Abstract
Tumors in neuroblastoma group offer one of the best models for investigating the biologically significant relationship between their genetic/molecular properties and morphologic manifestations. Over the past decades, histopathological analysis according to the International Neuroblastoma Pathology Classification (INPC) has provided invaluable information for determining the prognosis and therapy stratification for the patients with this disease. Now we are facing a great challenge for developing more efficient and less toxic treatment modalities for the patients with aggressive neuroblastomas. For this purpose, neuroblastoma pathology research needs to shift its gear to the direction of identifying “prognostic factors” to defining “actionable/druggable targets” in close collaboration with molecular biology, based on the concept of “precision medicine.”
Keywords
Actionable/druggable targets, ALK mutation/overexpression, Alternative lengthening of telomere, ATRX, Ganglioneuroblastoma, Ganglioneuroma, Intermixed, International neuroblastoma pathology classification, MYC family driven neuroblastoma, MYC protein, MYCN protein, Neuroblastoma, Nodular, Telomere maintenance and elongation, TERT
Introduction
Neuroblastoma is often used as a collective term for all types of peripheral neuroblastic tumors of neural crest origin and includes Neuroblastoma, Ganglioneuroblastoma, and Ganglioneuroma. Historically tumors in this group were described as “enigmatic” because of their unique and often unpredictable clinical behaviors, such as spontaneous regression, tumor maturation, and aggressive progression refractory to therapy. These clinical features are now considered to be closely associated with their genetic/molecular characteristics . It is also noted that tumors in this group offer one of the best models for investigating the biologically significant relationship between their genetic/molecular properties and morphologic manifestations .
Pathology Diagnosis
Whenever feasible, it is recommended to obtain biopsied/surgically resected samples before starting chemotherapy/irradiation therapy for pathology evaluation. Determining the histologic/biologic characteristics of the tumors is critical for patient stratification and appropriate protocol assignment at the time of diagnosis. International Neuroblastoma Pathology Classification (INPC) is applied only to the tumor specimen obtained before starting chemotherapy/irradiation therapy . After chemotherapy, tumor samples, especially of biologically/clinically unfavorable cases, show acute chemotherapy effects including a large area of necrosis and extensive hemosiderin (intracellular iron storage complex) deposition. Cytologic/morphologic changes of the tumors after chemotherapy, which could mainly represent an epigenetic phenomenon, are often not reliable for predicting clinical behaviors of the individual tumors. We should conduct further studies on recurrent tumors, as they could demonstrate different genetic/molecular properties of the tumors at the time of diagnosis.
At the surgical pathology gross bench, the priority should be the securing of enough samples for histological examination. For determining biological characteristics of the given tumor, it is critical for saving the snap-frozen material for molecular tests. Diagnosis by touch preparations is recommended for MYCN and other analyses by the FISH (fluorescence-based in-situ hybridization) test.
Immunohistochemical stainings often used for confirmation of the neuroblastoma diagnosis include neuronal markers (NSE, PGP9.5, Synaptophysin, Chromogranin, CD56, etc.) and so-called neuroblastoma marker (NB84) . However, those markers are not specific for neuroblastoma; for example, those neuronal markers are usually positive for Ewing’s/PNET. Markers for the neural crest tumors with neuronal and neuroendocrine differentiation, which include TH (tyrosine hydroxylase) and Phox2b , are more specific for neuroblastoma diagnosis. Between these two neural crest markers, Phox2b is more stable and can be used for the staining of the bone and bone marrow samples after decalcification . In our experience, Phox2b is more sensitive than TH and positive for all neuroblastomas including undifferentiated subtype . In contrast, TH is positive only for sporadic cells or even becomes negative in some of the tumors in the undifferentiated subtype. It should be noted that both Phox2b and TH are positive for pheochromocytomas and paragangliomas .
International Neuroblastoma Pathology Classification (INPC)
Histologic Categories and Subtypes
The International Neuroblastoma Pathology Committee defines four categories in this group of tumors: each is characterized by the grade of neuroblastic differentiation and the degree of Schwannian stromal development . They are Neuroblastoma (Schwannian stroma-poor), Ganglioneuroblastoma, intermixed (Schwannian stroma-rich), Ganglioneuroma (Schwannian stroma-dominant), and Ganglioneuroblastoma, nodular (composite, Schwannian stroma-rich/stroma-dominant and stroma-poor). It is believed that all Ganglioneuromas are once Neuroblastomas in their early stage of tumor development. The maturation sequences from Neuroblastoma to Ganglioneuroma are prompted by the cross talk between neuroblasts and Schwannian cells, comparable to the embryologically well-defined relationship in neural crest development toward ganglion structure of the autonomic nervous system . The cross talk seems to be supported by various signaling pathways including trkA/nerve growth factor (NGF) signaling and Nrg1/ErbB signaling, etc. .
Neuroblastoma (Schwannian stroma-poor) – NB: Tumors in this category include three subtypes: They are: undifferentiated, poorly differentiated, and differentiating. Those tumors are characterized by the typical growth pattern of neuroblastic cells forming groups or nests demarcated by thin fibrovascular stromal septa where limited or no Schwannian cell proliferation is observed.
