Case 2 -

Neuroblastoma, Stroma-Poor, Differentiating D. Ashley Hill, M.D. Mihaela Onciu M.D.

Case History: A 14-month-old girl presented with an abdominal mass. CT scan showed a 7.0 x 6.5 x 5.0 cm nodular left adrenal mass with focal calcifications. The mass did not cross the midline. Bone marrow biopsies were normal. A left adrenalectomy was performed. Gross Examination: The specimen consisted of a nodular mass covered with a thin fibrous membrane. A portion of adrenal gland was present at one aspect. Sections showed a relatively homogeneous pink-grey mass with focal calcifications. A portion of the tissue was snap frozen in liquid nitrogen and stored at -80oC for biologic studies. Microscopic Examination: Sections showed a tumor composed of neuroblasts showing a range of differentiation from poorly differentiated cells with round nuclei, speckled chromatin and minimal cytoplasm to ganglion cells with large nuclei, dispersed chromatin, large nucleoli and abundant eosinophilic cytoplasm. The tumor cells were embedded in a large amount of pink fibrillary neuropil. More than 5% of the cells showed synchronous nuclear and cytoplasmic cytologic differentiation toward ganglion cells. Mitotic figures and karyorrhectic cells were infrequent. A small amount of spindle cell stroma was noted focally. Adherent lymph nodes at the periphery of the mass showed involvement by neuroblastoma. Special Studies: Fluorescent in situ hybridization for the MYCN gene was normal showing two copies of the gene. A DNA index showed the tumor was hyperdiploid. Diagnosis: Neuroblastoma, stroma-poor, differentiating (Favorable histology) Discussion Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Its wide range of morphologic appearances and biologic behavior make it one of the more complex neoplasms to diagnose, classify and treat. Because NB and related tumors are derived from neuroblasts, the primitive cells that populate the sympathetic nervous system, these tumors are typically located in sites where sympathetic nervous system elements are found, the adrenal gland and paraspinal locations. The International Neuroblastoma Pathology Classification (INPC) (the Shimada system) is the current system for stratifying tumors into pathologic risk categories and takes into account both the age of the patient as well as the histopathologic characteristics of the patient's tumor (Table 2.1-2.3). Once the pathologic classification of a patient's tumor is defined, additional prognostic factors including MYCN amplification status, DNA index and clinical stage are used to assign treatment strategies. Gross Handling Depending on the degree of differentiation within a given tumor, the gross appearance may vary from the soft grey-red, hemorrhagic poorly differentiated, stroma-poor tumors to moderately firm yellow-tan fibrous stroma-rich tumors. Like with many other pediatric tumors that are treated by cooperative group protocols, the appropriate handling of the tissue received from the operating room is critical to both the risk assessment of the patient and the eligibility for enrollement into a protocol. An appropriate division of tissue for both histologic classification and biologic studies is an important function of the pathologist. Table 2.4 summarizes our recommendations for the management of a neuroblastic tumor in the gross room. INPC Classification Once it is apparent that one is dealing with a neuroblastic tumor, there are a series of steps to be taken in the appropriate classification of the neoplasm including the assessment of: 1) stroma; 2) degree of differentiation; 3) proliferation and apoptosis measurement and 4) age of the patient. Stroma: Neuroblastic tumors consist of two main cell populations, neuroblasts/ganglion cells and Schwannian cells that form stroma. Stroma is represented histologically by a spindle cell component seen in increasing amounts in neuroblastic tumors as they differentiate. The spindle cells show characteristics of Schwann cells both microscopically and immunohistochemically. It is important to recognize the difference between neuropil, the pink fibrillary material representing the collection of cytoplasmic processes of the neuroblasts, and the spindle cell component referred to as stroma. The first diagnostic decision point is which of the four main categories of neuroblastic tumors the tumor