinical outcome [24,25]. The prognostic effect of ploidy pattern is lost in children over the age of two years, possibly because “DNA index >1” tumors in older children typically have a number of structural abnormalities.
(3) Segmental chromosome aberrationsVarious segmental chromosomal aberrations have been reported as factors predictive of poor prognosis of [26- 29]. Chromosome 1pLOH (loss of heterozygosity), reported in 25-35% of cases, is often associated with MYCN amplification. Most deletions are located distal to 1p36, where possible tumor suppressor genes or genes controlling differentiation of neuroblasts could be located. Chromosome 11qLOH, identified in 35-45% of cases, is often associated with older patients, and rarely occurs in combination with
The presence of any of these chromosomal alterations (1p deletion, 11q deletion, and 17q gain) was reported to add prognostic information to the age and stage of neuroblastoma patients without
ALK is a receptor tyrosine kinase, expressed in the developing sympathoadrenal lineage of the neural crest. Historically,
Two mechanisms associated with telomere abnormality are reported in this disease: They are TERT (telomerase reverse transcriptase) overexpression and ALT (alternative lengthening of telomeres) phenotype. TERT is the protein component of telomerase, and higher levels of expression are observed in both MYC-driven neuroblastoma (neuroblastoma overexpressing either n-MYCN protein or c-MYC protein: please see MYC subtype in Subgrouping of Unfavorable Histology Neuroblastomas and Precision Medicine below) and non-MYC-driven neuroblastoma. In the former case,
Most tumors with ATRX loss due to
Since pNTs are biologically/clinically heterogeneous, patients with this disease are divided into different risk groups. There are 2 major risk-grouping systems: One is the COG Risk Classification System (Currently in the process of revision) and the other is the INRG (Inter-national Neuroblastoma Risk Group) (Table 2). Both systems use a combination of various prognostic factors, such as clinical stage, age at diagnosis, histopathology and molecular/genomic properties. Historically, the COG Risk Classification System (distinguishing low-, intermediate-, and high-risk group) has contributed to actual patient stratification and protocol assignment [51,52]. In contrast, the primary purpose of the INRG (distinguishing very low-, low-, intermediate-, and high-risk) is to facilitate the comparison of risk-based clinical trials conducted in different regions and countries [4]. By using the COG Risk Classification, approximately 90% of survival rate is expected by surgery alone for the patients in the low-risk group, and a similar survival rate is also expected by biopsy plus non-aggressive chemotherapy for children in the intermediate-risk group. In contrast, the survival rate of the patients in the high-risk group remains approximately 50% despite currently available high-intensity multimodal therapy.
Table 2 . International Neuroblastoma Risk Group (INRG) consensus pretreatment classification schema [4].
INRG Stage | Age (month) | Histologic category | Grade of tumor differentiation | MYCN | 11q Aberration | Ploidy | Pretreatment risk group | |
---|---|---|---|---|---|---|---|---|
L1/L2 | GN maturing; GNB intermixed | A | Very low | |||||
L1 | Any, except GN maturing or GNB intermixed | NA | B | Very low | ||||
Amp | K | High | ||||||
L2 | <18 | Any, except GN maturing or GNB intermixed | NA | No | D | Low | ||
Yes | G | Intermediate | ||||||
≥18 | GNB nodular; neuroblastoma | Differentiating | NA | No | E | Low | ||
Yes | H | Intermediate | ||||||
Poorly differentiated or undifferentiated | NA | |||||||
Amp | N | High | ||||||
M | <18 | NA | Hyperdiploid | F | Low | |||
<12 | NA | Diploid | I | Intermediate | ||||
12 to <18 | NA | Diploid | J | Intermediate | ||||
<18 | Amp | O | High | |||||
≥18 | P | High | ||||||
MS | <18 | NA | No | C | Very low | |||
Yes | Q | High | ||||||
Amp | R | High |
Pretreatment risk group H has two entries. 12 months=365 days; 18 months=547 days; blank field=“any”; diploid (DNA index ≤1.0); hyperdiploid (DNA index >1.0 and includes near-triploid and near-tetraploid tumors); very low risk (5-year EFS >85%); low risk (5-year EFS >75% to ≤85%); intermediate risk (5-year EFS ≥50% to ≤75%); high risk (5-year EFS <50%). GN, ganglioneuroma; GNB, ganglioneuroblastoma; Amp, amplified; NA, not amplified; L1, localized tumor confined to one body compartment and with absence of image-defined risk factors (IDRFs); L2, locoregional tumor with presence of one or more IDRFs; M, distant metastatic disease (except stage MS); MS, metastatic disease confined to skin, liver and/or bone marrow in children <18 months of age; EFS, event-free survival. Note: This table is the same as Fig. 2 in Cohn SL et al. (2008) The International Neuroblastoma Risk Group (INRG) Classification System: An INRG Task Force Report
It is noted that most of the tumors in the high-risk group are UH with unfavorable clinical and genomic/ molecular factors. Those high-risk or UH tumors are also heterogeneous and associated with different molecular backgrounds (see Genetically/molecularly defined factors above). Based on the fact that neuroblastoma can offer one of the best models for investigating biologically significant relationship between molecular alterations and their morphologic characteristics, Ikegaki et al. recently proposed four subgroups in UH neuroblastomas for precision medicine (Fig. 4). They are MYC subgroup (over-expressing MYCN or MYC protein), TERT subgroup (TERT overexpression due to genomic abnormalities), ALT subgroup (Alternative Lengthening of Telomere due to ATRX loss) and Null subgroup. These subgroups are immuno-histochemically identified by their protein makers that could be potential therapeutic targets [53].