- 1.
Neuroblastoma, undifferentiated subtype—NB-UD is rare and supplementary procedures, such as immunohistochemistry and/or molecular tests, are required to establish the diagnosis. The proliferating cells are uniformly primitive without clearly recognizable neurite production ( Fig. 1.1A ). Neuroblasts in this subtype usually do not express higher levels of a favorable marker trkA (high-affinity NGF receptor) and do not have a potential for differentiation. Accordingly, tumors in this subtype are considered as biologically unfavorable. The nuclear morphology of NB-UD cells often exhibits a vesicular euchromatic (transcriptionally active chromatin) appearance.
Figure 1.1
Categories and Subtypes of Peripheral Neuroblastic Tumors: (A) Neuroblastoma, Undifferentiated subtype (NB-UD); (B) Neuroblastoma, Poorly differentiated subtype (NB-PD); (C) MYCN amplified tumor showing the appearance of NB-PD with a high MKI (Mitosis-Karyorrhexis Index), (inset: neuroblastic cells having prominent nucleolar formation); (D) Neuroblastoma, Differentiating subtype (NB-D) (inset: typical differentiating neuroblasts with both cytoplasmic and nuclear enlargement); (E) Ganglioneuroblastoma, Intermixed (GNB-I); (F) Ganglioneuroma (GN) [inset: completely mature ganglion cell covered with satellite cell ( arrow )]; (G) Ganglioneuroblastoma, Nodular (GNB-N) composed of two distinct histologies (clones)—Ganglioneurmatous tissue (left) and neuroblastomatous nodule (right).
- 2.
Neuroblastoma, poorly differentiated subtype—NB-PD is composed of neuroblasts having varying amounts of neurite production with or without HomerWright rosette formation. This is the most common histological form among the peripheral neuroblastic tumors. Less than 5% of tumor cells have cytomorphologic features of differentiating neuroblasts (see blow c. NB-D). Nuclear morphology of the NB-PD neuroblasts is often described as salt-and-pepper (sprinklings of heterochromatin and a few inconspicuous nucleoli) ( Fig. 1.1B ). It is interesting to note that some tumors in this subtype, and more in the undifferentiated subtype, show the presence of some prominent nucleoli (nucleolar hypertrophy), especially when MYCN oncogene is amplified ( Fig. 1.1C ). Tumors in this subtype are either biologically favorable or biologically unfavorable. Biologically favorable tumors show spontaneous regression or cellular differentiation/tumor maturation. As mentioned above, the latter seems to be supported by the cross talk between neuroblastic cells and Schwannian stromal cells; those neuroblasts express higher levels of trkA and actively recruit Schwannian stromal cells. In contrast, biologically unfavorable tumors seem to have lower levels of trkA expression, do not recruit Schwannian stromal cells, and do not have a potential of differentiation/maturation. Amplified MYCN is known to downregulate trkA expression. However, many MYCN nonamplified tumors can also express lower trkA levels, and considered to be biologically unfavorable as well.
- 3.
Neuroblastoma, differentiating subtype—NB-D, is a tumor usually characterized by abundant neurite production. More than 5% of tumor cells show cellular differentiation and have an appearance of differentiating neuroblasts ( Fig. 1.1D ). Those differentiating neuroblasts are defined by synchronous differentiation of both the nucleus (enlarged, eccentrically located with a vesicular chromatin pattern and usually a single prominent nucleolus) and the cytoplasm (eosinophilic/amphophilic with a diameter or twice or more of the nucleus). A Nissl substance can be seen in the periphery of the cytoplasm. Majority of the tumors in this subtype are biologically favorable. However, some of the patients with differentiating subtype of neuroblastoma still have a poor clinical outcome (please see Prognostic Grouping section below).
Ganglioneuroblastoma, Intermixed (Schwannian stroma-rich) —GNB-I: Tumor in this category contains well-defined microscopic nests of neuroblastic cells in a background of naked neurites that are intermixed or randomly distributed in the ganglioneuromatous tissue. Those microscopic nests represent the areas where neuritic processes produced by the neuroblasts are not incorporated in the cytoplasm of Schwannian stromal cells. By definition, more than 50% of tumor tissue in this category should have a ganglioneuromatous appearance where ganglion cells are individually embedded in abundant Schwannian stromal cells. These microscopic nests are composed of a mixture of neuroblastic cells in various stages of differentiation, often dominated by differentiating neuroblasts ( Fig. 1.1E ). Some apoptotic cells may be seen in the nests as well. Presence of these microscopic nests is considered as a sign of the lagging behind of tumor maturation toward ganglioneuroma and the tumors are biologically favorable, leading to an excellent prognosis of the patients.