belongs to based on the amount and distribution of stroma. Neuroblastomas are by definition stroma-poor, less than 50% of the tumor is composed of stroma. Ganglioneuroblastoma, intermixed and ganglioneuroma are stroma-rich and stroma-dominant respectively and are distinguished by the amount of the residual neuroblastic component. Ganglioneuroblastoma, nodular tumors may be either stroma-poor or stroma-rich and the diagnosis of this neuroblastic tumor type depends on the configuration of and relationship between the neuroblastic and stromal components. The subclassification of neuroblastomas detailed below also applies to the neuroblastoma component of nodular type ganglioneuroblastoma. Ganglioneuroblastoma (GNB) and ganglioneuromas (GN) are discussed in the next section. Differentiation: Once a tumor is placed into the NB category, the next assessment to be made concerns the degree of differentiation. Undifferentiated neuroblastomas are malignant small cell tumors with no morphologic evidence of neuroblastic differentiation visible on routine histologic staining. These tumors do not show rosettes or neuropil. Ancillary techniques such as immunohistochemistry, cytogenetics or electron microscopy are required to demonstrate the neural phenotype. The cells of an undifferentiated NB typically have larger nuclei than poorly differentiated NB and may have prominent nucleoli and sharp nuclear membranes but with no cytoplasmic expansion. Tumors containing cells with the latter features have been termed "large cell neuroblastomas" and are associated with MYCN amplification. The differential diagnosis of undifferentiated NB includes primitive hematopoietic neoplasms, Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), solid alveolar rhabdomyosarcoma (ARMS) and undifferentiated sarcoma. In contrast to the undifferentiated tumors, poorly differentiated neuroblastomas are typically recognizable as neuroblastic tumors on routine hematoxylin and eosin stains. These tumors are composed of uniform round cells with speckled chromatin, indistinct nucleoli and ill-defined cell borders. The extensive cytoplasmic processes are visible as pink fibrillary material referred to as neuropil. Homer Wright pseudorosettes are commonly present. Differentiating neuroblastomas typically have abundant neuropil and by definition 5% or more tumor cells showing cytologic differentiation toward ganglion cells. For a neuroblast to be classified as differentiating, synchronous nuclear and cytoplasmic differentiation must be seen. Nuclear differentiation is characterized by an enlarged, eccentric nucleus with a vesicular chromatin pattern and usually a single prominent nucleolus. Cytoplasmic differentiation is recognizable by increasing amounts of eosinophilic or amphophilic cytoplasm. A good rule of thumb is that the amount of cytoplasm in a differentiating neuroblast is twice the greatest dimension of the nucleus. A Schwannian-stroma component may be seen but does not comprise more than 50% of the tissue and does not form a sharply defined zone around a nodule of neuroblastic cells. Occasional neuroblastomas contain tumor giant cells with or without nuclear pleomorphism. These cells are not to be regarded as ganglion cells. Giant cells without pleomorphism are more commonly seen in clinically favorable groups whereas tumor giant cells with pleomorphic bizarre nuclei are associated with clinically unfavorable groups. Mitotic-karyorrhectic index: The mitotic karyorrhectic index (MKI) is defined as the number of tumor cells in mitosis and in the process of karyorrhexis with the denominator being 5000 tumor cells. Karyorrhectic cells show condensed and fragmented nuclear material usually associated with dense eosinophilic, condensed cytoplasm. Cells with condensed nuclei that are not fragmented are not counted. The MKI should be assessed on different sections and regions of tumor and reflect the average for all the sections. Shimada et al have noted that high cellularity tumors have 700 to 900 cells per 400x high power field (HPF), moderately cellular tumors have 400-600 cells per HPF and tumors with low cellularity and abundant neuropil have 100-300 cells per HPF. The MKI is measured and classified as follows: 1) low MKI with <2% (<100/5000) mitotic and karyorrhectic cells; 2) intermediate MKI with 2-4% (100-200/5000) mitotic and