Continuation of fluoropyrimidine treatment with S-1 after cardiotoxicity on capecitabine- or 5-fluorouracil-based therapy in patients with solid tumours: a multicentre retrospective observational cohort study.

BACKGROUND: Capecitabine- or 5-fluorouracil (5-FU)-based chemotherapy is widely used in many solid tumours, but is associated with cardiotoxicity. S-1 is a fluoropyrimidine with low rates of cardiotoxicity, but evidence regarding the safety of switching to S-1 after 5-FU- or capecitabine-associated cardiotoxicity is scarce. PATIENTS AND METHODS: This retrospective study (NCT04260269) was conducted at 13 centres in 6 countries. The primary endpoint was recurrence of cardiotoxicity after switch to S-1-based treatment due to 5-FU- or capecitabine-related cardiotoxicity: clinically meaningful if the upper boundary of the 95% confidence interval (CI; by competing risk) is not including 15%. Secondary endpoints included cardiac risk factors, diagnostic work-up, treatments, outcomes, and timelines of cardiotoxicity. RESULTS: Per protocol, 200 patients, treated between 2011 and 2020 [median age 66 years (range 19-86); 118 (59%) males], were included. Treatment intent was curative in 145 (73%). Initial cardiotoxicity was due to capecitabine (n = 170), continuous infusion 5-FU (n = 22), or bolus 5-FU (n = 8), which was administered in combination with other chemotherapy, targeted agents, or radiotherapy in 133 patients. Previous cardiovascular comorbidities were present in 99 (50%) patients. Cardiotoxic events (n = 228/200) included chest pain (n = 125), coronary syndrome/infarction (n = 69), arrhythmia (n = 22), heart failure/cardiomyopathy (n = 7), cardiac arrest (n = 4), and malignant hypertension (n = 1). Cardiotoxicity was severe or life-threatening in 112 (56%) patients and led to permanent capecitabine/5-FU discontinuation in 192 (96%). After switch to S-1, recurrent cardiotoxicity was observed in eight (4%) patients (95% CI 2.02-7.89, primary endpoint met). Events were limited to grade 1-2 and occurred at a median of 16 days (interquartile range 7-67) from therapy switch. Baseline ischemic heart disease was a risk factor for recurrent cardiotoxicity (odds ratio 6.18, 95% CI 1.36-28.11). CONCLUSION: Switching to S-1-based therapy is safe and feasible after development of cardiotoxicity on 5-FU- or capecitabine-based therapy and allows patients to continue their pivotal fluoropyrimidine-based treatment.

New amplified and highly expressed genes discovered in the ERBB2 amplicon in breast cancer by cDNA microarrays.

Amplification of the ERBB2 oncogene at 17q12 has been well documented in breast cancer and has been shown to contribute to a poor clinical outcome. However, systematic surveys of copy number and expression levels of all genes within the 17q12 region have not been performed. Here, we used cDNA and comparative genomic hybridization microarray technologies to undertake a broad survey of genes involved in the 17q12 amplification in breast cancer. A chromosomal region-specific cDNA microarray containing 217 expressed sequence tag (EST) clones from 17q12 was constructed and used for parallel analysis of gene copy numbers and expression levels in seven breast cancer cell lines allowing direct identification of genes whose expression is elevated because of an increase in copy number in this chromosomal region. The copy number and expression survey identified 12 transcripts that showed a consistent pattern of increased copy number and expression in three or more of the 17q12-amplified cell lines. As expected, these included ERBB2 as well as the GRB7 and MLN64 genes previously shown to be coamplified with ERBB2. In addition, five other known genes and four uncharacterized ESTs were also found to be consistently activated by amplification in these breast cancer cell lines. Amplicon mapping by fluorescence in situ hybridization revealed a minimal common region of amplification containing four highly expressed genes, ERBB2, GRB7, MLN64, and an uncharacterized EST 48582. Furthermore, several other genes, although not located in the minimal common region of amplification, showed a correlated pattern of amplification and expression indicating that they might play a role in breast cancers with the 17q12 amplification. In conclusion, parallel analysis of gene copy number and expression levels by cDNA microarray can be used to directly identify candidate target genes involved in amplifications. Our results show that the 17q12 amplification in breast cancer leads to the simultaneous elevation of expression levels of several genes.

