A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification.

UNLABELLED: The overall purpose of this study is to provide proof of concept for introducing the anthracycline epirubicin as an effective, biomarker-guided treatment for metastatic colorectal cancer (mCRC) patients who are refractory to treatment with oxaliplatin-based chemotherapy and have TOP2A gene amplification in their tumor cells. BACKGROUND: Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin in patients with breast cancer and thus could be an alternative option for patients with CRC and amplified TOP2A gene. We have previously analysed the frequency of TOP2A gene aberrations in CRC and found that 46.6% of these tumors had TOP2A copy gain and 2.0% had loss of TOP2A when compared to adjacent normal tissue. The TOP2A gene is located on chromosome 17 and when the TOP2A/CEN-17 ratio was applied to identify tumors with gene loss or amplifications, 10.5% had a ratio ≥ 1.5 consistent with gene amplification and 2.6% had a ratio ≤ 0.8 suggesting gene deletions. Based on these observations and the knowledge gained from treatment of breast cancer patients, we have initiated a prospective clinical, phase II protocol using epirubicin (90 mg/m2 iv q 3 weeks) in mCRC patients, who are refractory to treatment with oxaliplatin. METHODS/DESIGN: The study is an open label, single arm, phase II study, investigating the efficacy of epirubicin in patients with oxaliplatin refractory mCRC and with a cancer cell TOP2A/CEN-17 ratio ≥ 1.5. TOP2A gene amplification measured by fluorescence in situ hybridization. A total of 25 evaluable patients (15 + 10 in two steps) will be included (Simon's two-stage minimax design). Every nine weeks, response is measured by computed tomography imaging and evaluated according to RECIST 1.1. The primary end-point of the study is progression-free survival. TRIAL REGISTRATION: Eudract no. 2013-001648-79.

The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer.

BACKGROUND: Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown. RESULTS: We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of 'DNA methylation entropy' to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples. CONCLUSION: Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy.

The glutamate transport inhibitor DL-Threo-ß-Benzyloxyaspartic acid (DL-TBOA) differentially affects SN38- and oxaliplatin-induced death of drug-resistant colorectal cancer cells.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer death globally and new biomarkers and treatments are severely needed. METHODS: Here, we employed HCT116 and LoVo human CRC cells made resistant to either SN38 or oxaliplatin, to investigate whether altered expression of the high affinity glutamate transporters Solute Carrier (SLC)-1A1 and -1A3 (EAAT3, EAAT1) is associated with the resistant phenotypes. Analyses included real-time quantitative PCR, immunoblotting and immunofluorescence analyses, radioactive tracer flux measurements, and biochemical analyses of cell viability and glutathione content. Results were evaluated using one- and two-way ANOVA and Students two-tailed t-test, as relevant. RESULTS: In SN38-resistant HCT116 and LoVo cells, SLC1A1 expression was down-regulated ~60 % and up-regulated ~4-fold, respectively, at both mRNA and protein level, whereas SLC1A3 protein was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-ß-Benzyloxyaspartic acid (TBOA)-sensitive, UCPH101-insensitive [(3)H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration-dependently augmented loss of cell viability induced by SN38, while strongly counteracting that induced by oxaliplatin, in both HCT116 and LoVo cells. This reflected neither altered expression of the oxaliplatin transporter Cu(2+)-transporter-1 (CTR1), nor changes in cellular reduced glutathione (GSH), although HCT116 cell resistance per se correlated with increased cellular GSH. DL-TBOA did not significantly alter cellular levels of p21, cleaved PARP-1, or phospho-Retinoblastoma protein, yet altered SLC1A1 subcellular localization, and reduced chemotherapy-induced p53 induction. CONCLUSIONS: SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while attenuating that induced by oxaliplatin. These findings may point to novel treatment options in treatment-resistant CRC.

TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.

An explorative analysis of ERCC1-19q13 copy number aberrations in a chemonaive stage III colorectal cancer cohort.

BACKGROUND: Platinum-based chemotherapy has long been used in the treatment of a variety of cancers and functions by inducing DNA damage. ERCC1 and ERCC4 are involved in the removal of this damage and have previously been implicated in resistance to platinum compounds. The aim of the current investigation is to determine the presence, frequency and prognostic impact of ERCC1 or ERCC4 gene copy number alterations in colorectal cancer (CRC). METHODS: Fluorescent in situ hybridization probes directed at ERCC1 and ERCC4 with relevant reference probes were constructed. Probes were tested in a CRC cell line panel and in tumor sections from 152 stage III CRC chemonaive patients. Relationships between biomarker status and clinical endpoints (overall survival, time to recurrence, and local recurrence in rectal cancer) were analyzed by survival statistics. RESULTS: ERCC1-19q13 copy number alterations were observed in a single cell line metaphase (HT29). In patient material, ERCC1-19q13 copy number gains (ERCC1-19q13/CEN-2 ≥ 1.5) were detected in 27.0% of specimens, whereas ERCC1-19q13 deletions (ERCC1-19q13/CEN-2 < 0.8) were only detected in 1.3%. ERCC1-19q13 gain was significantly associated with longer survival (multivariate analysis, HR: 0.45, 95% CI: 0.20-1.00, p = 0.049) in patients with colon tumors, but not rectal tumors. No ERCC4 aberrations were detected and scoring was discontinued after 50 patients. CONCLUSIONS: ERCC1-19q13 copy number gains occur frequently in stage III CRC and influences survival in patients with colon tumors. Future studies will investigate the effect of ERCC1-19q13 aberrations in a platinum-treated patient population with the aim of developing a predictive biomarker profile for oxaliplatin sensitivity in CRC.