karyorrhectic cells; and 3) high MKI with > 4% (>200/5000) mitotic and karyorrhectic cells. There is a high correlation with high MKI and MYCN amplification. These tumors are also typically undifferentiated or poorly differentiated as MYCN amplification leads not only to increased proliferation and apoptosis, but also a block in differentiation. INPC Prognostic Groups The criteria above are then combined with the patient's age into the INPC system. Within the NB category, all undifferentiated tumors, tumors with high MKI and tumors in patients greater than 5 years of age are considered poor prognosis or "unfavorable histology." Poorly differentiated NB and differentiating NB with intermediate MKI in patients greater than 1.5 years are also considered poor prognosis. Conversely, poorly differentiated NB and differentiating NB with low or intermediate MKI in patients < 1.5 years are "favorable histology." This algorithm is summarized in Tables 2.2 and 2.3. The relevance of this prognostic classification is visible in the event free (EFS) and overall survival (OS) of the two prognostic groups. Patients with favorable histology tumors have an EFS of 90.4% and an OS of 97.8% whereas patients with unfavorable histology tumors have an EFS of 26.9% and OS of 35.6% (p <0.0001). Caveats About Histologic Typing Prognostic evaluation ideally is performed on primary, pre-treatment tumor specimens. In cases with disseminated disease or large, unresectable masses, the pathologist may receive a small biopsy from a metastatic source for confirmation of diagnosis. Histologic evaluation of metastatic tumor is valid for INPC prognostic typing provided that the specimen is of adequate size. Small biopsies can create some problems when tumors are heterogeneous (e.g. nodular GNB). Correlation with clinical and radiographic studies is often helpful in resolving this situation. A different scenario is the case where surgical resection of the tumor occurs after an initial course of chemotherapy. Chemotherapy appears to induce a number of effects on the appearance of the tumor. Post-treatment specimens often have necrosis and calcification that may obscure the tumor cells. In addition, post-chemotherapy specimens often show a greater degree of differentiation than present prior to therapy. The histologic change is not necessarily reflected in a change in the biologic properties of the tumor. For this reason, post-treatment specimens cannot be used for prognostic evaluation. We recommend that in this instance, a diagnosis of "post-treatment neuroblastoma" with a descriptive microscopic comment be used so as not to provide a source of confusion to the treating oncologists and surgeons. Ganglioneuroblastomas (GNB) GNB are divided into two prognostically important categories. GNB, intermixed represents a Schwannian stroma-rich tumor that has a ganglioneuromatous component greater than 50% of the tumor and contains well-defined microscopic nests of neuroblastic cells distributed or intermixed within the ganglioneuromatous tissue. The neuroblast nests usually contain differentiating and mature ganglion cells in abundant neuropil. Intermixed GNB have a 93.2% event free survival and 100% overall survival. The other type, GNB nodular, as originally described, is a composite tumor characterized by the presence of a single, grossly visible, hemorrhagic neuroblastic nodule (stroma-poor component) within a background of intermixed GNB or GN (stroma-rich or stroma-dominant component). Pathogenetically, this represents the development of a malignant clone within an otherwise differentiating tumor. Microscopically the two components have an abrupt demarcation with a pushing border or even pseudocapsule between the enlarging neuroblastic component and the ganglioneuromatous component. Variants of the GNB, nodular subtype have recently been incorporated into the classification scheme and include tumors with large single or multiple nodules and stroma-rich/dominant tumors with no nodules but with stroma-poor neuroblastic tissue in a metastatic site (Table 2.5). The proportion of neuroblastic tissue to stroma is not relevant to the diagnosis of the nodular subtype. In GNB with large single nodules, the neuroblastic component may become so large that it is difficult to identify the stroma-rich/dominant portion by gross examination. Careful