CGH, cDNA and tissue microarray analyses implicate FGFR2 amplification in a small subset of breast tumors.

Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH). However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small-scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM-52 breast cancer cell line harbors several high-level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2) gene has been localized. High level amplification of FGFR2 in SUM-52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40-fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue) experiments revealed high-level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on http://www.esacp.org/acp/2001/22-4/heiskanen.htm

From chromosomal alterations to target genes for therapy: integrating cytogenetic and functional genomic views of the breast cancer genome.

A vast number of recurrent chromosomal alterations have been implicated in cancer development and progression. However, most of the genes involved in recurrent chromosomal alterations in solid tumors remain unknown, despite the recent substantial progress in genomic research and availability of high-throughput technologies. For example, it is now possible to quickly identify large numbers of differentially expressed genes in cancer specimens using cDNA microarrays. Integration of this "functional genomic view" of the cancer genome with the "cytogenetic view" could lead to the identification of genes playing a critical role in cancer development and progression. In this review, we illustrate how the combination of three different microarray technologies, cDNA, CGH, and tissue microarrays, makes it possible to directly identify genes involved in chromosomal rearrangements in cell line model systems and then rapidly explore their significance as potential diagnostic and therapeutic targets in human primary breast cancer progression.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer.

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

Multiple genes at 17q23 undergo amplification and overexpression in breast cancer.

Studies by comparative genomic hybridization imply that amplification of the chromosomal region 17q22-q24 is common in breast cancer. Here, amplification and expression levels of six known genes located at 17q23 were examined in breast cancer cell lines. Four of them (RAD51C, S6K, PAT1, and TBX2) were found to be highly amplified and overexpressed. To investigate the involvement of these genes in vivo, fluorescence in situ hybridization analysis of a tissue microarray containing 372 primary breast cancers was used. S6K, PAT1, and TBX2 were coamplified in about 10% of tumors, whereas RADS1C amplification was seen in only 3% of tumors. Expression analysis in 12 primary tumors showed that RAD51C and S6K were consistently expressed in all cases in which they were amplified and also in some tumors without amplification. These data suggest that 17q23 amplification results in simultaneous up-regulation of several genes, whose increased biological activity may jointly contribute to the more aggressive clinical course observed in patients with 17q23-amplified tumors.

Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis.

BACKGROUND: Studies by comparative genomic hybridization (CGH) have shown that chromosomal region 17q23 is amplified in up to 20% of primary breast cancers. We used microarray analyses to measure the expression levels of genes in this region and to explore their prognostic importance. METHODS: A microarray that contained 4209 complementary DNA (cDNA) clones was used to identify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal protein S6 kinase (S6K), and to localize it to the 17q23 region. Northern and western blot analyses were used to measure S6K gene and protein expression, and an enzymatic assay was used to measure S6K activity. Tumor tissue microarray analysis was used to study amplification of S6K and the HER-2 oncogene, another 17q-linked gene, and the relationship between amplification and prognosis was analyzed. The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are two-sided. RESULTS: S6K was amplified and highly overexpressed in MCF-7 cells relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary breast tumors, and a statistically significant association between amplification and poor prognosis (P =.0021) was observed. Amplification of both S6K and HER-2 implied particularly poor survival (P =.0001). CONCLUSIONS: The combination of CGH information with cDNA and tissue microarray analyses can be used to identify amplified and overexpressed genes and to evaluate the clinical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and may adversely affect the prognosis of patients with this amplification.

Amplification and deletion of topoisomerase IIalpha associate with ErbB-2 amplification and affect sensitivity to topoisomerase II inhibitor doxorubicin in breast cancer.

