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Deletions in metastatic colorectal cancer with chromothripsis
Summary. Aim: In our previously reported study, we found a correlation between DNA massive fragmentation and increased progression free survival (PFS) in metastatic colorectal cancer (mCRC), but not overall survival. The aim of this study is to find overlapping deleted genome regions in selected mCRC patients with chromothripsis and detect possible cause of increased PFS, and find new genes or combinations, involved in colorectal cancer oncogenesis. Materials and Methods: 10 mCRC patients with chromothripsis receiving 5-fluorouracil, oxaliplatin, leucovorin (FOLFOX) first-line palliative chemotherapy between August, 2011 and October, 2012 were selected for this study. Microarray analysis was performed using the Infinium HumanOmniExpress-12 v1.0 formalin-fixed paraffin-embedded (FFPE) BeadChip kit (Illumina). BeadChip was scaned on HiScan (Illumina). Analysis was performed by GenomeStudio software (Illumina) and R version 3.1.2. Copy number variation and breakpoints on the chromosomes were analyzed using the DNA copy package. Results: Eight deleted tumor suppressor genes (ROBO2, CADM2, FAT4, PCDH10, PCDH18, CDH18, TSG1, CTNNA3) and four deleted oncogenes (CDH12, GPM6A, ADAM29, COL11A1) were identified in more than half of patients. In 70% patients’ deletion in COL11A1 was detected. Deletion of MIR1269, MIR4465, MIR1261 and MIR4490 in patients with longer time to progression was observed. Four patients (40%) with PFS over 14 months, presented with NRG3 deletion (oncogene, еpidermal growth factor receptor (EGFR) ligand) what could possibly decrease proliferation of cancer cells via decreasing EGFR activation. Conclusions: Multiple chromosomal deletions (MIR1269, NRG3, ADK) in mCRC patients with chromothripsis are associated with better response to first line palliative FOLFOX-type chemotherapy and increased PFS.
Submitted: July 9, 2019. Correspondence: E-mail: firstname.lastname@example.org Abbreviations used: CRC — colorectal cancer; EGFR — epidermal growth factor receptor; FFPE — formalin-fixed paraffin-embedded; FOLFOX — 5-fluorouracil, oxaliplatin, leucovorin; mCRC — metastatic colorectal cancer; OS — overall survival; PFS — progression free survival.
According to cancer multiple hit hypothesis, cells acquire multiple mutations and chromosomal rearrangements step by step during cancerogenesis. Colorectal cancer (CRC) development often includes chromosomal instability leading to amplifications and deletions of large DNA segments. Copy number variations — deletions and amplifications — such as mutation of APC, RAS, p53, DCC are recently described in CRC oncogenesis. The impact of these events on survival and treatment outcome in CRC are majorly unclear. In 2011, Stephens et al.  reported chromothripsis that is observed in 2–3% cancers — a massive chromosome fragmentation occurred as a single catastrophic event leading to chromosomal shuttering in one or more chromosomes and consequent random repair . Chromothripsis can drive the development of cancer through several mechanisms such as deletion of tumor suppressor genes and activation of proto-oncogenes. Simultaneous loss of multiple tumor suppressor genes located on different chromosomes can lead to cancer formation and further progression. The prevalence, impact on prognosis and metastasis formation of chromothripsis is not clearly known. Incidence of chromothripsis is from 1.3% in multiple myeloma , 6.6% in acute myeloid leukemia  to 33% in osteosarcoma  and 52.6% in metastatic CRC (mCRC) . Chromothripsis has been associated with poor survival rates in aggressive subtype of breast cancer, multiple myeloma and medulloblastoma [2, 5, 6]. The prognostic and predictive role of chromothripsis in CRC is still unclear. In our previously reported study , we found a correlation between DNA massive fragmentation chromothripsis and increased progression free survival (PFS) in mCRC, but not overall survival (OS). In mCRC with chromothripsis, cancer genome harbours multiple deletions that could lead to better response to 5-fluorouracil, oxaliplatin, leucovorin (FOLFOX) type first line treatment and increased survival. The aim of this study is to find overlapping deleted genome regions in selected patients with chromothripsis and detect possible cause of increased progression free survival. Also, one of our interests is to find new genes or combinations, involved in CRC oncogenesis.
MATERIALS AND METHODS
Study population. 19 mCRC cancer patients who received chemotherapy in Clinic of Oncology of Pauls Stradins Clinical University Hospital (Riga, Latvia) between August, 2011 and October, 2012 were selected in our study, reported previously . The study was performed with approval from the ethics committee of Riga Stradins University and written informed consent was taken from each patient who underwent the study. Tissue samples were previously acquired as part of series of routine diagnostic and pathological analyses at the Hospital. Ten of 19 previously analyzed patients with mCRS was detected with chromothripsis phenomena. In this study we analyzed only patients with chromothripsis. Clinical characteristics of 10 patients are shown in Table 1. All ten patients received FOLFOX type first line chemotherapy. One patient received liver and peritoneal metastasis cytoreductive surgery. After progression 8 patients received irinotecan containing second line chemotherapy; two patients underwent best supportive care. Only two patients (20%) received third line therapy. One patient underwent hepatic surgery for colorectal metastases after discontinuation of second line chemotherapy, one patient — salvage transcatheter arterial chemoembolization of CRC liver metastases by irinotecan-eluting microspheres. We obtained data on clinical follow up until December, 2016. The patients had 4 to 48 months of follow up. mPFS in ten patients was 14 months (range 4–26 months).