attention to the peripheral portions of the lesion or septa between tumor nodules to identify a GNB, intermixed or GN component is required. In the past, GNB, nodular was uniformly given an unfavorable prognosis. Recent studies, however, applying the INPC system for classifying NB (age of the patient, differentiation and MKI assessment) to the neuroblastic nodule(s) of the tumor have shown that not all GNB, nodular behave poorly. If the neuroblastic nodule(s) in the tumor are classified as favorable histology, then the GNB, nodular is classified as favorable. If any neuroblastic nodule(s) in the tumor is classified as unfavorable then the tumor is considered unfavorable. The EFS and OS for GNB, nodular with favorable nodules is 86.1% and 90.5% respectively whereas the EFS and OS for GNB, nodular with unfavorable nodules is 32.2% and 33.2%. Ganglioneuromas (GN) GN are divided into two categories, GN, maturing and GN, mature. All GN are by definition Schwannian stroma-dominant being composed predominantly of stroma containing Schwann cells and fibrous tissue with a small component of either differentiating neuroblasts (maturing ganglion cells) or mature ganglion cells scattered throughout the lesion. Although these tumors originated from neuroblasts, they are fully differentiated and benign. The gross examination shows a well circumscribed, sometimes encapsulated, rubbery yellow-tan homogeneous mass. A common presentation for GN is a posterior mediastinal mass found incidentally on chest x-ray in an older child or adolescent. GN are also found in the retroperitoneum and adrenal gland. They can be associated with production of peptide hormones such as vasoactive intestinal peptide resulting in watery diarrhea. Gastrointestinal ganglioneuromatous polyps can be seen in juvenile polyposis, MEN type 2b syndrome and PTEN-associated disorders. Some additional families have been described with an as yet unnamed syndrome of ganglioneuromatous polyps and multiple cutaneous lipomas. Differential Diagnosis The differential diagnosis of NB is limited when there is sufficient differentiation to show malignant, uniform round cells in a background of neuropil with Homer Wright rosettes. Undifferentiated NB, on the other hand, by definition provides little evidence into its histogenesis. Primitive hematopoietic neoplasms including megakaryoblastic leukemias, ES/PNET, solid alveolar rhabdomyosarcoma and undifferentiated sarcoma may have similar undifferentiated features. A broad immunohistochemical panel including vimentin, neuron-specific enolase, PGP 9.5, synaptophysin and chromogranin, CD99, desmin, myogenin, and hematopoietic markers is helpful in most cases (Table 2.6). Electron microscopy to demonstrate neurosecretory granules or neural processes is helpful. Elevated urinary catecholamines often help confirm the diagnosis. The differential diagnosis of a GN may be considered when given a small biopsy specimen for intra-operative consultation. Biopsies containing only the stroma portion of the lesion may be mistaken for Scwannoma or neurofibroma. The identification of ganglion cells usually directs one to the appropriate diagnosis. Alternatively, non-representative biopsies showing stroma with or without ganglion cells may miss a less well-differentiated neuroblastic component in a nodular GNB. In the latter instance, consideration of the radiographic features of the lesion may provide clues to the homogeneity or heterogeneity of the neoplastic process. One other point to make about GN is that they are often associated with lymphoid aggregates. It is important to recognize these aggregates as lymphocytes rather than poorly differentiated neuroblasts. Neuroblasts are typically twice the size of lymphocytes. Clinical Staging The International Neuroblastoma Staging System (INSS) is detailed in Table 2.7. Unlike many other tumors, microscopic residual disease does not affect staging although a margin assessment is a standard element of a complete report. Assessment of lymph nodes submitted separate from the primary tumor and bone marrow examination are the most important pathologic components to the clinical staging process. Stages 1, 2A and 2B are considered localized or low stage disease and Stages 3 and 4 represent high stage disease. Stage 4 special (4S) represents a special category for infants less than 1 