For this study the TaqMan arrays were customised with 96 gene targets, which allowed up to 4 samples to be run per card (two ports per sample). Further background information on TaqMan arrays can be found in Appendix 3.
46
The design of the array was based on the array used in the pilot study (SL study)(275), which had been customised with 91 putative prostate cancer progression markers (Figure 2.1). The majority of the prostate cancer-related genes in the SL study had been identified as important prostate carcinogenesis genes in a study by Narayanan and Keedwell (286), which analysed published microarray data using artificial neural networks. Other genes had been identified through an extensive literature search or as a result of previous work in the department (287, 288).
The ABI manufacturing control 18S ribosomal RNA (18S) was also included on the array in addition to four other housekeeping genes that had been documented as showing stable expression in prostate cancer tissues between different stages and grades (289): hydroxymethylbilane synthase (HMBS), TATA-box binding protein (TBP), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and succinate dehydrogenase complex flavoprotein subunit A (SDHA).
The new array (BB array)(Figure 2.2) included the same genes (including housekeeping genes) as the SL array except for the following changes:
Ten genes were removed, that had been found to show p-values of >0.5 in the independent samples t-tests (for the comparison of the GS ≥7 and <7 group and/or recurrent and non-recurrent group) in the SL study (275). As a p-value of >0.5 suggested that these genes were unlikely to be useful in LR models, they were excluded from the array to provide space for other genes of interest. The excluded genes were CDH2, CTNNA1, HSPA5, KCNN4, MMP9, RBM12, SFPQ, TGM2, HSP90B1 and WIF1.
The genes ERBB2, C3, HPX, MKI67, MT1X, MUC1, PLAU, PTEN and TF were selected for the BB array as replacements for these genes. On theoretical grounds, ERBB2, MK167 MUC1, PLAU and PTEN were highlighted as putative prostate cancer progression markers following a review of current literature.
For instance, MUC1 encodes a transmembrane glycoprotein, which has been strongly associated with clinicopathological parameters of aggressive disease and poor prognosis in several types of cancer including prostate cancer (290-292). As MUC1 is an anti- adhesion molecule, it has been proposed that it may enhance the invasive and metastatic potential of cancer cells (292), for example by reducing cadherin-1 and
47
integrin-mediated cell adhesion (293, 294). In prostate cancer, studies have found MUC1 mRNA and protein overexpression to correlate with aggressive clinicopathological features and poor prognosis (290, 291).
The remainder of the novel genes on the new array (C3, HPX, MT1X and TF) were highlighted as genes of interest based on previous work in our department. For example, hemopexin (HPX) was identified as a putative marker of high grade prostate cancer (GS ≥7) in a serum proteome study (295). A further tissue microarray study showed significant differences in hemopexin expression across benign, low grade (<7), high grade (≥7) and metastatic tissue (295). Hemopexin is a serum glycoprotein which transports heme from the bloodstream to the liver for metabolism and iron recovery (296). Despite the fact that the role of hemopexin in cancer is not fully elucidated, numerous proteomic profiling studies using serum/plasma samples or other body fluids have identified hemopexin as being significantly up- or downregulated in various types of cancer including prostate cancer (297, 298).
The full names of each gene incorporated on the SL and BB array can be found in Appendix 2, where they have been grouped according to their putative roles in cancer initiation/progression.
The proportion of the total number of genes on the BB array within each category are illustrated in Figure 2.3.