Topoisomerase IIalpha (topoIIalpha) is a key enzyme in DNA replication and a molecular target for many anti-cancer drugs called topoII inhibitors. The topoIIalpha gene is located at chromosome band 17q12-q21, close to the ErbB-2 oncogene (HER-2/neu), which is the most commonly amplified oncogene in breast cancer. Because of the physical proximity to ErbB-2, copy number aberrations may also occur in the topoIIalpha gene. These topoIIalpha gene copy number aberrations may be related to the altered chemosensitivity to topoII inhibitors that breast cancers with ErbB-2 amplification are known to have. We used fluorescence in situ hybridization to study copy number aberrations of both topoIIalpha and ErbB-2 in nine breast cancer cell lines and in 97 clinical breast tumors, which were selected for the study according to their ErbB-2 status by Southern blotting. TopoIIalpha-protein expression was studied with Western blot and sensitivity to doxorubicin (a topoII inhibitor) with a 96-well clonogenic in vitro assay. Two of the five cell lines with ErbB-2 gene amplification (SK-BR-3 and UACC-812) showed amplification of topoIIalpha. In MDA-361 cells, ErbB-2 amplification (14 copies/cell) was associated with a physical deletion of topoIIalpha (four copies of chromosome 17 centromere and two copies of topoIIalpha). The topoIIalpha amplification in UACC-812 cells was associated with 5.9-fold-increased topoIIalpha protein expression and 2.5-fold-increased sensitivity to the topoII inhibitor, doxorubicin, whereas the deletion in MDA-361 leads to decreased protein expression (45% of control) and a 2.4-fold-increased chemoresistance in vitro. Of 57 ErbB-2-amplified primary breast carcinomas, 25 (44%) showed ErbB-2-topoIIalpha coamplification and 24 (42%) showed a physical deletion of the topoIIalpha gene. No topoIIalpha copy number aberrations were found in 40 primary tumors without ErbB-2 amplification. TopoIIalpha gene amplification and deletion are common in ErbB-2-amplified breast cancer and are associated with increased or decreased sensitivity to topoII inhibitors in vitro, respectively. These findings may explain the altered chemosensitivity to topoII inhibitors reported in ErbB-2-amplified breast cancers.

Characterization of topoisomerase II alpha gene amplification and deletion in breast cancer.

Topoisomerase IIalpha (TOP2A) is a key enzyme in DNA replication and a molecular target for many important anticancer drugs. TOP2A is amplified or deleted together with amplification of the closely located ERBB2/HER-2/neu oncogene in breast cancer. We characterized the copy number aberrations of TOP2A and ERBB2 in 136 primary breast tumors by FISH. Among the 70 primary tumors with ERBB2 amplification, amplification of TOP2A was found in 29 (41%); 30 tumors (43%) showed a physical deletion of TOP2A; and the copy number for TOP2A was not altered in 11 tumors with ERBB2 amplification (16%). No TOP2A gene aberrations were identified in 65 primary tumors without ERBB2 amplification. Fiber FISH revealed that simultaneously amplified ERBB2 and TOP2A were not present in the same amplicon, because repetitive tandem repeat-like signals of ERBB2 and TOP2A were in separate DNA fibers. The deletion of TOP2A (seen in the MDA-361 cell line and in 31 primary tumors) was interstitial, spanning less than two megabases of DNA. Mean copy numbers of TOP2A (2.4 +/- 0.6 for TOP2A vs. 4.9 +/- 1.1 for chromosome 17 centromere) suggest that the deletion of TOP2A occurs before polyploidization of the genome. Eight primary tumors with high-level ERBB2 amplification showed a new type of intratumoral heterogeneity; two different cell clones with either high-level amplification or deletion of TOP2A were found adjacent to each other in the same tumor. These results indicate that amplification of the ERBB2 oncogene is followed by complex secondary genetic aberrations, which lead to amplification or deletion of the TOP2A gene in a majority of tumors. Genes Chromosomes Cancer 26:142-150, 1999.