year of age who have a localized primary tumor and disseminated disease limited to the skin, liver and bone marrow. Metastatic tumor in a bone marrow biopsy cannot involve more than 10% of the biopsy specimen. These patients have a favorable prognosis despite the presence of metastatic disease. Biologic Factors NB are tumors that may regress, may show spontaneous or treatment-induced maturation or may grow aggressively in spite of therapy. A number of clinicopathologic, cytogenetic and molecular genetic factors have been implicated in the pathogenesis of this tumor, however, the complex relationships between these factors remain largely unknown. Table 2.8 summarizes the notable good prognosis and poor prognosis features in NB. MYCN amplification, seen in approximately 22% of tumors, is one of the most important prognostic factors and predicts an aggressive clinical course. Even with risk-adapted intensive therapy, patients with MYCN-amplified NB have a significantly decreased overall survival compared with non-amplified NB (42% vs. 74% respectively) (Mora et al). Morphologically, MYCN-amplified NB show markedly increased proliferation, apoptosis and vascularity and decreased differentiation and lymphocytic infiltration compared with non-amplified tumors. MYCN-amplified NB have larger nucleoli. They are also more likely to be diploid or near diploid and have other chromosomal abnormalities involving gain of 17q and loss of 1p36 regions, whereas non-amplified tumors are characterized by mitotic defects often resulting in whole chromosome gains without structural abnormalities. Because some non-amplified tumors also have loss of heterozygosity (LOH) at locus 1p, some investigators have postulated that 1p deletion may precede the development of MYCN amplification either deleting a gene that regulates MYCN expression or one that mediates cell death in the presence of elevated MYCN expression. An assessment of MYCN amplification status and DNA ploidy is a key component to the risk group and protocol assignment schema for Children's Oncology Group protocols (Table 2.9). Fluorescence in situ hybridization (FISH) is the method of choice for measuring MYCN amplification and can be done on touch preps (preferred), frozen or paraffin embedded tissue. Amplification is defined as > 10 MYCN gene signals at 2 p 24 in an interphase nucleus. FISH assays should include cohybridization with a probe to the centromeric or long-arm of chromosome 2 in order to distinguish low-level amplification from hyperdiploidy. The significance of low-level amplification is uncertain. Table 2.1 Summary of the histologic features of the neuroblastoma subtypes Undifferentiated neuroblastoma (NB, stroma-poor, undifferentiated) Requires immunohistochemistry or electron microscopy to rule out other small round cell neoplasms Clinical information such as urinary catecholamine values helpful Unfavorable histology always Poorly differentiated neuroblastoma (NB, stroma-poor, poorly differentiated) Recognizable as neuroblastoma with neuropil +/- Homer Wright rosettes < 5% of cells showing synchronous nuclear and cytoplasmic differentiation toward ganglion cells Consideration of MKI and age important in further prognostic classification (see Table 2.2) Differentiating neuroblastoma (NB, stroma-poor, differentiating) Typically abundant neuropil and neuroblasts showing a wide range of differentiation including some mature or nearly mature ganglion cells > 5% of neuroblasts showing synchronous differentiation Consideration of MKI and age important in further prognostic classification (see Table 2.2) Ganglioneuroblastoma, nodular (GNB, nodular or composite type) Composed of two recognizable and localized clones: neuroblastic and stromal Prognostic classification dependent on grading of neuroblastic component (see Table 2.2) Classic type: Macroscopically visible, circumscribed neuroblastic nodule surrounded by stroma Variant types: Multinodular: Two or more macroscopically visible neuroblastic clonal nodules in a ganglioneuromatous background (see Table 2.5) Large nodular: Ganglioneuromatous background difficult to appreciate grossly because of overgrowth of a large neuroblastic nodule. Microscopically, a thin rim of GN is seen at the periphery of the nodule or as the trabecular portion between neuroblastomatous nodules (see Table 2.5) No nodule: