Mechanisms of Topoisomerase I (TOP1) Gene Copy Number Increase in a Stage III Colorectal Cancer Patient Cohort

Abstract

BackgroundTopoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH).MethodsNine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN-20) or chromosome 2 (CEN-2). Tissue sections from 154 chemonaive stage III CRC patients, previously studied with TOP1/CEN-20, were analyzed with TOP1/CEN-2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and time to local recurrence (LR; rectal cancer only) were determined.Results TOP1 aberrations were observed in four cell line metaphases. In all cell lines CEN-2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN-2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN-2≥1.5). One hundred and three patients (68.2%) had TOP1 gain, of which 15 patients (14.6%) harbored an amplification (TOP1/CEN-20≥2.0). TOP1 gene gain did not have any association with clinical endpoints, whereas TOP1 amplification showed a non-significant trend towards longer TTR (multivariate HR: 0.50, p = 0.08). Once amplified cases were segregated from other cases of gene gain, non-amplified gene increases (TOP1/CEN-2≥1.5 and TOP1/CEN-20<2.0) showed a trend towards shorter TTR (univariate HR: 1.57, p = 0.07).Conclusions TOP1 gene copy number increase occurs frequently in stage III CRC in a mechanism that often includes CEN-20. Using CEN-2 as a measurement for tumor ploidy levels, we were able to discriminate between different mechanisms of gene gain, which appeared to differ in prognostic impact. TOP1 FISH guidelines have been updated.

Topoisomerase‐1 gene copy aberrations are frequent in patients with breast cancer

“…CEN-2 has previously been shown to reflect chromosomal ploidy levels in colorectal cancer and may therefore be a valid reference for TOP1 in colorectal cancer as CEN-2 is largely unaffected by independent numeric aberrations. 14 The TOP1/CEN-2 probe mix was applied in independent hybridizations and as a validation of the hybridizations and scoring we found a close correlation between the TOP1 signals counted either with the TOP1/ CEN-20 or the TOP1/CEN-2 probe mix (r 5 0.94, p < 0.0001, data not shown). Using the TOP1/CEN-2 ratio in the validation cohort we found a ratio $ 1.5 in 27.0% of the patients (data not shown).…”

“…The cut-off of 2.0 was chosen as this approach has previously been applied when analyzing TOP1 gain in colorectal cancer (CRC). 14 A total overview of the FISH data is provided in Supporting Information Table S1a. A fluorescense microscope (Olympus BX61) was used for visualization of the signals. Signals were enumerated in nonoverlapping nuclei with well-defined nuclear borders.…”

Assessment of the topoisomerase I gene copy number as a predictive biomarker of objective response to irinotecan in metastatic colorectal cancer

“…However, keeping in mind that the Top1 protein rather than the TOP1 gene status is the target of irinotecan it may be wrong to focus solely on focal gene amplification as the only cytogenetic change that potentially associates with irinotecan sensitivity. Gain of the CEN-20 signal counts was previously demonstrated to appear frequently with concurrent gain of the TOP1 signal counts [22,29] suggesting that substantial amounts of information may be lost when only reporting the ratio estimates. The level of Top1 protein expression may partly be a result of a TOP1 dosage effect caused by different cytogenetic changes resulting in TOP1 gain.…”

“…A certain separation of a predictive and prognostic parameter will only be identified by survival analysis when samples from a randomized clinical trial with a relevant control arm are used [9,32]. There is controversy of the prognostic impact of copy number alterations on 20q [22,26,27,29,33], but in a study by Xie et al [34], copy number gain of the 20q11.21-q13.33 region, which includes the TOP1 locus, was associated with prolonged OS in stage III colon cancer. However, the tumor samples used in the study by Xie et al all originated from a clinical trial, where patients were randomized between 5FU alone and 5FU plus irinotecan [35].…”

DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer

“…The gene-to-centromere ratio is a surrogate measure of the gene-to-chromosome relationship, and ratios of 1.5 suggest the presence of at least one extra gene copy per disomic chromosome set. Because the broad 20q gains may in some cases overlap with the centromere region, CEN20 is not ideal for normalization (52). For this reason we chose to include the uncorrected raw TOP1 gene copy number data in the statistical analyses.…”