Diane Rushlow

Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies.

BACKGROUND Retinoblastoma is the childhood retinal cancer that defined tumour-suppressor genes. Previous work shows that mutation of both alleles of the RB1 retinoblastoma suppressor gene initiates disease. We aimed to characterise non-familial retinoblastoma tumours with no detectable RB1 mutations. METHODS Of 1068 unilateral non-familial retinoblastoma tumours, we compared those with no evidence of RB1 mutations (RB1(+/+)) with tumours carrying a mutation in both alleles (RB1(-/-)). We analysed genomic copy number, RB1 gene expression and protein function, retinal gene expression, histological features, and clinical data. FINDINGS No RB1 mutations (RB1(+/+)) were reported in 29 (2·7%) of 1068 unilateral retinoblastoma tumours. 15 of the 29 RB1(+/+) tumours had high-level MYCN oncogene amplification (28-121 copies; RB1(+/+)MYCN(A)), whereas none of 93 RB1(-/-) primary tumours tested showed MYCN amplification (p<0·0001). RB1(+/+)MYCN(A) tumours expressed functional RB1 protein, had fewer overall genomic copy-number changes in genes characteristic of retinoblastoma than did RB1(-/-) tumours, and showed distinct aggressive histological features. MYCN amplification was the sole copy-number change in one RB1(+/+)MYCN(A) retinoblastoma. One additional MYCN(A) tumour was discovered after the initial frequencies were determined, and this is included in further analyses. Median age at diagnosis of the 17 children with RB1(+/+)MYCN(A) tumours was 4·5 months (IQR 3·5-10), compared with 24 months (15-37) for 79 children with non-familial unilateral RB1(-/-) retinoblastoma. INTERPRETATION Amplification of the MYCN oncogene might initiate retinoblastoma in the presence of non-mutated RB1 genes. These unilateral RB1(+/+)MYCN(A) retinoblastomas are characterised by distinct histological features, only a few of the genomic copy-number changes that are characteristic of retinoblastoma, and very early age of diagnosis. FUNDING National Cancer Institute-National Institutes of Health, Canadian Institutes of Health Research, German Research Foundation, Canadian Retinoblastoma Society, Hyland Foundation, Toronto Netralaya and Doctors Lions Clubs, Ontario Ministry of Health and Long Term Care, UK-Essen, and Foundations Avanti-STR and KiKa.

Patterns of missplicing caused by RB1 gene mutations in patients with retinoblastoma and association with phenotypic expression.

We have analyzed RNA from retinoblastoma patients and unaffected carriers with various RB1 gene mutations to determine the patterns of missplicing and associations with phenotypic expression. Most sequence alterations in or in the neighborhood of conserved splice signals that we tested resulted in simple exon skipping (15 mutations) or intron inclusion (new acceptor AG‐sites, four mutations) as expected. Two mutations resulted in skipping of a neighboring exon (exon 11), a complex pattern indicating competition for correct lariat formation. We observed no activation of a cryptic splice site but found that a recurrent missense mutation in exon 7 creates a new splice site (two families). RT‐PCR analysis enabled us to confirm the presence and to characterize the transcriptional consequences of gross insertions and deletions in the RB1 gene in six patients, including two patients with mutational mosaicism. We also used RT‐PCR analysis to search for unknown mutations in 15 patients and identified three oncogenic point mutations deep in introns. Two of these mutations are recurrent thus indicating that, despite the vast extent of the introns of the RB1 gene, few bases are effective targets for oncogenic mutations. When analyzing associations between phenotypic expression (16 families) and mutational consequences we observed no link to the presence or absence of a premature termination codon in the mutant transcript. However, the location of a mutation relative to the splice sequence has a strong and consistent influence on phenotypic expression. Hum Mutat 29(4), 475–484, 2008.

Using RB1 mutations to assess minimal residual disease in metastatic retinoblastoma.