Primary tumor has features of GNB intermixed or GN but a metastatic site shows neuroblastoma (see Table 2.5) Ganglioneuroblastoma, intermixed (GNB, Schwannian stroma-rich, intermixed) > 50% of tissue is ganglioneuromatous stroma with interspersed groups of differentiating neuroblasts Favorable histology always Ganglioneuroma, maturing and mature (GN, Schwannian stroma-dominant) Bulk of tumor is Schwannian stroma with individual maturing or mature ganglion cells Favorable histology always Table 2.2 Classification of Neuroblastoma: Age linked Prognostic Effects Differentiation MKI < 1.5 yr 1.5 - 5 yr > 5 yr Undifferentiated Low XXXXX XXXXX XXXXX Intermediate XXXXX XXXXX XXXXX High XXXXX XXXXX XXXXX Poorly differentiated Low OOOOO XXXXX XXXXX Intermediate OOOOO XXXXX XXXXX High XXXXX XXXXX XXXXX Differentiating Low OOOOO OOOOO XXXXX Intermediate OOOOO XXXXX XXXXX High XXXXX XXXXX XXXXX Good Prognosis: OOOOO Poor Prognosis: XXXXX Shimada H, Umehara S, Monobe Y et al. International neuroblastoma pathology classification for prognostic evaluation of patients with peripheral neuroblastic tumors: a report from the Children's Cancer Group. Cancer 2001;92:2451-61. This material is used by permission of John Wiley & Sons, Inc. Table 2.3 Revised INPC Classification System Peuchmaur M, d'Amore ES, Joshi VV et al. Revision of the International Neuroblastoma Pathology Classification: confirmation of favorable and unfavorable prognostic subsets in ganglioneuroblastoma, nodular. Cancer 2003;98:2274-81. This material is used by permission of John Wiley & Sons, Inc. Table 2.4 Key steps in handling neuroblastoma specimens If you are receiving a primary tumor resection: Weigh and measure. Inspect external surface for residual adrenal gland (if retroperitoneal primary) and adherent lymph nodes. Ink external surface. Bivalve the specimen in the longest axis to show the largest surface area of tumor. Photograph cut surface (instant photo, printed digital photo, photocopy using a leak-proof bag, or diagram all work well). Describe gross appearance of the tumor (homogeneous or heterogeneous, presence or absence of necrosis, calcification, grossly visible lymph nodes,and hemorrhagic nodules in an otherwise homogeneous background). Take at least two 1 cm3 pieces of viable tumor (if tumor is heterogeneous, sample each area and label accordingly) and place in sterile tissue-culture medium (RPMI 1640 for cytogenetics, MYCN, ploidy and chromosome 1p analysis). Take an additional two 1cm3 pieces of viable tumor and snap freeze to be kept at -70oC for molecular studies. Make 10 touch preparations fix them according to your FISH protocol in your laboratory. Store at -20oC if necessary. Take sections for histologic examination and document location on photograph or diagram At least one section per centimeter largest dimension Sample areas with differing gross appearances Sample tumor with respect to margins Sample periphery of grossly hemorrhagic nodules Sample tumor with respect to adrenal gland (if present) Sample adherent lymph nodes (if present) If you are receiving a biopsy specimen: Ask for as much tissue as possible and handle it as expeditiously as possible Ensure that you have a large enough sample for histologic classification Touch preparations can be made from needle biopsy specimens or from larger pieces and are useful for fluorescent in situ hybridization (FISH) for MYCN amplification Save a small amount in sterile culture medium for MYCN Southern blotting, DNA ploidy analysis, cytogenetics, etc… Snap freeze a small amount at -70oC for future biologic studies Save a small piece in glutaraldehyde for electron microscopy if you suspect an undifferentiated neuroblastoma Table 2.5 Ganglioneuroblastoma: Classic and variant types Peuchmaur M, d'Amore ES, Joshi VV et al. Revision of the International Neuroblastoma Pathology Classification: confirmation of favorable and unfavorable prognostic subsets in ganglioneuroblastoma, nodular. Cancer 2003;98:2274-81. This material is used by permission of John Wiley & Sons, Inc. Table 2.6 Immunohistochemical stains useful in the diagnosis of neuroblastoma Antibody Source (clone) Utility NSE (A0587)* DakoCytomation, Carpenteria, CA Positive in most if not all cases, including undifferentiated NB Synaptophysin DakoCytomation Granular cytoplasmic positive in most cases with poorly