Molecular cytogenetic mapping of 24 CEPH YACs and 24 gene-specific large insert probes to chromosome 17.

Defining boundaries of chromosomal rearrangements at the molecular level would benefit from landmarks that link the cytogenetic map to physical, genetic, and transcript maps, as well as from large-insert FISH probes for such loci to detect numerical and structural rearrangements in metaphase or interphase cells. Here, we determined the locations of 24 genetically mapped CEPH-Mega YACs along the FLpter scale (fractional length from p-telomere) by quantitative fluorescence in situ hybridization analysis. This generated a set of cytogenetically mapped probes for chromosome 17 with an average spacing of about 5 cM. We then developed large-insert YAC, BAC, PAC, or P1 clones to the following 24 known genes, and determined refined map locations along the same FLpter scale: pter-TP53-TOP3-cen-TNFAIP1-ERBB2-TOP2A- BRCA1-TCF11-NME1-HLF-ZNF147/CL N80-BCL5/MPO/SFRS1-TBX2-PECAM1-DDX5/ PRKCA-ICAM2-GH1/PRKAR1A-GRB2-CDK3 /FKHL13-qter. Taken together, these 48 cytogenetically mapped large-insert probes provide tools for the molecular analysis of chromosome 17 rearrangements, such as mapping amplification, deletion, and translocation breakpoints in this chromosome, in cancer and other diseases.

Tissue microarrays for high-throughput molecular profiling of tumor specimens.

Many genes and signalling pathways controlling cell proliferation, death and differentiation, as well as genomic integrity, are involved in cancer development. New techniques, such as serial analysis of gene expression and cDNA microarrays, have enabled measurement of the expression of thousands of genes in a single experiment, revealing many new, potentially important cancer genes. These genome screening tools can comprehensively survey one tumor at a time; however, analysis of hundreds of specimens from patients in different stages of disease is needed to establish the diagnostic, prognostic and therapeutic importance of each of the emerging cancer gene candidates. Here we have developed an array-based high-throughput technique that facilitates gene expression and copy number surveys of very large numbers of tumors. As many as 1000 cylindrical tissue biopsies from individual tumors can be distributed in a single tumor tissue microarray. Sections of the microarray provide targets for parallel in situ detection of DNA, RNA and protein targets in each specimen on the array, and consecutive sections allow the rapid analysis of hundreds of molecular markers in the same set of specimens. Our detection of six gene amplifications as well as p53 and estrogen receptor expression in breast cancer demonstrates the power of this technique for defining new subgroups of tumors.

Increased copy number at 17q22-q24 by CGH in breast cancer is due to high-level amplification of two separate regions.

Studies by comparative genomic hybridization (CGH) have defined a chromosomal site at 17q22-q24 that is often overrepresented in breast cancer, neuroblastoma, and several other tumor types. Due to the limited resolution and dynamic range of CGH, it remain unclear whether this gain reflects high-level amplification of small subregion(s) or low-level gain of most of the distal 17q. We used 32 physically mapped 17q probes to construct more accurate copy number profiles for 14 breast cancer cell lines by interphase fluorescence in situ hybridization (FISH). Six cell lines (43%) showed an increased copy number of the 17q-22q24 region by CGH, and seven (50%) by FISH. FISH copy number profiles had a substantially higher dynamic range than did CGH profiles. FISH revealed two independent, highly amplified regions (A and B) at 17q23, separated by about 5 Mb of non-amplified DNA. These regions were distinctly telomeric from the ERBB2 gene locus. However, region A was often co-amplified with ERBB2, whereas B was amplified in cell lines that showed no ERBB2 amplification. We conclude that distal 17q gains recently discovered in breast cancer by CGH are due to high-level amplifications of two different regions at 17q23. This chromosomal region has previously been reported to undergo allelic loss and therefore was thought to harbor a tumor suppressor gene. The present FISH data provide support for the presence, and a starting point for the positional isolation, of 17q23 genes whose upregulation by amplification may play a role in the progression of breast cancer and many other tumor types.