Molecular resistance fingerprint of pemetrexed and platinum in a long-term survivor of mesothelioma

Abstract

Background: Pemetrexed, a multi-folate inhibitor combined with a platinum compound is the first-line treatment of malignant mesothelioma, but median survival is still one year. Intrinsic and acquired resistance to pemetrexed is common, but its biological basis is obscure. Here we report for the first time a genome-wide profile of acquired resistance in the tumour from an exceptional case with advanced pleural mesothelioma and almost six years survival after 39 cycles of second-line pemetrexed/carboplatin treatment.

Methodology and principal findings: Genome-wide analysis with Illumina BeadChip Kit of 25,000 genes was performed on mRNA from pre-treatment and post-resistance biopsies from this individual as well on case and control samples from our previously published study (in total 17 samples). Cell specific expression of proteins encoded by selected genes were analysed by immunohistochemistry. Serial serum levels of CA125, CYFRA21-1 and SMRP levels were examined. TS protein, the main target of pemetrexed was overexpressed. Proteins and genes related to DNA damage response, elongation and telomere extension and repair related directly and indirectly to platinum resistance were overexpressed, as the CHK1 protein and the genes CHEK2, LIG3, POLD1, POLA2, FANCD2, PRPF19, RECQ5 respectively, the last two not previously described in mesothelioma. We observed a down-regulation of leukocyte transendothelial migration and cell adhesion molecules pathways. Silencing of NT5C in two mesothelioma cell lines did not sensitize the cells to Pemetrexed. Proposed resistance markers are TS, KRT7/ CK7, TYMP/ thymidine phosphorylase and down-regulated SPARCL1 and CDKN1B. Moreover, comparison of the primary expression of the sensitive versus a primary resistant case showed multi-fold overexpressed DNA repair, cell cycle, cytokinesis, and spindle formation in the latter. Serum CA125 and SMRP reflected the clinical and radiological course and tumour burden.

Conclusions: Genome-wide microarray of mesothelioma pre- and post-resistance biopsies indicated a novel resistance signature to pemetrexed/carboplatin that deserve validation in a larger cohort.

Figure 1. Thoracic computer tomography (CT) throughout the disease course.

A large mediastinal tumour as well as thoracic wall infiltration was seen on the left side (CT at the same level). For details see Methods.

Figure 2. Pyrimidine metabolism in tumour versus normal and at acquired resistance.

Genes involved in pathways of DNA metabolism and production in tumour versus normal (red corresponds to overexpressed and dark green correspond to down-regulated) and in acquired resistant tumour (in ovals, all were overexpressed). Abbreviations: 2.1.3.2 =  aspartate transcarbamylase 3.5.2.3 =  dihydroorotase 6.3.5.5 =  CAD; carbamoyl-phosphate synthetase 2 3.1.3.5 =  NT5C; 5′-nucleotidase 2.7.7.7 =  POLA2; DNA polymerase alpha subunit B 2.7.7.7 =  POLD1; DNA polymerase delta subunit 1 1.17.4.1 =  RRM2B; ribonucleoside-diphosphate reductase subunit M2 2.4.2.4 =  TYMP; thymidine phosphorylase.

Figure 3. Genes changed more than 2-fold after resistance.

Expression of genes that changed more then 2-fold in the post-resistance tumour, compared to initial tumour. Down-regulated genes are shown to the left (30 in total, two not shown) and the overexpressed to the right (15, three not shown). Green bars represent the relative gene expression before and red bars after acquired resistance.

Figure 4. Tumour before resistance and at acquired resistance.

Immunohistochemical images (x40) seen before treatment (top row) and five years later after acquired resistance (bottom row). Three of the experimental markers that were hypothesised to be important for mesothelioma aggressiveness as well as resistance, showed increased protein staining after acquired resistance. Chk1 staining increased from below 5% to 50% of the cells, TYMS staining from below 1% to 25% of the cells while nuclear staining of NQO1 increased from 20 to 70% of the cells. Two novel putative resistance markers, KRT7/CK7 and TYMP both gene and protein was significantly increased at resistance. SPARCL1 showed a significant decrease in gene expression, but its encoded protein, hevin, showed only a slight decrease in protein staining intensity.

Figure 5. Fold-change of gene expression between a primary resistant mesothelioma with only six months survival and the primary sensitive case with almost six years survival.