differentiated neuroblasts Staining may be focal, use with chromogranin Chromogranin A (LK2H10(2)) Chemicon International Granular cytoplasmic positive in most cases with poorly differentiated neuroblasts Staining may be focal, use with synaptophysin PGP 9.5 (13C4) Biomeda, Foster City, CA Similar to synaptophysin and chromogranin, perhaps increased sensitivity, not specific for neuronal cells S100 protein (S100) DakoCytomation Useful for identifying Schwann cells and their precursors in stroma CD44 DakoCytomation Strong diffuse membrane staining associated with non-MYCN-amplified tumors; Negative in MYCN-amplified tumors Vimentin (V9) BioGenex, San Ramon, CA Positive in undifferentiated neuroblastomas but typically negative in poorly differentiated and differentiating neuroblasts CD45 (PD7-26 & 2B11) Dakocytomation Negative in NB, helpful in differentiating from lymphoma CD99 (O13) Signet, Dedham, MA Negative in NB, helpful in differentiating from ES/PNET Desmin (33) BioGenex, San Ramon, CA Negative in NB, helpful in differentiating from solid ARMS and ectomesenchymoma * polyclonal; NB, neuroblastoma; ES/PNET, Ewing sarcoma/primitive neuroectodermal tumor, ARMS, alveolar rhabdomyosarcoma Table 2.7 International Staging System for Neuroblastoma Stage Features 1 Localized tumor with complete gross excision Ipsilateral, nonadherent lymph nodes are negative for tumor (lymph nodes adherent to the tumor and removed intact with the primary tumor may be positive) 2A Localized tumor with incomplete gross excision Ipsilateral,nonadherent lymph nodes are negative for tumor (lymph nodes adherent to the tumor and removed intact with the primary tumor may be positive) 2B Localized tumor with or without complete gross excision Ipsilateral, nonadherent lymph nodes are positive for tumor Contralateral, nonadherent lymph nodes are negative for tumor 3 Unresectable tumor infiltrating across the midline (tumor originating on one side infiltrating across midline beyond opposite border of vertebral column or midline tumor extending bilaterally beyond both borders of vertebral column or contralateral lymph node involvement rendering tumor unresectable) 4 Any primary tumor with dissemination to distant lymph nodes, bone, bone marrow, liver, skin and/or other organs except as defined in Stage 4S 4S Localized primary tumor (Stage 1, 2A or 2B) in an infant < 1 year of age with dissemination limited to skin, liver and/or limited involvement of bone marrow (<10% or total nucleated cells are neuroblasts) Brodeur GM, Pritchard J, Berthold F et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993;11:1466-77. Table 2.8 Prognostic factors in neuroblastoma Low Risk High risk Stages 1, 2, or 4S Stages 3, 4 Favorable histology Unfavorable histology Age < 1 year Age > 1 year Non-adrenal primary (thorax) Adrenal primary MYCN non-amplified MYCN amplified Hyperdiploid DNA content Diploid DNA content No chromosome 1p abnormalities LOH 1p36 Normal 17q content Gain 17q content LDH < 1500, normal ferritin LDH %gt; 1500, elevated ferritin VMA/HVA ratio > 1 VMA/HVA ratio < 1 CD44 present CD44 absent TRKA expression high TRKB expression high VMA, vanillylmandelic acid; HVA, homovanillic acid; LDH, lactate dehydrogenase; LOH, loss of heterozygosity Table 2.9 Children's Oncology Group risk group stratification for childhood neuroblastoma* INSS Stage Age MYCN status Histopathology DNA ploidy Risk Group 1 0-21 y Any Any Any Low 2A/2B < 1y >= 1y** >= 1y >= 1y Any Non-amplified Amplified Amplified Any Any Favorable Unfavorable Any - - - Low Low Low High 3 < 1y < 1y >= 1y** >= 1y >= 1y Non-amplified Amplified Non-amplified Non-amplified Amplified Any Any Favorable Unfavorable Any Any Any - - - Intermediate High Intermediate High High 4 < 1y < 1y >= 1y Non-amplified Amplified Any Any Any Any Any Any - Intermediate High High 4S < 1y < 1y < 1y < 1y Non-amplified Non-amplified Non-amplified Amplified Favorable Any Unfavorable Any > 1 = 1 Any Any Low Intermediate Intermediate High *All patients less than 21 years of age ** Recent studies suggest that this age cutoff for the low risk group can be increased to 460 days for children with non-amplified MYCN and Stages 1-3 tumors (London WB et al, J Clin Oncol 2005;23:6459) Recommended Reading: Shimada H, Ambros IM, Dehner LP, Hata J, Joshi VV, Roald B. 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