Underpinning the repurposing of anthracyclines towards colorectal cancer: assessment of topoisomerase II alpha gene copy number alterations in colorectal cancer.

OBJECTIVE. We propose a repurposing strategy where anthracyclines are reintroduced to a subgroup of patients with metastatic colorectal cancer with the highest likelihood of response. In breast cancer, DNA topoisomerase II alpha gene (TOP2A) alterations predict incremental benefit of anthracyclines, but this association has not been investigated in colorectal cancer. Frequency analysis of TOP2A gene alterations in colorectal cancer and the association with prognosis are evaluated and the challenges of using a TOP2A/CEN-17 FISH probe combination are addressed. MATERIAL AND METHODS. Formalin-fixed, paraffin-embedded material from 154 stage III colorectal cancer patients included in the RANX05 clinical trial was retrospectively assessed for TOP2A gene alterations using FISH. The TOP2A/CEN-17 ratio as well as the TOP2A gene copy number alone was used to define gene alterations and associations between gene status and outcomes were analyzed. RESULTS. TOP2A gene gain was a frequent finding with 9.8 % having a total of ≥4 TOP2A copies per cell. According to the TOP2A/CEN-17 ratio, 10.5 % had TOP2A gene gain. Polysomy or gain of the centromere region of chromosome-17 was not as frequent as reported in breast cancer. No prognostic characteristic of TOP2A was identified. CONCLUSION. TOP2A gene gain is present in numbers relevant to identify a subgroup of patients who may benefit from anthracycline therapy. Based on the present findings, we will initiate a prospective clinical trial designed to evaluate this hypothesis in patients with metastatic colorectal cancer who have failed 5-fluorouracil and oxaliplatin chemotherapy.

TOP1 gene copy numbers in colorectal cancer samples and cell lines and their association to in vitro drug sensitivity.

OBJECTIVE: A positive relationship between topoisomerase-1 (TOP1) protein and sensitivity toward the TOP1 inhibitor irinotecan has been reported in patients with metastatic colorectal cancer (mCRC). In this study, we analyzed TOP1 gene copy number variation in tumor tissue from CRC patients and CRC cell lines with different sensitivities to the TOP1 inhibitor SN-38 and oxaliplatin. MATERIAL AND METHODS: A TOP1 gene probe with a chromosome 20 centromere (CEN-20) reference probe was applied on normal mucosa and on tumor tissue from 50 stage III CRC patients. Additionally, associations between TOP1/CEN-20 ratio and in vitro sensitivity to SN-38 (irinotecan) and oxaliplatin were tested on 10 CRC cell lines. Results. In the malignant epithelium, 84% of the samples demonstrated an increased TOP1 gene copy number and 64% had an increased TOP1/CEN-20 ratio compared with the non-affected mucosa. Sixteen (32%) of the tumors had a ratio of ≥ 1.5 and 9 (18%) of these had a ratio of ≥ 2.0. A positive association was observed between the TOP1 gene copy number and the TOP1/CEN-20 ratio and in vitro sensitivity toward SN-38, but not toward oxaliplatin. CONCLUSIONS: A large fraction of the clinical samples demonstrated increased TOP1 gene copy number and increased TOP1/CEN-20 ratio. The cell line study suggested an association between TOP1 gene copy number or TOP1/CEN-20 ratio and sensitivity to irinotecan but not oxaliplatin.

Predictive biomarkers with potential of converting conventional chemotherapy to targeted therapy in patients with metastatic colorectal cancer.

The availability of systemic chemotherapy regimens for the treatment of patients with metastatic colorectal cancer (mCRC) is based on the results from large prospective, randomized studies. The main chemotherapeutic drugs used in treatment of mCRC are the fluoropyrimidines (5-fluorouracil (5-FU); capecitabine) in combination with either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI). The objective response rate to either combination is approximately 50%, where no significant differences with regard to progression free survival or overall survival have been observed. Interestingly, a number of preclinical and clinical studies have indicated lack of full cross resistance between oxaliplatin based and irinotecan based treatment. Therefore, it is possible that certain mCRC patient subpopulations would benefit more from one drug combination rather than the other. To address this clinical problem there has been much focus on development and validation of predictive biomarkers for these three drugs. Here, we present a thorough review on the current status of predictive biomarkers for 5-FU, oxaliplatin and irinotecan treatment of mCRC patients. The overall conclusions were as follows: Several promising biomarker candidates were identified, notably thymidylate synthase for 5-FU, topoisomerase I for irinotecan and ERCC1 for oxaliplatin. However, these candidates warrant further analysis, where assay performance and clinical trial design should be in focus.

Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations.

BACKGROUND: DNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38. METHODS: Three SN-38 resistant (20-67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated. RESULTS: Two of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings. CONCLUSIONS: This study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.

Mechanisms of topoisomerase I (TOP1) gene copy number increase in a stage III colorectal cancer patient cohort.

BACKGROUND: Topoisomerase 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). METHODS: Nine 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.