Distinct Molecular Variants Research Articles

Abstract 3447: FSTL5 improves prognostic subclassification of medulloblastoma

Abstract Current integrated genomic approaches indicate distinct biological variants in medulloblastoma. Comprehensive molecular classification strategies utilize cytogenetic or immunohistochemical biomarkers to refine risk stratification. Novel complementary markers may ameliorate outcome prediction particularly in intermediate or high-risk medulloblastomas. We combined transcriptome and DNA copy-number analysis for 64 primary tumors. Bioinformatic tools were applied to investigate marker genes of molecular variants. Differentially expressed transcripts were evaluated for prognostic value in the entire screening cohort. Immunohistochemical markers were used to determine molecular subtypes in adult and pediatric medulloblastoma samples (n=235). Immunopositivity of FSTL5 was correlated with molecular and prognostic subgroups for 235 non-overlapping medulloblastoma samples on two independent tissue microarrays (TMA). Unsupervised cluster analyses of transcriptome profiles revealed four distinct molecular variants: WNT, SHH, Group C, and Group D. Stable subgroup separation was achieved using only 300 most varying transcripts. Specific distribution of clinical and molecular characteristics was noted for each cluster. Notably, Group C tumors were exclusively present in pediatric medulloblastomas as determined by immunohistochemistry. Delimited expression patterns of FSTL5 in each molecular subgroup were confirmed by quantitative real-time PCR. FSTL5 transcripts were most up-regulated in Group C and Group D tumors with unfavorable prognosis, whereas WNT medulloblastomas showed marked down-regulation. Immunopositivity of FSTL5 identified a large proportion of patients (84 of 235 patients; 36%) at high risk for relapse and death in particular in patients with WNT/SHH-independent tumors. Multivariate analysis revealed that FSTL5 immunopositivity constitutes an independent prognostic marker in pediatric and adult patient cohorts (p<0.0001). Importantly, adding this biomarker to comprehensive outcome prediction schemes substantially reduced the prediction error of the model. Comprehensive analyses of transcriptome and genetic alterations unravel four distinct disease variants. By addition of FSTL5 immunohistochemistry, existing molecular stratification schemes can effectively be complemented and sub-classification of WNT/SHH-independent tumors substantially optimized. This approach may ultimately define clear risk groups to individualize treatment intensities in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3447. doi:10.1158/1538-7445.AM2011-3447

Molecular biology of medulloblastoma: bridging the gap between research and practice

Evaluation of: Northcott PA, Korshunov A, Witt H et al. Medulloblastoma comprises four distinct molecular variants. J. Clin. Oncol. DOI: 10.1200/JCO.2009.27.4324 (2010) (Epub ahead of print).Medulloblastoma, the most common primary pediatric malignant brain tumor, is a molecularly heterogeneous disease with different developmental origins, distinct phenotypes, diverse biological behavior and contrasting clinical outcomes. The current clinico-radiological risk classification fails to take account of this heterogeneity and resultant prognostic variability. It is widely accepted that dysregulation of normal developmental processes constitutes a key mechanism of tumorigenesis in at least a subset of medulloblastomas. Several attempts at biological classification have successfully identified distinct subgroups with subgroup-specific gene signatures, demographics, histologic subtypes and metastases; among these, tumors involving the wingless and sonic hedgehog signaling pathways have been the most reliably and consistently identified. However, such integrative genomic approaches have limited applicability in the clinic owing to the need for fresh frozen tissues and elaborate molecular biology tools. A novel four-antibody approach to subgroup medulloblastoma using immunohistochemistry on archival specimens as proposed by Northcott et al. appears extremely promising as it can be applied in any basic neuropathology laboratory across the globe. There is a compelling need to integrate assays of molecular biomarkers performed on archival specimens into stratification schemes for medulloblastoma alongside clinical and pathologic outcome indicators to refine risk stratification for individualizing therapy.

Protein A – a new ligand for human C-reactive protein

Phosphorylcholine (PC) is a classical ligand of C-reactive protein (CRP), a clinically important acute phase protein. In search of new ligands, CRPs were affinity-purified from several pathological samples, which exhibited distinct molecular variants induced in different diseases. Both glycosylated and non-glycosylated CRPs showed calcium-independent differential-binding to Staphylococcus aureus cell-surface Protein A. CRP possesses separate binding sites for Protein A and PC with different binding constants. We have demonstrated that Protein A is another ligand in addition to PC establishing an extended definition of CRP. Protein A binding may impart immunomodulatory roles of CRP in combating microorganisms or other foreign materials.

