Spreadsheet: 15c9e399-e406-42e4-99e7-10bd6bced00b
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Supplementary Table 5: HAND2 Methylation (Illumina methyl27 array data) in Set 1 (Bergen Set) and detailed associated molecular characteristics of the individual tumours. | Supplementary table S5: HAND2 Sequencing ([NAME] sequencing) The 5’ untranslated region up to intron 1 of HAND2 (NM_021973.2) was analysed for genetic variants using an ABI 3730xl DNA analyzer (Applied Biosystems).  Sequencing was performed by GATC Biotech and included gDNA from 17 normal samples and 55 endometrial cancer cases (nd, not done due to lack of DNA).  PCR amplification of a region that included the promoter region, transcription start site (TSS), translation start site (TLS) and exon 1 was performed in 3 PCR reactions using KAPA2G Robust HotStart ReadyMix (KK5701) PCR kit.  Primers were designed to amplify the region starting at nucleotide 7 (+1 TSS) to 584 bp into intron 1 (+2071 bp downstream of TSS; see [NAME]. 1E).  Genomic co-ordinates for the region amplified were chr4: 174,451,372 to chr4: 174,449,301. Primer3 was used to design primers (http://frodo.wi.mit.edu).  Primer sequences can be found in supplementary information.  [NAME] detection in sequencing traces was performed using [NAME] DNA Variant Analysis Software. Primer sequences as follows: Amplicon 1 Forward ATGGAGATCTTGCTGGGAAA, [NAME] (55°C), Amplicon 2 Forward GGCTTGGATTCCCGAGAC, [NAME] (55°C), Amplicon 3 Forward CTGTCCTACAGCCCCGAGTA, Reverse CATAGCCAAGGAAAAATTGGA (52°C),  HGVS (Human Genome Variation Society), MS (Mutation surveyor). 0, variant absent; 1, variant present. | Unnamed: 2 | Unnamed: 3 | Unnamed: 4 | Unnamed: 5 | Unnamed: 6 | Unnamed: 7 | Unnamed: 8
Tissue microarray sections (5 µm) of formalin fixed paraffin embedded tissue were stained for Estrogen Receptor α (ERα), Progesterone Receptor (PR), p53 and ARID1A, as previously published (Engelsen IB et al, AJOG 2008; [NAME] et al, PNAS 2009; [NAME] et al, [NAME] 2012 in press). Slides were incubated with antibodies against ER α ([NAME] M7047), PR (Dako M3569), p53 (Dako M7001) and ARID1A (Biosite AT1188a). A staining index was calculated assessing the product of staining intensity (0-3) and area (0 = no staining, 1 = <10%, 2 = 10-50% and 3 = > 50%), as described in previous publications (Engelsen IB et al, AJOG 2008; [NAME] et al, PNAS 2009). Negative ARID1A expression was defined if no tumor cells were stained (score index=0), although positive stromal staining was present in the same sample. For ERα and PR, low expression corresponded to score indices 0. High p53 expression corresponded to score indices > 4. | Sample | Amplicon 1 |  | Amplicon 2 |  | Amplicon 3
DNA ploidy was assessed by flow-cytometric method in fresh, ethanol-fixed tumor tissue as previously described (Wik E et al, AJOG 2009). In cases of 2 identified cell populations, tumors with DNA index > 1.0 were defined as aneuploid. |  | Variant 1 | [NAME] | Variant 1 | [NAME] | Variant 1 | [NAME] | [NAME]
PTEN was sequenced in genomic DNA, as previously described (Dutt A et al, PNAS 2008; [NAME] et al, PNAS 2009). |  | 356het_delG (MS);        NM_021973.2:c.-582delG (HGVS) | 575dupA (MS);                 NM_021973.2:c.-363dupA (HGVS) | 932G>GT (MS);      NM_021973.2:c.-6G>T (HGVS) | 992het_delC (MS);          NM_021973.2:c.54delC (HGVS) | 1252dupA (MS); NM_021973.2:c.314dupA (HGVS) | 1517G>GA (MS);          NM_021973.2:c.579G>A (HGVS) | 1597delG (MS);               NM_021973.2:c.659delG (HGVS)
Other mutations were detected in genome-amplified DNA using a mass-spectrometry based single base extension technique (Sequenom, Inc.) as previously described (Thomas RK et al, Nat Genet 2007). A set of 373 mutations in 26 (THERE ARE 28 IN THE ATTACHED SUPPL INFO) oncogenes ([NAME]) was investigated in 56 primary lesions. | Tumour | 0 | 0 | nd | nd | 0 | 0 | 0
5 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
10 | Tumour | 0 | 0 | 1 | 0 | nd | nd | nd
14 | Tumour | 0 | 0 | 1 | 0 | 0 | 0 | 0
30 | Tumour | 0 | 0 | 1 | 0 | 0 | 0 | 0
31 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
32 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
37 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
38 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
40 | Tumour | 0 | 0 | 0 | 1 | 0 | 0 | 0
41 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
45 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
51 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
63 | Tumour | 0 | 0 | 0 | 0 | 1 | 0 | 0
75 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
78 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
83 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
87 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
95 | Tumour | 0 | 0 | 1 | 0 | 0 | 0 | 0
96 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
101 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
111 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
113 | Tumour | 0 | 0 | 0 | 0 | 0 | 1 | 1
128 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
129 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
138 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
141 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
161 | Tumour | 0 | 0 | 1 | 0 | 0 | 0 | 0
162 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
172 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
177 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
201 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
202 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
206 | Tumour | 1 | 0 | 0 | 0 | nd | nd | nd
213 | Tumour | 0 | 0 | nd | nd | 0 | 0 | 0
225 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
234 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
235 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
244 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
247 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
256 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
272 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
293 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
306 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
325 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
328 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
334 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
367 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
394 | Tumour | 0 | 1 | 0 | 0 | nd | nd | nd
405 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
436 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
441 | Tumour | 0 | 0 | 0 | 0 | nd | nd | nd
443 | Tumour | 0 | 0 | 0 | 0 | 0 | 0 | 0
445 | Tumour | 0 | 0 | nd | nd | 0 | 0 | 0
456 | Tumour | 0 | 0 | nd | nd | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | 0 | 0 | 0
 | Normal | 0 | 0 | 0 | 0 | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | 0 | 0 | 0
 | Normal | 0 | 1 | 0 | 0 | nd | nd | nd
 | Normal | 0 | 0 | nd | nd | nd | nd | nd
 | Normal | 0 | 0 | nd | nd | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | nd | nd | nd
 | Normal | 0 | 0 | nd | nd | nd | nd | nd
 | Normal | 0 | 0 | nd | nd | 0 | 0 | 0
 | Normal | 0 | 0 | nd | nd | nd | nd | nd
 | Normal | 0 | 0 | 0 | 0 | 0 | 0 | 0
 | Normal | 0 | 0 | 0 | 0 | 0 | 0 | 0
 | Normal | 0 | 0 | 0 | 0 | 0 | 0 | 0