Identifying prognostic markers for high grade non-muscle invasive bladder cancer.

Abstract

Upon presentation, 70% of the bladder tumors are confined to the epithelial (stage pTa) and stromal layers (stage pT1) of the bladder. These non-muscle invasive tumors (NMI-BC) frequently recur, and 10-15% will eventually invade the detrusor muscle and may cause metastases. Patients with a high grade pTa or pT1
tumors are especially at risk for progression. The management of these tumors is characterized by the difficult choice between transurethral resection (TURB) in combination with drug instillation or total bladder removal by cystectomy. The aim of this study was to identify DNA copy number changes as biomarkers predicting tumor progression in high grade NMI-BC to aid decision making. A set of 18 DNA samples isolated from fresh-frozen high grade NMI-BC was hybridized on Illumina SNP arrays. Seven of these samples were from patients who later developed a muscle-invasive tumor. Analysis of alterations was done with Nexus software. Hierarchical clustering of significantly differing SNPs showed that tumors that progressed and those that did not clustered separately. A comparison of the differential alterations between tumor groups resulted in more than 300 chromosomal locations encompassing chromosome 1p, 2-6, 8-10, 12p, 13q, 17q, 18p, and 22q that were fit for inclusion in a validation assay. A custom Agilent 15K array was designed to include probes for these locations, supplemented with regions not frequently altered in bladder cancer as a control. We collected a retrospective series of 96 high grade NMI-BC paraffin-embedded tumors with a median follow-up of 5 years by collaborating with groups in Erlangen and Regensburg. Half of these samples were from
patients who developed a muscle-invasive tumor at a later time. DNA was hybridized against a reference DNA, consisting of a mixture of normal blood DNA samples, on these custom designed oligoarrays. Analysis of the array data is currently ongoing. The locations with the best independent prognostic value will be
used to design a definitive prognostic assay.

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LARGE DUPLICATIONS AT BALANCED
CHROMOSOME TRANSLOCATION
BREAKPOINTS WHICH COULD BE CAUSED BY
STALLED REPLICATION BUBBLES
Karen Howarth1, Juliet Beavis1, Scott Newman1, Elizabeth M. Batty1,
Jessica C.M. Pole1, Paul A.W. Edwards1
1. Hutchison-MRC Research Centre and Department of Pathology,
University of Cambridge, Hills Road, Cambridge, CB2 0XZ,
United Kingdom
It has been recognised for some time that reciprocal chromosome
translocation breakpoints are often complex. Most familiar are net
deletions of material at the breakpoints, up to megabases in extent.
We describe here the opposite phenomenondduplication of tens
of kilobases of sequence so that the same sequence is present on
both products of a translocation and the breakpoints overlap.
Breakpoints from three breast cancer cell lines were determined
by array painting. In array painting, chromosomes are isolated
by flow cytometry and hybridized to DNA microarrays. This
showed eighteen approximately balanced translocations. In six
cases, all breakpoint junctions were mapped to 2-kb resolution
using high-resolution array painting, FISH with fosmids and
PCR on sorted chromosomes. A total of four translocations, in
two of the cell lines, showed overlap of breakpoints, ranging in
size from 46 kb to 200 kb. By SNP analysis, the duplicated
sequences derived from the same chromosome. Of the remaining
twelve translocations mapped to lower resolution, two reciprocal
translocations showed large overlapping breakpoints of around
1.5 Mb. In most cases, knowledge of the overlap was necessary
for correct interpretation of gene targets. Several of the overlap-
ping breakpoints broke genes, and a fusion was formed between
CTCF and SCUBE2. These overlaps would generally be missed
by conventional FISH analysis. We propose that these duplicated
sequences could arise through errors at replication, possibly by the
production of stalled replication ‘bubbles’, rather than single repli-
cation forks. If translocations form during replication, the gains
we have seen could be the counterpart of the previously described
losses of sequence: of the two daughter cells formed, one would
show gain and the other loss at the breakpoint(s).
IDENTIFYING PROGNOSTIC MARKERS FOR
HIGH GRADE NON-MUSCLE INVASIVE BLADDER
CANCER
Angela A.G. van Tilborg1, Jelmar Quist1, Lucie C. Kompier1,
Maximilian Burger2, Arndt Hartmann3, Ellen C. Zwarthoff1
1. Department of Pathology, Josephine Nefkens Institute, Erasmus MC,
Rotterdam, the Netherlands
2. Department of Urology, University of Regensburg, Germany
3. Department of Pathology, University of Erlangen, Germany
Upon presentation, 70% of the bladder tumors are confined to the
epithelial (stage pTa) and stromal layers (stage pT1) of the
bladder. These non-muscle invasive tumors (NMI-BC) frequently
recur, and 10e15% will eventually invade the detrusor muscle and
may cause metastases. Patients with a high grade pTa or pT1
tumors are especially at risk for progression. The management
of these tumors is characterized by the difficult choice between
transurethral resection (TURB) in combination with drug instilla-
tion or total bladder removal by cystectomy. The aim of this study
was to identify DNA copy number changes as biomarkers predict-
ing tumor progression in high grade NMI-BC to aid decision
making. A set of 18 DNA samples isolated from fresh-frozen high
grade NMI-BC was hybridized on Illumina SNP arrays. Seven of
these samples were from patients who later developed a muscle-
invasive tumor. Analysis of alterations was done with Nexus soft-
ware. Hierarchical clustering of significantly differing SNPs
showed that tumors that progressed and those that did not clus-
tered separately. A comparison of the differential alterations
between tumor groups resulted in more than 300 chromosomal
locations encompassing chromosome 1p, 2e6, 8e10, 12p, 13q,
17q, 18p, and 22q that were fit for inclusion in a validation assay.
A custom Agilent 15K array was designed to include probes for
these locations, supplemented with regions not frequently altered
in bladder cancer as a control. We collected a retrospective series
of 96 high grade NMI-BC paraffin-embedded tumors with
a median follow-up of 5 years by collaborating with groups in
Erlangen and Regensburg. Half of these samples were from
patients who developed a muscle-invasive tumor at a later time.
DNA was hybridized against a reference DNA, consisting of
a mixture of normal blood DNA samples, on these custom de-
signed oligoarrays. Analysis of the array data is currently ongoing.
The locations with the best independent prognostic value will be
used to design a definitive prognostic assay.
54 Abstracts / Cancer Genetics and Cytogenetics 203 (2010) 44e65