Table 1. Clinical characteristics of patients (n = 10)
Note: CEA — carcinoembryonic antigen before chemotherapy; mts — metastases; nk — not known; mt — mutant; wt — wild type.
Genotyping. DNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples with QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quality was evaluated using the Illumina FFPE QC kit (Illumina, San Diego, CA, USA) by reverse transcription-polymerase chain reaction. DNA was restored with the Illumina DNA restoration kit (Illumina). Microarray analysis was performed using the Infinium HumanOmniExpress-12 v1.0 FFPE BeadChip kit (Illumina). BeadChip was scaned on HiScan (Illumina). Analysis was performed by GenomeStudio software (Illumina) and R version 3.1.2. (www.r-project.org). Copy number variations and breakpoints on the chromosomes were analyzed using the DNA copy package (bioconductor.org /pack-ages/release/bioc/html/DNAcopy.html). Statistical analysis. OS and progression free survival rates were estimated using the Kaplan — Meier method. The log-rank test was used to calculate any significant difference between the subgroups by univariate analysis. Significance levels were set at p < 0.05. All statistical analyses were performed by using MedCalc.
In 10 tumor samples we found multiple chromosomal fragmentations (> 100 breakpoints detected at one chromosome) — chromothripsis. The most affected chromosomes were chromosome 1, chromosome 2 and chromosome 6. We analyzed deleted regions which were overlapping in 5 or more patients (> 50%). Frequently deleted regions are shown in Table 2. Genes are identified, divided in five groups: 1) genes involved in proliferation or drug resistance, 2) possible tumor suppressor genes, 3) protocadherin/cadherin genes, 4) epigenetic regulators, and 5) genes of unclear function in oncogenesis. Overlapping deletions are seen in Table 3.
Table 2. Frequently deleted genes in study population (n = 10)
Note: COSMIC — Catalogue of Somatic mutations in Cancer.
Table 3. Overlapping deletions
Eight deleted tumor suppressor genes (ROBO2, CADM2, FAT4, PCDH10, PCDH18, CDH18, TSG1, CTNNA3) and four deleted oncogenes (CDH12, GPM6A, ADAM29, COL11A1) were identified in more than half of patients. In 70% patients’ deletion in COL11A1 was detected. Deletion of MIR1269, MIR4465, MIR1261 and MIR4490 in patients with longer time to progression was observed. Four patients (40%) with PFS over 14 months, presented with NRG3 deletion (oncogene) what could possibly decrease proliferation of cancer cells via decreasing epidermal growth factor receptor (EGFR) activation. Longer PFS was associated with deletion of MIR1269, MIR4465, MIR1261 and MIR4490.
Eight deleted tumor suppressor genes (ROBO2, CADM2, FAT4, PCDH10, PCDH18, CDH18, TSG1, CTNNA3) and four deleted oncogenes (CDH12, GPM6A, ADAM29, COL11A1) were identified in more than half of patients. In 70% patients’ deletion in COL11A1 was detected. It is reported previously that COL11A1 overexpression is associated with proliferation, migration and chemo-resistance to platinum in lung cancer . We can suggest that deletion of this oncogene could be associated with better survival due to slower progression and migration. In 50% patients’ overlapping deletion of MIR1269A was seen — overexpression of it promotes metastases and proliferation in hepatocellular carcinoma . In our study we observe deletion of MIR1269, as well as MIR4465, MIR1261 and MIR4490, in patients with longer time to progression. It might be beneficial for mCRC patients to harbor deletions of MIR genes because of decreased potential of proliferation and formation of metastases. 50% showed MAD2L1 deletion. Overexpression of MAD2L1 is reported in platinum resistant testicular germ cell tumors . Four patients (40%) with PFS over 14 months, presented with NRG3 deletion what could possibly decrease proliferation of cancer cells via decreasing EGFR activation. In previous publication we reported chromothripsis as a surrogate marker for increased mPFS and possible indicator for better response to oxaliplatin based treatment — chromothripsis was associated with mPFS 14 vs 8 months (p = 0.03), but association with OS was not detected . We observed that patients with multiple overlapping deletions have increased time to progression. There are two patients in our study group who are exceptional. Patient № 21 presents multiple overlapping deletions but has very short time to progression. This patient was diagnosed with bulky metastases in liver and lungs, discontinued fist line FOLFOX treatment due to poor performance status and toxicity and progressed in 4 months. Second exceptional patient № 8 had 26 months remission period but presented only one overlapping deletion (SSPO, gene with unknown function). Such a long PFS was observed due to fact that he underwent resection of primary tumor in colon and resection of metastases in peritoneum and liver (maximum cytoreductive surgery) before start of palliative chemotherapy. It is known that treatment strategy — surgery, chemotherapy intensity, targeted treatment, as well as patient related factors, such as performance status, comorbidities, burden of metastatic disease and tumor molecular characteristics, impacts time to progression and OS in mCRC patients. For further research it is important to have more homogenous patient group with similar clinical findings and treatment strategy.
Part of this work was supported by the State research program “Biomedicine for Public Health”.
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