To assess complete remission before subjecting nongermline metastatic retinoblastoma patients to an autologous peripheral stem cell transplant, we tested for patient-specific retinoblastoma tumor suppressor gene (RB1) mutant alleles in cerebrospinal fluid (CSF) and bone marrow. In 1 child with CSF and 1 with bone marrow metastases, allele-specific polymerase chain reaction (AS-PCR) detected the biallelic RB1 mutations specific to their tumors. The tumor of Child A was homozygous for R251X, and in Child B, it was homozygous for R358X. In Child A, the R251X mutation was detected in mutant controls diluted to 1:12,800 but not in CSF samples, corroborating clinical remission after chemotherapy. In Child Bs bone marrow, AS-PCR for R358X was strongly positive at the detection of relapse, and subsequent bone marrow samples corroborated clinical remission after chemotherapy. No mutant tumor RB1 alleles were detected in their harvested peripheral blood stem cells. Both children were deemed suitable candidates for supralethal-dosage consolidation chemotherapy followed by autologous peripheral stem cell rescue of the bone marrow aimed at curing their metastatic retinoblastoma. When Child A recurred, the mutant tumor RB1 allele was detected 3.5 months before conventional pathology detected retinoblastoma tumor cells in the CSF. Assaying tumor-specific RB1 mutations complements cytological and immunohistochemical assessment of retinoblastoma involvement of CSF and bone marrow. Tumor cells can be detected in numbers lower than possible by conventional methods. An early diagnosis of relapse may allow an early institution of new therapy. A prospective international multicenter trial of the rare patients with metastatic retinoblastoma would assess the role of molecular monitoring in surveillance for minimal residual disease and recurrence.

Needle core biopsies provide ample material for genomic and proteomic studies of kidney cancer: observations on DNA, RNA, protein extractions and VHL mutation detection.

The use of needle biopsies in basic research is increasing, and our study provides a comprehensive analysis of their adequacy in genomic and proteomic studies of kidney cancer. Frozen clear cell renal cell carcinoma (ccRCC) needle core biopsies and sections from core biopsies embedded in optimal cutting temperature (OCT) compound were used to extract DNA, RNA and protein. Their integrity was determined using genomic and proteomic analyses. VHL mutation testing was performed on ccRCC biopsies and corresponding tumors using bulk and laser capture microdissection (LCM) extractions for comparison. Adequate amounts of good quality DNA (5.8-13.3 μg/whole core, 0.6-2.7 μg/20 sections), RNA (2.9-11.9 μg/whole core, 0.5-1.3 μg/20 sections) and protein (137.4-444 μg/whole core, 39.9-74.1 μg/20 sections) were obtained from whole core and frozen sections of ccRCC needle biopsies, respectively. We observed VHL sequence mutations in 75% of ccRCC tumors and, in most cases, the same mutations were detected in both tumors and corresponding biopsies. Mutations observed by bulk extractions from tumors and biopsies were also detected by LCM without significant differences between both methodologies. ccRCC needle biopsies provide ample material for genomic and proteomic studies of kidney cancer. They are good representatives of their corresponding tumors for VHL mutation detection using both bulk and LCM extractions. LCM does not increase sensitivity of VHL mutation detection.

Adult ovarian retinoblastoma genomic profile distinct from prior childhood eye tumor.

changes in a typical case of AZOOR. We should note that care should be taken in evaluation of the COST line because its visibility is dependent on the intensity and direction of the laser light that reaches the photoreceptor layer. However, in patients with AZOOR, the COST line and the foveal bulge observed by OCT could help as indicators of early cone photoreceptor dysfunction in cases with minimal ophthalmoscopic and angiographic abnormalities.

MG-127 Residual disease monitoring in a retinoblastoma patient by pcr of a novel deletion breakpoint

Background Allele-specific (AS)-PCR for known point mutations is a high-sensitivity method to monitor for minimal residual disease (MRD) in metastatic retinoblastoma. However, this approach is difficult to apply to large RB1 gene deletions. Objective We describe a novel, high sensitivity molecular assay for MRD monitoring of a large RB1 deletion. Method The proband’s retinoblastoma RB1 deletion was identified using quantitative multiplex (QM)-PCR. Array comparative genomic hybridization (aCGH) was used to map the boundaries of the tumour RB1 deletion. Inverse PCR was then applied to capture the unique deletion breakpoint, and AS primers were optimised for high sensitivity surveillance for MRD. Result Full RB1 gene testing revealed no mutation in blood of this patient. Using QM-PCR, we found one RB1 mutation in the retinoblastoma tumour (del P- >11). aCGH confirmed an ~238 kb hemizygous deletion extending centromeric from RB1. The precise deletion breakpoint was determined using inverse PCR to amplify from the known two-copy flanking sequence to identify a PCR product in tumour DNA that was absent from the patient’s blood DNA. AS primers based on the breakpoint detected a tumour-specific PCR product with a sensitivity of 1 in 1000 cells. The original bone marrow (BM) prior to therapy was strongly positive, but all subsequent BM, CSF, and cells harvested for stem cell transplant have been negative for the tumour-specific marker. Conclusion aCGH followed by inverse PCR provides a cost- and time-effective way to map QM-PCR detected RB1 breakpoints for high sensitivity molecular surveillance for MRD.