Ganglioneuroma (Schwannian stroma-dominant) – GN: Tumors in this category are characterized by the presence of individually distributed ganglion cells in the Schwannian stroma ( Fig. 1.1F ). Neuritic processes produce by the ganglion cells are immediately enveloped by the cytoplasm of Schwann cells. Accordingly, there are no recognizable microscopic foci of naked neurites without Schwannian coverage. This category includes two subtypes: maturing and mature. The maturing subtype contains both maturing and mature ganglion cells, whereas the mature subtype contains only mature ganglion cells. The mature ganglion cells are surrounded by the satellite cells. The stromal tissue is usually well organized and shows the fascicular profile of Schwann cells bundled with perineurial cells. Ganglioneuroma is a biologically/clinically benign tumor. However, there are markedly rare cases where malignant Schwannoma develops in ganglioneuroma with or without irradiation therapy .
Ganglioneuroblastoma, Nodular (composite, Schwannian stroma-rich/stroma-dominant and stroma-poor) —GNB-N: Tumors in this category are characterized by the presence of grossly visible, often hemorrhagic and/or necrotic, NB nodule(s) (stroma-poor component), coexisting with GNB-I (stroma-rich component) or with GN (stroma-dominant component) ( Fig. 1.1G ). The term “composite” implies that the tumor is composed of biologically different clones.
Prognostic Grouping (Favorable Histology Vs. Unfavorable Histology)
INPC distinguishes two prognostic groups, Favorable Histology Group and Unfavorable Histology Group ( Fig. 1.2 ) . Tumors in the Favorable Histology Group are within a framework of age-appropriate tumor differentiation/maturation and age-appropriate mitotic and karyorrhectic activities. As for the morphologic indicators of tumor differentiating/maturation, the categories and subtypes described above are utilized. In other words, tumors in the Favorable Histology Group can demonstrate age-dependent differentiation/maturation from NB-PD to NB-D, then to GNB-I and finally to GN, based on the cross talk between tumor cells and Schwannian stromal cells. However, to observe tumor differentiation/maturation, it seems to take a certain amount of time; i.e., in vivo latent period. It is expected to take up to 18 months for those tumors of NB-PD subtype to become NB-D subtype, and up to 60 months to become GNB-I or GN. In contrast, tumors of NB-UD subtype in any age group, tumors of NB-PD subtype over 18 months of age, and tumors of NB-D subtype over 60 months of age are considered as having limited or no differentiating potential, and they are classified into the Unfavorable Histology Group.
Another morphologic indicator for predicting clinical behavior in this disease is mitotic and karyorrhectic activities of neuroblastic cells, and that is applied to tumors in the NB category . One of three MKI (Mitosis-Karyorrhexis Index) classes based on the activities is assigned to the given NB tumors: They are Low (<100/5000 cells), Intermediate (100–200/5000 cells), and High (>200/5000 cells) and their prognostic effects are also age-dependent. Low MKI tumors in the patients <5 years of age at diagnosis, and Intermediate MKI tumors in the patients <18 months of age at diagnosis are classified into the Favorable Histology Group. High MKI NB tumors in any age group, Intermediate MKI tumors >18 months of age at diagnosis, and Low MKI tumors >60 months of age at diagnosis are classified into the Unfavorable Histology Group. MYCN amplified tumors are typically associated with high MKI (please see Fig. 1.1C ) .
GNB-I and GN are always classified into the Favorable Histology Group , while tumors in the GNB-N category are classified into the Favorable Histology Group or Unfavorable Histology Group based on the characteristics of NB nodule(s) . For this purpose, the same criteria of age-linked evaluation for the grading of neuroblastic differentiation and the MKI class utilized for the prognostic distinction of NB tumors are applied to the NB nodule(s). It should be noted that making the correct diagnosis of GNB-N is often difficult by biopsy or partial tumor resection, since NB nodule could be hidden and not sampled for pathology examination. In that situation, it is recommended to add a disclaimer ”based on the review of limited material” in the diagnosis line after GN or GNB-I, Favorable Histology in the surgical pathology report. It is critically important since the clinical behavior of the given tumor would depend on the characteristics of NB nodule(s), if present .
In neuroblastoma, the patient’s age at diagnosis is one of the prognostic indicators. Historically, 1 year has been used as the cutoff mark. The prognostic contribution of age to the clinical outcome seems to be naturally continuous, and the survival rates of younger patients are always better than older patients in any age cutoff. Based on the Children’s Oncology Group (COG) Neuroblastoma study, London et al. reported the statistical evidence of an age cutoff greater than 1 year for risk stratification , and the COG is now in the process of moving the cutoff from 1 year (365 days) to 18 months (548 days). The age factor should be considered as a surrogate for other genetic/biologic risk markers. Although the INPC has an already built-in age cutoff point of 18 months, Sano et al. demonstrated that the INPC was able to add independent prognostic information beyond the prognostic contribution of age . In other words, the INPC clearly distinguishes two prognostic groups (Favorable Histology identifying a significantly better prognosis group than Unfavorable Histology) in different age groups, such as < versus >12 months; < versus >18 months, and < versus >24 months of age at diagnosis ( Fig. 1.3 ).
