This study examined the utility of stratifying children with medulloblastomas by a combination of refined histopathological classification and molecular cytogenetic evaluation.Detailed histopathological classification of tumors from a cohort of patients (n ‫؍‬ 87) composed mainly of children entered into the International Society of Pediatric Oncology (SIOP)/United Kingdom Children's Cancer Study Group PNET3 trial (n ‫؍‬ 65), included identification of the large cell/anaplastic phenotype. Fluorescence in situ hybridization was used to detect chromosome 17 abnormalities, losses of 9q22 and 10q24, and amplification of the MYCC and MYCN oncogenes.The large cell/anaplastic phenotype, which was present in 20% of medulloblastomas, emerged as an independent prognostic indicator. Loss of 17p13.3 (38% of medulloblastomas) was found across all of the histopathological variants, whereas MYCC/MYCN amplification (6%/8% of medulloblastomas) was significantly associated with the large cell/ anaplastic phenotype. Both of these genetic abnormalities emerged as prognostic indicators. Loss of 9q22 was associated with the nodular/desmoplastic medulloblastoma variant, whereas loss of 10q24 was found in all of the variants. Together with metastatic tumor at presentation, the large cell/anaplastic phenotype, 17p13.3 loss, or high-frequency MYC amplification defined a high-risk group of children whose outcome was significantly (P ‫؍‬ 0.0002) poorer than a low-risk group without these tumor characteristics.Combined evaluation of novel histopathological features and molecular cytogenetic abnormalities promises to allow stratification of patients with medulloblastoma, such that those likely to be cured will be spared the side effects of maximal therapy, which can be targeted at those with aggressive disease.

Among the variants of medulloblastoma in the current WHO classification of nervous system tumors, the desmoplastic variant, which has been reported to constitute 5%-25% of pediatric medulloblastomas, is defined by its nodular collections of neurocytic cells bounded by desmoplastic internodular zones. We have studied the frequency, morphological features and biological behavior of medulloblastomas in two contemporaneous SIOP/UKCCSG trial cohorts of children with medulloblastomas, CNS9102 (n = 315) and CNS9204 (n = 35), focusing on tumors with nodular and desmoplastic phenotypes. In children aged 3-16 years (CNS9102), the nodular/desmoplastic medulloblastoma represented 5% of all tumors, while in infants aged <3 years (CNS9204) this variant represented 57% of medulloblastomas. Using iFISH to detect molecular cytogenetic abnormalities in medulloblastomas with a nodular architecture, we demonstrated distinct genetic profiles in desmoplastic and non-desmoplastic (classic and anaplastic) tumors; in particular, abnormalities of chromosome 17 occurred in the latter, but not the former. Significantly different outcomes were demonstrated for classic, nodular/desmoplastic and large cell/anaplastic medulloblastomas in both cohorts. In conclusion, the nodular/desmoplastic medulloblastoma appears to have clinical, genetic and biological characteristics that set it apart from other variants of this tumor.

Histopathologic assessment of 273 non-desmoplastic medulloblastomas (MBs) from children aged 3 to 16 years and entered into the SIOP/UKCCSG (International Society of Pediatric Oncology/United Kingdom Children's Cancer Study Group) PNET3 trial revealed that 47 (17%) fulfilled criteria for the recently proposed anaplastic variant. In addition, an anaplastic phenotype was focally present in all 5 (2%) large cell MBs from this series. Children with large cell MBs had the worst outcome, but there was also a significant difference between the event-free and overall survivals of children with classic MBs and those with anaplastic MBs. While objective morphometric analysis confirmed that subjective evaluation of nuclear size and variability contributed to the separation of MBs into classic, anaplastic, and large cell variants, these cytologic measures were not themselves prognostic indicators. However, anaplastic and classic MBs also possessed significantly different mitotic counts/indices, and these measures of proliferation were related to survival. Significant prognostic indicators in a multivariate survival analysis were histologic variant, metastases at presentation, and subtotal surgical excision of tumor. Our study supports the concept of an anaplastic variant among MBs, demonstrating that it has clinical utility.

Diffuse Large B-cell Lymphoma (DLBCL) is classified into germinal centre (GCB) and activated B-cell (ABC) type by comparison with the phenotype of normal B-cells. Using single-color immunocytochemistry, tumors can be classified using a simple algorithm based on the expression of CD10, BCL6 and IRF4. This classification may have prognostic relevance and correlates with balanced translocations involving the immunoglobulin locus. However, the phenotypic differences between normal and neoplastic cells may be of greater relevance to understanding the pathogenesis of DLBCL and in developing effective diagnostic techniques. To investigate this we examined the co-expression of BCL6, IRF4 and FOXP1. These are key transcription factors that regulate the process of germinal centre and post germinal centre B-cell differentiation. Abnormal co-expression of these molecules would be expected to have major effects on the overall cellular phenotype. A multi-color immunofluorescence (MCIF) technique was developed that allowed the co-expression of these markers to be assessed in relation to the PAX5 positive B-cell population. The use of a multi-color technique allows the distinction between co-expression at the level of individual cells and differentiation within the tumor as a whole. We first determined the pattern of expression of these transcription factors in normal B-cells. In reactive lymph nodes the expression of BCL6, IRF4 and FOXP1 was almost mutually exclusive with only a small proportion of co-expressing cells. In a series of 61 DLBCL co-expression of both BCL6/IRF4 and IRF4/FOXP1 was found in 41/61 (67%) of the cases. In most of these cases the level of co-expression was greater than 50% of the PAX5 positive large lymphoid cells. Co-expression was not present in 11/61 (18%) of the tumors. In the remaining cases there was co-expression of either BCL6/IRF4 or IRF4/FOXP1. There was no correlation between the occurrence of co-expression of these combinations of transcription factors and the expression of CD10 or the classification into GCB and ABC phenotypes. In 16 of the cases the sample used was a small needle core biopsy in which assessment of nodal architecture was impossible. In these cases it was possible to confidently determine the presence of abnormal co-expression in 14/16 (87.5%) of the cases. One explanation for the aberrant co-expression of BCL6 and IRF4 in DLBCL would be the presence of a 3q27 rearrangement leading to dysregulation of BCL6 expression. However, in this series there was no correlation between BCL6/IRF4 co-expression and abnormalities of 3q27 detected by interphase FISH. These results show that in the majority of cases of DLBCL the key transcription factors regulating post germinal centre B-cell differentiation are expressed in combinations not seen in normal B-cells. This is likely to be a central element in the pathogenesis of these tumors. The ability to reliably identify these abnormalities by MCIF has potential value in improving the reliability of diagnosis of DLBCL when only small biopsy samples are available and it is likely that this approach can be extended to other types of lymphoma.