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Figure 2.1 Genes included on the SL array and their putative roles in cancer initiation/progression
Samples
ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDH2* CDKN1C CHM CRHR1 CTNNA1* A
CYP11A1 DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 B
HSPA5* HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB6 KCNN4* KLHL5 M M P2 M M P9* M NX1 M YC* NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC PLCL2 PLLP C
POP5 PRIM 2 PSCA PTGFR PVALB PVT1 RAP1GAP RBM 12* RHBDL1 SDHA SERPINB5 SFPQ* SLC44A2 PRELID3A TBP TBRG4 TERT TGFB2 TGM 2* HSP90B1* TRAFD1 TRIP13 VASH1 WIF1* D
ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDH2 * CDKN1C CHM CRHR1 CTNNA1 * E CYP11A1 DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 F HSPA5 * HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB6 KCNN4 * KLHL5 M M P2 M M P9 * M NX1 M YC * NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC PLCL2 PLLP G POP5 PRIM 2 PSCA PTGFR PVALB PVT1 RAP1GAP RBM 12 * RHBDL1 SDHA SERPINB5 SFPQ * SLC44A2 PRELID3A TBP TBRG4 TERT TGFB2 TGM 2 * TRA1 * TRAFD1 TRIP13 VASH1 WIF1 * H ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDH2 * CDKN1C CHM CRHR1 CTNNA1 * I CYP11A1 DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 J HSPA5 * HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB6 KCNN4 * KLHL5 M M P2 M M P9 * M NX1 M YC * NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC PLCL2 PLLP K POP5 PRIM 2 PSCA PTGFR PVALB PVT1 RAP1GAP RBM 12 * RHBDL1 SDHA SERPINB5 SFPQ * SLC44A2 PRELID3A TBP TBRG4 TERT TGFB2 TGM 2 * TRA1 * TRAFD1 TRIP13 VASH1 WIF1 * L ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDH2 * CDKN1C CHM CRHR1 CTNNA1 * M CYP11A1 DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 N HSPA5 * HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB6 KCNN4 * KLHL5 M M P2 M M P9 * M NX1 M YC * NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC PLCL2 PLLP O POP5 PRIM 2 PSCA PTGFR PVALB PVT1 RAP1GAP RBM 12 * RHBDL1 SDHA SERPINB5 SFPQ * SLC44A2 PRELID3A TBP TBRG4 TERT TGFB2 TGM 2 * TRA1 * TRAFD1 TRIP13 VASH1 WIF1 * P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 3 5 6 4 7 8 Port 1 1 2 2 3 4 Oncogenes
Tumour suppressor genes Apoptosis related Angiogenic
Invasion and metastasis related Metabolic
Differentiation and proliferation Detox
Others
Housekeeping genes
49
Figure 2.2 Genes included on the BB array and their putative roles in cancer initiation/progression
Samples
ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL C3* CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDKN1C CHM CRHR1 CYP11A1 A
DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERBB2* ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 B
HPX* HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB4 ITGB6 ITGB6 KLHL5 M KI67* M M P2 M NX1 M T1X* M UC1* NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC C
PLAU* PLCL2 PLLP POP5 PRIM 2 PSCA PTEN* PTGFR PVALB PVT1 RAP1GAP RHBDL1 SDHA SERPINB5 SLC44A2 PRELID3A TBP TBRG4 TERT TF* TGFB2 TRAFD1 TRIP13 VASH1 D
ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL C3 CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDKN1C CHM CRHR1 CYP11A1 E
DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERBB2 ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 F
HPX HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB4 ITGB6 ITGB6 KLHL5 M KI67 M M P2 M NX1 M T1X M UC1 NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC G
PLAU PLCL2 PLLP POP5 PRIM 2 PSCA PTEN PTGFR PVALB PVT1 RAP1GAP RHBDL1 SDHA SERPINB5 SLC44A2 PRELID3A TBP TBRG4 TERT TF TGFB2 TRAFD1 TRIP13 VASH1 H
ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL C3 CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDKN1C CHM CRHR1 CYP11A1 I
DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERBB2 ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 J
HPX HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB4 ITGB6 ITGB6 KLHL5 M KI67 M M P2 M NX1 M T1X M UC1 NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC K
PLAU PLCL2 PLLP POP5 PRIM 2 PSCA PTEN PTGFR PVALB PVT1 RAP1GAP RHBDL1 SDHA SERPINB5 SLC44A2 PRELID3A TBP TBRG4 TERT TF TGFB2 TRAFD1 TRIP13 VASH1 L
ABL1 ALCAM ALPG AM ACR ANPEP ANXA2 APOBEC3G ASB2 ATP10B BRCA2 18S ATP5M PL C3 CANX CD109 CD4 CD44 CD9 CDC42EP3 CDH1 CDKN1C CHM CRHR1 CYP11A1 M
DPT EDNRA EEF1A1 EFNA1 EFNA2 EFNA3 EFNA4 EFNA5 ERBB2 ERG EZH2 F11R FABP5 FADS1 GPM 6A GPR12 GPR19 GSTM 3 GSTM 5 GUCY1A1 HM BS HOXC6 HPN HPRT1 N
HPX HSPB1 INM T IRF7 ITGA2 ITGA3 ITGA5 ITGB1 ITGB4 ITGB4 ITGB6 ITGB6 KLHL5 M KI67 M M P2 M NX1 M T1X M UC1 NDUFAF1 NELL2 PNP NUP50 PCDH18 PGC O
PLAU PLCL2 PLLP POP5 PRIM 2 PSCA PTEN PTGFR PVALB PVT1 RAP1GAP RHBDL1 SDHA SERPINB5 SLC44A2 PRELID3A TBP TBRG4 TERT TF TGFB2 TRAFD1 TRIP13 VASH1 P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 Port 6 8 7 1 2 3 4 5 Oncogenes
Tumour suppressor genes Apoptosis related Angiogenic
Invasion and metastasis related Metabolic
Differentiation and proliferation Detox
Others
Housekeeping genes
50
Figure 2.3 Genes on the BB array, categorised according to their roles in cancer initiation/progression