The top figure shows the DNA repair genes found in , depicting a grave overexpression of these genes in the primary resistant case. Among them, CHEK1, FANCD2 and TYMS also seem to be important for acquired resistance (in ovals). Among the 32 top overexpressed genes (arbitrarily >6-fold), the 23 are involved in cell cycle, cytokinesis, and spindle formation, and several are known to be negative prognostic factors in other cancers. The marked differences in indicate which gene functions may be important for aggressiveness and intrinsic treatment resistance in mesothelioma. KRT7 and SDC1 genes that changed significally at acquired resistance were also >2-fold overexpressed in the aggressive case. NQO1, a putative treatment target where protein expression was increased in acquired resistance, was >2-fold overexpressed in the aggressive case.

Figure 6. Serum biomarker changes over time.

Ca125 and Mesothelin (SMRP) biomarkers in serum were elevated at diagnosis and decreased and increased according to the clinical and radiological regression and progression respectively. The straight horizontal dashed lines depict the maximum normal values of the two markers. CYFRA 21-1 is not shown.

Figure 7. Cartoon summarizing the most important findings related to pemetrexed-platinum resistance and tumour aggressiveness in post- versus pre-treatment biopsies.

All genes labelled red are significantly overexpressed except TYMS and CHEK1 where only the encoded protein overexpression was seen. Thymidylate synthase protein overexpression is a known resistance factor against pemetrexed and the TYMP gene/protein was overexpressed. Pemetrexed inhibits the folate enzymes TYMS, GARFT and DHFR. Overproduction of thymidylate and hypoxanthine can reduce the pemetrexed effect on all three enzymes and rescue tumour cells from pemetrexed toxicity. NT5C was overexpressed and encodes an enzyme, 5′, 3′-nucleotidase, a key enzyme for production of thymidylate and hypoxanthine. TYMS and the downstream metabolite 2-deoxy-D-ribose increase angiogenesis and tumour aggressiveness. POLA2 and POLD1 are important for DNA elongation, telomerase extension and cell survival, but also for repair, namely nucleotide excision repair (NER, POLD1 combined with LIG3) and base excision repair (BER, LIG3 with XRCC1) thus important for platinum resistance. Two novel DNA repair genes with undefined mechanism related to both drug and radiation resistance, RECQL5 PRPF19, were overexpressed. Damage response gene CHEK2 and Chk1 protein was overexpressed, both involved in delaying mitosis and facilitating DNA repair. Abbrevations: CHEK1/Chk1; checkpoint 1 kinase CHEK2; checkpoint 2 kinase dTDP; deoxythymidine di-phosphate dTTP; deoxythymidine tri-phosphate dUMP; deoxyuridylate IMP; inositol mono-phosphate LIG3; Ligase III NT5C; 5′, 3′-nucleotidase, cytosolic POLA2; DNA polymerase alpha subunit B POLD1; DNA polymerase delta subunit 1 TYMP; thymidine phosphorylase TYMS; thymidylate synthase.

Figure 8. Leukocyte transendothelial migration in tumour versus normal parietal pleura and at acquired resistance.

Leukocyte transendothelial migration were among the top down-regulated pathways. Here we show the tumour versus normal profile with overexpressed (red) and down-regulated (green) genes, and genes marked with oval were down-regulated post-resistance. Abbrevations: CDH5; cadherin 5, type 2 (vascular endothelium) CLDN1; claudin 1 CLDN5; claudin 5 CXCL12; chemokine (C-X-C motif) ligand 12 ESAM; endothelial cell adhesion molecule JAM2; junctional adhesion molecule 2 OCLN; occludin (EC:2.1.1.67) VCAM1; vascular cell adhesion molecule 1 VCL; vinculin.

Figure 9. Cell cycle changes tumour versus normal and acquired resistance.

Cell cycle was one of the KEGG pathways with most de-regulated genes both in tumour versus normal (23 genes- red correspond to overexpressed, dark green correspond to down-regulated) involved in all phases of the cell cycle (G1-S-G2-M). At acquired resistance, only CDKN1B (in oval) was down-regulated and CHEK2 was overexpressed (in oval). Abbrevations: CDKN1B; cyclin-dependent kinase inhibitor 1B (p27, Kip1) CHEK2; checkpoint kinase 2.