Top 10 overexpressed and down-regulated pathways in post- versus pre-resistance tumour in KEGG pathways map.

<p>Several interesting and important systems were deregulated after resistance. Note that overexpressed genes in GO metabolic process was 89 and in KEGG only 18. The reason is that KEGG includes new genes in a pathway only when several publications have confirmed it, and thus is more conservative but with a high level of evidence. Most pronounced were the metabolism and modification of DNA and RNA through pyrimidine and purine metabolism and aminoacyl-t-RNA biosynthesis. Interestingly, down-regulation of cell adhesion molecules and leukocyte transendothelial migration as well as cytokine-cytokine receptor interaction was pronounced, also an expression characteristic of mesothelioma versus normal pleura.</p

Genes changed more than 2-fold after resistance.

<p>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.</p

Serum biomarker changes over time.

<p>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.</p

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

<p>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.</p

Thoracic computer tomography (CT) throughout the disease course.

<p>A large mediastinal tumour as well as thoracic wall infiltration was seen on the left side (CT at the same level). For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040521#s2" target="_blank">Methods</a>.</p

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.

<p>The top figure shows the DNA repair genes found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040521#pone.0040521-Martin1" target="_blank">[7]</a>, 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.</p

Pyrimidine metabolism in tumour versus normal and at acquired resistance.

<p>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.</p

Change of immunohistochemical expression in tumour before and after resistance.

*<p>(−) indicates no change, (+) indicate increase and (--) indicate reduction.</p><p>Experimental markers are antibodies to proteins encoded by differentially expressed genes in mesothelioma or genes related to mesothelioma biology.</p

Tumour before resistance and at acquired resistance.

<p>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.</p