Approximately 90% of
It is noted that augmented expression of the MYC- family protein is characteristically associated with prominent nucleolar formation (nucleolar hypertrophy) that is considered as a putative site of MYCN/MYC RNA synthesis and accumulation (Fig. 6A-C) [54-58]. Due to the presence of prominent nucleoli, indicative of hyperactive rRNA synthesis and protein translation, MYC-driven neuroblastomas are morphologically distinguishable from the conventional neuroblastomas with so-called “salt- and-pepper” nuclei. In contrast to n-MYC overexpressing neuroblastoma, it is extremely rare to see the gene amplification in c-MYC overexpressing neuroblastoma [59]. In this regard, other molecular mechanisms for high c-MYC protein expression independent of the genomic amplification, such as the oncogene activation through enhancer hijacking by translocations, focal enhancer amplification near to the c-
The histologically defined, rare and very aggressive “large cell neuroblastoma (LCN)” is also included in this group (Figs. 4C, 6D) [62]. This is an extreme form of the MYC-driven neuroblastoma that demonstrates large cell appearance with bull’s eye-like enlarged vesicular nuclei containing one to few very prominent nucleoli supporting exceedingly higher levels of either MYCN or MYC protein expression. Their “euchromatin-rich” open nuclei suggest stemness of the tumor cells [63]. Though the nuclear morphology of LCN cells is often similar to that of differentiating neuroblasts, these two cell types should be strictly distinguished. The tumor cells in LCN have larger nuclei than conventional neuroblasts and their cytoplasm is similarly scant. In contrast, differentiating neuroblasts have abundant cytoplasm with a diameter of at least two times larger than that of the nucleus [64].
It is a highly formidable task to directly target the MYC-family proteins with small molecules [65]. Hence, many investigators have sought indirect approaches to down regulate MYC-family protein expression in tumor cells. The strategies currently under consideration include, but not limited to, transcriptional repression of MYCN/MYC genes by BET-bromodomain inhibitors [66, 67] and CDK (Cyclin-dependent kinase) inhibitors [68,69] or destabilization of MYCN by Aurora kinase A inhibitors [70], etc. We also reported that small molecules, such as CX-5461 (a potent RNA Pol I inhibitor) and Halofuginone (a potent protein translation inhibitor), could effectively down-regulate MYC-family protein expression at the level of hypertrophic nucleoli of the neuroblastoma cells by interfering rRNA synthesis and protein translation [33].
As mentioned above, telomere elongation by increased TERT (telomere reverse transcriptase) activity can be detected immunohistochemistry in both MYC-driven and non-MYC-driven neuroblastomas in any age group [71]. In this subgroup, non-MYC-driven tumors having
In the treatment of TERT overexpressing neuroblastoma, telomerase inhibitors can be considered. Imetelstat (GRN163L) [73] is a potent and specific inhibitor of telomerase by binding with high affinity to TERC (telomerase RNA component). Interestingly, sorafenib has been shown to synergize with Imetelstat to inhibit growth of mouse xenografts of human cancer [74]. Sorafenib is a FDA-approved kinase inhibitor [75], and its synergistic effect with imetelstat appears to be due to the p21 attenuating activity [74].
Most tumors with
Since ATRX loss is due to structural alterations in
Neuroblastomas without MYC-family protein overexpression, TERT protein overexpression and ATRX protein loss are tentatively placed into the Null subgroup. Without having any other known specific molecular abnormalities, we hypothesize that tumors in this subgroup may respond to treatment according to the current high- risk protocols.
Mutually exclusive relationships likely exist among tumors in the MYC subgroup, TERT subgroup and ALT subgroup. These subgroups constitute the vast majority of therapy-resistant and UH tumors of this disease. Thus, targeted therapies against MYC-family overexpression, TERT overexpression and ALT phenotype should improve outcome of patients with the high-risk neuroblastomas.
Towards the end of this review, we would like to express our cordial thanks to the fellow clinicians and researchers. In order to make any meaningful progress in this field of pediatric oncology, we really need a great deal of support/collaboration from our colleagues nationally and internationally. With a long history of the cooperative studies, we have identified prognostic factors clinically, histopathologically and genetically/molecularly, and keep improving risk grouping system. However, the survival rate of the patients with high-risk neuroblastoma, many of them are UH according to the INPC, still remains very low at the “far-from” satisfactory level. Now we are moving to a new stage of precision “pathway targeting” medicine and creating subgroups in the Unfavorable Histology Group by immunohistochemically identifying protein markers that are associated with aggressive clinical behaviors through different molecular mechanisms.
The authors have no conflict of interest to declare.