Gastroesophageal junction adenocarcinoma displays abnormalities in homologous recombination and nucleotide excision repair

Figures & data

Table 1 Characteristics of the patients who were included in the analysis

Patient	Age (years)	TNM stage	Grade
1	74	T3N0	Poorly differentiated
2	83	T3N1	Moderately differentiated
3	79	T3N1	Poorly differentiated
4	63	T3N1	Poorly differentiated
5	71	T2N1	Poorly differentiated
6 (female)	48	T1N0	Poorly differentiated
7	65	Unavailable	Unavailable
8	77	T3N0	Poorly differentiated
9	73	T3N1	Moderately differentiated
10	67	T4N1	Moderately differentiated
11	80	T3N0	Poorly differentiated
12	75	T3N1	Poorly differentiated

Abbreviation: TNM, tumor note metastasis.

Table 2 Summary of DNA repair genes analyzed with their average fold changes and P-values

Pathway and gene symbol	Assay	Average fold change	P-value	Number of tumors with alterations
Nucleotide excision repair
XPA	Hs00166045_m1	0.48	0.0004*	12↓
DDB1	Hs00172410_m1	0.92	0.207	5↓
DDB2	Hs03044953_m1	0.75	0.0435*	8↓
ERCC1	Hs01012158_m1	0.76	0.0644	9↓
ERCC2	Hs00361161_m1	1.15	0.162	2↓
ERCC3	Hs01554450_m1	1.08	0.478	1↓
XPF	Hs00193342_m1	0.710	0.004*	7↓
XPG	Hs00164482_m1	0.715	0.039*	10↓
XPC	Hs01104206_m1	0.633	0.009*	9↓
RPA1	Hs00161419_m1	0.900	0.234	6↓
RPA2	Hs00358315_m1	0.996	0.535	5↓
Homologous recombination
RAD51	Hs00153418_m1	3.039	0.073	5↑
RAD52	Hs00172536_m1	1.218	0.254	2↑
BRCA1	Hs01556193_m1	1.867	0.187	5↑
BRCA2	Hs00609073_m1	1.867	0.137	5↑
EME1	Hs00385890_m1	2.971	0.007*	6↑
MUS81	Hs00228383_m1	0.833	0.457	4↓
Nonhomologous end-joining
Artemis	Hs01052780_m1	0.98	0.127	6↓
DNAPK	Hs01016093_m1	1.49	0.485	2↓
XRCC5	Hs00221707_m1	1.00	0.602	5↓
XRCC6	Hs01922652_g1	0.86	0.267	4↓
ATM	Hs01112307_m1	0.98	0.467	5↓
ATR	Hs00354807_m1	1.37	0.652	2↓
Ligase IV	Hs00172455_m1	1.28	0.678	3↓

Note:

* Statistically significant genes, P#0.05.

Abbreviation: DNA, deoxyribonucleic acid.

Figure 1 Quantitative reverse transcription polymerase chain reaction analyses of messenger (m)RNA expression changes of homologous recombination genes. Data shown are the average of two separate quantitative reverse transcription polymerase chain reaction assays. Genes of interest were normalized to GUSB transcript levels and are presented as fold changes relative to normal matched samples. EME1 transcripts are increased in EAC samples.

Notes: *Statistically significant results with P-value ≤ 0.05.
Abbreviations: SEM, standard error of the mean; EAC, esophageal adenocarcinoma.

Figure 2 EME1 messenger (m)RNA expression in individual esophageal adenocarcinoma samples, showing three tumor samples display more than fourfold overexpression of EME1.

Note: Dotted line refers to fold change of 1.

Figure 3 EME1 immunohistochemistry in gastroesophageal junction adenocarcinoma. (A) Well-differentiated gastroesophageal junction adenocarcinoma shows uniform strong nuclear staining pattern with EME1 (250×). (B) EME1 staining intensity was noticeably less in the less differentiated areas (500×). Note the nuclear immunoreactivity for EME1 in a control of benign endometrial stroma (inset).

Table 3 Summary of Oncomine database search results of tumors with increased EME1 expression

Data set and cancer type	Number of samples	EME1 expression fold change	P-value
French brain
Anaplastic oligoastrocytoma	33	2.615	8.5E–5
Oligodendroglioma	33	2.3	8.24E–7
Murat brain
Glioblastoma	84	2.968	1.31E–7
Richardson breast 2
Ductal carcinoma	47	2.770	2.62E–9
TCGA breast
Invasive ductal carcinoma	47	2.165	2.08E–27
Invasive lobular carcinoma	47	2.071	1.71E–8
Pyeon multicarcinoma
Tonsillar carcinoma	84	2.162	4.28E–5
Ye head-neck
Tongue squamous cell carcinoma	38	2.172	3.02E–6
TCGA renal
Papillary renal cell carcinoma	88	2.380	3.06E–7
Compagno lymphoma
Diffuse large B cell lymphoma	136	2.459	1.50E–14

Note: Data available from: http://www.oncomine.org.³⁴

Abbreviation: TCGA, the cancer genome atlas.

Figure S1 Taqman endogenous control assay used to determine the optimal housekeeping gene. X axis shows samples used, Y axis showed Ct values of each gene.

Figure S2 Western blot analysis evaluating EME1 primary antibody in HeLa, WI-38, and SK-GT4 cell extracts. One major band can be seen at expected size of 65 kDa in both HeLa and SK-GT4 cell extracts. The band is present but much weaker in the WI-38 cell line.

Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Table S1 Summary of messenger RNA expression of homologous recombination genes in three tumor samples and their biological duplicates

Gene and tumor number	Average fold change	Biological replicate	Average fold change
EME1
4	4.5	T4 replicate	5.7
3	5.9	T3 replicate	3.3
2	1.9	T2 replicate	0.9
RAD51
4	7.6	T4 replicate	12.8
3	1.5	T3 replicate	1.5
2	0.5	T2 replicate	0.5
RAD52
4	0.7	T4 replicate	0.5
3	2.3	T3 replicate	2.1
2	0.4	T2 replicate	0.4
BRCA1
4	3.1	T4 replicate	1.5
3	5.2	T3 replicate	2.9
2	0.7	T2 replicate	0.5
BRCA2
4	4.2	T4 replicate	5.4
3	3.7	T3 replicate	1.7
2	0.8	T2 replicate	0.8
MUS81
4	0.8	T4 replicate	0.4
3	1.0	T3 replicate	1.0
2	0.3	T2 replicate	0.1

Table S2 Summary of results of EME1 messenger (m)RNA and EME1 protein expression in each of the individual esophageal adenocarcinoma samples

Sample number	EME1 immunohistochemistry score	EME1 mRNA fold change
1	2+2+	1.8
2	2+2+	1.9
3	2+3+	5.9
4	2+2+	4.5
5	Block not available	1.4
6	Block not available	1.1
7	3+3+	2.3
8	Block not available	2.5
9	2+3+	2.6
10	3+3+	1.1
11	3+2+	1.8
12	2+3+	8.2

Life Technologies (webpage on the Internet)Oncomine™ Research Edition2013 Available from: https://www.oncomine.org/resource/login.htmlAccessed February 2, 2014

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