Diagnostic Utility of PHOX2B in Primary and Treated Neuroblastoma and in Neuroblastoma Metastatic to the Bone Marrow

Abstract

Context.-Neuroblastoma (NB) is the most common extracranial tumor of childhood. Although most cases have a distinctive histology, a subset of primitive cases require immunohistochemical studies to distinguish them from other small round blue cell tumors of childhood. Immunohistochemistry is also used to detect small amounts of tumor metastatic to the bone marrow and in posttreatment samples with obscuring fibrosis, calcification, or inflammation. The transcription factor PHOX2B is essential for the differentiation and survival of sympathetic neurons and chromaffin cells, and therefore is highly specific for the peripheral autonomic nervous system.Objective.-To determine the diagnostic utility of PHOX2B immunohistochemistry as a marker of primary, treated, and metastatic NB.Design.-Neuroblastoma tissue microarrays were stained with PHOX2B, CD57, and synaptophysin. Arrays containing rhabdomyosarcoma, Ewing sarcoma, and Wilms tumor were stained with PHOX2B, and negative bone marrow samples were stained with PHOX2B and CD57.Results.-PHOX2B and CD57 were similar to synaptophysin in their ability to detect NB. PHOX2B and CD57 similarly showed robust staining in posttreatment NB and NB metastatic to the bone marrow. In contrast to the cytoplasmic staining pattern seen with synaptophysin and CD57, clear and strong nuclear PHOX2B permitted identification of individual tumor cells. PHOX2B staining was absent in all cases of rhabdomyosarcoma, Ewing sarcoma, and Wilms tumor, and in the negative bone marrow.Conclusions.-PHOX2B and CD57 are useful markers of NB. PHOX2B is specific for NB in its differential diagnosis with other small round cell tumors, and its nuclear staining may be helpful for accurate bone marrow tumor quantification.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group

“…The value of IC and IHC are both dependent on the quality of the sample, and the specificity and sensitivity of the antibodies used. For IHC, we advise using antibodies against at least 2 of the recommended antigens, endorsing the use of synaptophysin and chromogranin A and advocating the introduction of PHOX2B . For IC, we support the use of antibodies to GD 2, and commend the inclusion of a second antibody (eg, anti‐PHOX2B or anti‐CD56) to control for those rare situations in which GD 2 expression may be weak or negative .…”

“…For IHC, we advise using antibodies against at least 2 of the recommended antigens, endorsing the use of synaptophysin and chromogranin A 12 and advocating the introduction of PHOX2B. 22,23 For IC, we support the use of antibodies to GD 2,13 and commend the inclusion of a second antibody (eg, anti-PHOX2B or anti-CD56) to control for those rare situations in which GD 2 expression may be weak or negative. 38,39 We advocate the use of RTqPCR within clinical trials to quantify the level of the neuroblastoma mRNA tyrosine hydroxylase (the most widely evaluated mRNA target, and which has prognostic value in bone marrow 13 ) in all children in the minimal disease setting and in high-risk children at trial-specific disease assessment time points.…”

“…Highly specific target antigens for which IHC is unambiguous include synaptophysin, tyrosine hydroxylase, chromogranin A, and paired‐like homeobox 2b (PHOX2B). Additional frequently used markers include CD56 and PGP9.5 . When suspected, Schwann cells can be reliably detected by morphology and IHC for the S‐100 protein .…”

“…Additional frequently used markers include CD56 and PGP9.5. 11,[20][21][22][23][24] When suspected, Schwann cells can be reliably detected by morphology and IHC for the S-100 protein. 20 Less useful markers for the detection of neuroblastoma cells in the bone marrow are neuron-specific enolase (NSE) and NB84, neuronspecific enolase because it lacks specificity 25 and NB84 because it is rarely expressed by neuroblastoma cells in the bone marrow.…”

Examination of PHOX2B in adult neuroendocrine neoplasms reveals relatively frequent expression in phaeochromocytomas and paragangliomas

“…Neuroblastoma is a primitive tumour of sympathoadrenal lineage. Given PHOX2B's central role in determining this lineage, it is not surprising that it has been investigated as a diagnostic marker of neuroblastoma, in which it has been shown to be highly sensitive and specific as an immunohistochemical marker, including in decalcified bone marrow specimens, and in quantitative real‐time polymerase chain reaction panels as a marker of minimal residual disease …”