Human retinoic X receptor beta: complete genomic sequence and mutation search for ossification of posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the ligament. OPLL is a very common disorder, in fact it constitutes the leading cause of myelopathy among Japanese. In the previous report, we provided the genetic linkage evidence that the genetic susceptibility of OPLL mapped to HLA complex of chromosome 6. As a candidate gene approach, retinoic X receptor beta (RXR beta), assigned to chromosome 6p21.3 adjacent to HLA class II, was analyzed for a possible causality. To start screening for the molecular variants of RXR beta in OPLL subjects, we first obtained P1 phage genomic clones containing the entire human RXR beta and elucidated the genomic organization of the gene. The human RXR beta is composed of 10 exons spanning over 6.2 kb of genomic DNA. Sequence analysis of the promoter region revealed a GC-rich sequence without TATA motif. We have identified three distinct molecular variants, one was in exon 10 and two were in the intergenic region between RXR beta and collagen 11A2 (COL11A2). Two variants in the intergenic region, 3' end + 140 and 3' end + 561, exhibit statistically significant associations with OPLL in case-control study (p = 0.0028 for 3' end + 140 and p = 0.034 for 3' end + 561). These results indicate that the genetic causality of OPLL lies within or close to the RXR beta/COL11A2 locus.

Evidence for Two Antigenically Distinct Molecular Weight Variants of Prostaglandin HSynthase in the Rat Ovary

Two affinity-purified polyclonal antibodies have been generated that differentially recognize two mol wt (Mr) variants of prostaglandin H synthase (PGS) in the rat ovary: antibody-2 recognized PGS of 72,000 Mr (PGS72), and antibody-3 recognized PGS of 69,000 Mr (PGS69). Immunoblot analyses showed that PGS72 was rapidly induced by LH in granulosa cells of preovulatory (PO) follicles and was associated with the increased production of prostaglandins (PGs) obligatory for ovulation. PGS72 was low (negligible) in other ovarian tissues, including PO follicles, corpora lutea, and interstitium. In contrast, PGS69 was constitutively present in small antral and PO follicles (primarily in thecal cells), was unaffected by LH, and was found at higher levels in corpora lutea throughout pregnancy and in the ovarian interstitium. PGS69 (but not PGS72) was also detected by immunoblots in rat adrenal glands, heart, uterus, and kidney. Immunofluorescent localization of PGS72 and PGS69 to ovarian tissue sections confirmed the cell-specific distribution of PGS observed by immunoblot analyses of cell extracts. Immunofluorescent detection of PGS72 required methanol fixation, whereas PGS69 was also observed with paraformaldehyde fixation and Triton X-100 permeabilization, further suggesting biochemical differences in these molecules. Immunoreactive PGS69 in PO follicles, thecal cells, and granulosa cells was associated with low amounts of indomethacin-sensitive production of PGs by these tissues in vitro, which was unaffected by inhibitors of transcription or translation. In contrast, stimulation of PGs in PO follicles by LH in vitro correlated with the marked induction of PGS72, but not PGS69, and was sensitive to both transcriptional and translational inhibitors. Collectively, these studies provide the first evidence that the rat ovary contains two immunologically distinct forms and Mr variants of PGS, each of which is selectively regulated by hormones, localized to specific cell types, differentially sensitive to inhibitors of transcription/translation, and differentially solubilized for immunocytochemical localization.

Uniformity of glycans within molecular variants of alpha-protease inhibitor with distinct affinity for concanavalin A.

Human alpha 1-protease inhibitor contains four asparagine-linked carbohydrate chains per molecule. Three types of carbohydrate chains were released from the polypeptide backbone by hydrazinolysis: (a) biantennary (80%), (b) biantennary with an intercalated N-acetylglucosamine residue (14%), and (c) triantennary (6%). Using concanavalin-A-affinity chromatography, native and S-carboxymethylated alpha 1--protease inhibitor were fractionated into three distinct molecular variants which were shown to contain only one type (a, b, or c, respectively) of glycan per molecule. This and previous observations on other serum glycoproteins support the proposal of uniformity of glycan type within individual molecular variants of glycoproteins.